OXYGEN-OZONE THERAPY

A CRITICAL EVALUATION
 OXYGEN-OZONETHERAPY
A Critical Evaluation

by

VELIOBOCCI
Medical Doctor; Specialist in Respiratory Diseases and Haematology,
Professor of General Physiology at the University of Siena,
Siena, ltaly

Springer-Science+Business Media, B.V.

A C.I.P. Catalogue record for this book is available from the Library of Congress.

Printed Oll acid-free paper

1-0103-100Is
First published 2002

All Rights Reserved

ISBN 978-90-481-6008-2 ISBN 978-94-015-9952-8 (eBook)

DOI 10.1007/978-94-015-9952-8

© 2002 SpringerScience+Business Media Dordrecht

Originallypublishedby KluwerAcademicPublishersin 2002.
Softcoverreprint ofthe hardcover 1st edition 2002

No part of this work may be reproduced, stored in a retrieval system, or transmitted

in any form or by any means, electronic, mechanical, photocopying, microfilming, recording
or otherwise, without written permission from the Publisher, with the exception
of any material supplied specifically for the purpose of being entered
and executed on a computer system , for exclusive use by the purchaser of the work.
This book is dedicated to all patients with the hope
that oxygen-ozone therapy will benefit them .

Homines dum docent, discunt

(Humans leam as they teach)
Seneca (5 B.C. - 65 A.D), Epist., 7,8.
 COLLABORATORS

B . BIAGIOLI MD
Institute ofThoraeie Surgery, Cardiovascular and Biomedical Technologies
University ofSiena

E. BORRELLI MD
Institute ofThoracic Surgery, Cardiovascular and Biomedical Technologies
University ofSiena

A. DIADORI
Department of Ophthalmology, University of Siena

vi
 PREFACE

When I was about fifteen , my Biological Seiences teacher, Prof. N. Benacchio, lent
me a book by Paul de Kruif "The Microbe Hunters" and I remained fascinated by
infectious diseases. I was intrigued by the potency of virulent bacteria which are
constantly trying to invade our bodies and often overcome what today we call innate
and adoptive immunity. Indeed, shortly after that, I was struck by his tragic death
due to peritonitis. Later, while studying medicine (although medical knowledge in
the 1950s was almost primordial compared with today), I soon realised how the
various biological systems were wonderfully organised but at the same time frail and
how our life could end in a few minutes. Slowly it became obvious that our
"wellness" was the result of a dynamic and very unstable equilibrium between
health and disease . This unstable equilibrium could be broken forever if the body's
response could not reverse the pathological state . I stuck a sort of poster on the wall
of my room with these three words and connecting arrows :

HEALTH ~-? DISEASE -? DEATH

As I don 't believe in another world after death, it became obvious to me that we
should make every possible effort not only to delay death, but to try always to shift
the equilibrium to the left. In this book, I will try to show that this can be achieved,
as a last resort, even with ozonetherapy.
The progressive prolongation of the human life-span in the last fifty years is the
best evidence of the immense progress of biology and orthodox medicine. I have
been extremely lucky to work and contribute, albeit minimally , during this period .
Particularly after the 1990s, with the advent of molecular biology , the pace of
progress is so fast it is becoming more and more difficult to grasp the final practical
applications. It is actually disturbing that after the almost daily discovery of another
gene, one can jump to the conclusion that another disease can be cured . This is
practically unrealistic and unfair because diseases are not necessarily linked to a
single cause but rather to a number of factors . Moreover, it eventually disappoints
the hopeful patient. This remark is not intended to diminish the validity of any
discovery but simply to caution that there is always a great delay between the
observation at the laboratory bench and the therapeutic act at the patient's bed. This
exuberant enthusiasm is, however, comprehensible and, with even less justification,
also occurs in the field of natural medicine where progress has been painfully slow.
The bio-oxidative therapies, including oxygen-ozone therapy , are one of the least
known of the many branches of natural medicine, in comparison with acupuncture,
homeopathy and phytotherapy. Owing to a growing world-wide interest in natural
medicine , it is always correct to warn patients against the risk of the placebo effect
or worse of plain damage , as occurred in orthodox medicine with regard to
thalidomide and fenfluramine.

vii
viii

About a decade ago, I knew nothing about ozone except that I had heard about
the "ozone hole". From my chemistry course I remembered that it was a strong
oxidant gas but I did not know that ozone had been used since World War I as a
therapeutic agent against gaseous gangrene caused by anaerobic Clostridium. It was
by mere coincidence that, at a meeting on blood substitutes, a colleague asked me
why ozonization of blood ex vivo followed by reinfusion into the donor could be
useful for treating chronic viral hepatitis. It was also coincidental that this
conversation occurred when I was trying to se1ect an inducer of interferon (JFN) so
that we could obtain at will the release of endogenous IFN and possibly other
cytokines. We had examined hundreds of potential indueers but it proved very
difficult to find a eompound that would be effective, atoxic and possibly
inexpensive. Owing to the finding of my friend Ferdinando Dianzani, I remembered
that oxidising agents, sueh as periodate, galactose oxidase, etc., added to human
blood mononuclear cells (BMC) in vitro eould induee the release of huge amounts
of IFN y, particularly in the presence of a Ca 2+ ionophore. Thus, it was easy to
imagine that I) exposing blood to ozone eould aetivate immune cells, 2) after blood
reinfusion, BMC would horne in their mieroenvironments and start to release
eytokines, and 3) in a paraerine fashion, the cytokines eould switeh on a self-
arnplifying meehanism of immune aetivation. The idea was even more attraetive
beeause sinee 1981 (and against eurrent wisdom) I had proposed the existenee of the
physiologieal IFN response, i.e. the eoneept that IFN and other eytokines were
always produeed in traee amounts even during "health", thus priming leukocytes and
keeping the immune system in astate of eontinuous alert or "surveillance" against
pathogens.
The idea seemed so stimulating that I decided to explore it experimentally, even
though I realised that , for the few physicians who knew of its existenee,
ozonetherapy was in the eentre of a thick cloud of seepticism and ineredulity .
However, in Science, prejudiees ean save time, but they can also be detrimental. So I
started my relationship with ozone and I soon realised that sinee the medieal world
was very much eoneemed with the cellular formation of free radieals, the medieal
application of ozone would be strongly rejeeted. The oxygen (0 2 ) level in the
atmosphere reaehed the current levels of 2 1% about two billion years aga and all
living beings, in order to profitably use O 2 and to survive, have had to develop
numerous detoxifying meehanisms. However, in spite of these mechanisms, we all
suffer, more or less rapidly, the eonsequenees of "oxidative stress". There is no
doubt that many seemingly unrelated diseases are due to an exeessive produetion of
free radicals, and ozone is a master in generating them. This ereates a formidable
problem and offers a good reason not to use ozone in medieine. Indeed its toxicity
for plants and humans, partieularly for the respiratory traet in large eities, is very
weil known. This problem has dominated my mind for several years . Although more
reeently I have eome to realise that ozone toxieity ean be tamed, in this book I wish
to leave the question open so that the reader will deeide for his or herself on the
basis of several positive and negative arguments.
It took some time before we were able to handle ozone eorreetly. Firstly we tried
to understand how ozone works and how toxie it ean be. Then, in eollaboration with
a few c1inieians, we started some medieal investigations, although this proved to be
 ix

very frustrating, as most colleagues refuse to collaborate. However, I have been able
to witness incredible results particularly in patients with awful ulcers due to hind-
limb ischaemia. Unfortunately most of the c1inical data, although encouraging, are
based on anecdotes that may be exaggerated by the enthusiasm of the physician.
Today I can say that we have at least some ideas about the mechanisms of action of
ozone . Yet the Achille's heel remains the lack of controlled, double-blind c1inical
studies. For this reason, I feel that under any circumstance we must first take
advantage of conventional medicine and only when this fails or the patient refuses it,
can we seriously think about what ozonetherapy can do. Surprisingly, in spite ofthe
enormous progress of modern medicine, this occurs more frequently than one might
imagine .
A reasonable question is whether there is areal reason to write this book in
English . In 1999, I wrote a similar book in Italian because a few serious
ozonetherapists asked me to do so: no other book was available except an English
translation of abrief compendium written by Renate Viebahn and an overly
enthusiastic book entitled: "Oxygen Healing Therapies" written by the journalist
Nathaniel Altman . I am an admirer ofthe conciseness and elegance of good English
and, in spite of my best effort, I must apologize for my poor style. Yet my hope is to
provide a scientific and truly objective account of ozonetherapy for all English-
speaking physicians.
Although ozonetherapy was invented and has been practised in Germany during
the last fifty years, very little scientific work has been produced because it has been
mainly a private medical activity . Similarly, in Italy during the last eighteen years,
more importance has been given to the use of ozone in cosmetics and in creating a
profitable business selling instruments and accessories. I deplore the fact that the
mercantile aspect prevails over scientific endeavours and I have over-emphasised
that OUf new International Medical Ozone Society (IMOS) has no commercial
interest and strongly opposes the monopoly of a single ozone generator. We hope
that, in spite of economic difficulties, the enthusiasm and efforts of OUf members
will permit scientific progress.
The purpose of the book is to give the physician a practical idea about how
ozone works when comes into contact with body fluids and cells and how it can
elicit toxic effects if one disregards its powerful oxidising properties. It is not meant
to give simple medical recipes but rather to create a "forma mentis", i.e. the ability
to understand and adjust the therapy to the patient 's need. The book is also meant to
give an objective assessment of what is known and what we should try to learn
about ozonetherapy. Moreover many anguished people who daily ask me questions
or solicit advice via Internet may find satisfactory answers in this book .
A very ambitious goal is to provide sceptics and opponents with an objective
basis to discuss whether ozonetherapy deserves to be evaluated or disregarded. I
would be very grateful to anyone willing to open a dialogue or a collaborative effort.
Only after controlled clinical trials performed in various institutions will we be able
to decide whether ozonetherapy is really valid or worthless, as was demonstrated for
Laetrile and other drugs in the recent past.
Obviously I tried to do my best but the reader will find faults in this book. One
may be the repetition that ozonetherapv induces only a brief and calculated
x

oxidative stress not to be confused with the life-Iong endogenaus stress. I apologize
but I said this on purpose in almost every chapter because it is a crucial remark and
will help dispel the idea that "ozone is toxic any way you deal with it"
This book will suceeed if it is able to promote the e1ucidation of the issue : is
ozonetherapy therapeutic? I feel that the Medieal Establishment must take this
responsability because it would be deplorable to further neglect a possible treatment
for millions ofpatients, partieularly in poor eountries.
Velio Bocci
 ACKNOWLEDGEMENTS
It has taken almost two years to write and have this book ready for publieation.
However, without the help of several friends, it would have taken twenty years. I am
very grateful to Dr. Emma Borrelli and Dr. Julian Blaneo Gareia for typewriting the
manuseript and to Dr. Peter W. Christie, B.Se., M.Se., Ph.D., for the linguistic
revision . Mrs. Patrizia Marroeehesi, with her usual zeal and skilI, has taken eare of
the extensive bibliography. All ozonetherapists should be grateful to her for having
now a souree of information that was badly needed . Moreover she deserves the full
merit for having prepared the manuseript for printing . Dr. Carlo Aldinueei has been
very helpful during the preparation of ieonography. I would like to express my
gratitude to many eollaborators that, throughout the years, have worked with me in
studying the biologieal effeets of ozone .
I thank all Authors and Publishers for kindly allowing the publieation of their
data and diagrams . I gratefully aeknowledge the eneouragement and support given
by Mr. Colin Tongs, President of the Medical Seienees Teehnology Corporation,
UK, who aims to expand the use of ozonetherapy all over the world. A grant from
the Bank Monte dei Pasehi di Siena has partly helped us to develop the EBOO
methodology.
I am grateful to Mr. Peter Butler, Manager of the Biomedieal Unit of Kluwer
Academic Publishers for the enthusiastie support ofthe projeet.
Last but not least, I am deeply grateful to my wife Helen and to my children
Erica and Robert who, for many years, have been exeeedingly tolerant and patient
with me and my work.

xi
 FOREWORD

I have known Prof. Velio Bocc i for the last forty years and for some time, even if
pursuing different aims, we shared the research .on interferons. At that time, he
produced major contributions in a rather neglected area such as the study of
metaboIism and pharmacokinetics of these proteins. During the last decade his
interest shifted mainly to the analysis of the biological and clinical effects of ozone.
Although this seems a rather unusual subject, he once told me that his interest was
aroused by a fortuitous observation he connected to one of our previous discovery,
that interferon gamma eould be induced by some oxidants, particularly galactose-
oxidase. From this hint , with his eollaborators, he has clarified that ozone dissolves
in biological fluids and generates reactive oxygen speeies (ROS) of which hydrogen
peroxide is one of the most important. At the same time, other scientists have
clarified that hydrogen peroxide is a erueial physiological messenger and Velio has
been able to show how, indireetly, ozone ean elieit a variety of biologieal aetivities,
such as the induetion of eytokines, hormonal effects and the aetivation of
metabolism either by faeilitating oxygen transport and delivery or/and biochemical
pathways. Interestingly ROS activate also platelets and endothelial cells leading to a
tremendous amplification of effeets. Another stimulating and apparently paradoxieal
development is that ozone, one of the most potent oxidant, if properly used , can
induce the adaptation to a chronic oxidative stress , a phenomenon not really new ,
because we know that any stressful agent ean either kill an organism or allow the
induction of resistanee. The fact is praetically important because today this is a
possible way to reverse, or stabilise a chronie imbalanee between an excess of
oxidants and adefeet of antioxidants.
In the 90s ozonetherapy was, among several eomplementary approaches, still
immersed in a fog of magic but, thanks to these researches, it has now become
amenable to a truly scientific investigation. This is the only way to proceed if one
wants to validate a medical approach. Velio correctly says we cannot forget that
ozone is intrinsically toxic and, consequently, must be used with great precision and
care . He now has come to consider ozone as areal drug with all its inherent
advantages and disadvantages. Another surprising aspect is the one presented by the
therapeutic use of ozone in medicine, where the dominant dogma is that many
pathologies, if not generated, are maintained by an excessive production of free
radicals. Thus, as Velio admits, the idea of using ozone seems a crazy one and
indeed, most of the medical world is very much against it. However he maintains
that this dominant concept is born out either by a plain prejudice from lack of
knowledge, or by not recognising that ozone induces only-an exogenous, transitory
and calculated oxidative stress that represents an important way to counteract the
endogenous chronic oxidative stress. If he is correct, .this new idea may indeed help
to explain why ozonotherapy appears therapeutically useful. Moreover, the concept
that ozone is not a panacea for a11 illnesses has been well clarified and it is due to the
acti vation of several mechanisms relevant in disparate diseases.

XIII
xiv

I must say I appreciate that Velio, throughout the book, strives to be very
objective and critical. Although he is c1early in love with this story, he does not
spare his harsh criticisms on the many problems plaguing ozonetherapy, as he
believes that only by following a scientific approach, ozonetherapy may have the
chance to enter mainstream medicine. Indeed , so far, although medical results
appear promising and , in some cases, almost too good to be true, there is the
absolute need to perform randomised c1inical trials in appropriate institutions.
In conclusion, Velio's book, in my opinion, represents a serious attempt to
understand the fundamental basis of ozonetherapy and is a relevant step for
achieving further progress. Velio has already written a similar book for Italian
ozonetherapists but this is a completely new version with deepened scientific and
medieal backgrounds: as such it can be useful to all ozonetherapists, to physicians
and scientists that may be interested to know how ozone truly acts . There are also
some autobiographieal annotations that show Velio's personal involvement with this
problem and some ofhis disputes with other scientists. The present evidence, albeit
imperfect, may be helpful to advance this approach and hopefully to be useful to
many patients.

Rome, October 2001

Professor Ferdinando Dianzani
Professor of Virology and Dean of
Medical University "Campus Bio-Medico"
Rorne, Italy
 TABLE OF CONTENTS

CHAPTER 1 ABRIEF HISTORICAL REVIEW .. .. .. . . . . . . ..... ... .. .. ......... PAG .

CHAPTER 2 How I CAME TO STUDY OZONETHERAPY: AN ODYSSEY
WITH NO END IN SIGHT......... .. .. ........ .. .. .. .. .... .. ...... 7
CHAPTER 3 OZONE IN NATURE . . . . . . . . . . . . . . . ... . .. .. . . . ... . . . . . .. . ........ 31
CHAPTER 4 PHYSICO-CHEMICAL PROPERTIES OF OZONE . . .. .. ... ... . ... 37
CHAPTER 5 OZONE TOXICITY: BIOLOGICAL RISK, TOXICOLOGY AND
FIRSTAID . ....... . .. .. .... . .. . ... .. . .. .... ..... . .. . .. . .. ... .. .. . 41
CHAPTER 6 THE OZONE GENERA.TOR. .. . .. .. . . . . .. .. .. .. . .. ... . . . ... . .. . ... 43
CHAPTER 7 How TO CHECK OZONE CONCENTRATIONS . . .... . .. .... .... 47
CH .\PTER 8 SOLUBILITY OF OZONE IN WATER AND PREPARATION OF
OZONIZED WATER AND OlL. .. . . .. . . . . . .. . . . . . . . . . . ... . .. . . .. . " 51
CHAPTER 9 PRELIMINARY BASIS FOR UNDERSTANDING OZONE
REACTIvlTY AND THE POTENTIAL RISKS OF
OZONETHERAPY . . . . . . .. ... . .. . .. . .. . .. . .. . .. . . . . .. . .. . .. . .. . .. . " 57
CHAPTER 10 How IS OXIDATIVE STRESS ASSESSED? ......... ... .. ........ 71
CHAPTER 11 Is THERE ANY JUSTIFICATION FOR USING OZONETHERAPY
IN HUMAN PATHOLOGIES CAUSED OR WORSENED BY FREE
R\DICALS? . . .... . ... ... . . . ..... . . ... ... . .. . . .... ....... ... ...... 75
CHAPTER 12 THE ANTIOXIDANT SYSTEM AND THE DEFENCE SYSTEM
AGAINST OZONE . .. . . . . . . . . . . . .. .. .. .. . . ... .. . . . . . . . . .. . . . . . . ... . 79
1. Hydrosoluble antioxidants . .. .. . .. .. .. . .. ... .. .... .... .. .. . " 81
2. Liposoluble antioxidants . .. .. .. . .. .. .. . .. .. .. .. .. . .. .. .. .. 89
3. The enzymatic system .... . .. . .. . .. .. .. . .. .. . .. .. .. .. . .. .. . " 98
CHAPTER 13 How DOES OZONE ACT? ......... ....... .. ...... .. ... .. .. . .... . 109
CHAPTER 14 WHAT HAPPENS IN THE INTRACELLULAR ENVIRONMENT
AFTER BLOOD OZONIZ.-\TION? . . .. . . .. .. . . .. ... . .. . ..... . .. .. . 121
I . Erythrocytes .. . .. .. . ..... . .. .. . . . .. . . . ... ... .. .. . . ... . . .. . . . 121
2. Leukocytes and the immune system . . .... . ... . .. .. . . . . . . 132
3. Platelets, haemostasis and growth factor release .. . ... 151
4. Endothelial ceIls and the vascular system .... ... .. .. .. . 161
CHAPTER 15 ARE BLOOD LIPOPROTEINS OXIDIZED AFTER
OZONIZATION? . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " 171
CH .\PTER 16 How IS OZONE ADMINISTERED? ... . .. . . . . . . ... . .. .. . . .. . . ... " 173
1. Administration via parenteral routes . . . . . .. . .. . .. .. . . .. .. " 174
2. Basic concepts regarding topical applications. .. . . . .. .. " 177
3. Major and minor 0 3 autohaemotherapy (AHT) . . . . .. .. " 182
CHAPTER 17 EXTR\CORPOREAL BLOOD CIRCULATION VERSUS O 2-0 3
(EBOO) . . ... . .. . .. . .. .. .. .. . . . . .. . .. . .. . . . . . . . .. . . . . .. . . . . .. . .. " 189
CHAPTER 18 QUASI -TOTAL BODY EXPOSURE TO O 2-0 3 (BOEX).
(V. Bocci and E. Borrelli) .. ..... .. .. ....... ... ... .. .. .... ... . 199
CHAPTER 19 RECTAL INSUFFLATION OF O 2-0 3 (RI). ... . . . . .. . . . . . . . . . .. . 213

xv
xvi TABLE OF CONTENTS

CHAPTER20 THE POTENTIAL TOXICITY AND MUTAGENICITY OF OZONE. PAG. 223

CHAPTER2I SIDE EFFECTS AND CONTRAINDICATIONS OF
OZONETHERAPY .... . .. . .. ... ... ..... ... ... .. .. ... . .. .. . . .. ... . . 227
I . Can ozonetherapy interfere with conventional
treatment? ... .. ... . .. .... . ... ... ... .. .. .... . . .. " . . ... ,.. .. .. . . 228
2. Are there contraindications for ozonetherapy? . .. . .. .. 230
3. Does prolonged use of ozonetherapy give rise to
sequelae such as tumours, degenerative disease , etc.? ... 230
CHAPTER22 THE ADAPTATION TO CHRONIC OXIDATIVE STRESS (COS)... 233
I . The paradox of ozone as a drug and the concept of
the multivaried therapeutic response to COS . . .. . .. . ... ... 234
2. Is an antioxidant supplementation necessary? .. .. . .. .. " 237
CHAPTER23 How TO EVALUATE OZONETHERAPY? .. .. . .. . .. . ...... .. ... 241
CHAPTER24 OZONETHERAPY IN VARIOUS PATHOLOGIES .. . .. ... .. .. .... 243
I. Infectious diseases (bacterial, viral, fungal, parasitic) 246
2. Autoimmune diseases .... ... . .. .. ...... .... ... ..... .. . .. .. 271
3. Ischaemic diseases (hind -limb ischaemia, cerebral
and heart ischaernia, venous stasis) . .. .. .... ... . ..... .. .. .. .. 276
4. Retinal degenerative disorders .
(A. Diadori and V. Bocci) . .. .. .. .. .... .. .. ...... .... .. .. . 286
5. Dermatological diseases .. .. .... .. .. .. .. .. .... .. ... .... ... 296
6. Pulmonary diseases (emphysema, asthma, COPD and
ARDS) .. .... .. . .. . .. . ... .. . ... .. ... ... .... ... .. .. . . .. . .. . .. .. . . . 297
7. Renal diseases .. ..... ... ....... .. .... . .. ... . .. . .. . ... .. . .... 298
8. Haematological diseases . .... . .. .. .. .. .. .... ..... .. ... ~ ... 299
9. Neurodegenerative diseases. . . .. . .. . .. . .. . .. .. . . ...... .... 300
10. Cancer.. .. . . .. .. .. ... .... ...... . . ... .. . ..... . .. . ... .... ... 303
11. Orthopaedic diseases . The problem ofback-ache . ... 314
12. Chronic fatigue syndrome (CFS) and fibromyalgia . 322
13. Trauma/bum injuries and emergency surgery .
Ozonetherapy before transplantation or before elective
surgery . .. ..... .. . .. . ... ... .. . .. .. . . .. . ... .. . .. . .. . .. . ... ... ... . 324
CHAPTER25 How IMPORTANT IS THE PLACEBO EFFECT IN
OZONETHERAPY? . . ... . . ... .. . . ... .. . .. . . . .. . ... ... ... .. .. .. . .. " 327
CHAPTER26 OZONETHERAPY IN DENTISTRY. . . ... .. . .. ... . .. .. . .... ... ... " 331
CHAPTER27 OZONETHERA.PY IN COSMETOLOGY.. . ... .. ... . ... .. . .. . ... . 333
CHAPTER28 OZONETHER.\PY IN VETERINARY MEDICINE. . .. . ... .. .. .... 337
CHAPTER29 OZONE AS DOPING rN ATHLETES. .. .. .... ...... .. .. .. ... . .. .. 341
CHAPTER30 OZONE AS A REJUVENATING AGENT! . . .. . .. . .. .. . . .. .. . ..... 343
CHAPTER31 OZONE AS A DRINKING WATER DISINFECTANT . . .. .. . ... . .. 347
CHAPTER32 OZONE D1SINFECTION TO PREVENT NOSOCOMIAL
INFECTIONS .. .. .. . . .. . ... .. .. .. ... ... ... .. . .. ... . .... ... , . .. . ... " 349
CHAPTER33 CAN OZONE BE USEFUL FOR B.\NKEO BLOOO OR FRESH
FROZEN PLASMA? .. ... .. .. .. .. . .. ... . .. ... . .. . .. . ... .. . ..... .. " 351
CHAPTER34 Is THERE ANY WAYTO SUBSTITUTE OZONIZATlON OF
BLOOO? . . ... .. ... .. . . .. .. . ... . ... ... .. . .. . ... .. . ... .. . ... .. .. ... 353
 T ABLE OF CONTENTS xvii

CHAPTER 35 HYPERBARIC OXYGEN THERAPY (HOT) VERSUS

OZONETHERAPY . PAG. 359
CHAPTER 36 ACUTE CARE DURING OR AFTER OZONETHERAPY
(8. B IAGIOLI AND V. Boccr) '" . 363
C HAPTER 37 ORTHODOX MEDICINE VERSUS COMPLEMENTARY
MEDICINE : A CON FLICT THAT MAY BE RESOL VED WITH
APP ROPRIA TE RESEARCH . 365
CHAPTER 38 D OES OZONETHERAPY HAVE A FUTURE IN MEDICINE? . 369
CHAPTER 39 ApPENDIX : THE OPTIMIZED PROCE DURE OF OrAHT . 375
REFERENCES . " 381
INDEX . 429
 ACRONYMS

A- Semidehydroascorbate radical anion

AA Arachidonic acid
Aa Angina abdominis
ABI Ankle-brachial index
ABTS 2,2'-azinobis-(3ethyl-benzothiazoline-6-sulphonic acid)
AC Adenylate cyclase
ACD Citric acid-citrate, dextrose solution
ACE Angiotensin-converting enzyme
ACR American College of Rheumatology
ACTH Adrenocorticotrophic hormone
AD Atopic dermatitis
ADCC Antibody-dependent cellular citotoxicity
ADP Adenosine diphosphate
AEI Anion-exchange protein
AGE Advanced glycation end products
AgIl Angiotensin 11
AR Ascorbic acid
AHIT Autohomologous immunotherapy
AHT Autohaemotherapy
AIDS Acquired immune deficiency syndrome
AMP Adenosine monophosphate
AP Atmospheric pressure
AP-) Activator protein 1
APC Antigen presenting cells
APR Acute phase reactants
ARDS Acute respiratory distress syndrome
ARMD Age related macular degeneration
AT Antioxidant therapy
ATP Adenosine triphosphate
ATPase Adenosine triphosphatase
AZT Azidothymidine (zidovudine)
BALF Bronchoalveolar lavage fluid
BALT Bronchial-associated lymphoid tissue
bFGF Basic fibroblast growth factor
BGBP Butyl-glycobutyl-phthalate
BLS Basic life support
BMC Blood mononuclear cells
BOEX Body ozone exposure
BrdU 5-bromo-2'-deoxyuridine
Ca 2'-_ ATPase Ca 2 ' adenosine triphosphatase

xix
xx ACRONYMS

CaCI2 Calcium chloride

CAF Cell antiviral factor
cAMP Cyclic adenosine 3'-5'-monophosphate
CAT Catalase
CCK Cholecystochinin
Cd Cadmium
CD4 + Helper T Iymphocytes
CD8 + Cytotoxic T Iymphocytes
cDNA Complementary DNA
CDP Cytidine diphosphate
CE Energetic charge
CFCs Chlorofluorocarbons
CFS Chronic fatigue syndrome
CGMP Guanosine 3'-5'- cyclic monophosphate
CGRP Calcitonin gene-related peptide
CH 4 Methane
CI Chlorine
CMP Cytidine monophosphate
CNS Central nervous system
CO Carbon monoxide
CO 2 Carbon dioxide
CoA Coenzyme A
ConA Concanavalin A
CO PD Chronic obstructive pulmonary disease
CoQ Coenzyme Q (Ubiquinone)
COS Chronic Oxidative Stress
CPD Citrate -phosphate dextrose
CRH Corticotrophic releasing hormone
CRP C-reactive protein
CSF Cerebrospinal fluid
CTL Cytotoxic T Iymphocytes
CTP Cytidine triphosphate
Cu Copper
Cu/Zn-SOD Copper/Zinc superoxide dismutase
Cys-NO Cysteine nitrosothiols
Cyt Cytochrome
DAG Diacylglycerol
DALT Duct-associated lymphoid tissue
DCHA Docosahexaenoic acid
DEHP Di(2ethylesil) phthalate
DHA Dehydroascorbic acid
DHEA Dehydroepiandrosterone
DHLA Dihydrol ipoate
DIC Disseminated intravascular coagulation
DNA Deoxyribonucleic acid
DNase Deoxyribonuclease
 ACRONYMS xxi

DNTC Diffused noxious inhibitory control

2,3-DPG 2,3-Diphosphoglycerate
2,3-DPGM 2,3-Diphosphoglycerate mutase
2,3-DPGP 2,3-Diphosphoglycerate phosphatase
EO Alpha-tocopheryl radical
EBOO Extracorporeal blood circulation against O2-03
EC Energy charge
ECs Endothelial cells
EDCF Endothelium-derived contracting factor
EDCF-I Contraction factor I
EDHF EndotheIium-derived hyperpolarizing factor
EDRF Endothelial-derived relaxing factor
EF Elongation factor
EGF Epidermal growth factor
EH n-rocopherol (vitamin E)
ELISA Enzyme-Linked Irnmunosorbent Assay
eNOS Endothelial NO synthase
EP European Pharmacopea
EPA Eicosapentanoic acid
EPO Erythropoietin
EPR Electron paramagnetic resonance spin trapping
technique
ERG Electroretinogram
ESR Erythrocyte sedimentation rate
ET-l Endothelin-l
EVA Ethylen vinyl acetate
F2-lsoPs F 2- isoprostanes
FAD Flavin adenine dinucleotide, oxidized form
FADH 2 Flavin adenine dinucleotide, reduced form
FCS Fetal calf serum
FDA Food and Drug Administration
Fe 2+HFe J + Iron
FFP Fresh frozen plasma
FGF Fibroblast growth factor
FRAP Ferric reducing/antioxidant power
FRBM Free Radicals in Biology and Medicine
GABA Gamma amino butyrric acid
G3-PD Glyceraldehyde 3-P-dehydrogenase
G-6P Glucose-6 phosphate
G-6PD Glucose-6 phosphate dehydrogenase
GALT Gut-associated lymphoid tissue
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
GCSF Granulocyte Colony Stimulating Factor
GDP Guanosine diphosphate
GGT Gamma-glutamyl transpeptidase
xxii ACRONYMS

Glu Glutamate
Gly Glycine
GM-CSF Granulocyte-monocyte Colony Stimulating Factor
GMP Guanosine monophosphate
GRP Glucose-regulated proteins
GRPs Glucose-regulated proteins
GS Glass syringe
GSH Glutahione reduced form
GSH-Px Glutathione peroxidase
GSHT Glutathione transferase
GS-NO Gluthatione nitrothiols
GSSG Glutathione disulfide
GSSGR Glutathione reductase
GTP Guanosine triphosphate
GTPase Guanosine triphosphatase
GVDH Graft versus host disease
H.p . Helicobacter pylori
H2 Hydrogen
H 202 Hydrogen peroxide
H2S Sulphidric acid
HAART Highly active anti-retroviral therapy
HAV Hepatitis A virus
Hb Haemoglobin
HbCO Carboxyhaemoglobin
Hb0 2 Oxyhaemoglobin
Hbs Haemoglobin sickle cell
HBV Hepatitis B virus
HCV Hepatitis C virus
HDL High-density lipoprotein
HDV Hepatitis delta virus
HES Hydroxy ethyl starch
HETE Hydroxyeicosatetraenoic acid
Hg Mercury
HGF Hepatocyte growth factor
HIF-I Hypoxia induced factor-I
HIV Human immunodeficiency virus
HK Hexokinase
HLA Human leukocyte antigens
4-HNE 4-hydroxy-2,3-trans-nonenal
HO-I Haeme-oxygenase I (HSP 32)
H0 2 Hydroperoxy radieal
HOCI Hypoclorous acid
HOT Hyperbarie oxygen therapy
H-O -U Heat, ozone and ultraviolet light
HPLC High pressure liquid chromatography
Hr Hours
 ACRONYMS xxiii

HSPs Heat shock proteins

5-HT 5-hydroxytryptamine (Serotonine)
HUVECs Human vascular endotheiial cells
IA Intraarterial
IALT Internal-associated lymphoid tissue
Iat Intraarticular
IDis Infectious disease
ID Intradisc
IF Intraforarninal
IFN Interferon
IgE Immunoglobulin E
IgG Immunoglobulin G
IKB Protein blocking NFKB activity
IKK-a Protein kinase phosphorylating IKB
IKK-ß Idem
IL Interleukin
ILes Intralesional
IL-lRa Interleukin I Receptor antagonist
IM Intramuscular
IMOS International Medical Ozone Society
IOA International Ozone Association
IP} Inositol-I,4,5-trisphosphate
Ipe Intraperitoneal
IPL Intrapleuric
IU International Unit
IV Intravenous
KDa KiloOalton
KGF Keratinocyte growth factor
KI Potassium iodide
LA u-Iipoic acid (Thioctic acid)
LAK Lymphokine activated killer cells
Lb Lactobacillus
LO Leukocyte depletion
LOH Lactic dehydrogenase
LOL Low Oensity Lipoproteins
LE Lipid emulsion
LLFL Lung lining fluid layer
LMWA Low Molecular Weight Antioxidants
L-NAME NG-nitro-L-arginine methyl ester (Nos jnhibitor)
LOPs Lipid oxidation products
LPS Lipopolysaccharides
LTB 4 Leukotriene B4
M Mean
ß2M ß2Microglobulin
MALT Mucosal-associated lymphoid tissue
xxiv ACRONYMS

MB Methylene blue
MCP-l/JE Monocyte chemotactic protein I
MDA Malonyldialdehyde
MDR-MT Multi-Drug-Resistant-Mycobacterium Tubercolosis
MegaU I million units
MEM Minimum essential medium
MHb Methaemoglobin
MHC Major histocompatibility complex
Min Minutes
MIP-Ia Macrophage intlammatory protein la
MIP-Iß Macrophage intlammatory protein I ß
MM Muscularis mucosae
Mn Manganese
Mn-SOD Manganese-superoxide dismutase
MPO Myeloperoxidase
mRNA Messenger RNA
Mx Mxprotein (lFN marker)
Nz Nitrogen
NzO Nitrous oxide
Na/K-ATPase Na/K ATPase
Na ZS Z0 3 Sodium thiosulphate
NAC N-acetyl-cysteine
NAD Nicotinamide adenine dinucleotide, oxidised form
NADH Nicotinamide adenine dinucleotide, reduced form
NADP Nicotinamide adenine dinucleotide phosphate, oxidised
form
NADPH Nicotinamide adenine dinucleotide phosphate, reduced
form
NaHC0 3 Sodium bicarbonate
NaOCI Sodium hypochloride
NFKB Nuclear factor Kappa B
NGF Nerve Growth Factor
NH 3 Ammonia
Ni Nickel
NK Natural Killer
NMR Nuclear magnetic resonance
NO' Nitric oxide
NO"z Nitrogen dioxide
NO x Nitrogen oxides
NOs Nitric oxide synthase
NSAID Nonsteroidal anti-inflammatory drugs
°zOZ,· Oxygen
Anion superoxide
03 Ozone
102 Singlet oxygen
 ACRONYMS xxv

Or AHT Ozonated autohaemotherapy

OFSP Oedematous-fibro-sclerotic panniculitis
OH Hydroxyl radical
5-0H-dCyd 5-hydroxy-2'-deoxycytidine
8-0HdG 8-hydroxy-2' deoxyguanosine
8-0HG 8-hydroxyguanine
ONOO' Peroxynitrite
OSE Ozone in Science and Engineering
OSP Oxidative Stress Proteins
OSPs Oxidative shock proteins
OxLDLs Oxidized low-density lipoproteins
PAET Small autohaemotherapy
PAF Platelet activating factor
Pd Palladium
PDGF Platelet-derived growth factor
PEG Polyethylenglycol
PEG-IFNa Poliethylenglycol-Interferon a
PEG -IL2 Poliethylenglycol-Interleukin 2
PEG-SOD Polyethylenglycol-superoxide dismutase
PET Positron electron tomography
PF 3 Platelet factor 3
PF 4 Platelet factor 4
PFK Phosphofructokinase
PGI 2 Prostacyclin
PGs Prostaglandins
6PGD 6-phosphogluconate dehydrogenase
PGH 2 ProstagIandin H 2
PHA Phytohaemagglutinin
PHN Post-herpetic neuralgia
PI Proliferation index
Pi Inorganic orthophosphate
PIP 2 Phosphatidyl inositol 4,5-biphosphate
PK Piruvate kinase
PKC Protein Kinase C
PLA 2 Phospholipase A 2
PLC Phospholipase C
p0 2 O 2 partial pressure
POAD Peripheral occlusive arterial disease
POD Peroxidase conjugated with antibody
Ppase Phosphatase
Ppbv Parts per billion volume
Ppi Inorganic pyrophosphate
Ppmv Parts per million volume
PRP Platelet-rich plasma
PRCLs Primary root carious lesions
PS Physiological saline
xxvi ACRONYMS

PTG Protein thiol groups

PUFAs Polyunsaturated fatty acids
PVC Polyvinyl chloride
PVC-OEHP PVC-di(2ethylesil)phthalate
PV0 2 O 2 venous partial pressure
PWM Pokeweed mitogen
Q Ubiquinone
QIO Coenzyme Q (Ubiquinone)
QH 2 Ubiquinol
QTBE Quasi-total body exposure (Ba EX)
RA Retinoic acid
RANTES Regulated upon activation, normal T-cell expressed and
secreted (Chemokine)
RBP Retinol binding protein
RCTs Randomised clinical trials
ROA Recommended dietary allowances
RES Reticulo endothelial system
RI Rectal insuffiation
RIA Radio immuno assay
RNA Ribonucleic acid
RNAase Ribonuclease
RNI Reactive nitrogen intermediates
Ra· Alkoxyl radical
noo' Alkoperoxyl radical
ROOH Hydroperoxide
RaS Reactive oxygen species
RPE Retinal pigment epithelium
RPMI Roswell Park Memorial Institute (tissue culture
medium)
RS· Thiyl radical
RS·/RSO· Thiyl/sulphenyl radicals
RSNO S-nitrosothiols
RTLFs Respiratory tract lining fluids
S Streptococcus
S-59-UVA Psolaren S-59 UV A
S-O Solvent-detergent
SM Serum amyloid A
SALT Skin-associated lymphoid tissue
SC Subcutaneous
SCE Sister chromatid exchange
SO Standard deviation
SES Staphylococcal Enterotoxin B
Sec Seconds
SEM Scanning electron microscopy
SOOT Aspartate aminotransferase
 ACRONYMS xxvii

SGPT Alanine aminotransferase

SLE Systemic lupus erythematosus
SODs Superoxide dismutases
SRA Scavenger receptor A
TYz Half-life
TAS Total Antioxidant Status
Tat Trans-activator oftranscription (HIV protein)
TB ARS Thiobarbituric acid-reactive substances (Marker of
peroxidation)
TC 99 Technetium 99
TCA Thrichloro acetic acid
TEHT Tri -(2 etylesil trismellitate)
TEM Transmission electron microscopy
TGFa Transforrning Growth factor alpha
TGFß Transforming Growth factor beta
TIAs Transient ischemic attacks
TIL Tumour infiltrating lymphocytes
TNFa Tumor Necrosis Factor alpha
TRAU Transfusion-related acute lung injury
TRAP Total (peroxyl) Radical-trapping Antioxidant Parameter
TrX Thioredoxin
TrX Px Thioredoxin peroxidase
TrXR Thioredoxin reductase
TSP-I Thrombospondin
TxA 2 Thromboxane A 2 (active form)
ß-TBG ß thromboglobulin
TxB 2 Thromboxane B 2 (stable form)
UDP Uridine diphosphate
UTP Uridine triphosphate
UV Ultraviolet light
VA Visual acuity
VAS Visual analogue scale
VEGF Vascular endothelial Growth Factor
VIP Vasoactive intestinal peptide
Vitamin A Retinol
Vitamin E (EH) c-tocopherol
VLDL Very low density lipoprotein
WHO World Health Organisation
WMA World Medical Association
vWF von Willebrand factor
WBC White blood cells
WSC Work site concentration
XDH Xantine dehydrogenase
XO Xantine oxidase
 CHAPTER 1

ABRIEF HISTORICAL REVIEW

As often happens for many discoveries, two preliminary observations were made for
ozone suggesting the generation of a new gas: firstly, around 1785, Van Marum
observed that when oxygen was exposed to an electric discharge, it was activated
and reacted with mercury; secondly, in 180 I, Cruikschank noted that an unknown
gas could be formed during electrolysis. It is unclear if Christian Friederich
Schönbein was aware of these results since he was not able to follow a proper
Chemistry course .

Figure 1. Christian Friedrich Schönbein discovered ozone in 1840.

Sch önbein (Fig. I) was born on October 18, 1799 in the small town of
Metzingen, near Stuttgart in southern Germany . He was the first of eight children of
a humble dyer, who was obliged to do other jobs to provide a living for his family.
This seems to be the reason why Schönbein, only thirteen years old, started an
apprenticeship to become a practical chemist. Later, he worked in a chemical plant
near Erlangen but since he could not afford to enrol at the University, he tried hard
to become a self-taught man. However, he did manage to artend lectures by Faraday,
2 CHAPTER 1

Dumas, Ampere, Gay-Lussac, and was certainly inspired by their genial minds and
their experimental approaches. In Erlangen, he also established a great friendship
with Justus von Liebig (1803 -1873) who became a leading chemist and most likely
gave good advice to Schönbein when, in 1839, he presented a lecture at the Basel
Natural Science Society entitled "On the smell at the positive electrode during
electrolysis of water". Schönbein had perfonned research in both Physics and
Chemistry, namely electricity, polarisation and electrolysis. Working with voltaic
piles in the presence of oxygen, he noticed the emergence of a pungent gas with an
"electric smell" and, with good intuition , discovered a new form of oxygen also
defined as "active oxygen", In nature, ozone is produced during thunderstonns
owing to the electric discharge of lightning which catalyses the fonnation of ozone
from atmospheric oxygen.
In 1835, he was appointed Professor ofPhysics and Chemistry at the University
of Basel and subsequently made other discoveries. Most notably, he demonstrated
the usefulness of galvanic deposition of zinc to protect iron from corrosion and the
production of ammonia from air and water for making fertilizers . It must also be
remembered that he invented Nitrocellulose or "gun cotton", which however did not
reward hirn fmancially as dynamite did for Alfred Nobel. Schönbein was a very
productive scientist. Among bis 343 papers, the one published in 1861 reported that
qualitative analyses can be perfonned using filter paper, a new notion that marked
the very first description of chromatography. Later, he became interested in
biochemical processes, particularly the ability of hydrocyanide acid to stop meat
putrefaction; thus he succeeded in showing the possibility of conserving meat for a
long time. While conducting this study , he contracted a Bacillus anthracis infection,
most likely from perished meat, and he died in Baden-Baden on August 29, 1868. In
acknowledgement of his great scientific merits and outstanding contributions to his
University, he was buried in Basel. It is ironically sad that although Schönbein had
certainly envisioned the possibility that ozone could act as a strong disinfectant for
the syphilis and gonorrhoea pathogens, he was unable to take advantage of ozone
himself(Nolte, 1999).
At the opening ofthe International Congress ofOzonetherapy (Glaxo-Wellcome
Research Centre, Verona , March 11-13, 1999), I had the privilege of
commemorating the 200 lh anniversary of Schönbein's birth. Firstly, I tried to
emphasize that important discoveries often seem to be the result of a stroke of luck
or, as we say, of serendipity. In reality, this is not the case : Jenner, Sch önbein ,
Fleming, Furchgott, Isaacs, Levi-Montalcini, just to mention a few innovators, made
their crucial observations ,as a result of their insight in explaining an apparentIy
casual result occurring during their daily work in their particular fields .
Moreover, Schönbein realized that ozone existed everywhere in nature and he
observed that its concentration increased with altitude. Indeed, in 1853 in the
Austrian mountains, he made the first measurement of different sampies of air and
invented a simple ozonometer consisting of test paper activated with iodide and
starch .
In my talk, I pointed out that Schönbein, a pioneer of atmospheric chernistry,
could not envisage the relevance of the ozone layer (about 10 ppm) in the
stratosphere which , at about 20 Km from the earth 's surface , neutralises most
 ABRIEF HISTORICAL REVIEW 3

ultraviolet rays (bands C:100-280 nm and B:281-315 nrrr), thus minimising their
mutagenie effeets on living beings. Nor eould he imagine that some 100 years later,
human negligenee in allowing the release of ehlorofluoroearbons (CFCs) into the
atmosphere would eause partial destruetion of the ozone layer, the renowned "ozone
hole", due to ehlorine free radieals (Harris and Bishop, 1999). Molina and Rowland
(1974) were awarded the Nobel prize in 1995 for explaining the eatalytie ozone loss
in the stratosphere. Today, we are learning the hard way that the ozone
eoneentration, which used to be extremely low in the troposphere (about 0.03 ppm,
i.e. about 300 fold lower than in the stratosphere), is dangerously inereasing
everywhere and is now at exeeptionally high levels in large eities. Ozone, mixed
with nitrogen oxide (NOx), carbon monoxide (CO) and other acid ehernieals,
composes the photochemieal smog that is very toxie for the respiratory tract,
beeause there are not enough neutralizing substances for this murderous mixture . It
is also sad to see wonderful marble and bronze statues in Florenee or Veniee
deteriorating due to aeid smog. It seems we never do enough to preserve or restore
them.
If Sch önbein eould have participated in the Basel Symposium (October 21-22,
1999) organized by the International Ozone Assoeiation (IOA) to honour bis
memory, he would have been pleased to see how important ozone has beeome in
many industrial proeesses and in our daily life, i.e. in the treatment of waste and
drinking water . These teehnologies will become more and more important as there is
a growing need to purify and save water. Today, nobody doubts the versatile oxidant
and disinfectant properties of ozone . After the first installation of a drinking water
treatment plant in Holland (1893), there are now more than 3,000 munieipal
treatment facilities in the world (Chapter 31). Indeed, Rice (1999) concluded bis
leeture saying that "the future for ozone in this area is indeed bright".
Finally, it wouldn't be fair if I didn't mention that, besides looking for the
practical application of his diseoveries, Sch önbein tried to understand how physical
and ehemieal phenomena could be related to the fundamental laws of nature .
Although he never had the opportunity of serious theoretieal training, he must have
been greatly influeneed by the oceasional teaehing and eonversations with the
philosopher Schelling. He eonsidered this friendship "to be a gift from heaven" and
it eertainly helped hirn to realize our transitoriness and short-sightedness when he
humbly wrote "although nature appears to us by space and time as an infinite mass
of unrelated details, a eolourful mixture of astonishing diversity, this is only caused
by the terrible narrowness of our vision" (Nolte, 1999).
Some eleven years before Sch önbein's death , the chemist Werner von Siemens
invented and patented the so-ealled "super-induction tube", so that even today we
talk about Siemens'stube. This was an important step forward: it was realized that
ozone was a very reaetive, unstable and unstorable gas and that it had to be produced
"ex tempore" from oxygen and used at once. At first, ozonisers were used for
preliminary industrial applications and disinfection of water after it was shown that
ozone displayed broad and potent bactericidal activity . Several decades elapsed
before a valid and praetical medieal ozoniser was devised by the physieist Joaehim
Hansler (1908-1981). He founded a manufacturing company and the invention of a
simple, reliable ozoniser greatly enhanced the diffusion of ozonetherapy. The
4 CHAPTER I

availability of a photometer for the real-time measurement of ozone concentration

represented a crucial step for serious progress. It was certainly the lack of a safe
medical ozoniser in the pre-antibiotic era that prevented the medical profession from
taking advantage of ozone's bactericidal activity: it was used only during World
War I to treat German soldiers affected by gaseous gangrene due to anaerobic
infections. It remains unclear how a Swiss dentist, E.A Fisch (1899-1966) had the
first idea to use ozone as agas or ozonized water in his practice. By a twist of fate, a
surgeon, Dr. E. Payr (1871-1946), had to be treated for a gangrenous pulpite and
was surprised by the efficacy of the ozone treatment. He was so enthusiastic he
extended its application to general surgery and he reported the results at the 59 th
Congress of the German Surgical Society in Berlin (1935).· In his paper on the
application of ozone in surgery (Uber Ozonbehandlung inder Chirurgie), he wrote
"wh ich other disinfectant would be tolerated better than ozone? The positive results
in 75% of patients, the simplicity, the hygienic conditions and innocuity of the
method are some ofthe many advantages". In France, Dr. P. Aubourg also published
a paper on the medical application of ozone and he was one of the first (1936) to
propose the use of insufflation of O2-0 3 into the rectum to treat chronic colitis and
fistulae . However, the local application of ozone was difficult in the 1930s because
of the lack of ozone-resistant polyethylene tubing . I believe that Dr. Payr was the
first to inject a srnall volume of O 2-03 directly into the vein, giving rise to a
procedure that later fell into the hands of charlatans and became so dangerous it was
prohibited.
Most likely realizing the potential risk of embolization, Werhly and Steinbarth
(1954) proposed to col1ect blood in a special quartz ampulla and to expose it for a
short time to ultraviolet light (UV) in the presence of pure oxygen, followed by
reinfusion into the donor. This was a variation of autohaemotherapy used since the
1940s: 5-10 ml of blood were withdrawn and then, without any further treatment,
were reinjected via the intramuscular (IM) route into the donor as an aspecific
immunomodulator, Werhly and Steinbarth were the first to have exposed blood to
oxygen and ozone (produced by UV light), although this method (rarely used today)
is cumbersome, imprecise and risky because the ampulla must undergo difficult and
unreliable sterilization before it can be reused. In fact, hepatitis infection has been
transmitted by this method (Gabriel et al., 1996) and, moreover, the ozone
concentration remains uncertain. Similarly, the idea to treat blood with heat, ozone
and ultraviolet light (H-O-U) is a matter of opinion (Cooke et al., 1997) and it
remains difficult to say which agent, if any, is effective.
Dr. H. Wolff(I927-1980) started his own practice in the 1950s and he was able
to use areal medical generator. Thus he promptly realized that blood should be
exposed ex vivo in a dispensable, ozone-resistant glass bottle to a mixture of OrO..
with a precise ozone concentration. Wolff deserves the credit for having developed
the real ozonized autohaemotherapy (03-AHT) which, with some modifications, is
used today . Just before his death, he published a book (1979) on the various
applications of ozone in medicine.
However, one must bear in mind that, because of the lack of scientific and
clinical studies, the use of ozonetherapy has always encountered great scepticism
and the current situation varies in different countries. It is favoured in
 ABRIEF HISTORICAL REVIEW 5

underdeveloped or poor countries, such as Eastem Europe, Cuba, Mexico and South
America. It is accepted in the motherland Germany, Austria and Switzerland but it
has elicited very little interest at the university level. It is more or less tolerated in
Italy, France, England, Canada and in a few states of the USA, while it is severely
prohibited in other states. We will examine the many reasons for this opposition in
Chapters 16 and 38.
Almost every country has one or more scientific societies for the development of
oxygen-ozone therapy . In Germany, the first society was founded in 1972 by Wolff
and Hansler while in Italy a society was founded in 1984. A respectable scientific
society should aim to promote basic and clinical research, as weIl as the serious
preparation of ozonetherapists in the hope of eventually obtaining valid data that
will be accepted by official Medicine. It would have been good if this had been
achieved because it would have implied that ozonetherapy had an irreplaceable
treatment to offer. However, it is deplorable that most of these so-called scientific
societies have mainly commercial interests linked to the manufacture of a generator
and accessories (often not even of good quality). For this reason , in 1999, we
founded the International Medical Ozone Society (IMOS , Italy) which has no
mercantile relations and tries honestly to promote the progress of ozonetherapy.
 CHAPTER2

HOW I CAME TO STUDY OZONETHERAPY:

AN ODYSSEY WITH NO END IN SIGHT

"Absit iniu ria verbis "

Titus Livius (59 B.C. - 17 A.D.)
(Words are not intended to be offensive)

This ehapter has little seientifie relevanee but the reader may be interested to know
why I started this investigation and the type of problems that I have eneountered.
The most eritieal problem was whether ozonetherapy was a worthwhile undertaking
at all. Some twelve years ago, the leading idea was that ozone will transfer some
energy to the blood, henee to the body. However, this seemed nebulous and rather
simplistie, resembling faneiful ideas typieal of natural medieine. A seeond
formidable problem was to understand why the proponents of ozonetherapy,
represented by physicians and naturopaths but also by unqualified people that ean be
eategorized as charlatans, were so enthusiastic while the opponents, represented by
allopathie physieians, exeellent biochemists and pathologists, eonsidered
ozonetherapy worthless and toxic . As often happens, both groups might have been
partly right and partly wrong and it was challenging to find the truth . Yet it was
obvious that the path of ozonetherapy would be bumpy and uphill.
In the prefaee, I mentioned that I eame aeross ozonetherapy by chance and I was
attraeted by the idea that ozone might aet as a eytokine indueer, as had been
demonstrated for other oxidants (Novogrodsky et al., 1977; Antonelli et al., 1985;
Dianzani et al, 1985). For some time, we had searched for an interferon (IFN)
indueer that needed to be effeetive, non-tolerogenic, non-antigenic and atoxie and
we wondered if ozone would be suitable. After Isaacs and Lindenman had
diseovered IFN in 1957, it took about 25 years before it eould be tested elinieally
and the amounts produeed by buffy-eoat leukoeytes were so small to make a
eontrolled clinical trial almost impossible. One sehool of thought was to produee
large amounts of IFN in vitro to be administered as an exogenous eompound and
this beeame feasible only with the advent of reeombinant technology; another was to
induce the production of endogenous IFN and other cytokines with some substance
(polynucleotides, lectins , etc.). Although vaecines are no more important than
antisera , I thought that the latter idea was rational and praetieal. Unfortunately,
however, the available indueers were toxie and thus the approach was abandoned in
the 1980s. It has turned out (Bocei, 1990a,b, 1991a,b, 1992a,b,c, 1993) that
exogenous administration of eytokines does not render them a "wonder drug" and
there is now a reconsideration of immunologieally specific indueers (Krieg and
Wagner, 2000 ; Tazulakhova et al., 2001) .

7
8 CHAPTER2

In June 1988, it appeared worthwhile to test the effieaey of 0 3-AHT because it

was a simple , rapid and apparently atoxie procedure: although only a small
percentage of leukoeytes became activated, after reinfusion these cells eould horne
in various lymphoid microenvironments, priming or activating neighbouring cells
after releasing cytokines. This process resembles the physiologieal IFN response
(Boeci 1981c, 1988b) that keeps the immune system in astate ofmild activation ,
possibly useful in chronic viral diseases. The only minimal , if any, increase of
cytokine plasma levels was considered an advantage because eytokines are paracrine
honnones and when present in the circulation can generate serious side effects
resembling a flu-like syndrome.
It took a long time for Dr. Luana Paulesu and myself to master a precise and
reliable technique to handle the gas mixture of oxygen-ozone, after we had attended
the careless demonstration of a "famous" ozonetherapist. All the steps of 03-AHT
were carried out with great approximation: the volume of blood, the volume of gas,
the 0 3 concentration, the timing of exposure of blood to the gas, the period of
reinfusion. 1 was told there was no real need to be precise and that an "expert eye"
could decide, by the changing eolour, when the blood was weil oxygenated and
ready for reinfusion. Although we have tried hard to standardise every detail of the
procedure, even today I know that some ozonetherapists boast about perfonning an
03-AHT treatment in 6 rninutes instead ofthe canonical 35-40 min,
We evaluated several teehnical procedures and, after many trials and errors, we
selected a procedure that remains valid today and simulates the so-called minor
AHT : a glass or silieonated syringe (10 ml volume) eontaining a precise volume of
human blood or serum (5 ml) was connected via a two-way stopcock with another
syringe that had just been used to collect an equal volume of the gas mixture (5 ml
of0 2-03) at a precisely detennined 0 3 concentration (from 5 to 100 ug/ml). The gas
was immediately aspirated into the first syringe and the tap was closed so that 5 101
of blood could interact with 5 ml of gas. Use of the I :1 ratio was found to be easy
and practical because we eould then test different ozone eoncentrations for each ml
of blood (for example I ml blood interacting with I ml gas containing 40 or 60
ug/rnl of ozone). The blood was immediately mixed with the gas in a standard way,
gently to avoid foaming. For how long? The "famous" ozonetherapist told us that a
few seconds were enough but we were not convineed; as physiologists we knew that
venous blood (p02 - 38 mm Hg) cireulating in the pulmonary capillaries becomes
oxygenated (p02 - 98 mm Hg) in about one second, but this happens because blood
is exposed to alveolar air (p02 - 100 mm Hg) in a layer of 3-5 microns .
Bearing in mind that blood is a very viscous fluid and assuming that we exposed
a 5 micron layer of blood to the gas phase for each rotation of the syringe along its
longitudinal axis, we calculated that it would take at least 80 rotations, to be
eompleted in one or (better) two minutes , to adequately mix the blood and gas.
Obviously different volumes (up to 250 ml) in different containers, such as an
ordinary glass (500 ml) bottle (used for the so-called major 0 3-AHT), will take a
longer time. In fact, by perfonning a gas analysis every minute, we have shown
(Bocci , 2000) that p02 increases progressively from about 38 to 500 mm Hg only
after 10 min (plateau phase) . One must consider that both gases in a bottle cannot
dissolve imrnediately in the water phase, as occurs for the normal 5 litres
 How I CAME TO STUDY OZONETHERAPY 9

volume/min of blood exposed to air over some 100 m2 of the alveolar space.
Moreover, as we shall discuss in Chapters 4 and 13, oxygen and ozone not only have
different solubility coefficients, but ozone never reaches an equilibrium since, on ce
dissolved, it reacts IMMEDIATELY with various blood substrates. However, it is
perhaps necessary to re-emphasize that not all ozone dissolves at onee in the wbole
volume of blood owing to the surface constraint.
It is distressing to note that our advice is disregarded by either incompetent or
careless ozonetherapists, either to be quick or because by using the wrong AHT
system a long delay will allow blood coagulation in the reinfusion tubing. I never
miss an opportunity to condemn this sort of malpractice because this is one of the
countless difficulties of ozonetherapy.
Coming back to our initial experimental problem, we made the hypothesis that
ozone will activate some leukocytic cells (probably monocytes and lymphocytes
nonnally defined as peripheral blood mononuclear cells, BMC).
When ozonized blood is reinfused into the donor, BMC will horne in several
microenvironments (likely in the lung, spleen, Iymph nodes and bone marrow) and
will find the ideal habitat to synthesise and secrete cytokines in the following hours.
In vitro, we could imitate this situation reasonably weIl in a thennostat where
sterility, temperature, humidity, p02, pC0 2 and pH are closely regulated. The
obvious limitation is that incubation is time-limited because of the progressive lack
of nutrients and increase of catabolites. To make a long story short, I will say that
we tested different conditions and various incubation times. After that, sampies were
centrifuged and the supematant was stored frozen until Luana could measure the
IFN activity by biological assay. This is a long, tedious procedure but if it is unable
to defme IFN types , it assurnes that any measured IFN molecule is biologically
active . It took several months of hard work but eventually we demonstrated that
ozone can induce the production of IFN, possibly type y according to a preliminary
characterization. IFN was released by either isolated BMC or leukocytes in either
whole blood or in buffy coats treated with ozone at concentrations from 2 to 108
ug/rnl of gas per ml of cell suspension or blood. Wemade a few interesting
observations: isolated BMC, resuspended in tissue culture medium, responded only
at very low (2 ug/ml) 0 3 concentrations while leukocytes in whole blood produced
more IFN when exposed to an ozone concentration of 42 ug/ml and progressively
less at higher 0 3 concentrations. The kinetics of IFN release was similar to that
measured using an extremely active stimulator, Staphylococcal Enterotoxin B
(SEB), but ozone appeared to be a far weaker inducer. Air or oxygen tested as
control gases were ineffective. At that time, although we could not yet grasp the
mechanism of action, we realized that increasing ozone concentrations inhibited the
process and reduced viability because of toxicity. These were the very first results
showing that ozone could act as a cytokine inducer (Bocci and Paulesu, 1990); for
better or for worse, they have been followed by many others in various cell types
(Beck et al., 1994; Arsalane et al., 1995; Takahashi et al., 1995; Jaspers et al., 1997).
Being sure to have made an original observation, I wrote areport that was submitted
to the Scandinavian Journal of Immunology. We remained very disappointed by the
harsh critical comments of two referees who considered ozone a toxic gas and
difficult to measure; as a consequence, they invalidated OUf results. At this point, I
10 CHAPTER2

apologise to the reader for the necessity of a short digression . Besides the weil
known aphorism "publish or perish", any scientist has the right, if not the duty, to
inform the scientific community at large of herlhis new results . However,
competition has become very fierce and the space in internationally renowned
journals is very limited . Moreover, although the anonymous referee system, meant
to critically evaluate the paper and eventually to perfect it or reject it, is generally
positive and useful, it is not fool-proof. There are clamorous examples of excellent
papers of Nobel prize calibre that have been rejected in the first instance . This was
certainly not the case of our paper but nonetheless we feit that the prejudice against
the use of ozone had no justification. In deciding to evaluate the biological effects of
ozone , perhaps naively, I had not anticipated the beginning of an endless ordeal. As
a matter of fact, we could have published our first report immediately in the
Newsletter the so-called scientific Italian Society of Ozonetherapy published every
3-4 months . But we refused to do that because the Newsletter is virtually unknown,
printed only in Italian (therefore useless for the English-speaking scientific
community), without an editorial committee and worst of all published to support a
commercial enterprise . In synthesis, to publish in this Newsletter would be
equivalent to burying a result. Once again, I take the opportunity to emphasize
another chronic problem with ozonetherapy: scientific data have been very
scarce, ofpoor quality, and at best published in obscure journals that nobody reads.
Thus the "impact factor" is zero.
With rare exceptions duly reported in the References, it seems as if ozonetherapy
has been performed on a desert island without any contact with official Medicine.
This situation initiated a vicious circ1e in which scepticism was fed by scarce and
unreliable data often presented without any control.
In conc1usion, the refusal of our first paper was not altogether negative because
we became weil aware of the obstac1es to overcome and that we could not succeed
easily. We then submitted the manuscript to "Haernatologica" which, although not
considered a first-class journal, is sponsored by the Italian Haematological Society,
has a serious editorial committee, publishes in English and is regularly reported in
Current Contents, Life Sciences. In this case, the referees comments were objective
and, after a modest revision and addition of some data, the paper was published in
1990 (Bocci and Paulesu).
This was only a small step ahead because every day I could perceive the ostracism
ofthe medical world. There were several reasons for that: firstly, the toxicity of ozone,
which in Summer is the daily concern of the mass media on account of increased
ozone levels in cities with consequent pulmonary pathology; secondly, the widely
accepted and hardly disputable concept that several diseases and ageing are due to free
radicals; thirdly, ozone is a master in generating free radicals; fourthly, a profound
disbelief that ozonetherapy is beneficial, due to a lack of a serious data in conjunction
with vaunting, triumphant claims; and fifthly, the widespread derision of ozonetherapy
as a sort of"panacea" able to eure all human iIlnesses.
Therefore, it appeared neeessary not only to publish experimental papers but to
objectively clarify whether there was any benefit from the ozonetherapeutie
approach and to pursue any possibility to prove or disprove it with controlled
clinieal studies. Sinee 1991, I have tried to break the isolation and the aseertainment
 How I CAME TO SrUDY OZONETHERAPY 11

that, albeit with some good reasons, ozonetherapists were held in contempt. By
painfully searching and analysing the scarce and usually poor literature, I tried to
distil what seemed relevant and to present it in brief reviews . This was a frustrating
time because lieft myself open to criticism. Yet I paid attention to the criticism
since in most cases the objections were substantially correct. At the same time, I
found it beneficial to leam from our mistakes and I hope that the reader will be
willing to participate in this sort of catharsis .
In 1992, I tried to give a new interpretation to the empirical use of ozonetherapy
in chronic viral diseases, particularly chronic hepatitis and recurrent herpes
infections . The new hypothesis was based on our experimental results showing the
induction of IFN and possibly of other cytokines. If it was correct, in addition to
improving the general metabolism, repeated treatments of OrAHT might have
enhanced priming and activation of the immune system, a fundamental requirement
for recognizing and destroying virally infected cells. The editor of Medical
Hypotheses found the article interesting and, after minor editorial revision,
published it (Bocci, 1992a). I remained somewhat disappointed by the lack of
response by infectivologists because the availability of effective immunomodulators
was meagre and I was proposing to evaluate an approach able to trigger a
physiological cascade of events "with a more comprehensive activation of the
immune network. However, I carmot forget Mr. W. Martin's (Fairhope, Alabama,
USA) letter, received on November 1992. He was somehow related to Dr. Helen
Coley Nauts, daughter of Dr. W.B. Coley, who in 1906 had proposed the use of
mixed bacterial toxins (now known to elicit release of several pro-inflammatory
cytokines) to treat cancer patients. Mr . Martin urged me to continue even though he
wamed that it may take a long time to get things going : Dr. H. Coley was able to
establish the Cancer Research Institute only in 1955 but the FDA was still
maintaining a ban on Coley's mixed toxins, which had to be tested at the Children's
Hospital in Beijing.
After gaining good experience in handling ozone from 1991 to 1993, we
managed to publish two papers in "Lyrnphokine and Cytokine Research" (LCR):
one showing the induction of tumour necrosis factor a (TNFa) and the other of
IFNy and IFNß, Interleukin-6 (IL-6), IL-2, and Granulocyte-Monocyte colony
stimulating factor (GM-CSF) (Paulesu et al., 1991; Bocci et al., 1993a). In the 1993
paper, we attempted to define conditions for optimal induction of cytokines,
particularly in regard to anticoagulants: in cornparison to citrate, which chelates the
plasma Caz+, heparin enhances the production of cytokines by leukocytes in whole
blood. Actually the addition of 5 mM CaClz strengthens the process but causes a
modest increase in haemolysis . It was suggested that OrAHT could be potentiated
using heparinized and Ca2+-supplemented blood in patients with chronic viral
hepatitis. I was recently informed by a group of ozonetherapists working in New
York that this formulation was found to be very effective, but regretfully 00 clinical
or virological data could be provided. Later on in our study ofHIV patients, we did not
use the CaH addition since it might have enhanced platelet aggregation (Chapter 14).
I gratefully acknowledge the help of Dr. L.B. Lachman, who, as chief editor of
LCR, was extremely helpful during the revision phase of these two papers. At that
12 CHAPTER2

time, we began to examine some biochemical parameters, such as reduced

glutathione (GSH) , ascorbic acid and glucose levels, to gain some insight into the
ozonization process (Bocci et al., 1993b). A few months later, we could also
measure the release of transforming growth factor ß 1 (TGF-ß 1) from ozonized
blood (Bocci et al., 1994b), a finding that could explain why the healing of torpid
ulcers in hind limb ischaemia patients was enhanced by 03-AHT.
Armed with these new experimental data, I naively thought that a critical
revision ofthe 03-AHT procedure would be useful. However, the paper was rejected
by "Transfusion Medicine " on the basis of the following referees reports :
Referee 1.- "This review of autohaemotherapy after treatment of blood with ozone is I)
speculative in that it predom inantly presents hypotheses rather than established facts, 2)
preliminary in that it presents no evidence for or against efficacy , and 3) subjective in
that it draws heavily on work already pnblished by an author while failing to critically
review or present a eoncise overview of work by others in this area" .
Referec 2.- " I entirel y agree with the author that an impartial assessment of the use of
ozone therapy is required . However, on read ing the reappraisal 1 found that the author
did not provide any quantifiable data which could be used in an assessment. The
following examples illustrate my view :
Which viral infections were successfully treated? What types of neoplasia were cured?
What kind of cutaneous infections were treated ? What were the apparently good results
in how many ofpatients treated? "

The referees were right but how could I discuss significant clinical data when
ozonetherapists at best report only a few anecdotes . My hope to elicit interest in the
approach so as to promote clinical trials in other institutions was shattered.
Eventually I restructured the manuscript and, only after a lengthy revision , it was
published in the "Journal International Medical Research" (Bocci , 1994b), but again
it evoked very little interest.
In mid- I994, the spread of the HIV epidemic was of great concern and, except
for the minimal efficacy of azidothymidine (AZT), there was no therapy. On the
other hand, Oxylist and the popular mass media propagandized the claim timt
ozonetherapy was "curing" HIV patients. Charlatans without any medical
qualifications in Canada, USA, Mexico, Caribbean Islands, etc. were attracting
desperate patients by promising that two weeks of therapy eonsisting of direet
intravenous (IV) injeetion of O 2-03 at a eost of 8-10,000 US dollars would make
them HIV-negative. I feit that this was a shameful exploitation. Nonetheless , the
potent virucidal activity of ozone and the recently discovered induction of cytokines
raised the question as to whether an appropriate programme of OrAHT perfonned
in the hospital at no expense would be of some use to patients in Italy. After
evaluating the pros and cons, I prepared a c1inical protocol to be submitted to the
Health Authorities as weil as to patients in order to obtain their informed consent.
Both the risk and toxicity of the treatment appeared nil on the basis of the extensive
evaluation of OrAHT by Jacobs (1982) . Hoping to receive a comment or advice, I
sent the programme to Prof L. Montagnier, whom I had met at an IFN meeting in
Siena, to Prof R. Gallo, to Prof. A. Fauci, to several ltalian infectivologists and also
to Prof E. Guzzanti, then Minister of Health. To my dismay, no one answered
except Prof Anthony Fauci, director of the National Institute of Allergy and
 How I CA ME TO STUDY OZONETHERAPY 13

Infectious Diseases, Bethesda, Maryland, USA. Although he had doubts about my

proposal, I greatly appreciated bis comments and willingness to help,
"Thank you for your letter of June 20 and thank you for your kind words
concerning my lecture on pathogenesis of AIDS at the recent biotechnology meeting in
Florence. From your letter it is clear to me that you are interested in the evaluation of
the biological and clinical aspects of ozone therapy . Over the past few years there have
been a number of individuals in the United Statcs who have been interested in pursuing
ozone therapy in HIV infection . However, to be honest with yo'u there has been very
little interest on the part of funding agencies or on the part of established investigators
in pursuing this line of research . I agree that from the data that I have seen, the therapy
appears to be generally free of side effects. However, the data indicating that it would
be of any use in HIV is really rather scanty . Your work on the induction of cytokines by
ozone is interesting . One of the problems wilh the induction of TNF by ozone is the
potential for the induction of HIV expression in surrounding cells. You may be aware
that my own laboratory and a number of other laboratories have demonstrated that
certain cytokines including TNF can potently induce HIV expression from latently
infccted cells . In this regard I would be concerned about the possibility of actually
inducing HIV virus.
With regard to the possibility of pursuing these types of trials, I will pass the
infonnation that you sent me on to our Division of AIDS (DAIDS). DAIDS is
responsible for the conduct of clinical trials that are funded by our Institute . I certainly
cannot promise you anything in this regard since I know that in the past they have
refrained from pursuing studies on ozone therapy . Nonetheless I will forward the
material to them. Dr. Steven Schnittmann is one of the major figures in DAIDS in the
conduet of clinical trials. If he has any interest in pursuing this with you I am sure that
he will contact you directly.
I am sorry that I cannot be more helpful to you on this matter. However, as you
mentioned in your letter there are a number of other therapeutic approaches that are
bcing activcly pursued wilh a higher priority than ozone therapy including the IL-2
studies that you mentioned in your letter to me. I wish you luck in your endeavors".

Shortly after, I met Dr. Schnittmann at an HIV Congress in Strasbourg but, as I

expected, he was too busy with bis own programmes.
Only in July 1995 with another physician and a group of virologists of our
University, we were able to perform an accurate evaluation for almost 7 months of
12 patients in the pre-AIDS phase who had refused other forms of therapy. Very
briefly, we found that even up to 50 consecutive (twice weekly) 03-AHT sessions
were unable to significantly reduce the viral load but did induce a modest increase of
T Iymphocytes CD4 ", However, no side effects were noted, while most patients
experienced a sense of weil being and opportunistic infections became rare and most
important, contrary to the darkest expectation, only one patient had an increase of
the viral load and we stopped the therapy immediately.
The critical appraisal of ozonetherapy for HIV infection was published (Bocci,
1994a) in "Mediators of Inflammation" as a "Therapeutic speculation" via the
understanding of a colIeague, Prof. LL. Bonta (Erasmus University, Rotterdam). The
experimental results were published in "Clin, Microbiol. Infec ." (Bocci et al., 1998c)
with the blunt title "Lack of efficacy of ozone therapy in HIV infection" imposed by
the Editor (see Chapter 24).
Pursuing my idea of informing the academic world about ozonetherapy and
achieving a colIaborative effort, I submitted a manuscript entitled "Ozone: a mixed
14 CHAPTER2

blessing ..." to the Editors of two important journals, but to no avail. The answers
follow :
" Immunology Today " Septemb er 1994. "Many thanks for your manuscript
eoneeming the therapeutie effieaey of autohaemotherapy as a elini eal treatment. At the
moment I must decline your offer for the reason you have alluded to in your letter : i.e.,
that the data so far, though striking, are still aneedotal . Given the pressure on spaee in
"Immunology Today" , until data from 11 large c1inical trial ure availahle we eould
not prioritise this subjeet for diseussion. My apologies for being the bearer of
diseouraging news on this occasion , however, onee more data ean be added to the initial
studies I would be interested to hear from you again " .

And then :
"British Medieal Journal" April 1995. "Thank you for sending us your paper, whieh
I regret we do not wish 10 publish . Allhough what you had to say is undo ubtedl y
interest ing, we are not inlerested in 11 review whlch is really just 11 selection of
anecdotes, If there is now sueh enthusiasm around, why don ' t you iniuate rigorous
studies of ozone therapy in single eonditions, whieh eould then be reported in medieal
journals and would do mueh more for your eause than one of experien ees gained by
private mcdical practitioners" .

Even my appeal to the Deputy Editor did not serve the purpose:
"Thank you for your letter appealing against our deeision to reject your discussion
of ozone . Before devoting mueh space to sueh a topie we would need a hody of sound
research on wh ich to base such a dehnte. In the ease of hyperbarie oxygen at least
there was more than aneedolal reports to go on. I am sorry 10 disappoin l you".

In contrast, it was much too easy to publish reports on the occasion of the XII
Congress of the International Ozone Association (IOA) in Lilie (Bocci, 1995a) or in
the Ozon-Handbuch edited by Dr. R. Viebahn (Bocci, 1995b). Each year, this book
collects the best publications in German but unfortunately remains unknown.
Obviously there is no criticism in the small ozone circ1e and because contributions
are so few, almost everything is accepted. This attitude does not favour competition
or progress and if the reader scrutinizes some of the abstracts of the Lilie
Symposium he/she will remain astonished by the contents.
As a further dreary example, in 1994 I presented two papers at the National
Congress ofOzonetherapy organized by the Italian Society (SIOOT) in Rome. With
the exception of a pompous ceremony and the demonstration of only one type of
ozone generator produced by the firm owned by the president, it was the usual
fanfaronade . Worst of all, I was told that the papers were going to be published later
(?) in a journal that nobody reads but in fact I never saw them. The journal is
probably kept in a cellar with old wine!
The paper entitled "Ozone: a mixed blessing ..." was eventually published in
"Forschende komplernentarmedizine" (Bocci, 1996d) but once again it received
little attention because the journal focuses on other approaches of natural medicine .
In September 1995, the Russian Ozone Society organized an international
Congress at Nizhni Novgorod and I was invited to give the keynote speech . When
we visited the hospital, I was impressed that ozonetherapy was used everywhere.
Certainly as a disinfectant, ozone was useful in infections and in awful war traumas
and bums (conflict in Chechnya) but, in other cases, such as the questionable
 How I CAME TO STUOY OZONETHERAPY 15

infusion of weakly ozonized saline, it was probably little more than a placebo
(Chapter 34) . As in Cuba, it was sad to see that a great country was so impoverished
to have to rely mostly upon ozone, even though the Russian doctors seemed satisfied
with the c1inical results. On returning to Italy, I thought it would be interesting to
write a short letter "Ozonetherapy has come of age in Russia" and I sent it to the
editor of"Nature" in October 1995. The response was:
"Thank you for your leiter, but I am afraid we cannot publish it in its present form.
The difficulty is this. As you yourself say in your letter, there is a prejudice against
ozone therapy in the West, and the reason is simple: there has been a lot of quackery
and there have been no convincing demonstratlons to the contrary. I know that
there are many readers who would take your description of what the Russians are doing
as proof, but there are many other readers who would remain sceptical , saying that they
would have been more convinced if somebody (Iike you) were to describe some
particular circumstances in which data have shown that ozone therapy can be benefic ial.
If there is something that sticks in your mind from the conference you went to that
would have that effect, it would be worth putting it in a letter . You will, I am sure, know
that letters in "Nature" should be as short as possible ".

At this point, I gave up and I sent the paper to Dr. S. Peretyagin who, 1 believe,
translated it into Russian and published it in a local journal (1996b). In June 1996, I
went back to the University of Nizhni Novgorod because in the meantime they had
received a grant from the Soros Foundation. The plan was to investigate whether
ozonized erythrocytes labelIed with TC99 remained in the circulation or were quickly
removed by the spleen (see Chapter 14). We performed good preliminary
experiments in about a week and then lieft, hoping that they would continue to
examine the actual half-life by testing Cr 51-labelled erythrocytes, Yet I later heard
that they could not carry out the experiments owing to insurmountable difficulties.
In 1996, the Italian Ministry of Health ratified a 1992 decree that stated that
ozonetherapy was still an uncertain medical practice and could only be investigated
in university clinics and large hospitals. On reading the decree, I realized that the so-
called Superior Council of Health did not have the slightest idea about
ozonetherapy, but being a Superior Council they didn't need any information. Worst
of all, the council left ozonetherapy in a vacuum for private physicians and thus
allowed a very equivocal way ofpractising ozonetherapy.
As a matter of fact , no comprehensive review of the topic had ever been
published and I spent considerable time in collecting all available data, in tenns of
biochemistry, immunology, toxicology and clinical data . I thought that I had done a
useful job when I submitted it to the editor of "Pharmacology and Therapeutics"
(New Haven University), a good journal specializing in pharmacological reviews. I
was rather hopeful that the present work would be as well appreciated as two
previous reviews of mine (Bocci, 1981b, 1987b). The foIlowing reply by a highly
qualified referee proved that ozonetherapy was anathema in the U.S .A. :
"Dr . Bocci admirably attempts to put forth a rationale for ozonated
autohemotherapy, e.g., exposing ex vivo human blood to agas mixture including ozone,
followed by reinfusion . As one of a group of investigators who has studied the
biopathologies of ozone toxicity for over two decades , who has been a member of the
Europcan and American Free Radical Societies for over a decade, and who is a
practici ng physician , I cun envision no rationale for the mcdicul upplicution of this
archaic practice, which I would put in the same cutegory as the 18th und 19th
16 CHAPTER2

century practices of bloodletting. Indeed, bloodleUing, exposing the blood to ozone,

lind thcn reinfusing the products of the "ozonutlon" are even more harbnric, to
this revicwer. It is comforting to know that direct EV infusions of ozone have been
prohibited by the European Soeiety for Ozone Therapy. Certainl y, studi es of hydrogen
peroxide infu sions earried out in the early 1990s in animals and selected human s
reached similar conclusions. It is truly amazing how few animal experimentation trials
have been produced for the number of poor human intervention uneontrolled studies!
The review is broad-based and in some measure scholarly, but piecemeals bits of
non rele vant seienee to outright quaekery in far 100 many places . If the title werc " From
Quackery to Toxi city " rather than " From Natural Medicine to Phannaeology" there
might be a more rational basis for publieation in a scientifie journal. This reviewer has
no doubts that infusions of ozonized blood contain lots of bioactive substan ees
(aIthough the ozone itself will have quiekly rcacted with biomoleeular targets), rnany of
which ean be expectcd to eause effeets on eells (e.g., 4-hydrox ynonenal, phosphoeoline
ozonides and aldeh ydes , ete .), and that thcse substanees might havc benefieial or
delctcrious influenees on inflammatory immune processes depending on dose and vigor
of host responses . It is grotesque to think that an y Western World drug ReguIating
Agency would condone infusing the hodgepodge nf ozonized products to trcnt
diseases , aIthough it is probable that the produets would initiate and/or modulat e a wide
speetrum of inflammatory-immune processes to varying degrees. These lipid ozonation
produets have already been shown to aet as ozonation signal transduetion moleculcs in a
number of epithelial eell preparations . Infus ions of toxic lipids and endotoxins would
predietably have similar effeets to that of ozone exposures to blood. and all would
undoubtedly induee various dysfunetional eytokine aetivations, probably in a dose-
response manner".

I found these comments too general and I appealed to the Editor for a further
evaluation, but to no avail because two associate editors answered as folIows:
Editor 1.- " I too have considerable reservations. This is fringc medieine whose rationale
is diffieult to justify. On oeeasions such as this, one often tries to think of a ploy when a
change of titlc or emphasis might render a review aeeeptable but on this occasion I
believc we would be stretching the eredulity of our readers by publishing on the topic .
" Pharmacology aud Therapeutics" has built up an enviable reputation as a high quality
review journal and I bclieve we must proteet that position. 1 would emphatically reject
the rcview" .
Editor 2.- " I have read the review of the paper by Dr. Boeei . 1 agree with your decision
to reject it. I think it would be quite inappropriate for a journal such as P&T 10 embark
on this kind of topie . It may weil be that it requircs serious seientifie examination, but
ours is a review journal, not an experimental journal, and so P&T is not the relevant
publieation . Ir a time comes whcn ozone is prnved to be un effective therapeutic
agent, then a review ofthe evidenee in P&T might weil be welcome, but until such time
comes I think we should steer elear nf unything thut sounds quuckery and thereforc
could bring the journal into disrepute" ,

As further confinnation of a sort of American crusade against ozonetherapy, I

would like to report the judgement of a distinguished American professor of
Physiology and Medicine, who wrote me in February 1997:
" As is the case for most Ameriean scientists, 1 remain extremcly skeptical (doubtful)
about any conceivable usefulness of this type of therap y. If you want to activate
cytokines, or other subtle aspcets of the inflammatory-immune response, thcre arc better
ways of doing so than exposing blood to ozonc ."

I don't agree at all about this last remark; indeed, although there are many ways,
none is good . Anyway I must infonn the reader that this time I didn't give up and I
 How I CAME TO STUDY OZONETHERAPY 17

submitted the manuscript to ."Journal of Biological Regulators & Homeostatic

Agents". The editor and referees were at first somewhat perplexed but, after a final
discussion on the phone, the editor decided to publish it provided he could add bis
own commentary at the end of the paper. This is interesting to read it:
" The dilemma of exposing or burying a complementary medical approach . (J. Biol.
Regul. Homeost. Agents 10:54, 1996)ln this issuc Bocci describes the present state of
the art of ozonetherapy.
Bocci undertook the study of a possible biological action of ozone by sheer
coincidence when he was looking for cytokine inducers and linked the oxidant
properties of ozone to our findings (I) that galactose oxidase could induce interferon-j
production. Bocci et al found that ozone can indced act as a mild cytok ine inducer via
production of a powerful intermediate such as hydrogen peroxide. There is now
considerable evidence that redox reactions regulate signal transduction and particularly
hydrogen peroxide can activate the transactivating factor NFKB (2).
Bocci's review attempts to organize a framework into which the heterogenous blood
cell components undergoing the action of the reactive oxygen species generated by
ozone can be orderly put and may explain a number of biological actions and perhaps
therapeutic activity. On this basis the review deserves some credit because it tries to
find a scientific foundation to an empirical procedure, although it is clear that Bocci is
only bcginning to explain a few mechanisms of action . The possibility that ozone may
act as a bioregulator by either enhancing cell metabolism , or cytokine production , or
upregulating the synthesis of antioxidant enzymes is interesting and needs to be
precisely confirmed. If the latter observation will prove to be true, ozone could become
a unique drug capable of inducing astate of oxidative adaptation long sought after for
improving or stabilizing degenerative diseases.
Owing to the fact that ozone , as a strong oxidant, can be toxic, considerable
attention has becn devoted to show that toxicity can be checked and minimized if one
knows, on one hand, the exact dose and concentration of ozone and, on the other hand,
the antioxidant properties of the substrate , i.e., human blood to be treated. Surprisingly,
if one observes a stoichiometric relationship between ozone and blood, toxicity appears
to be ncgligible and it is interesting that patients report a sense of weil being and no side
effects during treatmcnt.
As far as claims of therapeutic activity are concemed, I am trying to have an open
mind because skepticism and prejudice in Science can bc bad advisers. The fact that
ozonetherapy is apparently useful in unrelated pathologies such as chronic hepatitis and
ischemic disordcrs does not bother me because ozone, by acting either upon leukocytes
or erythrocytes can obviously elicit different biological activities. Instead what is really
coneeming is the present state of elinical results mostly uncontrolled or simply
anecdotal and I agree with the eritical remarks raised by Bocci about the poor quality of
the work carried out during the last decades .
Are therapeutic effects really true and due to ozone, or are they due to a placebo
effect partl y elicited by oxygen alone or by nenrotransmitters and honnones released by
the neuroimmunological network following the ozonated autohemotherapy? On the
other hand Bocci refers to this potential therapeutical approach as "complemcntary" or
"natural" Medicine, a tenn that encases other unconventional types of treatment such as
homeopathy , acupuncture, without mentioning pranotherapy and similia . Here in my
opinion the situation is different since ozone has clear and demonstrable
pharmacological effccts and Bocci, who is one of the world's leading experts on the
pharmacology of cytokines , is certainl y weil -qualified to critically face this dilemma .
I have preferred to expose this work with all its controversial aspects to the scrutiny
of the scicntific community hoping that by doing so possible advantages or
disadvantages will bc definitively clarified . On the basis that some human diseases are
not yet properly treated by convcntional Modieine the publication of this review may be
of service to Medicine .
References
18 CHAPTER2

I. Antonelli G, Blalock JE. Dianzani F. Generation of a soluble IFN-gamma

inducer by oxidation of galactose residues on macrophages. Ccll Immunol 1985;
94:440-6.
2. Suzuki YJ, Ferman HJ, Sevanian A. Oxidanis as stimulators of signal
transducnon. Free Radical Biol Mcd 1997; 22 : 269 -85 .
Ferdinando Dianzani
Co-Editor of JBRHA

If I remember correctly, it was in the spring of 1997 that Dr . A. Balkanyi,

president of the Swiss Society of Ozonetherapy, organized an international congress
addressing the impact of natural Medicine on Cancer therapy. He asked me to talk
about "Ozonetherapy and cancer", a very challenging task because the literature is
old, scanty and anecdotal (Zabel, 1960 ; Mattassi, 1985). At first, I was uncertain but
the issue was so stimulating and open to speculation, particularly considering the
relationship between oxygen levels and tumour growth, neoplastic suppressor
factors and cytokine induction, oxygen levels and angiogenesis etc., that I could not
refuse. The Congress was held in Interlaken. The weather was glorious, so it gave
me again the joy of admiring the majesty of the Jungfrau and of having long early
morning swims in the wonderful pool of the Grand Hotel Victoria. I discussed the
rather exciting, yet theoretical, possibilities of ozonetherapy but I was ashamed that
the available clinical data were only a cloud of smoke (see Chapter 24) . There was
only a small discussion at the end of my talk and I feit that the promoters of other
approaches, namely anthroposophie medicine and homeopathy, regarded
ozonetherapy as a second-rate newcomer. As is seen among different religions, there
is also a tendency to sectarianism among the various branches of natural medicine.
A few months later, I heard that the manuscript that Dr . Balkanyi had asked me to
prepare before the meeting would not be published in the foreseen volume. This
drawback was annoying because I now had to find an editor or to leave the paper in
a drawer to get mouldy. I hate to do that because we put so much heart in our papers
they become like children that eventually have to be married. Even considering its
intrinsic weakness, I submitted the paper to 'The Cancer Journal". But the Editor
could not publish it owing to the lack of clinical data and I could understand his
reason:
" I have carefully read your paper on ozonatcd major autohaernotherapy, twice , with a
two weeks interval, bcfore taking a decision . I am sOITY but we cannot publish this
article in "The Cancer Journal " and I shall explain the reason for this . The jnurnal is
ready to puhlish unorthodox research lind unusual or nnn-ucademic therapeutic
methods which usually do not find their WIlY into other perindicals. But bcing a
journal devoted to cancer research we want to publish results lind hypotheses hased
on results . We admit empirical results and der ived speculations. But if we consider
your paper, I could not find any results conccrning cancer patienls. What [ expected was
a scries of canccr patients who have received ozonated rnajor autohaemotherapy and
were objectively improved, if not cured . I did not find either experimental cancer
results. I am sorry for this rebuttal for which I hope you will not be angry " .

What I cannot understand is why some physicians (see Chapter 24) who claim to
have treated hundreds of cancer patients with spectacular results do not publish their
results at least as "best case series".
Believing that ozonetherapy can be beneficial particularly in elderly patients
where chemotherapy makes Iife unbearable, I submitted the article entitled
 How I CA ME TO STUDY OZONETHERAPY 19

"Ozonetherapy as a possible biological response modifier in cancer" to "Forschende

Komplernentärmedizin". After several pleasant discussions with Dr. Klaus Linde, it
was published (Bocci, 1998d) followed by an artic1e by Prof. Beck et al. with the
welcome provision that the two referees Prof. E. Ernst and Prof. K. Zänker would
also publish their critical comments at the end .
They are worth reading because Prof. Ernst is a world authority in the field of
natural medicine.
" Zu den Arbeiten von Bocci und Beck et a\. Forsch Komplementärmed 5 76 (\998)
Kommentar E. Ernst
Rather than tediously addressing each debatable statement in detail, I will attempt to
provide a general , critical and hopefully construct ive comment to the articles by Bocc i
and Beck et a\.
Bocci's paper is a focused review of ozonetherapy for cancer. The author presents
the theoretical background why ozonetherapy might work. He is objective in that he
points out that, at present , the proposed mechanisms are nothing more than speculation.
He is honest in that he states that many often-voiced opinions on the subject 'are
worthless' and that 'there is no serious evidence that ozonetherapy can be beneficial to
cancer patients'. He is realistic in that he acknowledges that only RCTs (randomised
clinical trials) can answer whether ozonetherapy is useful at all. Yet Bocci is uncritical
when he states that ozonetherapy has 'practically no side-effects' (see below) .
Beck et a\. also elaborate on speculative mechanisms of action. But in doing this
they are far less critical and seem to imply firstly that these mechanisms are highly
plausible and secondly that, because of their plausibility, one should conclude that
ozonetherapy works c1inically. Beck et a\. also admit that no c1inical trials exist, but
they play down the importance of this fact. They suggest that this void is excusable
since there are no funds available .
In complementary medic ine we are often faced with a depressing lack of funds. Yet
there arc dozens of RCT of, for instance, acupuncture. Acupuncture is also not backed
by the pharmaceutical industry . Furthermore , it is not entirely true to say that no RCT of
ozonetherap y exists. I know of at least one which , ironically, was even published (as an
abstract) in this very journal [I].
Both articles have one thing in common : they are narrative reviews in which the
authors present those bits of the total evidence that best fit their, hypothes is. Negati ve
data, it seems, are discretely omitted. The above-mentioned trial [I) represents such
negative data, but there is much more. For instance , Diehm and Rechtseiner dedicate
more than 30 pages to a powerful criticism of ozonetherapy [2]. They give an overview
of clinical studies (all uncontrolled ), offer a biting critique of the plaus ibility of the
speculat ive mechan isms of action, and provide a summary of the safety data .
For obvious reasons the safety issue of ozonetherapy is of utmost importanee. In
both articles, the risks of ozonetherap y are minimised. Yet several reports of serious
complieations exist . Schmitt, for instance, reports five fataloutcomes [3]. Those who
find it hard to access German doctoral dissertations might still be able to read the Lancet
where recently 2 cases of hepatitis have been reported [4]. Proponents will, of course,
counter that these cases are mere examplcs of improper use of ozonetherapy. I would,
howevcr, insist that even if this were so, it would then represent an important safety
issue that must not be swept under the carpet .
In my view , the most worrying aspect of these articles is the fact that Beck et a\.
defend ozonetherapy referring to the physician's freedom of prescrib ing ('arztliche
Therapiefreiheit'). Surely this much treasured privilege is not applicable to a treatment
with no solid evidence for efficacy and worrying evidence of potential hann to the
patient.
'Narrative or unsystematic reviews , as they are called, are a proven danger to health'
[5]. I support this rather provocat ive statement by Andrew Vickers and leave it to the
reader to decide to what extent it applies to the above articles [6].
20 CHAPTER2

So what could be a constructive way ahead for ozonetherapy? Firstly, somcone

should publish a systematic review of this treatment including the pros and cons.
Sccondly, on thc basis of such a review , one could fonnulatc a few promising and
testable hypotheses . Thirdly, one could document a few cases in the form of a 'best casc
series'. If this is possible, it might change the negative attitude of opinion leadcrs wh ich
Bocci laments. Fourthly, randomizcd trials should bc carried out by teams consisting of
proponents and informed opponents of ozonetherapy. Such trials need careful planning
and adapting to the rcsearch qucstion (e.g , should they be carried out versus a placebo
or a standard treatment?). Fifthly, thc results of such investigations might require
replication. Sixthly, thorough research should address, in parallel with the abovc
activities, the safety issue. This rnay seem like a long and stony road to go down, but in
the interest of our patients there must bc no short cuts and no double standards in
making as sure as possible that we are doing more good than harrn [6]. As it stands now
I'm not convinced that ozonctherapy fulfils this most elernentary criterion.
References
1 Kraft K, Stcnkamp E, Vetter H: Effect of autohernotherapy with ozonc on
cardiovascular risk factors in mildly hypertensive patients, Forsch Kornplementarmed
1995 ;2 :352 .
2 Diehm C, Rechsteiner Hl : Wer hat Rech!'! München, Zuekschwcrdt, 1987 , pp8-39.
3 Sehmitt H: Zur Ozontherapie. Med Dissertation, University of Marburg, 1982 .
4 Gabriel C: Transmission of hepatitis C by ozonc enrichment of autologous blood .
Lancet 1996;347:541.
5 Vickers Al, Smith C: Analysis of the evidence profile of the effcctivcness of
complementary therapies in asthma. Compl Ther Med 1997;5 :202 -209.
6 Ernst E: Systematische Reviews losen traditionelle Ubersiehtsarbeiten ab. Perfusion
1997;5 :157 .
Prof. Dr. E. Ernst
Department of Complemcntary Medicine
Postgraduate Medical School
University of Exetcr
25 Victoria Park Road
UK-Exeter. EX24NT"

I feit that Prof. Emst's comments, although instructive, were not totally correct
and I asked to publish my counterpoint as folIows :
"Antwork der Autoren . Forsch Komplemcntärmed 5:78 (1998)
Antwort Bocci
I regret that Prof. Ernst did not have the opportunity to read my prcvious monograph
[I], in wh ich the problern of the potential toxicity of ozone was exhaustively discussed .
At least four deaths have been due to foolish direct intravenous administration of gas
(02-0J) in spite of the fact that, since 1984, this route has been prohibited. The cases of
hepatitis are not pcrtinent because all ozonctherapeutic procedures are carried out with
disposable material and the two cases in question were duc to a different approach (UV
treatment of blood in contaminatcd glass container) that has nothing to do with
ozonetherapy.
My personal expericnce based upon ample biological and clinical results is that , if
ozone is used properly in a concentration up to about 70 J.lg/ml pcr g of blood , there are
neither acute nor ehrenie (up to 60 sessions) toxic effects. The only problem is
rcpresentcd by a progressive deterioration of venous access due also to previous
prolonged therapies.
On the othcr hand, intramuscular injection of 02-0J (when used for the treatment of
hernial disc) is very painful for a few minutes but, iuterestingly, pain elicits analgesia
subsequently and appears to alleviate back pain in somc patients .
As far as cancer is concerned, either as minimal residual disease or metastatic
cancer in patients with a good Karnovsky's index refusing radio- or chemothcrapy, a
'best case se ries' should be cvaluated as soon as possible using ozonated
 How I CAME TO STUDY OZONETHERAPY 21

autohaemotherapy with the options of either rectal ozone insuffiation or body exposure
[I J. I could not agree more with Prof. Ernst about the need of standardizing the
procedure, the schcdule, the tolerance and the relevance of RCT, either versus a
standard treatment or/and versus O 2 autohacmotherapy. There is indeed the compelling
need of assessing the importance of the placebo effect because
psychoneuroimmunologic effccts are most likely involved with this approach.
Physicians practicing complementary medicine, bclieving that their treatment is
effective, feel that randomization is unethical. Most of them do not care about the
possibility of a 'placebo effect' and actually they welcome it. This attitude is totally
wrong and prevents acceptance of ozonetherapy. On the other hand , results obtained
with a positive, well-conducted randomized pilot trial could be published by a peer-
reviewed jeumal and stimulate confirmatory studics.
I am grateful to Prof. K. Zanker for his constructive criticism and I would like to
add that the possibility of an autovaccination (section I) is becoming more substantial
after the demonstration [2J that ozone induces the expression of heat shock proteins
(HSPs). The interesting and novel aspect is that HSPs chaperone the antigenic repertoire
of tumour cells and that the HSP-tulJlour antigen complex can elicit a potent T-cell
response against the autologous tumour [3J.
Finally, I take the opportunity to say that the future of ozonetherapy is in our hands
and until the time that we will produce biological and clinical data according to the
standard set by orthodox medicine, this approach will remain in the hands of
practitioners and quacks .
References
I Bocci V : Ozone as a bioregulator. Pharmacology and toxicology of ozonetherapy
today . J Biol Regul Horneost Agents 1996 ;10:31-53 .
2 Su WY , Gordon T : In vivo exposure to ozone produces an increase in a 72-kDa heat
sho ck protein in guinea pig . J Appl Physiol 1997;83 :707-711.
3 Tamura Y, Peng P, Liu K, Daou M, Srivastava PK : Immunotherapy of tumors with
autologous tumor-derived heat shock protein prcparations. Science 1997 ;278 :117-120."

In January 1995, I sent some of our publications to the editor of the prestigious
joumal "Free Radicals in Biology and Medicine" (FRBM), one of the most expert
chemists on ozone and asked if he was interested in examining a review on
ozonetherapy. He readily answered:
" As far as writing a review on the use of ozone for therapy, I am skeptical. The little
reading 1 have done in this field suggests to me that there is a great deal of bad science
going on here . Ozone is toxic, no matter how you deal with it . Do you have
publications in this field that would argue otherwise?"

On reading it I was a little bit shocked: I could not agree more about the "bad
science" but I found the sentence about toxicity overly dogmatic. He was certainly a
good chemist but was not reasoning as a biologist: what is true in Chemistry is not
necessarily correct in Biology and Medicine. A few days later I replied to hirn:
"I never doubted that FRBM is an excellcnt journal and I mentioned that ozone therapy
has been badly used by quacks or totally inexpert physicians. I regret that you , as the
vast majority, arc taking a skeptical position towards a properly performed ozonated
autohemotherapy. If you remember, I was saying that ozonc can be highly toxic and
indeed any drug is. All depends on selccting the therapeutically active and minimally
toxic dose . Fortunately, although the therapeutic window is narrow, we can obtain this ,
thanks to onr highly integratcd antioxidant systems. By the same token , NO and H20 2
can defend us from bacteria. I learnt that in Biology one should never be dogmatic.
Finallv I would like to assure vou for what it mav serve, that ozone toxicitv can be
cntirely controlled and if we ~sc judicious doses we can achieve a clinic~1 benefit
22 CHAPTER2

without any side effeet. But oncc again I repeat that ozone must be carefully used as a
drug knowing what we are doing .'

I deeided not to submit a review to FRBM. But after almost three years. In
Oetober 1997, I reeeived a letter from the editor that truly surprised me:
" Dear Prof. Boeei,
I very mueh cnjoyed your review on ozone in J. Biol. Regul. Homeost. Agents .lQ3 I -53
(1996). I wonder if you would be kind enough to send me a reprint. I had always
thought that " ozone therapy" was a very bad idea. but you re viewcd the literature
beautifully.'

This eneouraged me and I sent him the following letter:

" I enjoyed reading some of the papers on oxidative stress and defence mechanisms in
plants and I fee I that the idca of Forum presentations is a good olle because it is ablc to
rcally focus the attention of the readers on a partieular topie.
In our previous correspondenee I eannot forget your sentenee "o zone is toxic any
way you deal with it". I agree that ozone is toxic , like many other drugs, but in Biology
and Medieine dogmatism is dangerous . Besides the providential activity of man)'
oxidants produced by our cells such as H2 0 2, HOCI , NO , etc . it is beeoming vcry clcar
that some crucial pathways are physiologically activated by oxidants. Cells. animals and
plants can produce and then control them by virtue of a versatile and potent antioxidant
system, particularly when wc can apply, as in the casc of ozonetherapy, a "transicnt and
calculated oxidative stress" . Moreover it is becoming evident that, providing time and
suitable conditions, Iiving creatures ean upregulatc the antioxidant systcm thus
becoming tolerant to an oxidative stress that otherwise will deslroy them. This situation
is of great interest and raises the possibility, until now unthinkablc, to either understand
mechanisms apt to block oxidative stress or/and to possibly stabilizc the progression of
degenerative diseases in humans , I have discussed this exciting possibility of " oxidative
stress adaptation" 01' "oxidative prcconditioning" in two reeent papers (Med .
Hypotheses 46 , 150-4 , 1996 and J. 8iol. Regul . Homeost. Agents 10,31-53 , 1996) and I
hope you reeeived the lalter. It may be the time has eome to evaluate the other side of
the coin 01'. in other words , whether ozone, whcn used properly, ma y display useful
aetivities that could be exploited for treating some human diseases , when orthodox
therapies have proved to be ineffective.
I would be grateful if you call consider my suggestion on a future topie that may be
entitled : may ozone have useful therapcutic acti vity ? 01' may we tame ozone toxicity
and may we deline its therapeutic index?
Obviously this is jusl a very prcliminary suggestion that I am afraid will be ver)'
hard to swallow by almost all Ameriean scientists . However, just in ease )'011 are
interestcd, I would be glad to write a general overvicw.
Thanks for your attention."

The Editor kindly answered me:

"Thank you for your letter of October 7. I am glad YOII enjoycd our FORUM on 07.01lC
and defense mechanisms in plants , I am always interested in good ideas for FORUMs
but ozone is scary to me . I know therc arc papers that argue that ozone therapy has some
merit, but I have always been uneasy with this eonccpt. Unlike hydrogen peroxide, our
body does not produce ozone. So it is not an oxidant that we producc and lcarn to
regulate . It is true there is some cross tolerance of one oxidant for another, but I am not
sure that the damage ozone docs would be overcome and make it worth whilc trying to
develop cross toleranee by the use of ozone itsclf.
I would, on the other hand , be willing to entertain a very short " m in i-review" from
you 011 the subject of the bcneficial cffccts of ozone thcrapy. By a mini-review, I mean
something limited to 10-12 pagcs. typcd double spaced, including all figures and
references . I would get it reviewed by sorne people that I think might be critical, and I
 How I CAME Ta STUDY OZONETHERAPY 23

would welcome suggestions from you of reviewers to use as weil . If you are interested
in pursuing an article along those lines, just let me know when you think it might be
ready."

On reading it, I became enthusiastic and I thought that the golden opportunity to
start a dialogue had appeared. Thus I replied:
"Many thanks for your lctter of December 16 commenting on ozone toxicity. I share
your concern only in part and it has always struck me the fact that in Europe, millions of
people have undergone ozone therapy and there is not one significant note of
dissatisfaction, provided of course one uses ozone with care , I do not think that all these
paticnts are absolutely uncritical. The ozone concentration is the critical point and
whoie human blood should not be exposed to concentrations higher than 80 ug/ml per g
of blood. Unfortunately, in the past, too many experiments have becn performed using
washed erythrocytes suspended in saline and this unphysiological situation, due to the
lack of crucial antioxidants , has been totally misleading .
Although I have still somc reservation , on the whole, my personal experimental and
clinical experience compels me to go ahead until, hopefully , I can find what is really
good or bad. It is true that so far cells do not seem to produce 0 3 (one never knows
insofar NO has taught us!) but phagocytic cells produce all the RaS that 0 3 can
generate . The problern can arise when our powerful antioxidant defense system is
impaired but we have means to check it.
Thus I am glad that your vicw is now somewhat more open and you will be willing
to examine a minireview. As an afterthought , I feeI that your decision is a wise one
bccause my first idea of a FORUM now appears premature in the sense that biologieal,
and above all clinical work, on the topic is scanty and rather poor. However, if
evcntually you can acccpt thc paper , it may be good bccausc somehow it might kindIe
critical comments and hopefull y some interest . If you wish, you can even make abrief
introduction with your reservations, like the one Prof. Dianzani wrote on my 1996
review,
In conclusion, I am grateful for this opportunity and I promise you that the
minireview will be as critical as possible . After all our research work is to find the truth!
I will do my bestto send it to you at the end of next May, or slightly before, if 1
ean.
Thanks again"

I started irnmediately to work so that the minireview was submitted by the end of
March 1998. I really tried my best to explain that although ozone is intrinsically
toxic, a large part of the ozone dose is quenched by the blood' s antioxidant system,
as shown by many experimental data . I went a little further to clarify that too little
ozone can be useless (placebo effect), that too much can be toxic (easily assessed
with several parameters) and that we need to use the ozone concentration that
transitorily just overcomes the antioxidants and allows the generation of messengers
necessary for triggering biological activities. I was optimistic that, at long last, we
could open a frank and constructive dialogue between supporters and detractors of
ozonetherapy. Shortly after (May I, 1998), I received a laconic response:
"Your manuscript was sent to three referees who are expert in the field. I regret that,
after evaluation of their comments , I cannot accept your paper for publication in Free
Radical Biology & Medicine . In fact, the referee comments were so strongly negative
that I feel I cannot consider any revised version of this paper.
This is one of those situations in pubfishing research that, although distasteful to the
author as weil as to the editor, inevitably occurs from time to time. Although I cannot
consider your paper for FRBM, I wish you success in getting it published elsewhere.
With warm regards,"
24 CHAPTER2

My answer was:
"Dear Editor,
Many Ihanks for your letter of May I informing me that thc minirevicw on
ozonetherapy (MS#569-R-97-wp) has been rejected . Of course I feel depressed but
angry as weil for this , The reason is easily explained : on one hand there are thousands
of patients and physieians performing ozonetherapy and it seems that patients are like
guinea-pigs and doetors are crazy and practising Nazi medieine. On the other hand there
are the opponents of this therapy and they may weil be right, but they also may be
extremely biascd because they have no real practical experienee. In an)' event they
remain anonymous but , as I am sure they have the courage of Iheir convictions, they
ought 10 express their opinions openly as has happen cd in the Laneet rccently when the
editor published a long review (Linde et al.; Are the c1inieal effeets ... 350 :R34-43,
1997) on homeopathy that is still far from being surely effective and at the same time
two signed commentaries (see pages 824, 825), one of whieh had the title : Homcopathy
trials: going nowhere.
Opening up this problem has been extremely positive.
Would it be possible to do the same for ozonetherapy in FRB&M'!
I would be very grateful for your final dceision regarding this.
Whatever you decide, I wnuld greatly appreciute receiving und to be able to rcud
the strongly negative commcnts of the three referces. I fccl thnt it is fair thut I reut!
them as I muy Icarn something useful from thern ."

By rnid-July, I had not yet received a reply and I begged hirn to send the
referees' cornrnents:
"Dear Edilor,

I 6/07/19RR
In my e-mail leiter of June 26 I said I was eagerly waiting 10 read the three strongly
negative eomments of the referees in regard to Ihe rejeeted minirevicw. As to datc I
have not yet reeeived them , I am asking again if you would be so kind to forward thcm
to me . Probably you do nol agree but I think it is the right of the Author to read Ihem
and the duty of the editor 10 provide thcm. Otherwise the whole system is very unfair."

After four days, his answer was:

" Dear Dr. Bocci ,
Yes, we nonnally do send eomplete referee opinions to authors, whether we aeeept 01'
rcject an artiele. However, in this case I have deeided not 10 do so . The reason is that the
opinions were short and basically state that the work is not believed aud has nol been
tested by true eontrolled trials , All the referees are truly outstanding namcs in ozonc and
YOIl would know and respeel thern . The opinions would do you no good in rewriting,
and probably wnuld offend you and just prolong the agony.
Your article, as you know, is on a hot lopie of eonsiderable eonlroversy. I have
taken some editorial leeway in deeiding how to handle it, and this is what I have done. I
have tried 10 act for the besl interests of the journal.
Sorry, but this one just did not work oul weil for you . Try us again with something
else on ozone and we'lI give it auother go ."

After this, there was nothing I could do except give rny final cornments to the
Editor :
" Many thanks for your e-mail lctter of July 20, where you explained your reasons for
nol sending me the referees' answcrs . However, even if cxtremely negative and even if
offensive, as you suggested they are, I would have preferred to read thcm . The main
objcctive of thc minirevicw was in fact 10 show that ozone therapy is a ver)'
controversial issue and I pointcd out pros and eons clearly. I still believe that in nOI
 How I CAME TO STUDY OZONETHERAPY 25

publ ishing it, you may have acted for the interest of the journal but certainly you have
not played a useful role for clar ifying whcther a medical treatment is valid or not.
Obviously, neither you, nor the referees have appreciated the importance of an open and
objective discussion."

The reader may find the detailed account of the correspondence between the
Editor and myself rather distasteful. It seems fair to clarify that my intention was
neither to open a fruitless dispute nor to scorn the Editor, who acted in the exclusive
interest of his journal. The point that cannot be missed is that a chemist and a
medical biologist could not reach mutual comprehension.
I never doubted that his statement "ozone is toxie ..." was correct in so far as
lipid peroxidation and ozonization products are tested either in tissue culture or are
examined in the context of the delicate respiratory system . What I was trying to
advocate is that blood is a much more ozone-resistant "tissue", Even more important
is that reinfusion of ozonized blood into the patient implies five crucial
consequences:

(I) DILUTION of oxidized lipids, lowering their concentration to

pharmacological but not toxic levels . Obviously this depends upon the
ozone dose .
(2) NEUTRALISATION due to enormous amounts of antioxidants in
plasma and extracellular fluids .
(3) DETOXIFICATION (rarely observable in vitro) due to the interaction
with many cell types more or less endowed with detoxifying enzymes.
(4) EXCRETION of oxidized lipids into the urine and bile.
(5) BIOACTIVITY without toxicity . Submicromolar concentrations of
these "toxic" compounds can act as physiological messengers able to
reactivate a biological system with a therapeutic effect.

Would it not be better that the ehernist, the biologist and the physician cooperate
to ascertain ifthis is correct?
In retrospect, I was not overly upset because another paper of mine had been
rejected , but I found the rejection of an open discussion extremely unscientific;
indeed , I thought that one advantage of a FORUM would have been to air the
opposite opinions in public .
This episode also made me wonder why in so many countries there are many
mad men like me and millions of human beings who, by undergoing ozonetherapy,
have to behave like laboratory mice, and why the saviours are unable to stop this
disgrace if it is such. Moreover, the whole story may serve to inform proud
ozonetherapists, who believe they are providing wonderful therapy, that
conventional medicine ranks them as charlatans.
As I deeply trust my aim to c1arify the value , if any, of ozonetherapy, I decided
to go on hoping that sooner or later someone would share my enthusiasm. The
unlucky minireview was purposefully revised for submission to "Perspectives in
Biology and Medicine" printed by the University of Chicago. The title itself " Is
ozonetherapy therapeutic?" was meant to be an invitation to objecti vely discuss the
issue. Ozone had become one of the most controversial gases . Indeed, I liked to
26 CHAPTER2

compare it to Janus gerninus, the Latin God with two faces : ozone is protective in
the stratosphere and toxic in the troposphere, hannful to the lungs while valuable for
vascular and infectious diseases, damaging at high concentrations and stimulating at
low doses . The editor did not approve the comparison with Janus and I had to adapt
the text to his Iiking (Bocci, 1998b). Outing this period, two English physicians (one
of German origin) visited my lab and, in telling me that they could practise
ozonetherapy in England, they urged me to publish a paper in an English medical
journal that was fairly broadly based and had an editor open to new perspectives.
Mindful of past experience, I prepared a less fanciful text than the previous one and
asked the question: does ozonetherapy have any future in medicine?
The editorial process was lang and slow ; I was asked to include all possible
clinical data and I obliged the request since the editor correctly intended to infonn
even the G.P . of all possibilities offered by ozonetherapy (Bocci, 1999a). After the
publication of these two articles, I expected to receive plenty of reprint request
cards , as used to happen in the 1980s when I was working on the metabolism and
pharmacokinetics of IFN. The requests numbered about 40, i.e. almost nothing in
view of the fact that the papers were reported in the index of the weekly Current
Contents. They were from Cuba , Poland, Germany but very few from the USA and
England. Perhaps two were from ltaly! To me, this meant that ozonetherapists either
do not read or are not interested. Yet this is not surprising, busy as they are to work
in their private clinics! Obviously orthodox physicians or scientists were either
uninterested or considered ozonetherapy to be rubbish.
However, the reader of this book should know that I haveri't told this story to be
pitied, To the contrary, I have chosen this unusual subject by my free will, I will
pursue it and I will not miss any opportunity to remind ozonetherapists that if we
don 't begin to work SERIOUSLY we will remain only CHARLATANS. To make
the argument c1ear, Ireport the reply of the deputy editor of the New England
Journal of Medicine. This first class journal had published a review article on
hyperbatic oxygen therapy, a procedure that is absolutely life-saving for some
diseases but which, in my humble opinion, could be substituted by ozonetherapy for
many other diseases, with the advantage of simplicity, rapidity, minimal cost and
most likely enhanced efficacy (Chapter 35) . In the USA , there are many facilities
but in Italy there are too few hyperbaric chambers, one of which recently exploded
in Milan, killing several people. Unfortunately, it is rare to find a physician who
honestly and intelligently thinks that the two approaches could weil be
complementary.
For this reason, I proposed to submit an article explaining the state of the art of
oxygen-ozone therapy. The response was :
" I am SOfTY, but a review on oxygen-ozone thcrapy would not be right for our
journal. Our rcviews on therapies foeus on Ireatments which are in general use and for
whieh there is a substuntial hmly of scientiflc evidence. Our c1inieal readers do not
want rcviews on methods that are insufflciently resenrched, even if they ure
promising, Thank you for your interest in our journal ."

What can I say as a btief conclusion? I must admit that occasionally I feit
discouraged, but not too much because, perhaps wrongly , I have caught a glimpse of
how ozone works (Chapter 13). It is hard to understand why other scientists do not
 How I CAME TO STUDY OZONETHERAPY 27

even want to think about it, and apriori reject the idea as non sense, when many
patients have gratified me immensely just by saying how well they feel doing
ozonetherapy. I can only think that my 47 years of lab work and reading so many
good papers have helped me to examine this problem from several angles . I shall
discuss later on how today we can reconcile this old and "barbaric" approach with
the fantastic progress and promises of modem medicine. I had no reason to be
depressed because several objections raised to my endeavours are indisputable and it
is up to us to find the correct solution. However, on the whole about 15 experimental
papers have been published fairly easily , although some were published after a long
struggle. They are easily traceable in the scientific literature even though, as fashion
goes, my "impact factor " is low . It was indispensable to publish because if the
problem is not exposed to the criticism of the medical world , we will forever remain
in limbo.
I would like to thank all the editors who, with more or less reluctance, have
published our papers but I am particularly grateful to Prof. I.L. Bonta and Prof. F.
Dianzani for their generous and constructive decisions. In brief, the outlook for the
future remains dreary but at the same time challenging.
Finally, I must confess I have been surprised that the worst offences I received
have come from the Italian Society of Oxygen-Ozone Therapy (SIOOT). In
November 1998, without being asked , I was nominated President of the
International Congress of Ozonetherapy to be held the following March at the
Glaxo-Welcome Research Centre in Verona. At first, I was puzzled but I decided to
accept when I received the written assurance: firstly , to deliver the commemoration
of Schönbein ; secondly, to give the Schönbein opening lecture and to receive the
related award; thirdly, and most important, to organize and moderate a round table
entitled "The ozonetherapy dilernma", for which I had perrnission to invite four
dist inguished American scientists who oppose the use of medical ozone, as well as
one German, one Cuban, one Italian and one Japanese who favour its use. I had
dreamt that we would be able to open a dialogue that would have important
consequences. I must mention that the organizer, who was president of SIOOT,
signed a document stating that travel expenses would either be anticipated or
reimbursed to the lecturers. After a couple of months, I was informed that , with
regard to the scientific exhibition, only one firm producing ozone generators would
be allowed to be present and two competing firms, one German and one Italian,
would be forbidden unless they paid an exorbitant fee. I must add the not irrelevant
detail that the only firm allowed to exhibit its instruments was owned and directed
by the president's wife.
Although I was not responsible for the administrative part, I was indignant at this
plot because the Congress was officially organized by the scientific society and not
bya commercial enterprise; ifthe latter had been the case , I could not have objected.
Therefore, I wrote letters to the president and to the SIOOT Committee affirming
that any respectable scientific society could not impose such grave discrimination.
As I explain at length in Chapters 6 and 7, the future of ozonetherapy depends at
least in part on the reliability and validity of ozone generators. Therefore, a truly
scientific Society has the duty to present all possible instruments to the participants.
I firmly believe that monopoly is wrong and that we can improve the quality and
28 CHAPTER2

reduce the price of instruments only by fair competition . Exercising my patience, I

wrote a letter trying to change this poor planning, which in the end would discredit
everyone . Even though the programme had been printed a few days earlier, I
considered resigning . However, I discarded this possibility because it would have
been eagerly accepted, things would possibly have got worse, the round table would
have gone astray and nobody would have been able to oppose the monopoly . It was
frustrating to realise that the much boasted "World Congress of Ozonetherapy" for
the next millennium was not considered to be a memorable scientific event but
instead a trivial mercantile business. I then wrote to the SIOOT President stating that
I would not resign , that the Schönbein award should be given to the University of
Siena to be used for research purposes only as I would not accept it and that, in the
opening speech , I would amply criticize the shameful discrimination and the blatant
use of the congress to seil the President's instruments . Certainly it was not a good
start for a Congress! Needless to say, I fell into disgrace but I surely said everything
I had promised to say at the Congress, even though I was despised the whole time.
The round table was very stimulating and at least one important point emerged very
c1early: that the ozone toxicity noted in tissue cultures cannot be equated at all to
what we are doing with blood ex vivo. At least Prof. C. Cross, being a physician ,
could realize the difference and honestly appreciate the results obtained by Dr.
Werkmeister in patients with severe ulcers (Chapter 24).
Throughout the years, I have participated in innumerable Congresses with grcat
pleasure and scientific reward . Yet the Verona Congress was the black sheep, a
really tertible three days for me, even though I was glad not to have shrunk from my
respons ibilities . Another typical episode was that the agent for a German instrument
was not allowed to participate freely and had to be escorted by the police to enter! I
apologized to the foreigners speakers for this exceptional tension and I was gratified
by the empathy of many participants. There were also unpleasant sequelae . To spite
and annoy me, the SIOOT President blocked the printing of all abstracts that I had
reviewed and prepared for publication. However, this also had a boomerang effect
on him because the registered participants had paid the fee and wanted the abstract
book. Moreover, in spite of my insistence, he has never reimbursed the travel
expenses of three American scientists and, in the same vein, never paid the
Schönbein award to my University. The whole affair has been so disgraceful and has
no equal in any other scientific meeting . I decided to tell this incredible story
because the reader must know what is behind ozonetherapy in Italy. Some two
months later, SIOOT expelled me as a dishonourable rnember, an expulsion that J
instead consider a great honour. At my age, I have become aware that, in the
medical field, there is always a huge business behind the development of drugs but I
have never encountered such indecent behaviour. This was the straw that broke the
camels back and, with many other colleagues in September 1999, we founded a
new society. By majority vote, it was named the International Medical Ozone
Society (IMOS , Italy) with the idealistic goal of creating a world organization to
promote and develop ozonetherapy. In my opinion, this is unrealistic for the time
being. Some six years ago, I organized a meeting with the representatives of Russia,
Germany, Holland , Spain, Austria , etc. and I was shocked when Prof. E.G. Beck
(Gennany) said that he could accept only an aggregation of German-speaking
 How I CAME TO STUDY OZONETHERAPY 29

countries! Nonetheless, our non-profit society has scientific purposes only and
forbids any commerciallinks.
The reader must be informed that the truly International Ozone Association
(IOA) has been in existence for at least three decades, organizes a world Congress
every two years, has many members and is dedicated to supporting industrial
applications of ozone and water disinfection. It also publishes a good scientific
journal "Ozone in Science and Engineering" (OSE), which is the only one known
for ozone and has an impact factor of 1.085 (2000). The only problem is that until
very recently, the editors chose to steer clear of medical ozone . This has been a
serious but comprehensible drawback. I am sure that a reliable International Journal
of Ozonetherapy devoted to publishing serious controlled biological and c1inical
papers would be a great advantage for the cause of medical ozone, in contrast to
worthless newsletters printed only for mercantile purposes. Yet how and from where
could we rely on a good stream of valid papers? This is yet another vicious circ1e
that cannot be solved if ozonetherapy is carried out by physicians in private practice.
To do proper research, we need government support, but in both Germany and Italy
there has not been the will to help. Only during the last few years has there been a
slight opening for some approaches ofnatural medicine.
I greatly appreciated that Mr. N. Naef, president of the 10A Comrnittee for
Schönbein celebration in Basel (October 1999), invited me to give a lecture on
"Ozone in Medicine". On that occasion, r was fascinated by the keynote lecture
delivered by Dr. Molina.
Unfortunately my talk was scheduled to be the last one, which was too bad
because many people were in a hurry to leave (me too), and very few physicians
were present. After a very lengthy revision (one referee asked for more c1inical
evidence and less biochemistry while the second one wanted to expand the
biochemical part) , the editor accepted the manuscript for .OSE, saying that it was
time to publish a paper on ozonetherapy (Bocci , I999b; Bocci et al., 200Ia). The
XV rOA Congress will be held in London at the Imperial College (September 2001)
and we will have a day-Iong session on ozonetherapy. For the future , Dr. R. Viebahn
and r will try to convince the new president Mr. R. Lowndes and the new Editor Mr.
B.L. Loeb to allow some space in the journal for a few reliable papers on
ozonetherapy.
Thus , although it will be very difficult, I will erase the awful Verona story from
my mind . However, I will still have to deal with ozonetherapists without any
scientific interest or with those boasting about great uncontrolled results just to
acquire a good name and earn more money. Obviously, I cannot prevent that a few
unprofessional persons taint the field and these are the worst lot because they will
discredit ozonetherapy. At least for now, our society is armed with several good
intent ions: the primary objective is to implement each year effective theoretical and
practical teaching courses for serious physicians; in 2001, after the previous exciting
experience, I will direct the third course at my University. Another two courses will
be held in northern (Padua) and southern (Bari) Italy. Moreover, we will organize
periodic meetings to update selected topics . r organized the 1si IMOS Congress at
my University in November 2000 with reasonable success.
30 CHAPTER2

I believe that good training is crucial, so that the ozonetherapist will understand
what he/she is doing and will be able to change the dose , schedule, etc. depending
on the patient and the disease. It is disheartening when , during a meeting or too
frequentlyon the phone, a physician asks me abruptly: what ozone dose should I
give in such a disease? It is as if he is asking for a recipe to make a tart; indeed ,
several physicians openly criticised my previous book (in Italian ) because it did not
provide recipes. It is most unfortunate that, in the past, a physician could become an
ozonetherapist in a few hours provided he bought a certain instrument.
The second ambitious project is to establish and keep up to date precise and
complete regulations on how an ozonetherapy clinic should be set up, organized and
run. Hopefully, this project will soon be presented to the Italian Ministry of Health
for discussion and approval.
If we succeed in this endeavour, it may be hard on inexpert and superficial
ozonetherapists at first, but eventually it will be a great improvement because some
clinics today are of substandard quality. A third goal will be to introduce
ozonetherapy in every hospital as a free treatment for vascular and infectious
diseases, which are, in my opinion, the war-horses of ozonetherapy. This will be a
difficult project, but certainly not impossible if we can find favourable politicians
and unprejudiced hospital directors. University clinics appear to be a farfetched
target, mostly because they have no money to support expensive clinical trials .
However, if we can take the first step we may in time obtain the badly needed
clinical results: if they are positive, we will move ahead rapidly and if they are
negative, we can conclude that ozonetherapy is only a type of placebo . Fourthly, we
will favour fair competition among the manufacturers of ozone generators. So far, I
have been gratified to see that our simple philosophy is weil accepted and enhan ces
improvements of the apparatus.
I have summarized an ambitious but not unrealistic programme, but as things go
it is not impossible that I will also be expelIed from the new Society. If I am lucky,
my odyssey in the field of ozonetherapy will continue and although we may fall into
new ambushes (the ferocious Scylla and the treacherous Charybdis), we can hope
one day to happily land on Ithaca or, in other words , to be gratified by the extensive
use of ozonetherapy for all patients.
 CHAPTER3

OZONE IN NATURE

Ozone is naturally present in the atmosphere surrounding the earth . In the

stratosphere between 20 and 30 Km from the earth's surface , there is an ozone layer
that may reach a maximal concentration of 10 ppmv (parts per million I :106) at
about 22 Km. The ozone layer is extremely important because it absorbs most of the
ultraviolet (U V ) radiation «290 nm ) emitted by the sun and damaging biological
systems : UV rays include band A (316-400 nm), mainly responsible for suntan, and
bands Band C (from 100 up to 315 nm) which are far more mutagenic. Ifthey reach
the earth's crust, they can enhance skin carcinogenesis during Summer; indeed, it
has been estimated that a 10% drop in the level of stratospheric ozone could cause a
25% increase of skin carcinomas and melanomas.
With the progressive increase of atrnospheric oxygen, biological systems started
to evolve rapidly some two billion years aga (Kasting, 2001) . Since then, the ozone
layer has evolved as a crucial protective agent. In the past, the ozone concentration
in the stratosphere has certainly varied according to solar activity but probably was
regulated by the UV emission itself. Thus ·increased UV irradiation was balanced by
an increased ozone concentration able to maintain the protective effect. The
following reaction indicates that ozone is continuously formed by the action of short
wavelength solar radiation on molecular oxygen :

O 2 + UV « 242 nm) --~) 0 + 0

2 O2 + 2 0 ) 203

While UV emission favours the ozone-forming reaction, the natural dissociation

of ozone and particularly nitrogen oxides (NO x) and chlorine control ozone
abundance by destroying large amounts of ozone through a catalytic ehain reaetion
meehanism (Crutzen, 1971; Johnston, 1971).
Stolarski ( 1999) mentioned that by 1972 the idea that ehlorine destroyed ozone,
which dated to the early 1900s, was revived but that sporadic volcanic emissions of
chlorine did not seem important. Then Molina and Rowland (1974) developed the
CFC ozone theory : CFCs are compounds that are insoluble in water, unreactive and
do not absorb visible light. Industrial CFCs were developed in the 1930s and used as
foam-blowing agents and refrigerant fluids, replacing ammonia and sulphur dioxide,
both toxie and flammable. CFCs were very effective as refrigerants but, being so
stable, they progressively aceumulated in the atrnosphere without anybody knowing
their fate or worrying about environmental problems. Unfortunately, it tumed out
they were slowly transported by ascending currents to the stratosphere above the

31
32 CHAPTER3

ozone layer. There, these molecules were destroyed by short-wavelength radiation,

freeing chlorine atoms that started the catalytic ozone-destroying cycle. One single
chlorine atom can destroy thousands of ozone moleeules before being transported
back into the troposphere. Excessive destruction of ozone that is no longer
counteracted by increased ozone synthesis is the reason for the famous "Antarctic
ozone hole", which covered an area about 3.5 times larger than the USA in 1998
(Fig. 2). Ozone depletion takes place over Antarctica in Winter because the
extremely low temperature (down to -80°C) allows the extraordinary fonnation of
thin ice clouds in which ice crystals "act ivate" chlorine that destroys ozone more
efficiently than anywhere else (Molina, 1999). The CFC theory also explains how
NO x are transported up into the stratosphere by nitrous oxide (NzO) and, together
with sulphate particles from volcanic eruptions and the possible influence of the II
year solar sunspot cycle , make the forecast for ozone recovery difficult. However,
the international effort to halt ozone depletion by phasing out CFCs has been a great
success (Prather et al., 1996) and there is hope that the hole will disappear by 2050
(Schrope, 2000).

Figure 2. The ozone hole (indicated by arrow) over the Antarctic as seen hy a satellite.

Chaotic and short-sighted human activities in the last 7-8 decades have led to a
dangerous environmental disorder, not only for stratospheric ozone but also for
tropospheric ozone . The troposphere extends from the earth's surface to the
tropopause, which ranges from 8 Km above the poles to 17 Km over the equator .
The average tr0f-0spheric amount of ozone ought to be far less than 40 ppbv (parts
per billion 1:10 ), i.e. far less than that in the stratosphere. Yet in large metropolises
 OZONE IN NATURE 33

like Mexieo City, but also ' in Florenee and Milan, ozone ean reaeh toxie
eoneentrations during Summer. Anthropogenie emissions, mainly of NO x = NO +
N0 2 but also of methane (CH 4 ) , earbon monoxide (CO) and sulphurie eompounds,
have eaused a progressive inerease of the ozone eoneentration up to 100 ppbv or
more (Zimmermann, 1999). A large series of intereonneeted reaetions (Ehhalt,
1999) leads to the formation of NO , N0 2, hydroxyl radieal (OH-) and
hydroperoxyradieal (H0 2- ) , whieh aet as eatalysts and maintain a reaction chain
recycling OH·:

CO + OH· ) CO 2 + H
H+0 2 ) H0 2-
NO· + HO/ ~ NO/ + OH·
N0 2• + UV ) NO· .+ 0
0+0 2 )~
N0 2·+ O 2 (+UV)~ 0 3 + NO-
NO·+0 3 ) N0 2·+02
H0 2• + 0 3 ) OB" + 20 2
CO + 0 3 ) CO 2 + O 2

CO + 20 2 (+UV) ~ CO 2 + 0 3
OH· + 0 3 ) H0 2- + O 2

In daylight eonditions, the NO x emissions derive (in deereasing order) from

industrial processes, vehieular traftic, soil exhalation, lightning, biomass burning in
tropical areas (Thompson et al., 2001) and air traffic (a small amount but expeeted to
inerease) (Johnston, I 97 I). Oxidation of methane or higher hydroearbons in the
presenee of NO x leads to the formation of peroxyl radieals (R0 2 -) that reaet with
NO·,

R0 2- + NO·--~) RO- + N0 2•
N0 2• + O 2 (+UV) ) 0 3 + NO·

thus eontributing to the produetion of ozone. Indeed, one NO x moleeule ean produee
hundreds of ozone moleeules before being destroyed by photolysis or removed by
deposition (Zimmermann, 1999).
At the street level , ozone has beeome the main toxieant not only for the
respiratory traet but also for the eyes and nose and to a lesser extent the skin. It
should be noted that it is the acidic mixture eomposing the photoehemieal smog that
potentiates the irritation of mueosae (Warren and Last, 1987) . Certainly ozone has
aequired a bad name beeause the daily information in mass media about its
concentration in the air has ereated ozone toxicomania. Thus it is obvious and
reasonable that the lay public begins to wonder why ozone, being toxic in the street,
should be used as a therapeutie agent. Moreover, ozone-induced lung inflammation
has beeome an important problem for health authorities, toxicologists and
respiratory disease specialists, as demonstrated by the weekly publication of 3-4
34 CHAPTER3

papers in important journals such as "Am. 1. Respir . Crit. Care Med .", "J. Appl.
Physiol .", "The Lancet", etc . (Aris et al., 1993; Kelly et al., 1995; Kinney et al.,
1996; Torres et al., 1997; van Hoofet al., 1997; Broeckaert et al., 1999; Frampton et
al., 1999; Foster et al., 2000; Cho et al., 200 I; Long et al., 2001) . Ozonetherapists
ought to be aware that, while there are practically no studies on ozonetherapy,
studies on ozone toxieity thrive , c1early indicating how much official medicine
disregards ozonetherapy and worries about toxieity . This trend is reinforced by
numerous high -quality studies of ozone toxicity in plants, which interestingly lend
support to the potential importance ofthe induction of ozone tolerance (Chapter 22).
There is plenty of evidence that acute and chronic inhalation of ozone is toxic for
the respiratory mucosa and there are indieations that asthmatic patients are at greater
risk than normal subjects (Hatch, 1991; Bayram et al., 2001) . It is more difficult to
decide if chronic exposure can predispose to lung cancer because ozone toxicity is
compounded by many other substances present in the photochemieal smog . Animal
studies do NOT support the idea that ozone is a pulmonary carcinogen (Witschi et
al., 1999). However, it is more than obvious that ozone shou1d never be inspired,
because the respiratory tract Iining fluids (RTLFs) have a negligible protective
capacity. Yet the point that needs to be stressed is that, on the basis of my
experience, dogmatic statements such as "ozone is toxie any way you deal with it"
cannot be accepted because human blood has a multi form and redundant system for
coping with ozone provided it is correct1y used .
It is almost needless to say that, in ozonetherapy c1inies, not even a trace of
ozone should be present and a suitable exchange of air must be assured. Although
we don 't have a highly developed sense of smell, it does help because our odour
perception threshold for ozone is about 0.01 ppmv (- 20 ug/rrr'), ten times lower
than the maximum work site concentration (WSC) of 0.1 ppmv (0.2 mg/rrr' = 0.2
ug /L) over a breathing period of one hour. According to the World Health
Organization (WHO), it is permissible to work for 8 hours when the ozone
concentration is 0.06 ppmv (120 ug/nr' = 0.12 ug /L). However, nobody should be
chronically exposed to this concentration.
It must be emphasized that, because of tolerance, olfaction cannot be considered
a reliable parameter and that a sensitive monitor analyzer with alarm should be
installed, if necessary. It is unfortunate that there are different ways of reporting
ozone concentrations in Europe and the USA. Hence Table 1 may be useful to avoid
confusion.

Tahle 1. Conversion values between 0 3 concentration expressed as ug/ml 01' ppmv.

0.1 ppmv = 0.2 mg/rn} = 0.2 ug /L = 0.0002 ug/ml

Therefore
1 ppmv = 0.002 ug/ml
1000 ppmv = 2.0 ug/rnl
10,000 ppmv = 20.0 ug /ml" = 0.42 mM
40,000 ppmv = 80 Ilg/ml* = 1.66 mM
*Usual concentrations of ozone used in O)-AHT Ülg/ml of gas per ml of blood)
 OZONE IN NATURE 35

Weber (2000) has reported the new environmental limits agreed upon by the
European Union. Until 20 I0, ozone levels may exeeed the threshold of 120 ug/rrr'
air during a maximum of 25 days per year . However, if the values exeeed 240 ug/rrr'
air for more than I hour, the population must be wamed and effeetive
eountermeasures must be taken at onee, e.g. the prohibition of vehieular traffic
(Kondro , 1999).
In eonclusion, ozone appears to be a eontroversial gas in nature . Similarly, while
it is weil aeeepted for water disinfeetion and industrial applieations, it is objeeted to
in Medieine.
 CHAPTER4

PHYSICO-CHEMICAL PROPERTIES OF OZONE

The word ozone derives from the Greek 06<0, whieh means "to give off a smell" . It
is an unstable gas of a soft sky-blue eolour, with a pungent, aerid smell already
perceptible at a eoneentration of 0.005-0.01 ppmv. The moleeu1e is composed of
three oxygen atoms (0 3) and has a moleeular weight of 48.00. It has a eyc1ieal
strueture assessed by the speetrum absorption in the infrared region, with a distanee
among oxygen atoms of 1.26 A. Figure 3 shows that ozone does not have a stable
strueture but exists in several mesomerie states in dynamic equilibrium.
, @~ @,
/0' ~ 0..... ~ 0'
.c >: ",,~ ~ <, <, < r >,> ~
8 \9- --9@ \0 --9/8 8\9- 0/

Figure 3. Structure anti mesomeric states ot ozone.

In the liquid state, ozone has a dark blue colour and, as seen in Table 2, has a
boiling point quite different from oxygen .

Table 2. The main constunts ofO, in comparison to O!.

Ozone Oxygen
Boiling point (at 760 mmHg) -111.9 °C -182 .96°C
Fusion point -192.7°C -218.4 -c
Molecular weight 48 32
Specifie weight ofthe gas in liquid form at 1.571 1.14
-183 °C
Solubility (crrr') in 100 ml water (at 0 °C) 49.0 4.89
0.02 M 1.5 x 10- 3 M

In the liquid and solid state, ozone is highly explosive. Among oxidant agents, it
is the third strongest (03 , E"=+2.076 V), after fluorine (Eo=+3.0353 V) and
persulphate (Szot, E"=+2.866 V) . It violently reaets with oxidizable organie

37
38 CHAPTER4

eompounds sueh as benzol, dienes and alkanes. Ozone is soluble in methanol and
CFC in equal volumes and, of interest from the medieal point of view , is far more
soluble in water than is oxygen (Table 2). Dipole moments for CO z, 0 3 and H20
eorrespond to 0, 1.8 and 6.1711 x 1030 Coulombs.m, respeetively.
Ozone is formed from oxygen via an endothermie proeess:

30 2 ~(--~) 203 - 68.400 cal.

For the produetion of medieal ozone, it is indispensable to use pure oxygen

for medicaI use.
As shown above, the reaetion is reversible and the dissociation veloeity from
ozone to oxygen depends on the temperature (Fig. 4). This means that ozone is a
metastable gas with a temperature-dependent half-li fe and it is hardly storable.

1i
80
70
60
50
<40

--
E
0)
:L
Ja

C 20
0
:0:- 15
ro
....
.-
C 10
Q) 9
U 8
C 7
0
o t.
Q) 5
C
0 ..
N
0

~~6O~mm~~~~m~~m~rn~
I I I I I
1 2 1 .. 5
Time (minutes and hours)

Figure 4. Thehalf-life of ozone depends 0/1 the temperature: it decreases as temperature

increases. At 20 "e. the half-life is 40 min. while at - 50 oe it is as long as 3 months !
Therefore, for the biomedieal applieation of ozone, it must be prepared ex
tempore and used at onee for treating the patient. This means that the ozone
generator must be situated near the patient, in a suitably aerated room so that even
 PHYSICO-CHEMICAL PROPERTIES OF OZONE 39

traces of ozone in the air are quickly removed. A monitor analyzer with an alarm
may be quite useful and is compulsory in Italy. If leakage of ozone occurs, the
generator must be tumed off and repaired before further use .
For industrial purposes only, ozone can be temporarily stored in stainless steel
tanks at low temperature and in a weil ventilated storehouse. For medical purposes,
it is not possible to transport ozone, even in the neutral glass bottle used for 0J-
AHT : besides the loss in concentration due to natural decomposition, ozone may
attack the tap closing the bottle under vacuum (unless it is a special ozone-resistant
one). I am aware that careless ozonetherapists load aseries ofbottles in the moming
to perform OJ-AHT 'during the day for domiciliary treatments, with questionable
results. It is also prohibited to add sodium citrate solution (as an anticoagulant) to
ozone in a bottle for later use ; in any case , the correct sequence would be :
anticoagulant-blood-ozone. In exceptional cases, ozone could be collected in the
typic al ozone-resistant polyethylene bag used to deliver the gas for rectaI
insufflation with a manual pump . Knowing the precise delay time, one could load
the bag with a higher ozone concentration that would decrease to the desired
concentration, say in one hour. The bag should be transported in an ice-box with ice
to control the temperature. However, if the ozonetherapist must perform domiciliary
treatments, he should use a reliable portable generator, thus resolving a11 problems.
 CHAPTER5

OZONE TOXICITY:
BIOLOGICAL RISK, TOXICOLOGY AND FIRST AID

It has been mentioned (Chapter 3) that inhalation of ozone ean be very deleterious to
the pulmonary system and possibly other organs, as demonstrated by a number of
studies (Mehlman and Borek, 1987; Lippman, 1989; Mustafa, 1990; Devlin et al.,
1991; Rahman et al., 1991, 1992; Pryor, 1992; Aris et al., 1993; Stevens et al.,
1995). Prolonged breathing of ozone eauses progressive toxieity whieh ean be
exemplified as folIows :

Table 3. Toxic effects ofgaseous ozone in humans.

0 3 concentrations in air (pymv) Toxic effects

0.1 Laehrymation and irritation ofupper
respiratory airways.
1.0-2 .0 Rhinitis, eough, headaehe, oeeasionally
nausea and retehing.
Predisposed subjeets may develop asthma.
2.0-5.0 (10-20 min) Progressively inereasing dyspnoea,
bronchial spasm, retrostemal pain.
5.0 (60 min) Aeute pulmonary oedema and oeeasionally
respiratory paralysis.
10.0 Death within 4 hours.
50.0 Death within minutes.

The DL so toxieity in the rat after ozone inhalation at a coneentration of 4-6 ppmv
is 4 hours. The toxicologieal effeets are worse if the subjeet breathes ozonized air
mixed with CO, NO x, aeid vapour, ete., as oeeurs in inhalation of very polluted air
(Warren and Last, 1987; Fujimaki, 1989; Sagai and Iehinose, 1991; Hateh, 1991;
Foster et al., 2000). This gaseous mixture may have a pH as low as 3 and eannot be
neutralized by the weak buffering and antioxidant eapaeity ofRTLFs ofthe mueosa.
It must be pointed out that the toxicity of ozone for the respiratory traet eannot
be extrapolated to blood owing to strikingly different anatomieal, bioehemical and
metabolie eonditions (see Chapters 12 and 14).
It is almost unnecessary to warn that workers involved in risky industrial
applieations must wear a suitable respirator, while the ozonetherapy clinic must be
equipped with an efficient exhaust fan, even though no leakage of ozone is allowed.
In ease of aeeidental ozone inhalation, the subjeet must lay down and rest in a weil

41
42 CHAPTER 5

aerated room and , if necessary , wear a mask and breathe humidified oxygen.
Chapter 36 will describe in detail the most urgent steps to be performed in an
emergency. However, I am not aware of any serious accident in a c1inic invol ving
the breathing of ozone.
Slow intravenous (IV ) administration of ascorbic acid and reduced GSH in 5%
glucose solution may help limit the damage (Samet et al., 200 I). Ascorbic acid ,
vitamin E and N-acetylcysteine (NAC) can also be administered by oral route, but as
we shall see (Chapter 13) this type oftreatment is more rational as a preventi ve than
curative therapy . In fact , the higher is the anti-oxidant status of biological fluids, the
lower is the possible oxidative damage .
 CHAPTER6

THE OZONE GENERATOR

It is unfortunate that ozone cannot be stored and thus that a standard ozone sampie is
not available. Ozone must be generated only when needed and used at onee. The
ozonetherapist must have an ozone generator that is safe , atoxic, reliable and
reproducible. The manufacturing industries must exercise the utmost eare in
providing these requirements, using the best ozone-resistant materials, such as
INOX 316 L stainless steel , pure titanium grade 2, Pyrex glass, pure Teflon, Viton
or medieal silicone, and avo iding polyvinyl chloride (PVC) or any other plastic
material containing additives that could be released due to ozone oxidation. Even
heavily anodized (>4 um) aluminium is not ideal and certainly inferior to stainless
steel. The photometer's performance must be checked against the c1assical
iodometrie method.
As acti vated charcoal is not very durable, the destructor must be filled with a
matrix incorporating Pd, Ni, Mn oxides maintained at about +70 "C by an electric
thermostat. This works very weil for years, as we have experienced during
extracorporeal cireulation with a eonstant flow of Or03. If the exhaust gas mixture
(0 2 +0 3) contains water vapour, it must be removed beeause it will spoil the
destructor. In this case, we use a first trap immersed in ice water and a second trap
filled with renewable silica gel.
Schematically, the medical ozone generator eonsists of 2-4 high-voItage tubes
connected in series to either a rheostat (in older models) or to an electronic
programme able to set up voltage differences between 4,000 and 13,000 volts (Fig.
5). For more technical details, one can read Stiehl ' s report (1998) . The energy from
the electric discharge allows the breakdown of oxygen moleeules into oxygen atoms
whieh, in the presence of an excess of oxygen molecules, form the three-atom ozone
moleeule. The generator is fed with pure medical oxygen and , at the supply nozzle, a
gas mixture eomposed ofno more than 5 % ozone and 95 % oxygen can be collected
at a slightl y hyperbaric pressure (Table 4). The synthesis of ozone requires an
energy of ~H=140 KJ/Mol while the decomposition of ozone is aceompanied by
energy release. As unused ozone cannot be dispersed into the environment, it
undergoes decomposition to oxygen by a eatalytie reaction inside the destructor
containing heavy metal oxides bound to a matrix.
For medieal purposes, air eannot be used; sinee it eontains about 78%
nitrogen (N 2) , variable amounts of highly toxie NO x will be formed . It should be
noted that when ozone is not present, or has been exhausted, the addit ion of air is not
toxic because the dissociation of nitrogen molecules into atoms requires an energy
far superior to that released by ozone.

43
44 CHAPTER6

r((
220 V...

Figure 5. A sehemaue view ofan ozone generator: 0 1 inlet, transformer, ozone destructor.
nozzle for collecting 0 ] + 0.1' Ozone is generated while oxygenflows througli the two tubes.

The ozone concentration is determined by three parameters:

1. The voltage : the final ozone concentration increases with the voltage, albeit in a
non-proportional manner.
2. The space between the electrodes: this serves to modulate a gradual increase of
ozone concentration.
3. The oxygen flow: this is expressed as a volume of Iitres per minute (L/min) and
normally can be regulated from 1 to 10 Llmin . The final ozone concentration is
inversely proportional to the oxygen flow; hence, per time unit, the higher the
oxygen flow, the lower the ozone concentration and vice versa (Table 4).

The double-Iogarithmic scale diagram (Fig. 6) shows that these parameters

interact in a complex way: by testing 3 different voltages (5, 7 and 14 KV) at a
constant dischargegap of I rum, three different curves are obtained and the maximal
ozone concentration of 100 ug/ml can be achieved only with the highest voltage .
However, as the oxygen flow normally varies between I and 10 L, at the lowest
voltage (5 KV), the maximalozone concentration with agas flow of 1 Llmin is
 THE OZONE GENERATOR 45

about 30 ug/ml . In general , ozone concentrations range between 2-3 and lOO-llO
ug/rnl, which is more than enough for medical applications (useful range 3-80
ug /rnl). One type of generator on the market delivers an oxygen flow of only I
Llmin, which is considered disadvantageous when collecting large volumes of gas
(50 ml in 3.1 seconds) from the nozzle . Clearly the ozonetherapist should follow a
serious teaching course and gain personal experience before using any apparatus.

Table 4. Some basic conceptsfor the ozonetherapist.

OrO] Osflow LW
(as relative percentages) (miimin)
100.0-0% +++
99.5-0.5% +++ +
95.0-5.0% + +++

Important criteria for calculatingthe ozone dose:

• Total volume ofthe gas (02+03)

• 0 3 concentration (ug/ml)
• Barometrie pressure (mmHg) if different from normal

The total ozone dose is equivalent to the gas volume (mi) multiplied by the
ozone concentration (J.tg/ml).

Example: volume: 100 ml; ozone concentration: 40 ug/ml .

The total ozone dose is: 100 x 40 = 4,000 ug or 4.0 mg.

100+-----~-==:::::::?"-I-c:=----_I_

14KV

10
:J'
~ 7KV
3
o~
SKV

0,1 1 10
Gas flow rate (Llm in)

Figure 6. Concentration /flow diagram . Different concentrations ofozone are generated at

three different voltages (5. 71lnd /4 kV) witli / 111m discharge gap. (Viebahn /999).
46 CHAPTER6

A good ozonetherapist with an inefficient ozone generator produces bad

ozonetherapy. Therefore, it is necessary that the instrument undergoes periodic
maintenance (every 100-150 hours work or each year), including a control by
iodometric titration of the photometer, to insure delivery of a prec ise ozone
concentration.
Today, generators continuously undergo conceptual and technical improvements
and I would like to warn the inexperienced physician that he/she should be weil
acquainted with all possible options before purchasing an instrument.
Finally , it would be useful if the manufacturers would produce a smalI, handy
generator of ozone concentrations of 2.5, 5, 10, 20 and 30 ug/rnl. This range is
suitable for treatments such as rectal insufflation, topical treatments, quasi-total
body exposure and preparation of ozonized water and oil for patients to use at horne,
under the supervision of a physician-ozonetherapist.
 CHAPTER 7

HOW TO CHECK OZONE CONCENTRATIONS

We must honestly admit that until the advent of a standardized measurement method
by photometry, a number of ozone generators did not yield a precise ozone
concentration and even anecdotal results were doubtful. Even today, I frequently
observe that ozonetherapists use poor, or obsolete, or unchecked instruments so that
any c1inical result remains questionable and cannot be reproducible. The fault is
with careless manufacturers, with irrexpert technicians unable to do a proper check ,
with the lack of constant controls by health authorities and, last but not least, with
the superficial preparation of the ozonetherapist, who should demand the most
effective instrument. To my knowledge, instruments are supposed to pass a serious
test and have a certificate of origin. Yet it is my experience that some producers may
even exhibit two certificates for a poor instrument!
I don 't want to discourage the reader but if we really want ozonetherapy to
progress, we must be very serious about the efficiency and reliability of ozone
generators. This is also the reason why only open and fair competition (and not a
monopol y) can permit a selection of the best instruments. In the course of this book ,
it will become c1ear why I consider the precision ofthe ozone concentration to be so
crucial. Indeed , if my interpretation is correct, ozone must be considered as areal,
although atypical, drug and as such its precise concentration is essential. As
indicated in Table 1 (Chapter 3), this can be expressed in different ways, but luckily
European generators give the concentration directly in terms of ug /ml . There are
either small (portable) or medium-size medical generators able to deliver ozone
concentrations up to either 70 or 110 ug/ml, respectively, quite sufficient for
medical use. Until now, portable instruments did not have a photometer and some ,
still in use, are unreliable. To tell the truth, until about 1995, the ozonetherapist had
to trust a table near the control panel where, depending upon the voltage and the gas
flow, he could select the desired ozone concentration. Table 5 gives a typical
example and shows that either low, medium or high concentrations could be
obtained by switching on either programme I, 2 or 3, respectively. Oxygen pressure
was set at 1.5 bar and the final ozone concentration was inversely proportional to the
gas flow (L/min). Table 5 also shows that the reproducibility of ozone
concentrations measured at different times is reasonably good , except in a few cases.
How were the data on the table determined?
The iodometric method has always been the standard method for the
determination of ozone ; the revised procedure of the IOA was reported by
Massehelein in 1996. When ozone reacts with buffered potassium iodide (KI),
iodine is generated and the solution acquires an amber colour which , upon reduction

47
48 CHAPTER 7

with a titrated solution of sodium thiosulphate (Na2S203) and astareh indicator,

allows the determination ofthe ozone eoneentration

0 3+ 2r + H20 --~) 12 + O2 + 20H-

12 + 2S20/- ) 2r + S4062-

The eoneentration of ozone in g/L equals 24 x volume of thiosulphate in L x

Nonnality of thiosulphate divided by the inlet volume of gas flow in L. Obviously
one needs a precise flowmeter and the control of temperature and atmospheric
pressure. Precautions and potential interference are weil defined; the detection limit
of the analytical procedure is 0.1 mg/L and the reproducibility is 2% of the
measured ozone concentration.

Figure 7. Iodometrie method to determine 0 3 concentration.

Above: the system with four bottles is used to trap ozone
and measure the gas flow witli a precise flowmeter, shown on the right .
Below : titration is performed with Na2S204

My friend Amaldo Cinquantini (Professor of Analytical Chemistry at Siena

University) and 1 have organized a service to check the efficiency of ozone
generators. With slight modifications, we adopted the system proposed by Maier and
 How TO CHECK OZONE CONCENTRATrONS 49

Kurzmann (1977), which is shown in operation in Figure 7. There are also

colorimetric (indigo-trisulphonate) methods based on photometrie measurements of
several dyes modifted after reacting with ozone. However, although sensitive, they
are not as precise as the iodometrie method.
If the generator has been iodometrically tested and the instrument is built with
ozone-resistant material , one can be reasonably confident of the validity of the
concentrations reported in Table 5.

Table 5. A typical set 0/03 concentrations. Bold typed values checked after one month.

Gas Gas volume 0 3 concentrations (ug/ml)

pressure Programmes
(bar) (Um in) (mi/sec) I II III
0.8 10.0 3.5 18 14 3230
0.7 8.6 4 15 33
0.6 7.2 5 18 37
0.5 5.8 6 21 4142
0.4 4.5 8 25 5049
0.3 3.1 11 3431 61 59
0.25 2.5 17 12 36 71 68
0.2 2.0 33 .3 16 4342 78
0.15 1.5 25 .0 20 51 8788
0.1 1.0 16.7 3330 72 70 110 105

However, the delivered ozone concentrations tend to decrease in time and with
frequent use and the instrument needs to be recalibrated. The worst problem I have
observed is that some portable generators built with inferior material (poor quality
anodized aluminium tends to be oxidized by ozone) can deliver ozone
concentrations corresponding to 30 1% or less of the nominal value. It is disgraceful
that there are about 500 such instruments still in use in Italy!!
The ozonetherapist who is unaware of all this can be in trouble and the effect of
the treatment is probably due to a placebo effect. However, "if we have to choose the
lesser of two evils, it is preferable that ozone concentrations decrease because
otherwise we could induce serious toxicity.
From what has been discussed, it is c1ear that we must have a reliable system,
able to measure the ozone concentration just before withdrawal. Luckily, we can
now do that by photometrie determination. The rOA recommends a procedure based
on the pronounced absorption of ozone within the Hartley band (between 200 and
300 nm) with a peak at 253 .7 nm. At this wavelength, UV radiation (mercury vapour
lamp) is linearly absorbed in a concentration-dependent fashion (in agreement with
Lambert-Beer's law) on being passed through a tube containing ozone (Wallner,
1998) . The system is quite sensitive and precise but tends to decay due to lamp
ageing . The spectrophotometric measurement at 600 nm (Chappuis band) is less
sensitive but more stable (Fig. 8).
50 CHAPTER 7

.)

6000
....
/ \
/ \
/ \
2000 / \
c: / \
Q)
u
~o l- V -,
200 250 Joo J50

c
u
r-r-------r------r------,-----...., b)
.Q
c:
~
UJ

o 500 550 600 650 700

Wavelenght (nm)

Figure 8. Typical absorption peaks ofozone at 253.7 III1l (a) and at 600 III1l (b), usefulfor
measuring the ozone concentration with a photometer

The ozonetherapist now has the advantage of eheeking on a digital display, in

real time, the ozone eoneentration in the gas volume flowing into the syringe during
withdrawal. However, at least for some generators, he must be eareful to avoid
modifieation of the gas flow beeause any inerease of the pressure will inerease the
ozone eoneentration. This faet must be taken into aeeount when we injeet 02-0J into
the paravertebral muscles (Chapter 24) beeause an ozone eoneentration exeeeding
20 ug /ml is very painful and eould elieit a vagal hypertone.
After having used ozone for many years, I am able to provide a few tips . Firstly,
I leamt that what is important is the immediate use of the gas and not so mueh small
ehanges of eoneentration. Seeondly, polypropylene silicon-coated syringes (gIass
syringes are impraetieal) must be used only onee . Finally, about every two months
the photometrie data should be eheeked against the iodometrie titration, whieh
remains the basie referenee method .
 CHAPTER8

SOLUBILITY OF OZONE IN WATER

AND PREPARATION OF OZONIZED WATER AND OlL

Chronie torpid ulcers, exposed dirty traumatie lesions, infected wounds, bums,
inseet stings, herpetic skin lesions, fungal infections, etc. are advantageously treated
with ozonized water and/or oil rather than gas beeause it is easy to apply a eompress
soaked with ozonized water/oil to any part ofthe body. Moreover, there is no risk of
breathing ozone, partieularly with a generator not equipped with a suetion pump
eonneeted to an ozone destructor.
In Chapter 4, it was pointed out that ozone is far more soluble in water than
oxygen (Table 2) and there is a simple, praetieal way of preparing ozonized water.
The system consists of a glass or ozone-resistant cylinder about % filled with
bidistilled water through whieh the gas mixture is bubbled eontinuously for at least 5
minutes. The unused ozone is converted to oxygen through the destructor. The
system is either sold incorporated in the generator or independently as shown in
Figure 9.

Figure 9. Systemfor the preparation ofozonized water . After suitable ozonization, the water
is collected by open ing the tap at the bottom ofthe cylinder.
D: destructor: R: reservoi r ofbidistilled water.

51
52 CHAPTER 8

Solution of ozone in water takes place according to the law defined by Henry in
1803: under ideal thermodynamic conditions, the saturation concentration of a gas in
water is proportional to its concentration. However, this is correct only if the water
is absolutely pure (bidistilled) and the temperature and ozone pressure remain
constant. Figure 10 is interesting because it shows that ozone saturation in pure
water is reached only after 5-6 minutes of continuous bubbling of ozone at three
different concentrations. The data plotted in the diagram fit a linear relationship.
This resuIt must be remembered because I do not agree with hurried ozonetherapists
who boast of performing the whole OJ-AHT in 6 minutes, incorrectly c1aiming that
blood equilibrates instantaneously with ozone, mostly because blood should not
undergo either bubbling or violent mixing with ozone.

100 ·

-E
CJ)
::L
.......-
L-
.....
Q)
CU
~
C
C
2
0 10
.~

CU
.....
L-
C
Q)
U
3
C
0
U
Q)
C
0
N
0

5 10 lS 20 25
Minutes

Figure 10. Concentration-time diagram showing three curves ofozone concentration in pure
bidistilled water after bubbling ozonefor IIp to 25 min at concentrations of 1) 80 ug/ml; 2) 42
ug/m! and 3) 20 ug/ml. Temperature at 22 oe. 752 Torr and column of water of42 cm. At the
highest ozone concentration. the saturation is 26% (=20.8 ug/ml) within 51/1in (Viebahn.
1999).
 PREPARAnON OF OZONIZED W ATER AND OlL 53

After reaching the plateauphase, the ozonized, bidistilled water can be used or
stored in a glass bottle tightly closed with a teflon cap, possibly in a refrigerator.
Ozone decomposition depends largely on the temperature, as shown in Figure 11. If
it is kept at +5 "C, the theoretical half-life (tll2) is about 110 hours, i.e. after this
period the initial ozone concentration of 20.8 ug/ml (26% of 80 ug/ml) would
decrease to about 10 ug /ml . This is of practical importance because ozonized water,
if maintained properly, can be used for at least 48 hours at the patient's horne for
domiciliary treatment.

100 0;:-_ _

...-..

---oc
o~

:;:;
.....co
l-

a5
U
10
C
o
U
Q)
C
o
N
o
I
30 2S 20 -c 1S -c 10 -c

o 100 200 300

Hours
Figure 11. The decompo sition ofozone dissol ved in btdistilled water depends on the
temperature (Viebahn. 1999).

On the other hand, the stability of ozonized water also depends on the ionic
content and pH of the water: as occurs for either deionized or monodistilled water,
even residual traces of ions markedly shorten the half-life (t1/2 of about 80 and 20
minutes, respectively), as shown in Figure 12. The latter two solutions are prepared
and used extempore.
54 CHAPTER8

10

8
.........
UJ
'-
::J
o
.c 6

~
f:- ..

Figure 12. Half-life ofozone solubilized in:

- bidistilled water (left CO/1I11ln).
- deionized water (middle column)
- monodisttlled watet: (right cO/1I11ln)
(Viebahn , 1999)

Therefore, the instability of ozone is due not only to its metastable nature but
mainJy to its high reactivity with ions and an array of organic molecules such as
those present in biological fluids , namely PUF As, compounds with sulphydryl
groups (-SH), several amino acids and carbohydrates. In such cases, ozone solubi lity
no longer follows Henry's law because as soon as ozone dissolves in water, it
encounters a moleeule and reacts immediately with it. As a corollary, if ozone is still
present in the gas phase, it will dissolve in the water phase but again it will react
instantaneously with other moleeules and disappear from the water. It folio ws that ,
in these circumstances, the plateau phase achieved in Figure 10 cannot be reached
and the unattainable equilibrium implies that, if ozone remains present in the gas
phase, an "infinite" amount of ozone can pass into the solution and reaet with
substrates. The situation is quite different for oxygen, which is far less reactive than
ozone and remains physically dissolved in the plasmatic water depending on the
temperature, pressure and its solubility coefficient. We will eome back to this point
in Chapters 13 and 20 because it is fundamental to understand why even very low
but stable ozone concentrations in the gas phase overlaying the tissue culture
fluid can be cytotoxic, while one volume of gas (Oz + 0 3 ) added to one volume
of blood for the preparation of ozonized blood is not. Cell biologists studying
ozone toxicity in vitro should ponder on this point.
At the beginning of the Chapter, it was suggested that ozonized water could be
successfully employed in various pathologieal eonditions. Interna I use is also
possible : an empirical and fashionable treatment of gastritis due to Helicobacter
 PREPARAnON OF OZONIZED W ATER AND OlL 55

pyloris consists of drinking a glass of mildly ozonized water in the morning before
eating (Chapter 24) .
After draining of the pus in purulent cysts, chronic osteomyelitis infections and
empyemas, they could be quickly rinsed with ozonized water to remove residual pus
and necrotic debris . Then, after draining the water, the cavity can be insufflated with
the Or03 gas mixture at least twice daily . I am aware of a few precise, albeit
anecdotal reports, in which' ordinary medication became desperately hopeless and
yet, with great patience and acuity, dedicated ozonetherapists were able to eliminate
serious infections. Gas insufflation must be performed in a few minutes in a well
ventilated room leaving the polyethylene (or Teflon) cannula clamped to prevent the
exit of gas. It is up to the ingenuity ofthe ozonetherapist to evaluate for each patient
the sequence and combination of gas, ozonized water and oil. Moreover, it is fairly
obvious that one should use a high (80 ug/ml) concentration of ozone during the
septic phase and to progressively lower it as soon as the infection subsides, to
prevent the inhibition of cell proliferation.
Decubitus and dystrophic ulcers tend not to heal spontaneously and require
prolonged treatment (Chapter 24). The use of gas (after appropriate bagging or in a
suitable container with or without slight decompression) or of a pack soaked with
freshly ozonized water are useful for cleansing, disinfection and stimulation oftissue
granulation but the treatment is usually suspended during the night. To avoid
possible deterioration, it was thought that the application of ozonized oil might keep
the lesion in either a "stand-by' situation or even enhance healing.
In the last decade, there has been a growing interest in the application of
ozonized oils. In Cuba, probably because of the lack of other pomades, ozonized oil
has been widely employed in torpid ulcers, bacterial, viral, fungal and parasitic
infections. As a natural preparation, ozonized oil is now available in several
countries but, to the best of my knowledge, neither chemical data nor a standard
preparation have been published. However, very recently, ozonized sunflower oil
(Oleozon) from Cuba was tested by Sechi et al. (2001) and it was found to have
valuable antimicrobial activity against all the micro-organisms tested. At our
University Hospital, we make our own preparation by bubbling O 2-03 in pure olive
oil for at least 30 min in a cooled bath . This is obviously a temporary solution and it
is becoming necessary that the hospital pharmacy make standard preparations. At
the IOA Congress in London (September 14-15, 2001), Miura et al. (2001)
presented an interesting and very useful report on the elucidation of the structure of
ozonized olive oil. Pure olive oil (sunflower oil is employed in Cuba) is ozonized by
bubbling O 2-03 gas through it for two days until it solidifies. Olive oil contains
about 80% oleic acid (18: 1 n-6) and, according to Miura (who works at the College
of Medical Technology, Hokkaido University, Sapporo, Japan), 1.0 g of oil can
absorb up to 160 mg of ozone. After ozonization, characteristic modifications were
assessed by observing a 13C-nuclear magnetic resonance (NMR) spectrum: new
carbon signals could be related to Criegee's ozonides peaking at around 105 ppm,
with complete disappearance of olefinic carbon signals at about 130 ppm . The
absence of carbonyl compounds and carboxylic acids, in conjunction with data
obtained by NMR spectrum, elemental analysis and high-performance liquid
chromatography (HPLC), led to the conclusion that prolonged ozonization results in
56 CHAPTER 8

exclusive formation of triozonides of triolein. The structure of triolein triozonide

proposed by Miura et al folIows :

Remarkably, no spectrum changes were noted in ozonized olive oil stored in the
refrigerator for two years . There is no real need to have asolid oil preparation,
except for commercial purposes and long stability. Indeed , the pathological
situations are so variable they require great flexibility; thus one can use fairly liquid
or very viscous ozonized olive oiI after keeping it in the cold .
How ozonized olive oil acts is open to speeulation. However, it seems likely that
when the triozonide eomes into eontact with the wound, the body temperature and
the presenee of serum favour its deeomposition to reaetive ozone, whieh readily
dissolves in water, generating H202 and a variety of oxidized eompounds. This may
explain the strong and prolonged disinfeetant aetivity, whieh however must be
tempered so as not to damage the living tissue. This reasoning implies that we
should have titrated preparations with either high, medium or low triozonide
eoneentrations to be used during either the inflammatory pus-rich phase I,
regenerating phase II or remodelling phase III, respeetively. In Chapter 16, these
phases have been related in some details to the rapidly changing cell types and to the
release of cytokines and growth faetors that modulate the complex healing proeess.
In the Department of Surgery, Chi ba-Tokushukai Hospital , Japan , Matsumoto et
al. (2001) tested the efficaey ofthe oil prepared by Miura et al. in intraetable fistula
and wounds after surgieal operations (acute appendicitis with peritonitis, intrapel vie,
abdominal and perianal abseesses, ete .). In aseries of 28 patients, the ozonized oil
proved to be fully effeetive in 27 eases, without adverse side effeets .
Finally, I will mention that there are several pharmaceutieal vehicles for
ozonized oil, sueh as capsules, pessaries, suppositories and collyriums, to be used in
various infections. The smell of ozonized oil is similar to rancid fat (henee
unpleasant) and capsules ingested by mouth, although distasteful, are tolerable.
Their therapeutic efficaey also remains to be demonstrated in eontrolled c1inical
studies. Today, we are c1early using ozonized oil in a very empirieal fashion, but
hopefully further studies will permit its rational applieation. I am eonvineed that
ozonized oil will become a very useful medical treatment, onee physieians and
nurses realize its therapeutie potentiality
In Chapter 13, I will try to explain how ozone works when dissolved in
biological fluids , generating a easeade of reaetive oxygen speeies (ROS) and lipid
peroxidation produets (LOPs), whieh help to c1arify the multiple biologieal aetions
and potential toxieity.
 CHAPTER9

PRELIMINARY BASIS FOR UNDERSTANDING

OZONE REACTIVITY AND THE POTENTIAL RISKS
OF OZONETHERAPY

Ozonetherapy is one of over sixty "non-conventional" or so-called "alternative",

"natural" or better "complementary" medical approaches (Astin, 1998; Zollman and
Vickers, 1999; Ernst, 2000). Most of them arise from either old folk tales or
philosophical postulations or oriental cultures . For this reason, some approaches
cannot be evaluated according to the eriteria of orthodox medicine. However, there
are notable exceptions : acupuncture , which is more than 3000 years old and whose
validity is being confirmed by clinical studies (Mayer, 2000) and possible risks
(Ernst and White, 1997); homeopathy, about 200 years old and under intense
scrutiny (Linde et al., 1996; Vandenbroucke, 1997); phytotherapy, particularly
regarding extracts of Hyp ericum perforatum (Linde et al., 1996; Vogel, 2001 ;
Shelton et al., 200 I) and Ginkgo biloba (Soholm, 1998; Curtis-Prior et al., 1999; Le
Bars et al., 2000 ; Drew and Davies, 200 I).
At first, ozonetherap y was based on the powerful bactericidal effect on anaerobic
bacteria but later expanded its objectives with the esoteric idea of being able to
transfer the energy released during ozone decomposition to the body. Today, we are
still at a primordial stage: firstly, we must demonstrate that, when properly used,
ozone is not toxic as most scientists believe ; secondly, that ozone can modulate
several cellular functions; and thirdly, that ozonetherapy has, beyond any doubt,
therapeutic efficacy in diseases where orthodox medicine fails.
However, a positive characteristic of ozonetherapy is that it can undergo
objective scientific investigation , so that any prejudice is unjustified . Unfortunately ,
empiricism has dominated the scene and the lack of basic studies has delayed an
understanding of the meehanism of action. The exciting hope is that, onee we have
clarified those mechanisms , we may be able to establish a rational basis for using
ozone in medicine .
Even if the reader is a physician, he may be unfarniliar with chemical reactions
or the present terminology . Thus it seems proper to present some basic information
in a eomprehensible form for an understanding of the nature of the compounds
generated during ozone decomposition.
This is a fairly easy task because so much has already been clarified regarding
the concept of oxidative stress as a significant contributor to tissue damage . The
problem started about two billion years aga with the progressi ve increase of oxygen
(0 2) in the atrnosphere (Dismukes et al., 2001; Kasting, 2001). In 1924, it was
clarified that molecular oxygen contains two unpaired electrons and thus is a

57
58 CHAPTER 9

diradical (. Q - Q.). The unpaired electrons are commonly symbolized by a point.

However, oxygen does not have the extreme reactivity of ozone (OJ) and, by a
stepwise reduction with four electrons, forms water:

Nonetheless, during the reduction of oxygen, as electrons leak out of the

transport chain during mitochondrial respiration (Cadenas and Davies, 2000), some
3-5% of O 2 generate ROS such as hydroperoxyl radical (H0 2°), superoxide anion
(0 2 hydrogen peroxide (H 20 2) and hydroxyl radieal (OW) :
0
-) ,

O 2 + e' + H ' ) H02°---~) H +- + 0 2 0

-

0 2 + 2H ' + e'
0
- ) H20 2+ e' ) OH- + OH o
OH + e' + H+
o
) H 20

Although ozone (0 3) is not a radieal molecule and schematically can be

represented as 0=0-0 with a paired number of electrons in the extemal orbital (see
Fig . 3, Chapter 4), it is far more reactive than oxygen but generates the same ROS
produced by biochemieal processes within the body .
During ozonization ofblood, as soon as ozone dissolves in the plasmatic water , it
can react with several substrates. One of them is reduced glutathione (GSH), which
gives rise to an anion ozone radieal that is very unstable and after protonation forms
o
OH :

GSH + OJ --~) GSH + OJ 0 1

0-

0 3 + H-
0
- ) OW + O 2

Now we must clarify what is a free radiea\.

Most biological molecules usually have two electrons spinning in the extern al
orbital and are stable. Free radieals are chemieal species that contain one unpaired
electron. Thus they are unstable and reactive, as they tend to abstract an electron
from a nearby atom or molecule.
Radieals can react with other molecules with different modalities:

• when two radieals meet, they combine their unpaired electrons and join to form a
stable covalent bond. A simple example is the hydrogen atom with one unpaired
electron (therefore a radical) : when it combines with another hydrogen atom, it
forms the diatornie hydrogen molecule :

• A radieal may transfer its unpaired electron to a stable molecule, acting as a

reducing radieal.
 OZONE REACTIVITY AND RISKS 59

• A radical may take an electron from another moleeule in order to form a pair and
to achieve a more stable state , acting as an oxidizing radical (electron
abstraction) ,

As a consequence, the non-radical species becomes a more or less reactive

radical that must remove an electron from another stable moleeule. This first step,
called initiation, tends to propagate as a chain reaction, where one radical begets
another (propagation). The process can reach a final step (termination) owing to
several chain-breaking compounds that block further propagation. During the
evolution of aerobic life, many compounds and enzymes composing the antioxidant
system have become speci alized in containing radical reactions. In an almost ideal
physiological environment such as plasma, radical reactions occur but are restrained
to a large extent. We have already noted that a possible reaction is the
transformation of hydrogen peroxide (H 202 ) into a hydroxyl radical (OH") when
traces of transition metals such as iron or copper are present in ionic form . The
reaction described by Fenton and Jackson (1899) proceeds as folIows :

Ions ofCu, Co and Ni can also participate in this type ofreaction (Rotilio, 2001).
Moreo ver, Haber and Weiss (1934) described another metal-catalyzed reaction with
the formation of Oll" :

Fe2+
O2. ' + H 202

As another example, OH· can also be formed during the gamma radiation usually
occurring during cancer treatment. The radiation splits one oxygen-hydrogen
covalent bond in water and generates two radicals, one with a single electron on
hydrogen and another with an impaired electron on oxygen:

H-O-H--~) H·+OH·

OH· is the most potent oxidant known to chemistry. It has an extremely short
half-li fe (10.9 seconds) and attacks the nearest moleeule in living cells, setting off
chain reactions. In doing so , OH· is neutralized to water:

Obviously we are concemed with what may happen when blood is exposed to
and mixed with the gas mixture O 2-03 . Haemolysis must be carefully avoided
because any trace of Fe 2+ released from haemoglobin switches on the Fenton
reaction (Winterboum, 1995) . We will come back to this point in more detail in
Chapter 12.
It has been c1arified that ozone can dissolve as any other gas in pure bidistilled
water and remain in solution for some time (Chapter 8). However, once ozone
60 CHAPTER 9

dissolves in the plasmatic water, it reacts instantaneously with several biological

compounds: in order of preference, lipids (particularly PUFA s), antioxidants (uri c
and ascorbic acid, GSH, alpha -tocopherol, bilirubin), cysteine-rich proteins and
carbohydrates .
PUFAs , particularly arachidonic acid (5, 8, 11, 14-eicosatetraenoic acid, 20:4),
are present in membrane phospholipids, triglycerides, lipopro teins, chylom icrons
and hydrophobie pockets of albumin (Goheen et al., 1986; Mehlman and Borek ,
1987; Pryor, 1993). The more a fatty acid side chain is unsaturated, the more it is
attacked by ozone during a weil characterized reaction known as lipid peroxidation.
Thi s is due to the fact that the presence of a double bond weakens the carbon -
hydrogen bond at the adjacent carbon atom .
The chem istry of lipid peroxi dat ion is complex and has been clarified by the
German chemist Criegee (1975) and by Pryo r (1992 , 1993, 1994; Pryor et al., 1995)
in the USA. In a Iipophil ic environment, the initial reaction with a PUFA is an
add ition of O, to the carbon-carbon doubl e bond, producing a trioxygen intermediate
(called I, 2, 3-trioxolane), which rearranges to an ozon ide as descr ibed by Criegee
(Fig. 13). In a hydrophilie environment such as plasma , one mole of an olefin and
one mole of 0 3 give rise to 2 moles of aldeh ydes and one mole of HzOz (Pryor,
1994),
H 0-0 H

:>
)(.0.)( (1)
H H R R'
\ / Cr iegee ozonide
C=C

:;>
0)
I!°
/ \ H
R R' /
R-C + R'-C-- (2)
Unsaturated
lipid \ \ OH
H OOH
Aldehyde Hydroxyhydroperox ide

/
R'-C\OH
H

....---
~
R'-C
I! ° + HJOJ (3)
\
OOH H

Net reaetion

I!° I!°
H H
\ / (4)
C=C + 0) + HJO -R-C + R'-C + HJOJ
I \ \ \
R R' H H

Figure l LSchematic series O[chemical reactions betwe en ozone and an unsaturatedfatty

acid, according to Criegee and Pry or. The reaction is summarized at (4) (Pry or et al.. 1995).

The stoich iometry of this reaction shows the produ ction of compounds with
biolog ical and cytoto xic activities. Lipid perox idation in plasma membranes may
result in chan ges in fluidity, alterations in the ion transport mechan ism with
distortion of signal transduction (Kourie, 1998), increased permeability and possibly
 OZONE REACTIVITY AND RISKS 61

membrane rupture. As even a trace of haemolysis can be easily monitored, we have

always taken advantage ofthis sensitive marker.
lt is instructive to describe in detail the various peroxidation steps (from 1 to V),
as shown in Figure 14. Free radicals, particularly OHo, can abstract a hydrogen atom
(HO) from a methylene group (-CH 2- ) of a PUFA, leaving behind (step I) an unpaired
electron on the carbon (-CH"-). The remaining carbon-centred radical undergoes
molecular rearrangement resulting in a conjugated diene (step 11). This compound,
which shows specific absorption at 234 nm, can combine with O2 to form a peroxyl
radical (step III). The latter is able to abstract a HO from an adjacent PUFA (step IV)
and starts a chain reaction that terrninates because of lack of substrate or, more
likely, chain-breaking antioxidants such as liposoluble a-tocopherol andlor
bilirubin. The lipid peroxide (-C-O-OH) formed at step IV is a fairly stable
compound but traces of Fe 2+ or Cu+ can catalyze its decomposition with the
formation of alkoxyl (ROO) and alkoperoxyl (ROOO) radicals , which can induce
further peroxidation.

Figure 14. The sequence ofreactions (from I to V) occurring during lipid peroxidation wuh
production ofmalonyldialdehyde and 4-hydroxy-2 .3 trans-nonenal.

Eventually, a complex mixture of low molecular weight aldehydic end products,

namely malonyldialdehyde (MOA), n-alkanals, 2-alkenals, 4-hydroxy-2,3 trans-
nonenal (4-HNE) and other 4-hydroxy-2,3-alkenals of variable chain lengths, may
be formed . The chemistry and biochemistry of these compounds has been
masterfully described by Esterbauer's group (1991). Irnportant contributions
regarding their pathophysiological implications have been published by several
authors (Kozumbo et al., 1996; Dei Corso et al., 1998; Hamilton et al., 1998;
Comporti, 1998a,b ; Oianzani, 1998; Kreuzer et al., 1998; Parola et al., 1998; Selley,
1998; Bosch-Morell et al., 1999; Parola et al., 1999; Kalinich et al., 2000; Poli and
Schaur, 2000) . 1f one thinks about the wealth and heterogeneity of lipids, glycolipids
and phospholipids present in plasma, it becomes bewildering to imagine how many
potent biologically active compounds are able to either activate or depress the
inflammatory and immune systems.
62 CHAPTER9

However, by and large 4-HNE:

can be considered the prototype of the LOPs and seemingly the most noxious .
In conclusion, it appears that even one moleeule of 0 3 , 01' eventually one OHo,
can result in the conversion of hundreds of PUFA chains into lipid hydroperoxides
and then into cytotoxic aldehydes. Luckily the reaction can terminate in several
ways , as ind icated by the following reactions in which RO symbol izes a free radical :

a) R0 2° + R02°--~) ROOR + Oz
b) RO/ + RO ) ROOR
c) RO + RO ) RR

Other possibitities of stopping the chain reaction are due to the activity of GSH
peroxidase (GSH-Px) and a crucial intracellular antioxidant such as GSH :

ROOH + 2GSH _ _GSH. Px~ GSSG + HzO + R-OH

Chain-breaking agents such as u-tocopberol (vitamin E, EOH) and/or bilirubin

embedded in membranes are also providential :

R0 2° + EOH ---~) EO° + ROOH

Moreover, lipohydroperoxides and aldehydes can be degraded by GSH-Px and

aldehyde dehydrogenases (Vasiliou et al., 2000).
Some time ago , we studied the kinetics of the fundamental reaction reported in
Figure 13:

using human plasma. By comparing it with ozonization of a physiological solution,

we could observe interesting differences reported in Figure 15.
The question ofthe relative importance of formation of ozonides on the one hand
and of H20 Z and OHo on the other remains uncertain : Pryor et al. (1995) have
established that the former are 80-90'1.. in the respiratory mucosa , but probably only
a small percentage in plasma, hence with tittle relevance. As we have shown in
Figure 15, H20 Z is readily demonstrable, mostly because it is fairly stable. The
presence of OHo in plasma has been shown by Byvoet et al. (1995) and Ueno et al.
(1998) by means of the electron spin resonance (EPR ) technique, which detect s
radicals by measuring the energ y changes that occur as unpaired e1ectrons relax after
alignment in response to a magnetic field. However, for reasons that will become
apparent when we evaluate the pleiotropicity of the antioxidant system, OHo
formation during an appropriate ozonization ofblood must be minimal.
 O ZONE RE ACTIVITY AND RiSKS 63

Saline Plasma

15 f
20000 1500

°2 15000 30 °2 1250
1000

': L
- 10000 20 - 750
500
5000 10

0 0 -0 0
_._ ... _
......... .... ..... ..... .... ..-
I- 250
0
50 ~g 50 ~g 1250
15 15000 30
1000
. !:
10 ! 10000 20 - I- 750
sc:
! . ................. ......................• 500 s
"'
-~
5 - 5000 10 CI>
c:
iJ 250 E
~ .2
2; 0 E
0 0 CI>
0':r;N" L:
o
70 ~g I- 1250
15 15000 30 -
I- 1000

10 10000 20 - 750
500
:
5 5000 10 -

0 ·0 0
.. ... 250
0
I

: t..
1250
30 100 ~g
15000

.
1000
.,'
10000 20 750
500
5 5000 10
i~
•...
250

0 0 0 ' I
'"
' I 'T 'I 'T 0
o 10 20 30 40 50 60 o 10 20 30 40 50 60
Time (min)

Figur e l i .Kinetics ofthe chemiluminescent signals and ofthe production ofHzOz after
exposing either sa line 01' human p lasma to O2 alone 0 1' to three concentrations of O, (50, 70,
100 ug/ml p er ml ofsolvent). The rapid production and decay ofH Z0 2 in plasma is
notewort hy.
64 CHAPTER9

Although OH" is short-li ved, when it triggers the lipid peroxidation chain
reaction, it can lead to alteration and breakdown of the cell membrane (Fig. 16), to
oxidation of enzymes with loss of activity and to profound damage to DNA.
Luckily, the cell has evolved a number of defensive reactions to neutralize OH".
However, before examining them , I will brietly return to the critical point that,
normally and continuously throughout life, the cell produces abundant amounts of
0/-, and thus H 20 2 and possibly OH·. It was already mentioned that mitochondria
produce O 2. ' because the electron carriers remain in the reduced form at low oxygen
pressure. Halliwell (1994, 1996) calculated that, even at rest, we produce about 5 g
of O," daily, which can increase to 30 g during intense physical activity.
NORMAL

-.
i~U~~U~1
Internal
I
Channel
"- Protein
/
Phosphol ipids

LIPID PROTEIN PHOSPHOLIPASE

PEROXIDATION CROSSLINKING ACTIVATION

H~nnnnO~H 1nfis-sn~O~ fi~. no.n.~(r~~

lo~oi1UH04l lolU'-sUlU loH1UUUxlJl
Figure lti.Possible mechanisms 0/ damage to the cell membrane during ozonization .
Even minimal damage , if irreversible, can lead to cell death.

During phagocytosis, activated phagocytes (neutrophils, eosinophils,

macrophages) activate NADPH oxidase (respiratory burst) (Badwey and Karnovsky,
1980; Chanoch et al., 1994; Babior, 2000), which is a mernbrane-bound enzyme that
catalyzes the production of O 2. - from O 2 and the reduced form of nicotinamide
adenine dinucleotide phosphate (NADPH):

Another important site of the formation of O2. ' is represented by cells at the site
of a transitory ischaemia followed by reperfusion. The enzyme xanthine
dehydrogenase (XDH) reduces nicotinamide adenindinucleotide (NAD) :

xanthine + H20 + NAD - - XDH~ uric acid + NADH + Hf

 OZONE REACTIVITY AND RISKS 65

Ouring isehaemia, XDH is rapidly eonverted by Caz+-dependent proteases into

xanthine oxidase (XO) . As a substrate, XO uses hypoxanthine supplied during the
reperfusion proeess following degradation of adenosine triphosphate (ATP) to
genera te OZO.:

Hypoxanthine + HzO + 20 2 --~) urie acid + 20zo· + 2H+

Hypoxanthine levels increase owing to ATP degradation in isehaemic tissues :

ATP ~ AMP ~ adenosine ~ inosine ~ hypoxanthine.

Oxidation of catecholamines, released in large amounts during ischaemia, is also

a source of O," :

H~ }-__ o)'
<:):
_+2~+~
0 2-)

Another bioehemical process leading to Ozo. formation is due to activation ofthe

arachidonic aeid cascade :

Arachidonic acid -----. Prostaglandin G2 -----. Prostaglandin H2 + e-

02 ··

The haeme protein cytoehrome P450, partieularly present in liver microsomes,

takes advantage of the reaetivity of the iron-O , complex for catalytie oxidation of an
array of xenobiotics and endogenous eompounds:

(RH) Fe 3+ + e---~) (RH) Fez+ + Oz--~) (RH) Feh + Ozo.

The fate of Ozo. must be discussed in more detail. The reaetion:

is ealled a dismutation because the substrate reacts with itself to give an oxidized
molecule (Oz) and a reduced compound (HzOz). At low concentrations of Ozo., the
66 CHAPTER9

rate of the spontaneous reaction is very slow at pH 7.4. Yet it can be greatl y
accelerated by the enzyme superoxide dismutase (SOD) discovered by McCord and
Fridovich (1969) because the enzyme requires only one molecule of O, This was a
0 ,.

real breakthrough and opened the field of reactive oxidant biology. During
evolution, to minimize damage to eukaryotic cells, the process of dismutation has
been compartrnentalized, so that now two enzymes with two different transition
metals are known : a manganese enzyme Mn-SOD is found in mitochondria and a
copper-zinc enzyme Cu/Zn-SOD is present in the cytoplasm. In mice, Mn-SOD
appears essential for life while Cu/Zn-SOD' mice can survive, since the animal
probably compensates for lack of the enzyme by using other enzymes (HalliweIl ,
1999a).
As a preliminary conclusion, one can say that it is HzOz (from 0/') that can act
as a second messenger to switch on various biological effects and there are many
other enzymes, namely glucose oxidase, urate oxidase, monoamine oxidase, etc .,
that can produce HzOz without the intermediary Ozo'. On the other hand , if HzOz is
unquenched it can be deleterious, particularly by the formation of OHo, if iron or
copper are present in a low valence state :

Obviously, toxicity can be prevented by the unavailability of catalytic iron ions

and this fact can be used, with considerable reservation, to explain the fashion of
chelation therapy (Ernst, 2000) .
A far more reactive form of oxygen is singlet oxygen ('Oz), which is not a free
radical because the two electrons are paired. It can be produced by several reactions:

a) spontaneous dismutation ofO/" : O," + o,': + 2H - ) 'o, + HzOz

b) the reaction of O," with HzOz: Ozo, + HzOz ) OH' + OH o + 'o.
c) the reaction: O," + H20 Z ) OH' + OH· + 10Z
d) during metabolism of hydroperoxides by the haeme protein cytochrome P450
and/or the enzyme prostaglandin-endoperoxide synthase:
2ROOH ) 2ROH + 'Oz
e) at least in vitro, neutrophils can produce 10Z by the reaction between HzO z and
an oxidized halogen: HzOz + ocr ' ) 'o, + HzO + CI'
In such a case, '0 2 can cooperate with HzOz and ocr in killing bacteria (Saran et
al., 1999). Otherwise, the importance of 10Z in vivo remains uncertain.
Neutrophils contain the enzyme myeloperoxidase in their phagosomes, which
catalyzes the oxidation of halide ions, namely CI', Br' and I', to hypohalous acids
using H20 Z as a substrate:

According to Saran et al. (1999), Pullar et al. (2000), chlorine atoms may have
an extraordinarily strong intluence in the killing ofbacteria.
 OZONE REACTIVITY AND RISKS 67

The ozonization of saline mayaIso generate ocr, which even at a concentration

of 10-20 11M is extremely toxie and can cause phlebitis when infused by the IV
route. Ozonization of saline is widely used in Russia, albeit at low levels, but it is
not recommended.
Another reactive species, formed in physiological and pathological conditions, is
nitrogen monoxide or nitric oxide (NO "). It acts endogenously as a vasodilator and
was first described by Furchgott and Zawadzki (1980) as "endothelial-derived
relaxing factor" (EDRF ). One must ponder on the fact that until 1987 nobody
believed that a toxic gas could be produced by cells and perform crucial functions .
NO synthase catalyzes the reaction :

Arginine + O2 + NADPH --~) NO· + citrulline + NADP+

The constitutive NO synthase present in the vascular endothelium and the central
nervous system (CNS) is a low-activity enzyme that makes small amounts of NO·
for signalling purposes. The inducible form (iNOS) is a high-activity (Ca 2 + and
calmodulin independent) enzyme produced mostly by phagocytes after stimulation
with bacterial lipopolysaccharides (LPS) andlor cytokines. The regulation of vasal
tone (Michel and Feron, 1997) is finely modulated by vasodilator moleeules such as
NO· and CO (another toxic gas!), as weIl as by an array of moleeules such as
endothelins, leukotriene (LTB4), thromboxane A2, noradrenaline, angiotensin 11,
etc. Moncada (1992) and Hobbs et al. (1994) have demonstrated that, after binding
to the receptor situated on the plasma membrane of vascular smooth muscle ceIls,
NO· activates guanylate cyclase. Using guanosine triphosphate (GTP) as a substrate,
guanylate cyclase forms cyclic guanosine monophosphate (cGMP), which is
responsible for smooth muscle relaxation and thus vasodilatation. We shall discuss
the relevan ce of this process to ozonetherapy in Chapters 14 and 24 with regard to
ischaemic vasculopathies, particularly noting that the formation of nitrosothiols with
cysteine and GSH (Cys-NO and GS-NO) produces prolonged vasodilatation
(Koppenol, 1998; Gaston, 1999; Kashiba et al., 1999).
Physiological amounts of NO· playa beneficial role in innate immunity because
of their bactericidal and antitumoural activities (Nathan, 1997; Wink and MitcheIl,
1998). Yet excessive production, following induction of iNOS, contributes to acute
and chronic inflammation and cancer (Miller and Grisham, 1995; Titheradge, 1999).
However, as shown by Landino et al. (1996) and Squadrito and Pryor (1998),
excessive amounts of'Nö" after combination with 0/':

generate peroxynitrite, OH· and nitrogen dioxide, which are powerful cytotoxic
oxidants .
The formation of peroxynitrite is controlled by SOD, which can lower the
concentration of O2. -, Cell damage by peroxynitrite can be lirnited because it is
scavenged mostly by the ubiquitous CO2 (for details see Squadrito and Pryor, 1998).
68 CHAPTER9

As we noted for ozone, the recurring theme is that either high or low amounts of
oxidants can be toxic or even beneficial, respectively.
It was purposefully shown that practically all the most reactive oxidants are
generated by aerobic respiration and that ozone, being a triatomic oxygen, does not
produce new toxic oxidants. If this conclusion is accepted, why should the judicious
and weil controlled use of ozone in medicine scare anyone? However, the usual
exception is the inhalation of ozone, particularly when associated with NO z• and
acidie eompounds. Also in this ease, the toxicity depends on the oxidant dose and
the time of exposure. Mustafa (1990) hypothesized that, when OJ and NO z• enter the
lung, they can generate several gas-phase reaetion products (NO J, NzOs, HNO z and
HNO J) that cause additive or synergistie toxie effeets on the minimally protected
respiratory epithelium. As we use only pure O 2 and OJ, these products are not
fonned during ozonetherapy.
In conclusion, a summary of the main points may be useful:

I) a volume ofgas (02+0J) is added to blood (1/1 volume) only once, at the desired
0J eoncentration.
2) When blood is exposed to OZ-OJ at the gas-liquid interface in a closed glass
container, both gases dissolve into plasmatic water. The ozone reacts
immediately with a myriad of organic molecutes, but certainly a good deal of
ozone is promptly neutralized by hydrophilie antioxidants. Nonetheless, a variety
of new compounds are forrned (ozonides, HzOz, hydroperoxides, free radieals
and aldehydes). These compounds are intrinsically toxie and generally have a
very short half-life (Table 6).

Table 6. The half-life ofnew compounds genera ted after the interactton ofozone
with biologicalfluids .

Molecular species
HO· I X 10'9
RO· 1 x 10- 6
ROO· 0 .1-7
1 X 10-3
I X 10-6 in the presence ofSOD
1 X 10,6
From 0.1 to - 5 sec
Fairly stable in HzO
2.5 sec in plasma
< I sec in blood
HOCI Fairly stable
O=NOO' 1-4
O2 >10 2

3) Ozonization of blood must be perforrned by continuous and gentle mixing (to

avoid foaming) for at least five minutes in order to avoid perilous concentration
 OZONE REACTIVITY AND R!SKS 69

gradients. The gas mixture should never be bubbled into blood . A kinetic
evaluation of the p02 values and formation of peroxidation and oxidation
produets has shown that the plateau phase is approached in no less than five
minutes .
4) To obtain a homogeneous stimulation ofblood components without any toxicity,
it is essential to know the exaet gas and blood volumes (the usual ratio is I : 1)
and the preeise ozone eoncentration (ug/ml). The latter multiplied by the gas
volume yields the ozone dose (e.g. 225 ml ofblood are exposed to 225 ml ofgas
with an 0 3 concentration of 40 ug/rnl. The total ozone dose is 9 mg).
5) In addition to free radicals with an extremely short half-life, the ozonized blood,
whieh is being promptly reinfused into the donor in the following 15-20 min.,
contains a variety of LOPs derived from peroxidation of PUFAs (Madden et al.,
1993; Serhan, 1994; Serhan et al., 1996; Morrow and Roberts, 1997; Parola et
al., 1999). Since they are subject to dilution, receptor-binding, metabolism and
exeretion, they do not appear to display any toxieity in patients . Moreover, they
reach in trace amounts all organs and can represent messengers triggering
multiform biological activities .
6) At least in theory , the ozonized blood may contain many other compounds,
particularly if ozonization is (wrongly) carried out with an ozone concentration
higher than 80 ug/ml of gas per ml ofblood and the oxidant status ofthe blood is
subnormal. It has been stated (Chapter 2) that "any Western world Drug
Regulating Agency should condemn the infusion of the hodgepodge of ozonized
products to treat diseases", It is known that classical pharmacology and orthodox
medicine have selected the controllable approach of "one drug-one receptor-one
effect" . Yet this approach is often oflimited effectiveness when dealing with the
complexity of some human diseases . Thus the heterogeneous composition of
ozonized blood, which can potentially initiate and/or modulate a wide spectrum
of biological processes, may be seen as both the weak and the strong point of
ozonetherapy. Instead of taking a skeptical position, it would be preferable to
evaluate if the infusion of "the hodgepodge of ozonized products" yields a
beneficial effect not otherwise obtainable with conventional medication.
7) The available literature clearly indicates that 4-HNE, 4-hydroxyhexenal and
malonaldehyde derived frorn the breakdown of lipid hydroperoxides are
eytotoxic, genotoxic and have other deleterious actions. However, most of the
results have been obtained in vitro with aldehyde concentrations higher than 1
~M in fairly static conditions. The LDso of 4-HNE administered intraperitoneally
in mice is 0.44 mM or 68.6 mg/Kg body weight (Esterbauer et al., 1991). It has
been suggested that 1000-fold lower concentrations (0.05-0.5 ~M) may acquire a
physiological and perhaps pharmacological significance in vivo (Esterbauer et
a1. , 1991; Dianzani, 1998). Besides the initial ISO-fold dilution occurring in
plasma and interstitial fluid, the metabolism of aldehydes is far more effective in
vivo owing to the wealth of enzymes (aldehyde and alcohol dehydrogenases,
aldose reductase and GSH transferases) present in different cells (Esterbauer et
al., 1991; Xie et al., 1998; Srivastava et al., 2000). Moreover, hepatic
detoxification and renal exeretion ofthese toxie produets may help to understand
why we do not observe either aeute or chronie toxicity in patients.
 CHAPTER 10

HOW IS OXIDATIVE STRESS ASSESSED?

The evidence that endogenous ROS production is involved in several inflammatory

and degenerative diseases has greatly stimulated the search for representative
biomarkers and for precise, rapid methods for their detection. As ROS attack a
variety of organic substrates, oxidative stress can be evaluated by measuring
reaction products of oxidative damage. Ozonetherapy acts as an added stress and it
is imperative to carry out tests to assess any negative influence of ozone. Critical
substrates are lipids, proteins, carbohydrates and ONA contained in blood and
biological fluids . In an extensive review, Oe Zwart et al. (1999) carefully analyzed
the pros and cons of current methods of testing biomarkers of free radical damage.
There is a great variety of lipids: saturated lipids, such as palmitic (16) and
stearic (18) acids, are hardly attacked because PUFAs are preferential substrates.
The concentration of PUFAs is higher in plasma than in interstitial fluids while it
varies greatly in Iymph in relation to the diet and intestinal absorption. PUFAs are
present in chylomicrons, triglycerides, phospholipids of various lipoproteins and are
also bound to albumin. Moreover, phospholipids and cholesterol are present in the
cellular plasma membranes but , as we shall discuss in Chapter 14, do not seem to
undergo peroxidation, at least regarding erythrocytes.
PUFAs are heterogeneous and are typically represented by arachidonic acid (20:4n-6),
oleic acid ( 18:ln-6), linoleic acid (18:2n-6), o-linolenic acid (18:3n-3), 5, 8,11 ,14,17-
eicosapentaenoic acid (20:5n-3) and 4, 7, 10, 13, 16, 19-docosahexaenoic acid (22:6n-3).
With three unsaturated bonds proximal to the aqueous environment, PUFAs are
presumably more susceptible to attack by OHo, and peroxidation will generate an
array of compounds: conjugated dienes, peroxy and alkoxy radicals, hydroperoxides,
MDA, 2 and 4-hydroxyalkenals (particularly 4-HNE) and the relative adducts with
low-molecular weight- thiol proteins and enzymes. Independently of the
cyclooxygenase enzyme pathway, peroxidation of phospholipid-bound arachidonic
acid leads to the appearance of aseries of bioactive prostagiandin F2-like
compounds denominated F2-isoprostanes (F 2-IsoPs) (Morrow et al. , 1992).
The types and magnitude of biological activity of compounds generated after
exposure of blood to ozone is unknown but this topic is beyond the scope of this
chapter. However, such an analys is should obviously be perfonned with great urgency.
Understandably, a great number of more or less precise and sophisticated
techniques, including the well-known thiobarbituric acid-reactive substrates
(T BARS) assays, have been discussed in relation to the evaluation of peroxidative
processes in body fluids, cells , tissues and urine (Esterbauer et al., 1991 ; Buege and
Aust , 1994; Esterbauer, 1996) (Table 7). MDA is the most commonly and easily
measured LOP . Combining with thiobarbituric acid (T BA), it fonns a chromogen

71
72 CHAPTER 10

with an absorption peak at 535 nm. Several aldehydes have been measured in the
urine, but unfortunately aldehyde excretion is strongly influenced by several factors
that make the measurement unreliable (Draper et al., 2000) .
Measurement of ethane (from omega-6 fatty acids) and pentane (from omega-3
fatty acids) in the exhaled air by gas-chrornatography is an interesting non-invasive
method to assess lipid peroxidation in vivo, provided technical pitfalls are avoided
(Sagai and Ichinose, 1980; Drury et al., 1997; Andreoni et al., 1999). Interestingly,
Nowak et al. (2001) have measured TBARS and H202 in expired breath condensate
as a marker of airway inflammation. Measurement of Frisoprostanes is becoming a
valuable approach (Morrow et al., 1995) because they are specific products of lipid
peroxidation; moreover, their assay as urinary metabolites allows their investigation
in large clinical studies (Roberts 11 and Morrow, 2000) . Several immunological
assays (ELISAs, RIA) for proteins modified by LOPs or also for F2 -isoprostanes
have been proposed and are under evaluation.
Proteins undergoing free radieal damage generate a variety of oxidation products
due to oxidation of phenylalanine and tyrosine (Verweij et al., 1982; Berlett et al.,
1991; De Zwart et al., 1999). Carbonyl derivatives of proteins may result from
oxidative modifications of amino acid side chains and are an excellent index of
oxidative free radical damage (Stadt man and Levine, 2000) . The protein thiol group
(PTG) measured in plasma sampies after ozonization (Hu, 1994) is very useful for
routine evaluation of clinical sampIes. After oxidation, proteins cannot be repaired .
However, serum albumin is rapidly removed from the circulation by RES (Retieular
endothelial system) uptake and the albumin pool is promptly replenished by hepatic
synthesis. Damage to endocellular proteins and enzymes is far more critical because,
besides the loss of functional activities, the accumulation of oxidized proteins before
degradation may disturb the microenvironment.
The chemical basis for the action of radicals on DNA is likely oxidation and
chemical modification of the bases . If not promptly repaired by endonucleases and
glycosylases, such point mutations will lead to mod ified protein structure, with
unforeseeable consequences.
Both mitochondrial and nuclear DNA can be attacked by ROS, but ozone is
unlikely to proeure DNA damage if properly used . On the other hand, an excessive
ozone dose , or the IV infusion of ozonized saline (Foksinski et al., 1999), or
exposure of isolated cells suspended in physiologicaI media without antioxidants
(Leist et al., 1996) to even very low ozone concentrations can induce genotoxicity. It
is unfortunate that all of these unphysiological data, instead of being interpreted as
laboratory exercises, have led some people to believe that "ozone is toxic any way
you deal with it".
Wiseman and Halliwell (1996) have carefully reviewed the problem of
measuring oxidative DNA damage, particularly regarding 8-hydroxyguanine (8-
OHG) and 8-hydroxy-2'deoxyguanosine (8-0HdG). As the latter compound may
yield falsely elevated values due to sampIe handling and processing, it was proposed
to measure another three major oxidation products, one of which is 5-hydroxy-2'-
deoxycytidine (5-0H-dCyd) (Wagner et al., 1992; Beckman and Ames, 1997).
In spite of a large number of available assays, De Zwart et al. concluded their
review by stating that "there is still a great need for additional research on the
 How IS OXIDATIVE STRESS ASSESSED? 73

applicability of biomarkers of free radieal damage in humans, especially of non-

invasive and early biornarkers" .

Tahle 7. Possible intermediate and.final products present in blood after ozonization.

Some compounds are cytotoxic, som e may act as chemical messengers
and some can he used as biomarkers of oxidation..

Compounds References
A variety of lipid hydroperoxides, Serhan, 1994; Serhan et al. 1996
peroxyradicals, conjugated dienes,
cycloperoxides, eicosanoids,
platelet activating factor (PAF),
lipoxin

Thiobarbituric acid reacting Esterbauer et al., 1991; Pryor et al.,

substances (TB ARS) including 1995; Dianzani, 1998; Comporti,
aldehydes, glycation-related 1998b; Dei Corso et al., 1998; Parola et
aldehydes al., 1998, 1999; Uchida, 2000

Roberts and Morrow, 2000

Ethane, pentane, H202 and TBARS Drury et al., 1997; Andreoni et al.,
1999; Nowak et al., 2001

Oxidized amino acids and proteins. Stadtman and Oliver, 1991; Stadtman
Protein carbonyl content and Levine , 2000

Products ofDNA oxidation (8- Wiseman and Halliwell, 1996

OHG ; 8-0HdG; 5-0H-dCyd)
 CHAPTER 11

IS THERE ANY JUSTIFICATION FOR USING

OZONETHERAPY IN HUMAN PATHOLOGIES
CAUSED OR WORSENED BY FREE RADICALS?

"It is better 10 stir up 0 question withoutdeciding it, than 10 deckle il without stlrring it up"
Joseph Joubert

The involvement of RaS in several human diseases is widely accepted, although it

remains uncertain whether increased RaS formation is the primary cause or an
epiphenomenon that simply contributes to progression of the disease (HalliweIl et
al., 1992, 2000; Ames et al., 1993; McCall and Frei, 1999; Fukagawa, 1999). An
example of the former possibility can be found in neurodegenerative processes
where adefeet of antioxidants appears to be the main culprit, such as a defective
SOD in amyotrophic lateral sclerosis and GSH in Parkinson's disease (Jenner, 1994;
Bondy, 1995; Simonian and Coyle , 1996; Rowland and Shneider, 2001; Ferri et al.,
200 I). On the other hand, viral infections, such as hepatitis C virus (HCV) or human
immunodeficiency virus (HIV) , begin with viral contamination; after the infection is
established, the virus changes the cell environment to its advantage and by various
mechanisms induces a persistent oxidative stress due to an imbalance between
antioxidants and pro-oxidants in favour of the latter (Larrea et al., 1998). The
etiology of autoimmune diseases (lupus erythematosus, rheumatoid arthritis,
multiple sclerosis, diabetes, Crohn's disease, etc.) remains undefined. However, it is
likely due to a primary infection which, in a genetically predisposed individual ,
leads to a deranged immune response: an abnormal, cytotoxic T cell clone develops
and, by recognizing an autoantigen on some cells, destroys them . Atherogenesis
appears somewhat atypical but one of the primary events appears to be oxidation of
Low Density Lipoproteins (LDL) which, via the scavenger receptor A (SRA) , are
internalized in endothelial cells as a first step in increasing the cholesterol content
(Berliner and Heinecke, 1996; Steinberg, 1997). In any case , once the primary event
is established, activation of phagocytes, of lipoxygenases, metal-ion release and
deterioration of mitochondrial electron transport chains enhance the formation of
O 2. ' , H20 2, OH·, NO·, ONOO' and HOCI, with tissue damage and cell death .
In rheumatoid arthritis patients , neutrophil granulocytes are attracted into the
synovia I fluid by chemotactic factors and contribute to joint injury. A similar
situation seems to occur in adult respiratory distress syndrome (ARDS): by releasing
RaS and serine proteinases, leukocytes inactivate o't-antiproteinase so that elastase
breaks down pulmonary elastic fibres. Ischaemia may favour the release ofFe 2+ into
the anoxic area so that, during reperfusion, formation of OH· by the Fenton type
chemistry can lead to irreversible tissue damage (Gutteridge and Halliwell, 1990).

75
76 CHAPTER 11

Oxidative stress has also been documented during strenuous physical competition (Ji,
1995; Sen, 1995), such as the marathon , and even in ageing, albeit in a milder but
continuous way. Hannan (1956) was the first to propose the free radical theory of
ageing by assuming that ROS continually damage lipids, proteins and DNA. It has
been weil estabJished that the common denominator in all these diseases is that, once
started, oxidative stress goes on all the time until death (HaIliweIl et aJ., 1992; Ames et
al., 1993; Yu, 1994, 1996; Meydani et aJ., 1995; Halliwell, 1996, I999b, 2001;
Beckman and Ames, 1998; Floyd, 1999; Fukagawa, 1999; Hamilton et al., 200 I).
Why is it that Jiving organisms that have been able to counteract the offensive
action of oxygen during evolution can tolerate and then succumb to oxidative stress?
It seems as if this chronic state induces a sort of anergy or a total inability to
reverse the situation. There may be some spontaneous remissions or quieter
phases, and administration of antioxidants (since they are not harmful) may
ameliorate or delay, but not reverse, the oxidative stress . Dietary restrictions (Yu ,
1996; Harnilton et aJ., 200 I) may be helpful , but they are difficult to implement and
can yield uncertain results in patients. While nobody can doubt the efficacy of the
disinfectant properties of ozone, everyone knows that ozone can be toxic . Therefore,
is it rational to propose ozonetherapy?
Dr. E. Payr (1871-1946) and Dr. E. Fisch (1899-1966) were the first to propose
the medical use of ozone. Yet, in those days, orthodox medicine had Iittle to offer
and their pioneering idea could be justified. Today , the situation is radically different
and the proposal of a medical therapy based on ozone seems to border on insanity.
Nevertheless, a large body of medical evidence produced during the last decade,
even though anecdotal and fragrnentary, compels our attention. One of the aims of
this book is to search for valid reasons to use ozone and to point out mistakes
and false claims, Most important, however, is to demonstrate that the toxicity
of ozone can be tamed and to evaluate whether ozonetherapy is truly
efficacious.
The hypothesis that ozone can reverse chronic oxidative stress (Bocci , 1996a,d,
1988c; Leon et al., 1998; Barber et al., 1999) when we know (Chapters 5 and 9) that
ozone is a master generator of ROS sounds paradoxicaJ. What then are the ideas
behind the proposal to verify or refute the validity of ozonetherapy?

I) The toxicity of ozone as it is used for 03-AHT can be weil controlled and there is
overwhelming evidence that is insignificant. The oxidative stress imposed on the
blood is "calculated" (on the basis of ozone/blood concentrations) and is
extremely "transitory". In fact, rather than talking of an acute oxidative stress ,
it would be preferable to introduce the concept of a "multivaried therapeutic
response following smaJl and repeated oxidative stresses" or simply of a
therapeutic "shock".
2) The therapeutic "shock" is exogenous, i.e. it occurs ex vivo in a precisel y
controlled situation and , as such , must not be added to the chronic oxidative
stress (COS ) occurring inside the celJ. It appears most unlikely that ROS
generated during the therapeutic "shock" can reach and damage leukocytic DNA .
3) The amounts of LOPs produced ex vivo during 0 3-AHT (nonn ally 225 ml of
blood + 225 ml of O 2-03, with 0 3 concentrations in the range 20-80 ug /ml ; total
 HUMAN PA THOLOGIES DUE TO FREE RADICALS 77

doses ranging from 4.5 to 18 mg 0 3) are under control. The "therapeutic

response" is generated by RaS and LOPs derived from plasma. Reinfusion
occurs in 20-30 min and involves a 600- to 700-fold dilution because about 7 ml
of ozonized blood re-enter the circulation and are mixed with about 5 L of
plasma every minute . The final LOP plasma levels depend on diffusion into
about 10 L of extracellular fluid, as weil as on receptor binding, catabolism and
renal filtration. As a consequence and as we have observed, LOPs disappear
rapidly from plasma in vivo. Therefore, the therapeutic "shock" has a unique
pharmacokinetic characteristic: reinfusion of ozonized blood involves only a
transient pulse of ROS that can exert a stimulus at variance with the constant
RaS levels, as observed in patients with degenerative-inflammatory diseases .
4) Another critical difference between endogenous and therapeutic "shock" is that
the intermittent schedule of 03-AHT appears to induce an upregulation of
antioxidant enzymes which, in comparison to the administration of antioxidants,
is one possible way of reversing chronic oxidative stress . Evidence for this is
discussed in Chapter 22. The therapeutic "shock" may weil induce peculiar
oxidative stress proteins (OSl'), such as haeme oxygenase (HSP32) or other
chaperones, able to display therapeutic activities in vasculopathies, viral
infections and cancer.
5) Great care must be taken in ensuring that patients undergoing 03 -AHT have a
plasmatic total antioxidant status (TAS; Miller et al., 1993) within the normal
range (1.3-1.8 mM). Depletion of antioxidants can be suspected after anorexia ,
malnutrition, cachexia, chemotherapy, etc. Therefore, bearing in mind the axiom
"primum non nocere" (first, do not harm), the basic TAS level must be checked.
If it is below the normal value, standard pharmaceutical supplements must be
administered daily for at least two weeks before starting ozonetherapy. In
Chapter 12, the evidence that plasma antioxidants constitute a strong defense
aga inst ozone will be provided.

It is possible that, in future, gene therapy or the use of stem cells may work
miracles and make ozonetherapy truly obsolete. But when?
 CHAPTER 12

THE ANTIOXIDANT SYSTEM

AND THE DEFENCE SYSTEM AGAINST OZONE

Since the fonnation of the earth, for almost three billion years, there was no oxygen
in the atrnosphere . Praeticall y all the O 2 produced by early photosynthetie baeteria
eaused the oxidat ion of Fe2+ to Fe 3+ , with preeipitation of ferrie oxides in the sea .
Once the reserve of Fe 2+ was exhausted, cyanobaeteria, having developed the ability
to split water to obtain more energy, started to "pollute" the air with oxygen, which
slowly reached its current levels about two billion years aga (Dismukes et al., 200 I;
Kasting, 2001). In the meantime, baeteria had to face the option of either dying or
remaining anaerobic or adapting to the oxygenated environment. AIthough the
oxidation of substrates allowed the release of more energy (hence the possibility of
synthesizing more ATP ), it also raised the problem of evolving mechanisms of
protection against O 2 tox icity . Indeed , oxygen probably created one of the first
paradoxes on earth when the advantage of oxidation had to be paid for by either cell
damage or the development of antioxidant defences.
Today in Biology, we have plenty of these contrasting examples magically
represented by the Chinese symbol Yin-Yang (Fig . 17). Ozone started to be
synthesized as a protective agent and only recently has become a controversial gas,
as discussed in Chapters 3 and 9.
Here , we wish to evaluate the eomposition of the antioxidant system and how it
works to neutralize or limit damage indueed by RaS . At first sight, the system
appears to be composed of many parts , which on superficial examination appear
redundant: the aggressive power of RaS seems to have stimulated the cell to
"invent" various types of control that, by operating together or in tandem, maximize
the effieaey. However Ames et al. (1993 ) have estimated that the number of
endogenous oxidative hits on DNA per cell per day is up to 100,000 in the rat and
about 10,000 in the human, who has a lower metabolie rate . On this basis, the
antioxidant system does not seem to be perfect. Luckily, the cell is capable of
repairing most of the oxidative damage by means of glycosylases, whieh excise
oxidized bases from double-stranded DNA , and phospholipase A2, which c1eaves
lipid peroxides from phospholipids (Beckman and Ames, 1998). Oxidized structural
proteins and enzymes can be eatabolized and synthesized ex novo. There are a
number of eompounding factors, such as genetic background, gender difference,
type of diet and quantity of ingested food , age and life-style, that render indiv iduals
differently sensitive to RaS . As an extreme example, there are two typical diseases,
Wemer syndrome and progeria, in which very high levels of oxidized proteins are
highly correlated with premature ageing . This scenario, which helps explain ageing ,

79
80 CHAPTER 12

would seem to forbid the use of any extra oxidative insult, i.e. ozonetherapy. Yet,
onee again, we should bear in mind that this medical approach is based on an
extremely transitory and calculated oxidative stress imposed ex vivo and tamed
by antioxidants in plasma (Chapter 9).

Yin Yang

Oncogenes Antioncogenes
Hormones Chalones
Proteinases Antiproteinases
Oxidants Antiox idants
Insulin Glucagon
Thrombin Antithrombin 111
Norepinephrine Acetylcholine
Glutammic acid GABA
Thromboxane Prostacyclin
Angiogenin Angiostatin
Endothelin NO
CD4+, Thl CD4 +, Th2
IL-2, IL-12, IFNy IL-4, IL- 10, TGFßl

Figure 17. The phenomenon ofcontraposition.

The fin- fang. Examples of antagonism between biological effectors .

The system has been classified into two modalities:

l. preventive antioxidants, sueh as enzymes (SüD, GSH-Px, eatalase), earotenoids

and proteins that chelate transition metals;
2. seavenger antioxidants, namely vitamins C and E, urie acid, bilirubin , ubiquinol.

However, from a didaetie point of view, it seems easier to diseus s the antioxidant
enzymes separately frorn the hydrophilie and lipophilie antioxidant compounds
(Table 8).
 THE ANTIOXIDANT SYSTEM 81

Table 8. The antioxidant system .

NON ENZYMATIC ENZYMATIC

Hydro soluble Liposolllhie Chelating Protein s
Uric acid Vitamin E Transferrin Superoxide dismutases (SüD)
Ascorbic acid Vitamin A, Ferritin Catalase
Glucose, Cysteine, Caroteneoids Ceruloplasmin Glutathione peroxidases
Cysteamine, Coenzyme Q Lactoferrin Gluthatione redox system
Taurine, u-lipoic acid Haemopexin
Tryptophane, Bilirubin Albumin
Hystidine, Thioredoxin
Methionine Bioflavonoids
GSH Melatonin
Plasma proteins

I . HYDROSOLUBLE ANTIOXIDAl"fTS

1.1 Uric Acid

This compound is the final product of purine degradation and is excreted in the
urine. In humans, normal plasmatic levels range between 3 and 7 mg/dl (about 400
u M); owing to the poor solubility of urate, its increased level in the serum may
induce the deposition of urate crystals in the kidneys and joints (gout). Interestingly,
humans have levels of urate that are ten times higher than in prosimians; thus they
may have the painful drawback of gout, but they also have the protective advantage
of urate as a potent antioxidant. Uric acid is a very good scavenger of OH", O 2". ,
ROO· and ONOO· (Hooper et al., 2000); it prevents the oxidation of vitamin C and
thus helps conserve it. It can also bind transition metals. During ozonization, uric
acid is usually oxidized to allantoin, and like albumin behaves as a "sacrificial
moleeule", i.e. its oxidation spares other functional substrates.

Figure 18.Oxidation ofuric acid gives rise to allantoin.

Uric acid plays an important protective role in blood to be used for 03-AHT
(using a therapeutic concentration of ozone below 80 ug/rnl per ml of blood). The
contribution from urate to the Total (peroxyl) Radical-trapping Antioxidant
Parameter (TRAP) was found to be as much as 58 ± 18% (Wayner et al., 1987) .
 82 CHAPTER 12

Even though uric acid and bilirubin are moleeules to be excreted, they have great
value because of their antioxidant properties (Meadows et al., 1986; Meadows and
Smith, 1987).
100 Air 100
-0-

__ 2ppm I~~:---o- _ _--o

~ SO ~ SO
:;,R
~
---4ppm
C
..... Sppm
:'§ 60 :'§ 60
ro ..... 16ppm ro
o u
:.c 40 'e
:::l 40
5c
~ 20 20

o '---'-........L_"----'-_'---'-........L----l
o 15 30 45 60 75 90 105 120 15 30 45 60 75 90 105 120

Time (min)

Figure 19. Oxidation of'ascorbic acid and uric acid in human plasma samples exposed
forup to 120 min to either air 01' to increasing OZOl1e concentrations (2,4.8 and 16 ppm).
From Van der Vliet et al.. 1995,

150

100
•
~
•
50

••
0
0 20 40 60 80 100 120
0) (llg/mL)

Figure 20. Levels 0/ antioxidants in plasma ofwhole hlood reacted with low levels 01'
ozone: (0) bilirubin (10.3:1:1. 7 nmol/ml): (0) a-Toc (13.8:1: 1,5 nmol/ml); (.) uric acid (278
:1: 30 l1moI111l/); (0) ascorbic acid (50.2 :1:8,8 nmol/ml) . Control group (0 J.1g O/ml blood)
values are defined as 100 %, The initial concentrations are in parentheses. Values are
expressed as the AI :tSE of7 subjects. Significantly differentfrom control group (0 J.1g O./ml
hlood). '"p <0.05. "''''p <O.OI. (From Shinriki et al.. 1998).

There is convincing experimental evidence (Figures 19 and 20) obtained in three

different laboratories: in Canada (Wayner et al., 1987), in the USA (Cross et al.,
1992a; O'NeiII et al., 1993; Van der Vliet et al., 1995) and in Japan (Shinriki et al.,
1998). It is very c1ear that, depending on the concentration and time of exposure,
 THE ANTIOXIDANT SYSTEM 83

ozone is able to progressively reduce uric acid levels in plasma until exhaustion.
Cross et al. (1992a) proposed that a physiological function of uric acid in upper
RTLFs , which contain little ascorbic acid and albumin (Hatch, 1991), is to scavenge
inhaled ozone, thus protecting the epithelium. The findings of Shinriki et al. (1998)
are particularly relevant because they have perfectly imitated what happens during
blood ozonization. Contrary to what is normally thought , an ozone concentration of
I00 ug/ml per ml of blood practically exhausts the ascorbate while about 70% of
uric acid remains unoxidized.
In conclusion, serum uric acid levels are important in reducing oxidative
damage . In fact, Hooper et al. (2000) even suggested that low levels may predispose
an individual to the development of multiple sclerosis. Presumably, abnormally high
TAS levels may be present in hyperuricemic patients and it remains unchecked
whether hyperuricemia reduces ozone activity .

1.2. Ascorbic Acid (AH. Vitamin C)

It acts mainly as an antioxidant. It is a good scavenger of several ROS and has a

major role in the synthesis of collagen , because without it collagen is insufficiently
hydroxylated. The average concentration in human plasma ranges between 0.4 and
1.5 mg/dL , equivalent to about 50 ~M . Thus the concentration of ascorbic acid is 8-
16 times lower than that of uric acid and, according to Cross et al. (1992a), more of
the ozone reacts with uric acid than with ascorbic acid in human plasma .
Ascorbic acid is an essential vitamin because guinea pigs and primates have lost
the ability to synthesize it; they must acquire it from fresh fruits and vegetables . In
spite of the fact that a good deal of dehydroascorbic acid (DHA) is rapidly recycled to
ascorbic acid (Mendiratta et al., 1998a), we need an average daily dose of about 60 mg
(US/RDA). A lack of ascorbic acid in the diet for months causes scurvy, a deadly
disease described by Jacques Cartier in 1536. In 1753, James Lind, a Scottish
physician, advised that lemons should be included in the diet of sailors. Albert Szent-
Gyorgy identified ascorbic acid in 1933 and was awarded the Nobel prize in 1937.
Ascorbic acid has many virtues (Frei et al., 1989, 1991; Frei, 1999; Halliwell,
1994, 1999a,b; Berger et al., 1997; McCall and Frei, 1999).

• It scavenges O 2°', OHO, H0 2°, R0 2°, thiyl (RSO) and sulphenyl (RSOO) radicals,
NO°,10 2 , O=NOO', 0 3 •
• It slows down or blocks the formation of HOCI, acting as a substrate for the
enzyme myeloperoxidase.
• It inhibits lipid peroxidation by activated neutrophils, haemoglobin or
myoglobin-Hjö, mixtures and, acting as a competitive substrate , prevents haeme
breakdown, hence the release of Fe2+ that may favour OHo formation .
• By reducing o-tocopheryl radicals in lipoproteins and membranes, it regenerates
a-tocopherol.
• In the stomaeh, it can reduce carcinogenic nitrosamines to inactive compounds,
thus inhibiting carcinogenesis.
• It protects uric acid frorn OHo attack.
84 CHAPTER 12

• To some extent, it seems to protect against oxygen-derived species present in

cigarette smoke (Frei et al., 1991; Polidori et al., 200 I).
• According to Hatch (1991), RTLFs contain far less ascorbic acid than uric acid,
Thus ascorbic acid affords little protection against inhaled ozone .
• Last, but not least, by maintaining prolyl and Iysyl hydroxylases in an active
form (with iron in the reduced state), ascorbic acid allows the hydroxylation of
proline to hydroxyproline, which is indispensable for correct synthesis of
collagen.

All of these useful activities of ascorbic acid are due to the transfer of electrons
in one or two steps, with the formation of either semidehydroascorbate radical anion
o
(A , ) , a poorly-reactive radieal or dehydroascorbic acid (DHA) . The ascorbyl radieal
can be reconverted to ascorbate by NADH-semidehydroascorbate reductase while
dehydroascorbate either decomposes irreversibly to 2,3-diketogulonic acid or is
recycled to ascorbate by GSH-dependent enzymes.

H\H 2

H~f~~n
o OH
AH-
Figure 21. Two successive one electron ox idations transform ascorbate (AH) into ascorbyl
radical (A) and then into dehydroascorbic acid (DHA) . The reaction can be reversed hy
successive redu ction .

These compounds constitute an effective redox system and Figures 19 and 20

have c1early shown that oxidation of ascorbate depends upon the ozone dose.
McCall and Frei (1999) have emphasized the pleiotropic protection exerted by
ascorbate in plasma, where vitamin E appears less effective (Fig. 20) . At least in
part, the reactions of Figure 21 are reversible and the recycling of both
dehydroascorbic acid and ascorbyl radieal to ascorbate is extraordinarily rapid
(about 3 minutes) . Meister (1994), May et al. (1996) and Mendiratta et al. (I 998b)
have provided an important contribution to the c1arification of the cooperation
between GSH and ascorbate. The erythrocyte has an enormous capacity to
regenerate ascorbate because dehydroascorbic acid is rapidly taken up by facilitated
diffusion on the GLUTI glucose transporter and promptly reduced to ascorbate (up
to 1.5 mM) via a GSH-dependent reductase that oxidizes GSH to GSSG . The latter
compound will be recycled to GSH utilizing reducing equivalents from glucose and
the hexose monophosphate shunt (this Chapter). Moreover, thioredoxin reductase
(TrxR), one of the rnajor redox-regulatory molecules, contributes to the reduction of
DHA (Mendiratta et al., 1998b; Li et al., 2001) . If we had not developed this
 THE ANTlOXIDANT SYSTEM 85

mechanism ofrecovery, we would have needed a daily intake of AH' far higher than
60 mg and we would probably have to ingest about 100 g throughout the day to
maintain a plasma level around 50 11M. Levine et al. (1996, 1998) have shown in
healthy volunteers that, if the dietary intake is already saturating, further
supplementation will have no positive effect. This point needs to be discussed
because there are contrasting opinions and some physicians think it correct to
administer megadoses of ascorbate (10-20 g) daily . The pharmacokinetic study by
Levine et al. (1996) convincingly demonstrated that a dose of 400 mg pro die is
already oversaturating (plasma level of about 50 11M or 0.9 mg/dL) and therefore
megadoses appear useless because they are either poorly absorbed or mostly
elirninated in the urine.
Even more convincing is the analysis of the ascorbate gradient between the
cellular/extracellular environment, When ascorbate plasma levels are at a value of
50 11M, the ascorbate concentration in normal erythrocytes and lymphocytes is up to
1.5-3 mM (30-60 fold higher) and cannot increase further because the intracellular
concentration has reached the plateau (Tmax). The data beautifully illustrate that the
cell considers ascorbate to be very important as a protective agent. There may be
pathological conditions (viral infections, particularly HIV) where the transport
mechanism and/or the GSH-DHA cycle is impaired. However, in these cases, rather
than administering megadoses of ascorbate, it is better to specifically block viral
replication and restore the transport mechanism.
In pat ients with advanced cancer, a controlled double-blind trial with ascorbate
revealed no improvement in comparison with patients who received only placebo
(Moertel et al., 1985). Nonetheless, Linus Pauling's idea still lingers (Gottlieb,
1999) and it appears difficult to clarify the anticarcinogenic role of ascorbate alone ,
since there is always a contribution of other antioxidants.
Moreover, megadoses of ascorbate may have a drawback. At least in vitro,
ascorbate may become pro-oxidant (Halliwell, 1994, 1999a,b; Berger et al., 1997;
Carr and Frei, 1999; Frei, 1999). This may occur in the presence of transition metal
ions and generate O 2. ' , OH and H20 2 • The combination of ascorbic acid with FeH
O

causes lipid peroxidation, probably because:

Fe 3+ + AH'--~) Fe2+ + H-'- + A O

'

A which is a fairly unreactive species, may reduce another Fe3+:

O
' ,

Oxidation of AR may lead to the formation ofH 20 2 :

Consequently, the Fenton reaction can take place with formation of Oll":
86 CHAPTER 12

These reactions indicate that ascorbic acid administered to iron-overloaded

patients (haemochromatosis, ß-thalassemia) not treated with iron chelators
(desferrioxamine) can be toxic and carcinogenic, a problem weil reviewed by
Toyokuni (1996). Moreover, the potentially deleterious action of megadoses of
ascorbate can also be envisaged in hypercatabolic diseases such as cancer
(cachexia), AIDS and intensive chemotherapy (Carr and Frei, 1999). These
considerations do not detract from the antioxidant activities of ascorbate but only
warn about a possible risk . Before starting ozonetherapy, it is important to evaluate
the patient's TAS and, if necessary, a precautionary small dose of ascorbate can be
taken daily (Chapters 22 and 24).

1.3. Other Hydrosoluble Low Molecular Weight Antioxidants (LMWA)

In biological fluids, the reservoir and amount of glucose is substantial (about 60-100
mg/dL) and it can act as a OH· scavenger. There are also variable amounts of free
amino acids, but particularly cysteine can donate electrons and serves to reduce
oxidants. Methionine is readily oxidized.
Taurine can scavenge HOCI. The tripeptide GSH is scarce outside the cell
(Samiec et al., 1998) but it can react directly with several radicals (OH·, O 2•. , RS·,
R0 2· ) .

1.4. Plasma Proteins

A large reservoir of antioxidant capacity is attributable to the plasma proteins: in
plasma and in extracellular fluids, there are about 2 lOg and 500 g of proteins,
respectively. According to Wayner et al. (1985,1987), the changes in concentrations
of plasma antioxidants during peroxyl radical attack show that the first !ine of
defense is provided by the plasma -SH groups. Albumin, representing the bulk of
plasma proteins, is the main "sacrificial" target because ozone attacks the numerous
- SH groups and also histidine and tyrosine residues (Verweij et al., 1982; Berlett et
al., 1991; van der Vliet et al., 1995). Cross et al. (l992b) have emphasized the great
protective action of albumin. After human plasma was exposed to ozone for 6 hours,
there was no significant damage to either several enzymes (lactate dehydrogenase,
alkaline phosphatase, gammaglutamyl transferase and aspartate transaminase) or to
plasma lipoproteins, whereas exposure of single enzymes led to a rapid loss of
activity. An investigation using a solution of bovine serum albumin has shown a
potent inhibition of O,", H 20 zimd HOCI produced by activated human granulocytes
(Kouoh et al., 1999). In arecent study , we prepared a suspension containing
micelIes of arachidonic acid (AA) and either uric acid or AH- or human albumin ; we
then evaluated the capacity of one or a combination of these compounds to inhibit
the peroxidation of AA and the reduction of TAS values . Figures 22 and 23 show
the importance of albumin alone or combined with uric acid and AR (Larini and
Bocci, 2002, in preparation) .
 THE ANTIOXIDANT SY STEM 87

TAS
0 .6

0 .5

0 .4

"ee
Ci
0 .3
I/l
~
0 .2 -

0 .1

Jj2!fS "1c ! 2
03
~
0
~ i 1 ~ 2 lf S
J0
lf 2 !f S
J 0 J0
t
1 2 lf S jj2lfS

A 8 C o E F

Figure 22. Total antioxidant status 0/ six samples eontaining araehidonic acid (3 mg/ml).
Moreover:
sample B contains uric acid (5 mg/dl)
sample C eontains aseorbie acid (1 mg/d1)
sample D contains both uric and aseorbic acid
samp le E contains human albumin (40 mg/ml)
sample F contains uric and ascorbic acid and album in.
TAS values are significantly higher (" p <0.05. ** p <0.01. *** p <0.001) in samp les
eontaining either albumin alone 0 1' all three antioxidants. Sampies were either not treated
(control) 01' exposed to oxygen alone 0 1' to three ozone coneentrations (20. 40, 80 jlg/ml per
ml 0/ sampie).101' 10 min (Larini and Bocci, in preparatio n).

Both Wayner et al. (1987) and later Miller et al. (1993) developed methods to
measure the total antioxidant capacity of human plasma and blood. The acronyms
used are TRAP and TAS, respectively . With the TRAP method, it was shown that
contributions from urate (35-65%), plasma proteins (10-50 %) ascorbate (0-24%) and
vitamin E (5-10%) account for all of the peroxyl radical-trapping antioxidant
activity of plasma. With the TAS method , the antioxidant hierarchy in plasmatic
water was 43% for albumin, 33% for urate, 9% for ascorbate, 3% for vitamin E, 2%
for bilirubin and 10% for unknown antioxidants (probably cysteine, GSH, ß-
carotene, dihydrolipoate and ubiquinol). The TRAP method showed a chain-
breaking antioxidant activity, which allowed it to trap 0.7-1 mM ofradicals; this is
not much different from the range 1.30-1.77 mM plasma measured by the TAS
method .
88 CHAPTER 12

T9All S ~M

Ja

10
A 10

_d1_
Ja

'0
8
10

Ja

"_[jJ_rn_
C 10

': _ d .
Ja

lO

'0

El_
F ~ I_ cont rol
_ ----'='----..~
· ><'(9"" 20
c=- _
40
__'___""_
80

Figure 23. TBARS values (j1M) determined in the same six sampies described in Figure 22.
Interestingly lipid peroxidation is significantly (* p <O.05, ** p <O.OI, *** p <O.OOI) inhibited
by the presence 0/ either albumin alone or by all ofthe three antioxidants. Uric and ascorb ic
acids are less effective than albumin (Larini and Bocci, in preporation).

A third assay, the ferric reducing/antioxidant power (FRAP) assay proposed by

Benzie and Strain (1999) , uses antioxidants as reductants in a redox-linked
colorimetric method to measure the total antioxidant power ofbiological fluids .
Recently, the antioxidant capacity of LMWA in plasma was also measured by
the cyclic voltammetry method (Kohen et al., 2000; Chevion and Chevion, 2000)
and by combining the approaches of Wayner et al. (1985) and DeLange and Glazer
(1989) using R-phycoerythrin as an extemal probe (Ghiselli et al., 2000) . All of
these methods have shown the great antioxidant capacity of plasma and the
importance of this type of assessment for identification of conditions affecting the
oxidative status in vivo.
A couple of considerations are in order:
 THE ANTIOXIDANT SYSTEM 89

• Firstly, it is reassuring thar exposure of blood to the highest ozone concentration

(80 ug/ml per ml of blood), as occasionally occurs in 0 3 -AHT, lowers the TAS
value by no more than 30%. Notably, this value then recovers in less than 20 min
(Chapter 13). This is in keeping with the lack of acute and chronic toxicity, but
nonethe1ess critical parameters of oxidative stress should be monitored in all
patients undergoing ozonetherapy.
• Secondly, it is possible that some ozonetherapists using an ozone generator
without a photometer, or with a poorly controlled one, deliver lower than
necessary ozone doses and probably provide only a placebo treatment. This
remains a problem even though it does not proeure toxic effects .

2. LIPOSOLUBLE ANTIOXIDANTS

2.1. Vitamin E

cc-tocopherol (vitamin E, EH) is a very effective lipid-soluble antioxidant present in

cell membranes and lipoproteins. Acting as a chain-breaking agent, it can block the
propagation of lipid peroxidation of PUFAs in phospholipids. In Chapter 9 (Fig. 14,
phase III), it was shown that during peroxidation, a peroxyl radical ROO· abstracts
an electron frorn an adjacent acyl chain, with formation of a lipid hydroperoxide
(ROOH) . However, vitamin E can interrupt the chain reaction by forming an a-
tocopheryl radical (E·) that is fairly stable due to delocalization of the unpaired
electron :

EH + ROO· ~ ROOH + E·

Figure 24 shows the chemical structure with the OH group that, after donating
the electron, becomes 0·.

HO

Figure 24. Chemical structure ofvitamin E.

Vitamin E is a mixture of at least four tocopherols '(«, ß, y and 0), the a

component being the most active . The concentration in human plasma is normally
25-50 J.1M (Mecocci et a1. , 2000) . However , nature has provided a means to recycle
E· into EH by means ofthree reactions using ascorbate, GSH or ubiquinol (QH 2) :
90 CHAPTER 12

EO + ascorbate --~) EH + AO-

or

2EO + 2GSH -~) GSSG + 2EH

or

2EO + QH 2 --~) 2EH + Q (ubiquinone)

These examples iIIustrate the cooperation among various redox systems (Wefers
and Sies, 1988; Wang et al., 1996). The RDA of EH is 30 IU (10 mg) but even a
daily dose of 100 mg (300 IU) is probably weil tolerated (Kappus and Diplock,
1992). After extensively analyzing the role of vitamin E in heart disease, Pryor
(2000) suggested a supplementation of 100 to 400 IU daily as part of a "program of
heart-healthy behaviour that ineludes a fruit- and vegetables-rich diet and regular
exercise". As usual , some practitioners suggest the use of megadoses of 1200-2400
IU daily but these are likely to be superfluous if not outright deleterious: in the
absence of co-antioxidants such as ascorbate and ubiquinol, vitamin E associated
with LDL can behave as a pro-oxidant (Stocker et al., 1991; Bowry and Stocker,
1993; Frei, 1999) :

EH + LOOH --~) E· + La· + H 20

On this basis , it appears more rational to administer a lower dose of vitamin E

balanced with other SMWA.
The basic activity of vitamin E is certainly the trapping of peroxyl radicals
during peroxidation (Chow and Kaneko, 1979). It is an old observation that in
premature newboms with lack of vitamin E, hyperoxygenation enhances haemolysis
and retinal damage, so that it is imperative to administer the vitamin as soon as
possible. More recently, it has been discovered that, by inhibiting the translocation
of protein kinase C, cc-tocophercl can modulate enzymatic activity. This mechanism
may explain a number of effects of vitamin E on NO· activity, smooth museIe
proliferation and platelet aggregation . Together, these effects would maintain
vascular homeostasis and reduce the c1inical incidence of cardiovascular disease
(Keaney Jr et al., 1999).

2.2. Vitamin A (Retinol) and Carotenoids

In plants, there are about 600 carotenoids, some of which have vitamin A activity.
They are present in yellow and orange vegetables, milk and milk products, liver and
fish liver oil . They are polyenes containing an extended network of single and
double bonds (Fig . 25) .
The hydrophobic chain allows the molecules to absorb light and to inactivate
10 2, thiyl (RS·) and peroxyl R0 2• radicals. Carotenoids can be converted to yield
 THE ANTIOXIDANT SYSTEM 91

retinal (aldehyde form), retinol (alcohol) and retinoic acid, essential for vision and
maintenance of differentiated epithelia. In body fluids , they are always bound and
transported by retinol-binding protein (RBP). The mean values of these compounds
in plasma are : retinol, about 2.9 11M (interestingly centenarians have a mean value of
5.4!); n-carotene, 0.14 11M; ß-carotene, 0.77 11M; lycopene, 0.9 11M (Mecocci et al.,
2000; Polidori et al., 2001) . The RDA of retinol is about 5000 IV and exaggerated
daily supplementation (4-6 times the RDA) of retinol and ß-carotene for long
periods can be noxious and result in toxic effects.

Figure 25. Chemical stru cture ofß-carotene (above) and vitamin A (below) .

The antioxidant activity of these compounds is quite variable and is more

effective at low p02 values (10-30 mmHg) than at arterial values (p02 near 90-98
mmHg), when retinol and carotenoids can undergo auto-oxidation. Di Mascio et al.
(1989) have determined that, even at low plasmatic levels (0.7 11M), lycopene
(present in tomatoes) is more effective in quenching 10 2 than u -tocopherol (22 11M),
ß-carotene (0.5 11M) and bilirubin bound to albumin (15 JlM).
Dietary supplements of carotenoids seem to have little or no value in preventing
cardiovascular disease. In contrast to vitamin E and ubiquinol, ß-carotene does not
protect LDL from oxidation. Moreover, there does not seem to be a cooperative
interaction between ß-carotene and vitamin E (Tsuchihashi et al., 1995) .
Supplemental ß-carotene is also unlikely to be beneficial in reducing the incidence
of cancer in well-nourished populations (Mayne, 1996). Surprisingly, the intake of
tomato-based foods rich in lycopene has been found to be associated with
significantly lower prostate cancer risk (Giovannucci et al., 1995; Clinton, 1998;
Matos et al., 2000) and an old saw has jokingly been paraphrased as: a tomato a day,
keeps prostate cancer away!
It is weil known that retinol is indispensable for retinal function, particularly for
noctumal vision, and its deficiency is responsible for at least 100,000 cases of
childhood blindness in the world (Palace et al., 1999) . Age-related macular
degeneration in relation to ozonetherapy will be discussed in Chapter 24 in
comparison to the intake of retinol and possibly lutein (- 0.366 11M) and zeaxanthin
(- 0.133 11M), which are carotenoids present in spinach and collard greens
(Handelrnan et al., 1988; Mecocci et al., 2000).
92 CHAPTER 12

2.3. Coenzyme Q (Ubiquinone, Q[I)}

Ubiquinone is a Iiposoluble derivative with six to ten isoprene units . It is ubiquitous
and present in cell membranes and lipoproteins, particularly in high-density
lipoproteins (HOL). The isoprenoid tail makes QIO highly lipophilic and allows it to
diffuse into the hydrocarbon phase ofthe inner mitochondrial membrane. QIO acts as
an electron carrier between Complex I 01' II and Complex III of the respiratory
chain . As shown in Figure 26, Coenzyme Q exists in three states: the oxidized form
(Q) , the radical intermediate form (QW') and the reduced form (QH 2 , ubiquinol).
Coenzyme Q can be reduced by either NAOH 01' flavin adenine dinucleotide,
reduced form (FAOH 2) ; by either donating 01' taking electrons, it represents a good
redox system. Another important function of Q ~ QH 2 is to act as a scavenger of
free radicals. By blocking both the initiation and propagation phases ofperoxidation,
it minimizes damage to the proteins and phospholipids of all membranes. In this
regard, it may be more efficient than vitamin E (Stocker et al., 1991). Yet it remains
uncertain if QH 2 can reduce the cc-tocopherol radieal in vivo, areaction easily
carried out by both GSH and ascorbate.

Figure 26. The oxidisedform ofcoenzyme Q (Q.. ubiquinone) is reduced to afree radical
semiquinone (QH') hy the uptake ofa single electron. Reduction o/QH' hya second electron
yields ubiquinol (QH;).
 THE ANTIOXIDANT SYSTEM 93

The Coenzyme Q content in man is 0.5-1.5 g. A daily dose of 20 mg can yield a

blood level of about 20 ug/dl. after prolonged treatment. This may be useful in
ageing (Ames et al., 1993) and in degenerative diseases, in which a progressive
decline of Coenzyme Q has been observed in tissue levels due to impaired
endogenous synthesis (Linnane et al., 1995; Forsmark-Andree et al., 1997).

2.4. a-Lipoic Acid (LA, Thioctic Acid)

This compound was isolated from liver by Lester 1. Reed and I. C. Gunsalus in 1951
and is an essential component of pyruvate dehydrogenase and a-ketoglutarate
dehydrogenase, involved in carbohydrate metabolism.
LA has stimulated great interest as a thiol-replenishing and redox-modulating
agent. At pH 7.3 , LA is anionic and referred to as lipoate. Administered lipoate is
readily accepted as a substrate for bioreduction, with the formation of dihydrolipoate
(DHLA) containing two thiol groups. This process occurs at the expense of cellular
reducing equivalents present in NADH and NADPH, leading to an improvement of
intracellular reduced thiol status (Ziegler and Gries, 1997; Packer et al., 1997).

Figure 27. Structure oflipoic acid in the oxidisedform (ahove) and in the reducedform
(below) .

Figure 27 shows the structure of LA : C 6 and C S are linked to a reactive

disulphide bridge that can be reduced to dihydrolipoate. LA is covalently attached to
a specific lysine side chain of dihydrolipoyl transcetylase forrning lipoamide. The
lipoate-dihydrolipoate cycle in conjunction with vitamins C, E and NADPH favours
the continuous reduction of GSSG to GSH and thus plays a central role in the
antioxidant defense network (Sen et al., 1997; Sen et al., 1999). Indeed, LA-DHLA
couples are powerful scavengers of OH· , HOCI and other ROS (Scott et al., 1994).
Moreover, they can chelate several metal ions (Cu2+, Fe 3 \ Mn 2+, Zn 2+ and CD 2+).
LA is endogenously synthesized and thus is not an essential component "sensu
stricto". However, it may acquire therapeutic value in some diseases, namely
diabetes type H, diabetic polyneuropathy and especially hepatic pathologies due to
94 CHAPTER 12

mushrooms, alcohol , heavy metal intoxication and carbon tetrachloride (CCI 4 )

poisoning (Bustamante et al., 1998) . A beneficial effect of LA seems to occur at a
dose as high as 600 mg/day . Liver diseases (chronic viral hepatitis, primary biliary
cirrhosis) and other viral infections (HIV) involving oxidative stress mayaiso
benefit from the use of LA . Thus oral supplementation of LA may be useful during
treatment of chronic viral hepatitis with ozonetherapy (Chapters 22 and 24) .

2.5. Bilirubin
Bilirubin is a physiological constituent deriving from degradation (catalyzed by
haeme oxygenase) of the haeme group to biliverdin which is then reduced to
bilirubin through biliverdin reductase (Fig . 28). As the life span of human
erythrocytes is about 4 months, about 0.8% of the erythrocyte mass is broken down
every day in erythrocatheretic sites. Another relevant aspect of this catabolic
reaction is that a methene-bridge carbon is released as CO .

+ co

~NAOPH + W

~NAOP+

o HH H'l::l
."p-~
N
H
C
H
I
N
H
P
C
Hz
N
H
1-
CA
H
N
H

Figure 28. Haeme oxygenase degrades haeme to biliverdin (above) and carbon monoxide.
The uptake ofone electron reduces biliv erdin to bilirubin (below) .

Bilirubin is far less soluble in water than biliverdin and, in orde r to be

transported to the liver from the spleen and bone marrow, it is complexed to serum
albumin . 'Once attached to two glucuronate molecules (bilirubin-diglucuronide) in
the hepatocyte, bilirubin becomes water-soluble and is secreted into bile. The
normal plasmatic level of bilirubin is about I mg/dL , of which 0.8 mg is bound to
albumin and 0.1-0.2 mg are present as bilirubin-diglucuronide or bilirubin-sulphate.
Although bilirubin is a waste molecule, like uric acid, it is a very effective anti-
oxidant, while biliverdin is not:

2ROO· + bilirubin --~) 2ROOH + biliverdin

 THE ANTIOXIDANT SYSTEM 95

After rapid reduction, biliverdin is shuttled back to bilirubin . Therefore, bilirubin

behaves like vitamin E and, as a chain-breaking agent present in cell membranes, it
can effectively block the propagation of lipid peroxidation. It also inhibits the
oxidizing action of O=NOO- by oxidation to biliverdin (Minetti et al., 1998). On a
molar basis, the complex albumin-bilirubin is about a tenth as effective as ascorbate
and probably less than urate.
A critical issue is the likelihood that, during ozonetherapy, a trace of haemolysis
induced by ozone in old erythrocytes may cause the induction of haeme oxygenase
(HO-I) . This in turn may slightly increase levels of bilirubin and CO, which may
provide a protective effect and an improvement of hind limb ischaemia and other
pathologies (see Chapter 24).

2.6. Thioredoxin
Thioredoxin (Trx) is a ubiquitous protein with two redox -active cystine residues
within the active centre, -having the amino acid sequence -Cys-Gly-Pro-Cys
(Luthman and Holmgren, 1982). The Trx system is composed of NADPH, Trx, two
Trx reductases (TrxR) and Trx peroxidase (TrxPx) (Holmgren, 1989; Chae et al.,
1999; Mustacich and Powis, 2000; Tanaka et al., 2000) .

Inhlblted
apoptoBls

Antloxldanl ONA oynlhesla Gene Irenac"ptlon

Figure 29. Reactions andfunctions o(TJ:tR in the cell. TrxR utilizes NADPH to catalyse the
conversion ofoxidised (ox.) Trx into reduced (red.) Trx, and to reduce the oxidisedforrns Q(
ascorbate into reduced ascorbate. Reduced Trx provides reducing equivalents to (i) Trx
peroxidase......hich breaks down H202 to water; (ii) ribonucleotide reductase, which reduces
ribonucleotides to deoxyribonucleotides for DNA synthesis, and (iii) transcriptionfactors,
which leads to their increased btnding to DNA and altered gene transcription. In addiüon,
Trx increases cell growth and inhibit apoptosis. (From Mustaci cli and Powis, 2000).
96 CHAPTER 12

Oxidized Trx with a disulphide on its active site is reduced by NADPH and
TrxR, and the reduced compound can function as a protein disulphide reductase. The
availability of Se is a crucial factor determining the activity of TrxR in vitro and in
vivo (Mustacich and Powis, 2000) . TrxPx can reduce H20 2 and alkyl
hydroperoxides using e1ectrons provided by Trx , TrxR and NADPH (Chae et al.,
1999). The complex involvement of TrxR in biological functions is weil illustrated
in the scheme presented in Figure 29 (Mustacich and Powis, 2000) .
Trx and adult T-cellleukemia-derived factor ADF, first characterized by Tagaya
et al. (1988), are stress-proteins that can be induced by various stressors, namely
mitogens, cytokines, estrogen, viral infectious agents and oxidative stress like I-h02,
UV and probably also ozone (Makino et al., 1996; Sasada and Yodoi, 1999).
Trx l ADF appears to be a key regulator of intracellular signalling in the cellular
response against stresses, through the reduction/oxidation of protein cysteine
residues (Schenk et al., 1996; Sasada and Yodoi, 1999). It is not surprising that Trx
is not only strongly expressed in malignant celIs associated with viral infections but
also shows higher serum levels in patients with hepato-cellular carcinoma (Miyazaki
et al., 1999). It would be interesting to investigate whether ozonetherapy induces an
upregulation of the Trx system.

2.7. Biojlavonoids
These are compounds of vegetable origin (Gingko biloba, red wine, citrus fruits)
with either a polyphenolic or stilbene structure. These compounds have recently
become very popular and appear to be useful : they ean queneh PUFAperoxidation,
proteet capilIaries (vitamin P-like) and block the synthesis of prostaglandins and
leukotrienes by inhibiting both cyclooxygenase and 5-lipoxygenase (Laughton et al.,
1991). A diet rich in fruit and vegetables with the addition of a glass of genuine red
wine may be sufficient to prevent or delay cell damage by oxidative stress (Jang et
al., 1997; Fremont, 2000).

2.8. Melatonin
Melatonin is an indole (N-acetyl-5-methoxytryptamine) synthesized from
tryptophan via 5-hydroxytryptamine (serotonin) and secreted by the pineal gland
during the night (Fig. 30).
o
11
"'r.-----.-CH:CH: NH -C-CH.•

Figure 30. Structure ~lN-Acetyl-5-methoxYffyptamine 01' melatonin . lt derivesfrom

tryptophan via NsAcetylserotonin
 THE ANTIOXIDANT SYSTEM 97

It is a !ipophi!ie moleeule that rapidly enters the eell and may aceumulate in the
nucleus. Melatonin displays pleiotropie aetivities : it is an important eonveyor of
photoperiodie information to the animal kingdom, it influences haematopoiesis, it
modifies the !ife span in ageing miee and appears to modulate the immune response
(Reiter, 1991). It seems that non-pineal tissues , such as the retina and other organs,
can also synthesize and release melatonin into the blood . This is interesting because
melatonin is an endogenous free radical seavenger of OH", 10Z, HzOz and O-NOO'
(Tan et al., 1993, 2000; Marshall et al., 1996; Cuzzoerea et al., 1999). The
intracellular presence of melatonin may be eomplementary to many other
antioxidant components in maintaining ROS concentrations at minimal levels . It is
not known if ozonetherapy influences melatonin release. However, so many virtues
have been attributed to melatonin reeently that it would not be surprising if this
hormone, in conjunction with other faetors (serotonin and other hormones), is
responsible for the feeling of wellness reported by the majority of patients after 0 3-
AHT.

2.9. The Role 0/ Proteins Chelating Transition Metals

The main catalysts for the oxidative deterioration of macromolecules in biological
systems are iron and copper. This necessitates the safest possible storage of these
metal ions, which is insured by specialized binding proteins . Both ions are free
radicals beeause they have unpaired electrons in the extemal orbitals . In humans,
about two-thirds of the total iron (4 g) is linked to haemoglobin (Hb). Some 10% of
the iron is present in myoglobin while the remainder is bound to circulating
transferrin or is in intracellular storage proteins (ferritin and haemosiderin) or
enzymes.
In plasma, free iron should normally be absent because transferrin, a
glycoprotein (ß-globulin) of about 80,000 0, can bind 2 mol of Fe3+. However, it is
usually only partially saturated with iron. The metal is normally shuttled by
transferrin from the site of absorption and/or catabolism to various cells where it is
either stored in ferritin or bound to enzymes, myoglobin or Hb in the bone marrow.
A !ittle haemolysis is unlikely to yield free iron because Hb is promptly complexed
to haptoglobin and the complex is readily taken up by maerophages, thanks to the
scavenger reeeptor CDI63 (Kristiansen et al., 2001). Ferritin is not an absolutely
safe deposit either beeause I mol of protein can bind thousands of mol of iron and
because a pH value of about 6, present in hypoxie tissues or in the
microenvironment of phagocytes, can dissociate iron from the protein. Both
ischaemia and inflammation, which unavoidably lead to acidosis, ean thus favour
the release of iron and the produetion of ROS according to the following reactions

O," + Fe3+_ _~) Oz + FeZ'"

20zo,+ 2H-'- ) HzOz+ o,
Ozo, + Fe 2+ + 2H " ) HzOz + Fe3+
HzOz + Fe +2
) OH" + OR + Fe3+
98 CHAPTER 12

In addition to Fe2 +, Cu+ is also implicated in the fonnation of OH from H202 •

o

Ceruloplasmin, another plasma glycoprotein (a2globulin of 130,000 D), can bind up

to seven Cu+ molecules. Its protective function is due to either Cu ~ chelation or
scavenging of O," or, via ferro-oxidase activity, to catalysis ofthe oxidation ofFe2'
to Fe 3+. Nature has generously provided other physiological chelators, such as
lactoferrin, haemopexin and albumin (in man a total of about 430 g), which can bind
Cu t and neutralize OH- (HalIiweIl and Gutteridge, 1990). Albumin acts as a
kamikaze molecule and, once oxidized, will be recognized by the RES and broken
down . However, hepatic synthesis of albumin readily replenishes the pool.
Another phannaceutical iron chelator, desferrioxamine, is used in patients with
haemochromatosis and other haematological diseases in which it is essential to
lower excessive plasma levels ofFe2+ and Cu+.
Metallothioneins (MTs), which are sulphydryl-rich proteins, mayaIso be useful
because they bind heavy metals . In vitro, MTs display oxyradical scavenging
o
capacity and may block the fonnation of OH • Although they may have a
detoxifying role in Hg and Cd poisoning, their physiological importance rernains
unclear in humans. Postmortem analyses of tissues have revealed that only zinc and
cadmium are bound to MTs (Rotilio , 2001).

3. THE ENZYMATIC SYSTEM

So far we have reviewed the extracellular antioxidants which , particularly in regard
to ozonetherapy, represent the primary and most important defense against the
oxidative attack of ozone . However, the enzymatic system, mostly localized
intracellularly, is no less important. It serves to restore some oxidized SMWA and to
maintain internal homeostasis by protecting vital structures such as DNA, enzymes
and cell membranes.

3.1. Superoxide Dismutases (SOD) (see also Chapter 9)

This enzyme, discovered by McCord and Fridovich (1968, 1969) is present in all
aerobic organisms and catalyzes the conversion oftwo 0 2 into H202 and O 2 :
0
•

202 '+ 2H +-Mill~ H202+ O 2

0

The active site of the cytosolic enzymes in mammalian cells contains a copper
ion and a zinc ion coordinated to the side chain of a histidine residue . The reaction
develops in two phases during which one Cu 2t is first reduced to Cu l and then
oxidized to Cu 2+. It is noteworthy that the enzymatic reaction proceeds about 10,000
times more rapidly than spontaneous dismutation, particularly when the 0 2
0
'

concentration is very low .

Besides Cu/Zn-SOD, a dimeric protein of 32,000 D present in the cytosol,
nucleus and peroxisomes of all mammalian cells, there is a second isozyme, Mn-
SOD, which is a tetramer present in mitochondria. A third enzyme, Cu/Zn SOD
(EC-SOD), is present in plasma and extracellular fluids (Owry et al., 1996). The
 THE ANTIOXIDANT SYSTEM 99

enzymatic compartmentalization appears to be useful since the production of 0/- is

ubiquitous: it is released in mitochondria, microsomal and nuclear membranes, in
peroxisomes (owing to the presence of several oxidases) , in infected areas where
phagocytes undergo the oxidative burst (Badway and Kamowsky, 1980) and in
anoxic tissues during reperfusion for the activation of xanthine oxidase (Gutteridge
and Halliwell, 1990; McCord, 1995; Wang et al., 1998). The enzyme Mn-SOD is
essential for life, while EC-SOD becomes important during hyperoxia , as has been
shown in bacteria and knock-out mice (Carlsson et al., 1995; Halliwell, 1999 a).
There is also a striking difference in the intracellular-extracellular enzymatic
content : normal adults have an average SOD content in erythrocytes of 2583 V/g Hb
while the SOD in plasma amounts to 28 V/mI (Mecocci et al., 2000).
The beneficial integration between SOD and catalase is interesting because 0 2
0
•

is scavenged by SOD to H 20 2 ; however, unless rapidly scavenged by catalase, the

latter compound inactivates SOD because formation of OH o from H 202 inactivates
the imidazole moiety of histidine , which is the active site of the enzyme. On the
other hand, an excess of SOD activity in conjunction with depressed H202 reducing
enzymes can lead to excessive levels ofH 202, hence to cell damage .
Can we administer SOD to patients to prevent radical damage? Although there
has been considerable interest in developing a pharmacological intervention, the
results are only preliminary: after intravenous (IV) administration, effective plasma
levels cannot be reached because the enzyme has a short half-life . Perhaps this
problem could be overcome by conjugating SOD to polyethylenglycol (PEG-SOD),
as has been done for IL-2 and IFNu. However , there is the problem of
immunogenicity and; above all, the fact that a high SOD level is far more important
inside the cell than in plasma (Karlsson and Marklund, 1988; Greenwald, 1990).
Oral administration of SOD as a food supplement appears irrational owing to the
gastrointestinal digestion of the enzyme. Intra-articular administration has been tried
in order to reduce excessive ROS concentration in synovia I fluid of affected joints.
In chapter 24, the beneficial and somewhat enigmatic results obtained after intra-
articular administration of O2 + 0 3 will be discussed .

3.2. Cata/ase
This is a ubiquitous haeme protein particularly present in erythrocytes, kidney and
hepatic peroxisomes. It catalyzes the dismutation of H202 according to the reaction :

As a typical example of double control (Gaetani et al., 1994), peroxidases

catalyze a similar reaction in which H20 2 is reduced to H20 by a reductant:
peroxjdase

However, these enzymes have different affinities and specificities, at least as far
as H202 and hydroperoxides are concemed.
100 CHAPTER 12

H2 0 2 is not a free radieal and is a relatively strong oxidant with a long half-li fe in
comparison to other radieals (Chapter 9, Table 6). We must bear in mind that
ozonization of human blood ex vivo leads to a very transient production of H20 2 in
plasma: because H 2 0 2 is not an ionized molecule, any increase in extracellular water
causes a rapid passage through the plasma membrane into cytosol water, This
represents a signal of great physiological importance and can be interpreted as
the ozone second messenger because it can trigger various biological processes
(Chapters 13 and 34). However, it is also a risky phase because an excessive
increase of H 2 0 2 in the presence of a trace of Fe2 +can generate OH·.
Therefore, it is necessary to carefully control the ozone dose in such a way that it
allows generation in the plasma of an extracellular-intracellular gradient of H20 2 and
consequently an amount of H 20 2 in the cytosol able to reach a threshold level below
which there is no biological activation and above which may cause toxicity if H20 2
is not promptly reduced to H 20 + O 2 • In erythrocytes, catalase is important in
preventing haemolysis (Gaetani et al., 1996) .

3.3. Glutathione Peroxidases (GSH-Pxs)

There are two enzymes, one Se-dependent and another Se-independent, that catalyze
two reactions, one for H20 2 and another for lipid hydroperoxides:

2GSH + H20 2 --~) GSSG + 2H 2 0

2GSH + ROOH ) GSSG + H20 + ROH

As mentioned previously, the Se-dependent GSH-Px, present in the cytosol, has

less affinity for H 20 2 than catalase. The Se-independent GSH-Px acts preferentially
on hydroperoxides rather than H 20 2 . The amount of GSH-Pxs in normal adults has
been estimated at 0.112 nrnol NADPH/minlml (Mecocci et al., 2000).
Since 1973, when Flohe et al. and Rotruch et al. discovered the key role of
selenium (as Se-cysteine) in GSH-Pxs, the metal has been included among the
oligoelements and the supplementation of this micronutrient has become
fashionable . In the Western diet, a selenium deficit is unlikely as liver, fish, cereals,
yeast and Brazil nuts contain it as Se-methionine and Se-cysteine, both easily
absorbed by the gut. A sufficient daily intake is 50-200 ug . During prolonged
ozonetherapy, a small supplementation may be useful to support a possible increase
of GSH-Px synthesis during the stage of adaptation to chronic oxidative stress
(Chapter 22) .
On the other hand, a selenium deficit is responsible for Keshan's disease, a
serious myocardiopathy, the epidemiology of which was carefully analyzed in 1979
by the Chinese Academy of Medical Sciences. A dietary supplementation mayaiso
serve in anorexia, HIV-AIDS, cancer and Crohn's disease, particularly owing to
intestinal malabsorption. However, it is possible that an excessive dose (800-1000
ug) of selenium can be toxie.
 THE ANTIOXIDANT SYSTEM 101

3.4. Glutathione (GSH) RedoxSystem

In recent decades, there has been growing interest in GSH, the major low molecular
weight thiol compound in plant and animal cells. Known since 1888, it was
synthesized by Harington and Mead in 1935 as the tripeptide, L-y-glutamyl-L-
cysteinyl-glycine, the reducing group being -SH. The molecular weight is 307 and it
is hydrosoluble (Fig. 31).
SH..-
I
NH2 0 CH2 H

I 11 I I
HOOC - C- CH2 - CH 2 - C ~ N",,"""" CH - C _ .- N-CH2 - COOH
I I 11
H H 0

Figure 31. Chemical structure ofreduced glutathione (GSH) . The sulphydryl group is
indicated by the arrow

GSH is synthesized intracellularly in two consecutive steps, with the essential

presence of Mg 2-l and ATP. The first phase is catalyzed by y-glutamyl-cysteine
synthase:

L-glutamate + L-cysteine + ATP ~ y-glutamyl-L-cysteine + ADP + Pi

and the second is catalyzed by GSH synthase:

y-glutamyl-L-cysteine + glycine + ATP --~) GSH + ADP + Pi

The oxidized form glutathione disulphide (GSSG) is inactive, but it can be

recycled to the active form GSH. Degradation of GSH occurs via y-glutamyl-
transpeptidase and cysteinyl-glycine dipeptidase. Other reactions centred on the thiol
group lead to the formation of an array of compounds such as cysteine-protein
mixed disulphides, GSH-protein mixed disulphides, protein-mixed disulphides and
S-nitrosoGSH (GSNO). The S-thiolationldethiolation exchange reactions :

GSSG + Protein SH groups = GSH-protein mixed disulphides + GSH

are typical examples of the thiol interplay occurring during intracellular oxidative
stress .
The redox reactions are catalyzed by GSH peroxidases (GSH-Pxs) and GSSG
reductases (GSSGR) while the enzymes involved in thioether formation are GSH
transferases (GST) .
GSH is localized mainly in the mitochondria and cytoplasm, but it is also present
in the nuclear matrix . Although GSH is ubiquitous, the liver is the major source of
synthesis but high concentrations are present in the kidneys, erythrocytes, CNS,
102 CHAPTER 12

crystallin and in the bile as GSSG (Sies and Wendel , 1978). Intracellular levels vary
widely from 0.1 to 10 mM and no less than 95% is present as GSH . Human plasma
contains 5-50 11M of GSH (i.e. a 30-200 fold lower concentration than in cells ) and
is rapidly converted to GSSG.
After IV administration, the half-life of GSH is extremely short (about 1.6 min).
Since GSH does not penetrate into the cell, it is irrational to infuse it intravenousl y
hoping to increase the intracellular concentration where it is reall y needed.
In ARMD, diabetes and surprisingly in the elderly (centenarians), a signifieantl y
lower plasma GSH than in normal or young individuals (from 387 down to 127 ~l M)
has been repeatedly noted (Ceballos-Pieot et al., 1996a; Samiee et al., 1998;
Mecoeei et al., 2000) .
The main role of GSH and GSH-Px is to proteet the "internal milieu" from
possible damage due to exeess H20 2 , aceording to the reaetion :

The following examples show the impressive proteetive ability of GSH in the
eell (Sies, 1978; Meister, 1994; Wang and Ballatori, 1998; Cotgreave and Gerdes,
1998):

• Detoxi fieation of peroxides:

2GSH+ ROOH -~) GSSG + ROH + H 20

• DNA proteetion:

2DNA + 20H"'--~) 2DNA" + 2H 20 + 2GSH--~) 2DN A + GSSG

• Neutralization of 4-HNE :

4-HNE + GSH --~) 4-hydroxyalchenalglutathione + hemiaeetal

This reaetion is eatalyzed by GST isozyme 4-4 and allows inaeti vation of the
most toxie aldehyde of lipid peroxidation (Xie et al., 1998).
Moreover, by preserving the -SH group of several proteins (Ca 2 '-ATPases,
hormonal receptors), GSH maintains Ca 2+ homeostasis, thus preventing the
triggering of apoptosis. GSH also plays a role in signal transduetion and in gene
expression. Therefore, it is obvious that the eell needs to maintain a high
GSH/GSSG ratio . How is this done? There are several possibilities . Firstly, GSSG is
redueed through the eooperation of either aseorbate to DHA or vitamin E to (X -
toeopheryl. Secondly, the redueing power for GSSG is donated by NADPH
generated in the pentose phosphate pathway.
The aetivation of GSSGR (tla vinproteins) eatal yzes the reaetion between GSSG
and NADPH :
 THE ANTIOXIDANT SYSTEM 103

GSSG + NADPH + H+-~) 2GSH + NADP+

The conversion of GSSG to GSH is associated with dehydrogenation of glucose-

6-phosphate at C-l, areaction catalyzed by glucose-6-phosphate dehydrogenase
(G6PD). Successively, 6-phosphogluconate is oxidatively decarboxylated by 6-
phosphogluconate dehydrogenase (6PGD) to ribulose 5-phosphate. During this
three-step reaction, two NADP+ act as electron acceptors and yield 2NADPH. The
overall reaction is:

Glucose 6-phosphate + 2NADP ++ H20 ~ ribose 5-phosphate + 2NADPH + 2H+ + CO 2

The continuous provision of reducing equivalents to GSSGR by the pentose

shunt is essential, particularly in erythrocytes where the GSH/GSSG ratio must
remain very high to maintain erythrocyte viability and reduced iron in Hb. Genetic
deficiency of G6PD in erythrocytes (a sex-linked trait) markedly diminishes
NADPH availability, and the ingestion of either pamaquine, an antimalarial drug, or
broad beans (Vtcta fabai causes a serious haemolytic crisis and anaemia (Gaetani et
al., 1996) . Obviously, a simultaneous deficit of catalase turns the disease from bad
to worse . Deficiency of G6PD in women is fairly frequent in some Mediterranean
countries and this possibility must be taken into account before ozonetherapy,
because ozone may enhance haemolysis in these patients. However, in one ARMD
patient from Sardinia with a G6PD deficit of about 80%, we did not observe any
erythrocyte damage when using ozone at a concentration of up to 40 ug/ml per ml
blood . Nevertheless, this does not allow us to be anything less than cautious.

8 .,--------------TD.4

6 0.3

T ..... "'Y' :t'

••
....
•
..1
A'
:z:

.
0 ·..· •• 0.2
........ J. ~
....... Cl
'"
'"
Cl
- 0 - - GSH
2 0.1
......~ GSSG

o +---.-----,.---r---.----+ 0
o 25 50 75 100 m

Figure 32. The diagram shows the modest decrease 0/ erythrocytic GSH (and the increase
0/ GSSG) when human hlood is briefly exposed to ozone up to a concentration 0/100 ug/ml
(Shinriki et al.. 1998)
104 CHAPTER 12

If transmembrane transport of L-cysteine is impaired (HIV infection), the

intracellular level of GSH becomes low . Yet it does not make sense to attempt to
restore it by IV administration of GSH; it appears more efficacious and economical
to adrninister NAC orally (600 mg two-three times daily) (Bridgeman et al., 1991;
Hack et al., 1998). Ifnecessary, a similar strategy could be adopted during intensive
03- AHT.
Some important questions are : how much GSH is depleted in erythrocytes
exposed ex vivo to ozone? And how long does the depletion last?
Shinriki et al. (1998), very realistically, examined GSH and GSSG levels in
erythrocyte sampIes of blood exposed (I : I) to ozone concentrations from 25 to 100
ug/rnl per ml of blood (Fig . 32). Intracellular GSH was reduced by no more than
17% at the highest ozone -concentration and , as expected, there was an increase of
GSSG. We (Bocci et al., 1993b) also recorded a very small decrease of GSH in
erythrocytes after exposure to 42, 59 and 78 ug /rnl 0 3 per ml blood (Fig . 33 and 34) .
Most important is the fact that 20-30 min after 0 3 exposure, GSH levels retumed
to pre-ozonization levels, indicating the efficiency and perfect reversibility of the
process.

1000

800

E
......
(f)
CIl
0 600
E
0
c
cu
c
400
a;
o
200

c 42 78
OZ()f\E J,Jg/ml

Figure 33. Modification ofGSH levels in human blood after delivering one volume ot ozone
at concentrations 0/42 and 78 ug/ml with a syringe into the same volume 0/ blood. Control
sampies (c) were insufflated with air. GSH levels were determined after 30 sec. ofexposure.
Values are reported as M :±SDoffour blood samples. Variations were not statistically
significant.
 THE ANTIOXIDANT SYSTEM 105

_GSH mmm GSSG

1200

1080

960
E 840
01QI
Ö 720
8c 600
~
480
ihCl 360
240
120

0
C (B'42 59 78 C (F)42 59 78 C (842 59 78
Dose flQ/ml

Figure 34 Erythrocytic GSH and GSSG modifications 1 minute after exposing blood 0/
three healthy volunteers (B, Fand E) to Os-O, concentrations 0/42. 59, 78 ug/ml per ml 0/
hlood.

A brief summary folIows:

it has been shown which (and how many) antioxidant compounds are present in
the biological fluids and the cell, and how they ean neutralize ROS in hydrophilie
and Iipophilie environments.
The interaetion and eooperation among antioxidants, enzymes and the metabolie
system is eertainly useful as it allows their regeneration and an elevated antioxidant
status .
Three examples of cooperative interactions are:

1) among hydrophilie compounds, the GSH-aseorbate system aets as a rapid

donor of H+and electrons to neutralize peroxyl radicals and HzOz:

Aseorbate + 2ROO· ~ Dehydroaseorbic aeid + 2ROOH

Dehydroascorbic aeid + 2GSH ) Aseorbate + GSSG
HzOz + 2GSH ) 2H zO + GSSG
2GSSG + 4NADPH _GSH.RcI > 4GSH + 4NADP

2) among hydrophilie and Iipophilie eompounds:

2PUFAOO·+ 2EH ~ 2PUFAOOH + 2E· + Ascorbate ~ Dehydroascorbic acid+ 2EH

106 CHAPTER 12

3) among lipophilic compounds:

2EO + QH2 --~) 2EH + Q

or + bilirubin ) 2EH + biliverdin

A final example (Fig. 35) shows aseries of reactions switched on during

exposure ofblood to O 2-0 ).

OZONE =========::::l
Antioxidants

n
.e::

Albumin oxidation
PlASMA •
Lipid peroxidation

~..../~~ ~ Ib=-Sawen...

r
0a~ • HaOa C'" ~ 0a' + F.....
" Transferrln
~
~H
G-4-P NAI'O+) 2GSH ' \ + tIl°a

G) ( M'.R
HaO

6-PG NADPH \ GSSG /

~ +H+
Pentose
eyde
I ATP r
2.3 ooCPG t

Figure 35. The complex series of biochemical reactions elicited after the exposure of human
blood to ozone. After dissol ving into the plasmatic water, ozone reacts immediately with
hydrosoluble antioxidants (uric acid, ascorbic acid and albumin) and PUFAs. H202promptly
diffuses into the cells and activates a numb er ofmetabol ic pathways. However H202 is
rapidly reduced by the intracellular antioxidant system

The reader may be bored by this long excursion into the field of antioxidants, but
it was necessary to clarify how they act and interact outside and inside the cell. A
man of about 70 Kg consumes about 650 g of oxygen every day, so that in 70 years
he consumes about 17 tons of oxygen with an approximate production of 0.8 tons of
ROS . Most of them are freed intracellularly and they (particularly OH") act so
quicklyon adjacent biomolecules it is difficult to neutralize them in time . It is c1ear,
therefore, that the antioxidant system, even at its greatest efficiency, cannot entirely
cope with the continuous ROS bombardment. Thus ageing and progressive ceJl
degeneration are inevitable.

Therefore, any source of further ROS production, such as physical stress,

excessive feeding, acute or chronic infections, should be avoided. Ozone can be
inc1uded in the list, but once again I cannot agree with the famous sentence: "ozone
is toxic any way you deal with it" . Indeed, we are trying to use ozone as a drug in a
 THE ANTIOXIDANT SYSTEM 107

preeise and ealculated fashion, for an extremely brief period, hoping to aehieve a
multitude of biologieal effeets that ean lead to bloeking an infeetion, improving
oxygen delivery to anoxie tissue and upregulating the antioxidant system that may
paradoxically re-equilibrate the redox system. Thus, instead of thinking of
ozonetherapy only as a further oxidative stress, we hope to induce a multivaried
therapeutic response following small and repeated oxidative stresses.
In eomparison to the daily eonsumption of oxygen, how mueh ozone are we
using during therapy? At the highest dosage of 80 ug/ml per ml of blood for each
0 3-AHT (225 mI blood) , we are using 18 mg of Oj, two (or sometimes three) times
per week. In eonclusion, less than 54 mg of 0 3 against a minimum of 35 g of
endogenously produeed Oz•.. Moreover, the ozone exposure happens ex vivo and
lasts only a few minutes against the wealth of soluble antioxidants present in plasma.
In praetieal terms, most of the ozone dose is aetually neutralized by the soluble
antioxidants and the final aim is to induee the produetion of only a small surplus of
mediators (HzO z, LOPs, ete.) neeessary to trigger biologieal aetivities but readily
neutralized by several detoxifying mechanisms.
In eonclusion, it appears reasonable to use ozone as a drug and without toxie
risks if:

I. the ozone dose (eoneentration x total volume of gas) is preeise and proportional
to either the blood volume or to the site (gut lumen, muscie, etc.) where it is
applied . As any other drug, ozone has a therapeutie window whieh must be
known and strietiy observed (Boeei 1994a, 1996a,d, 1998b, I999a,b, 2000) .
2. To induee ozone toleranee, obeying the prineiple "start low, go slow", it appears
rational to use esealating doses.
3. Exposure of blood or an interna I site (exeept the pulmonary system,
eerebrospinal fluid (CSF), ete.) to ozone must be brief, transitory and oceur at
most 2-3 times per week. If deemed useful, an oral supplement of antioxidants
ean be administered daily .
4. The expression therapeutie "shock" in itself seems nonsensical and is really
meant to be a response to COS . Yet it does not exciude the fact that ozone
induces a transitory oxidative stress necessary to induce biological effects.
Thus, the stress must be adequate (not subliminal) to aetivate biochemical and
physiological mechanisms, but not excessive so that it overwhelms the
intracellular antioxidant system and eauses damage .
5. An excessive ozone dose or incompetence in manipulating the ozone can be very
deleterious. A very low ozone dose (below the threshold) is fully neutralized by
antioxidants and can produce only a placebo effect. It remains to be
demonstrated unequivocally that ozone does indeed have therapeutic activity .
 CHAPTER 13

HOW DOES OZONE ACT?

"Princtpium quant itate mtnimum, potestate autem maximum"

Aristotle (384 - 322 B,C.), Rhet ., 2
(A minimal amount ofa drug may have potent effects)

Trying to explain how ozone acts seems presumptuous but, after several years of
experimental work, some ideas have emerged and at least they can be used as
working hypotheses. For the sake of brevity, early ideas are not mentioned, because
they are either fanciful or unrealistic. Often data have been uncritically reported and
the primary source cannot be traced .
The gas mixture O2-03 is adrninistered by various routes : topical application on
skin and mucosae, parenteral injection and exposure to blood (see details in Chapter
16). In any event, ozone will come into contact with a film of water present on the
skin surface or in the interstitial fluids or in plasma . We reasoned that the most
precise, reproducible and easy system to study the interaction between the gas-water
phases , hence the reactions among biological compounds present in water and
ozone, would be the autohaemotherapy procedure : one volume of blood is exposed
in a closed ozone-resistant container (syringe, glass bottle) to an equal volume of gas
with varying ozone concentrations or oxygen only.

Figure 36. The scheme shows the variety ofcells and blood components susceptible to the
action % zone-ROS.

109
110 CHAPTER 13

Several parameters, such as gas pressure, temperature, blood mixing, time of

exposure and (if necessary) time of incubation, can be precisely controlIed so that
the interaction between blood and gas can be evaluated with almost stoichiometric
precision. However human blood, withdrawn from either healthy donors or patients,
represents a variable because blood is such a complex liquid tissue that one sampIe
is never equal to another. Figure 36 shows the cellular and plasmatic components
that, depending on their chemieal composition, can react with ozone differently .
Moreover, they have greatly different biologieal functions (0 2 transport,
procoagulant, oncotic, hormonal , immune activities, etc .) which are used to explain
the variety of effects in different pathologies.
Owing to the intrinsic and unavoidable variability of both normal and
pathological blood sampIes, we must always check a few basic parameters such as
number of erythrocytes, haematocrit value and plasma protein concentration . Before
and after ozonization, it is critieal to assess: a) TAS , b) PTG, c) TBARS and d)
haemolysis values. These are simple tests already mentioned in Chapters 9 and 10
and briefly summarized here to explain their meaning.

a) The TAS (total antioxidant status) is a simple and rapid method (Miller et al.,
1993 ; improved by Re et al., 1999) used to monitor the antioxidant status during
ozonetherapy. When 2,2'-azinobis-(3 ethylbenzothiazoline-6-sulphonic acid)
(ABTS) is incubated with aperoxidase and H2 0 2 , the relatively long-lived
radieal cation with ABTSO+ is fonned . A number of ROS induced by ozone
reacting with blood react rapidly with ABTS to form ABTS·+, which has a fairly
stable blue-green colour and is measured at 600 nm . Antioxidants in the plasma
cause suppression of this colour production to a degree which is proportional to
their concentration. In anormal European population the reference ranges
between 1.28 and 1.83 mmol /L plasma.
b) Measurement ofprotein thiol groups (PTG). Most ofthe plasma - S H groups are
present in albumin and the rnethod described by Hu (1994) is simple and
reasonably accurate to detect the effect of ozonization of blood. Interferences are
first removed by precipitation of proteins with trichloroacetic acid (TCA),
followed by procedure 2. Protein - SH groups are highly susceptible to oxidative
damage by ozone and the decrease of their level is easily detected and depends
upon the ozone dose.
c) The TBARS assay is based on measuring MDA with the TCA-TBA -HCI reagent
followed by butanol extraction as a practical index for determining the extent of
the peroxidation reaction before and after exposure ofblood to ozone (Buege and
Aust, 1994). Although the method has some limitations, it appears precise
enough for monitoring lipid peroxidation in plasma and is very informative.
d) The Hb detennination is carried out using 20 111 of original blood and an equal
volume of plasma collected after the ozonization . SampIes are mixed with 5 ml
of the c1assie cyanide-methaemoglobin reagent. Optical density, read
spectrophotometrically at 540 nm, is converted to haemoglobin according to a
standard curve and referred to as a percentage oftotal haemoglobin .
 How DOES OZONE ACT? 111

In particular circumstances we have also measured H202 and NO levels . In the

future we hope to include routine measurement of FrIsoPs and protein carbonyl
content because a spectrum of data is more informative than only one type of assay .
The analysis of the results of these tests has been enlightening and has allowed
us to envision a rapid sequence of events during either plasma or blood ozonization.
If the reader is not acquainted with this sort of work , he may find the schemes
reported in Figures 37 and 38 helpful : on the basis of experimental results (Figures
39 and 40), rapid reactions appear to go on simultaneously and successively between
0 ] ~ROS and biological substrates.

Figur e 3 7. The scheme helps to imagine the multiplicity ofsubstrates reacting with ozone
dissolved in the plasmatic water. Small circle s, triangles and squares sy mbolize hydrosoluble
antioxidants present in 100 ml of human blood (uric acid 4.5 mg/dL , ascorbic acid 1.5 mg/dl;
gluc ose 80 mg/dl; etc). Large albumin molecules (2700 mg/dL) exposing -SH groupsform a
cloud over the cell membrane and protect it. Molecules such as transferrin and ceruloplasmin
bind Fe 3 + and Cu + and prevent fo rmation 0/011. A LDL mole cule is shown on the left side .
The exogenous addition 0/4-8 mg ot ozone to j 00 ml 0/ blood is transitory and controlled by
antioxidants. In contrast, the endogenous production 0/ ROS is cont inuous and barely
quen ched by intracellular antioxidants.
112 CHAPTER 13

~ 03
ROS

/J

/ ' n ( I'<'5)P'

cAMP
1~C3 >+

Figure 38. The glyeoprotein domain 0/ a plasma membrane receptor emerges in the
extraeellularfluid and may undergo oxidation by ROS. Activation 0/ enzymatic activities
(adenylate eyclase: AC; phospholipase C: PLC; protein kinase C: PKC) and the opening 0/
ion channels result in eell depolarization and activation ofmetabolic pathways.
Diaeylglycerol: DAG; Inositol-Le.S-trtsphosphate: IP 3 ; Pro tein G: G; Cyclic adenosine
3 '-5 'nnonophospluue: cAMP.

Both oxygen and ozone present in the gas phase overlying the superficial layer of
blood (about 10 u) pass into solution in plasmatic water and this process of gas
solubilization goes on continuously when the blood is mixed gently in a glass bottle
for at least 5 min. Oxygen slowly equilibrates with the extracellular and intracellular
water and becomes bound to Hb until it is fully oxygenated (venous blood has a pOz of
about 30-40 mmHg) . However, as soon as it is dissolved, ozone reacts instantaneously
with several substrates, namely soluble antioxidants, albumin moleeules and more or
less accessible lipids, i.e. PUFAs. Lipoproteins and blood cells are suspended in
plasma but, as we shall see, are somewhat shielded from ROS attack.
As blood mixing goes on during the first five minutes, new layers of blood
become continuously exposed to O 2-0 3 ; ascorbic acid, uric acid, free cysteine, GSH
 How DOES OZONE ACT? 113

molecules and -SH groups of albumin undergo oxidation, as .shown by a progressive

decline of TAS levels in frequent measurements.
1,5 Plasma
e
;--e-----~>--------&-------------o

1,0

•
0,5

(/)

~ 0,0
::2:
E

Blood
1,0
"

0,5

0,0 -t---.--.------.----.--~-..---.--.,--r-- T-.,--r--.-.-_.-.,-.--.-.,I__,

o 5 10 15 20
Minutes

Figure 39. Kinetics oftotal antioxidant (TAS) levels in pla sma and in blood sampies offour
normal donors. Plasma and blood samples were exposedfor J min to either O2 (control 0) or
Os-O, witb ozone concentrations 0/ 40 (~ and 80 (~ ug/ml. It can be noted that antioxidant
levels are rapidly reconstituted only in blood sampIes. Statistically significant differences
(p<O.OJ) between O2 and ara] are indicated by the asterisk.

There is no doubt that these molecules represent the first line of defense against
the ozone which, at least in part, is destroyed in sacrificial reactions (Fig . 40).
Similarly, oxidized protein thiol groups (PTG) significantly decrease in an
approximate ozone dose-dependent fashion while oxygen is practically ineffective.
In contrast, PUFAs bound to albumin and other lipids undergo progressive
peroxidation and TB ARS values reach the highest level in plasma at the end of the
mixing period (Fig . 40) .
114 CHAPTER 13

~ 4
2;
~ 3
<:
III
I- 2

1,5
~
oe;;
CI)

>- 1,0
Ö
E
Ql
C1l
J:
0,5

a.o

1,5

~
.s 1.0

s
(J'J

0.5

0,0

0,50

I
(,!)
I-
o,
0.25

0,00 -j---l.--r--'--

Figure 40. Thirteen human hlood samp les were exposed to air (control), or Ob or OrO./
with ozone concentrations of40 anti 80 ug/mlfor J min . While TBARS. TAS and PTG Levels
vmy significantly (p<O.OJ) aft er ozon e exp osure. there is a negligihle increase ofhaemolysis
 How DOES OZONE ACT? 115

Are the phospholipids of the erythrocytic membrane attacked by ozone? This is

an important and vexed question, because in the past several papers (Goldstein and
Balchum, 1967; Freeman et al., 1979; Gomicki and Gutsze, 2000) have reported that
erythrocytes isolated from plasma, after washing and resuspension in physiological
medium, undergo structural change and intense haemolysis when exposed to ozone.
Uppu et al. (1995) showed that both proteins and PUFAs of isolated erythrocytic
membranes undergo simultaneous and competitive ozonization but they clearly
pointed out that this was intended to be only a model system. It is unfortunate that
some of these data have greatly contributed to the belief in ozone toxicity; in fact,
they are irrelevant and misleading because erythrocytes, deprived of plasma
antioxidants and their natural albumin shield, are obviously very sensitive to ozone.
The best proof of this erroneous belief is represented by Shinriki et al. (1998) and our
data (Bocci et al., 1993b, and see also Fig. 40): 1 rnl of citrated human blood exposed
to a concentration as high as 100 ug/ml yielded only 0.2% and 0.5% haemolysis,
respectively. Moreover, Shinriki et al. (1998) further demonstrated that TBARS
derive only from lipids present in plasma and not from erythrocytic membranes.
In agreement with these results, they noted only an ozone-dependent depletion (about
20%) of a-tocopherol in plasma (Fig. 41).

-
. 150 20-.--------------,

T T
T :;; 15
.. ..........•........~

.:
........~......... ~ 1 1 o
"
~100
~ ö- • :;; ;;:) .
~ _··..··..·..·..····..·0·· ..J

-a
GI
~ 10
.5-
g
b 50

......<>..... Plasma
~
== .. ö
...............
- - 0 - RBC membranes
•
o+ - - - - r - - - r - - - - r - - , - - - !
o
o
°
25 50 75 100 125 25 50 75 100
03hlg/mL blood) 3 (lIg/mL blood)

Figure 41.Modification 0/ a-tocopherol (left) and MDA (right) levels in plasma 01' in
erythro cytic (RBC) membranes after exposure 0/ human blood to ozone at concentrations up
to 100 ug/m! o(blood. No lipid peroxidation occurs in the RBC membranes because there is
neither a decrease 0/ a-tocopherol nor an increase 0/ MDA (Shinriki et al.. 1998).

Other studies by Galleano and Puntarulo (1995), Caglayan and Bayer (1995),
Mudd et al. (1997) and Mendiratta et al. (1998a,b) have clearly shown that when
erythrocytes are protected by plasmatic antioxidants, peroxidation of membrane
phospholipids is absent. In practical terms, it appears that only one (probably an old
cell) out of 200-400 erythrocytes undergoes breakdown, at least in part due to
manipulation rather than peroxidation. This does not exclude that some membrane
glycoproteins, acting as receptors, undergo oxidation, as has been hypothesized in
the scheme reported in Figure 38. At medium-high ozone concentrations (40-80
116 CHAPTER 13

ug /ml per ml ofblood), partieularly in heparinized blood, we have some evidenee of

an inereased Ca 2 + influx and possible aetivation of phospholipase C (Boeei et al.,
1993b).
Another erueial event during the initial exposure of blood to ozone is the
progressively inereased produetion of H20 2 • As shown in Figure 15 (Chapter 9), this
eompound inereases rapidly and then deereases just as rapidly beeause HzOz, an
unionized moleeule, diffuses into intraeellular water where it triggers several
bioehemieal pathways (Chapter 14). However, at the same time , H20Z undergoes
reduetion in both water eompartments owing to the presenee of GSH , catala se and
GSH-Px. The transitory presenee of H Z0 2 in the cytoplasm means that H20 2 acts as
one ofthe ozone ehemieal messengers and that its level is eritieal : it must be above a
eertain threshold to be effeetive but not too high to become toxie . In the partieular
experiments reported in Figure 15, the HZ0 2 half-li fe in plasma was estimated at
around 2.5 min and this eould be prolonged by the addition of eatalase and GSH-Px
inhibitors. In blood, the proeess of H2 0 2 generation, diffusion and reduetion must be
extremely rapid because even when adding inhibitors we have been unable to
reliably measure H 20 2 in the plasma isolated two minutes after blood ozonization.
The inability to measure H20 2 is eorroborated by the finding shown in Figure 39 and
the reader is strongly invited to examine it carefully. We evaluated the kineties of
TAS levels after treating 4 sampies of normal human blood and relative plasmas
with oxygen and with two ozone eoneentrations : 40 and 80 ug /ml (usual 1:1 v ratio) .
In the upper part of the diagram , the TAS values of plasma show that O2 is
ineffective, while ozonization eauses a significant deerease of 41% and 52%,
respectively, ten minutes after ozone addition. Afterwards, the deerease remains
static and the double coneentration of ozone does not appear to lower TAS levels
proportionally. We then tested the blood sampIes and we were so puzzled by the
results that we repeated the experiment twice to be really sure . As expeeted, oxygen
was without effeet while two levels of ozonization (40 and 80 ug /ml) eaused a
smaller deerease than in plasma (whieh was only 21 and 33%, respecti vely) after
one minute; then, surprisingly, the TAS values began to rise progressively and
retumed to the preozonization level within 20 minutes. A reasonable interpretation
of these results is that the presence of erythroeytes in the blood, in spite of
ozonization, is able to regenerate antioxidants and quiekly normalize TAS levels.
We owe very mueh to Galleano and Puntarulo (1995) and espeeially to May et al.
(1996) and Mendiratta et al. (I 998a ,b) for having c1arifted the power of erythrocytes
to rapidly reeonstitute the antioxidant reservoir (Chapter 14).
The reader is now kindly asked to examine the diagrams presented in Figure 40
where the TAS, PTG, TBARS and haemolysis values have been measured only one
minute after ozonization. The timing was chosen because in this set of experiments we
could complete the ozonization process very rapidly . For the sake of eoherence, it
must be c1arified that these experiments were carried out with the double syringe
method , in whieh mixing the blood with ozone, by rapid axial rotation of the syringe,
can be achieved within I min. In contrast , to avoid foaming , mixing a large volume of
blood in a 500 ml glass bottle takes a good 5 min. It seems correet to add that the
timing of the exposure of blood to ozone is a parameter adjusted to the system in use
and is optimized on an experimental basis : as an extreme example, during the
 How DOES OZONE ACT? 117

extracorporeal circulation ofblood against Or03 (Chapter 17), in each minute a blood
volume ofabout 80 ml runs outside thousands ofhol1ow capillary fibres (about 1.7 rn')
in which Or03 flows continuously at an ozone concentration as low as 4-llg/rnl.
The results of Figure 40 are interesting and appear complementary: once again,
oxygen is ineffective in comparison to the untreated 13 normal blood sarnples, but
ozone (40 and 80 ug /ml ) is able to significantly reduce both T AS and PTG levels.
There is no doubt that ozonization was effective because T.BARS increased up to 5
fold while, reassuringly, haemolysis was negligible. However, it must be added at
once that haemolysis increases steeply as soon as ozone concentrations become
higher than 100 ug/ml (see next chapter). It is regretful that until now the lack of a
pre cise experimental approach allowed a number of conjectures to thrive and to
insinuate false concepts in the ozonetherapist's mind. On the basis of imprecisely
defined biochemical measurements, Rokitansky's group (1981) claimed that
ozonization of blood led to peroxidation of the membrane phospholipids. The
successive relaxation of these molecules would have increased the fluidity of the
membrane, enhanced the erythrocytic deformability and the negative charge of the
membrane. All of these favourable changes would improve blood microrheology,
but are they true? And how can ozonization of small volumes of blood lead to a
generalized circulatory improvement? These claims have been refuted by
experimental data (Morgan et al., 1988) that actual1y showed a loss of
deformability and decreased filterability. As discussed in Chapter 14, there is
clearly the need to carry out further careful work on this problem. Another
unresolved issue is the role of LOPs, which are certainly generated during
ozonization , as shown by the significant increase of TBARS. It is not yet clear
which PUF As are the main contributors but they are probably those bound to
albumin and present in chylomicrons, since oxidation of LDL and of the membrane
does not seem to occur. In Chapter 9, we specified a large nurnber of LOPs that are
more or less toxic, more or less susceptible to be catabolized and excreted, and more
or less capable of binding to specific receptors all over the body and triggering a
number of biological responses. This remains an enigma to be solved and hopefully
this book will elicit the interest of other biologists and clinicians. In the meantime, it
seems useful to delineate some conclusions regarding several processes occurring
when blood is mixed in the presence of OZ-03 just prior to reinfusion into the donor:

I) Tfthe preparation is performed correctly, blood pOz rises from about 35 to 400
mmHg. Hb is fully saturated with Oz and about 1 ml of O 2 is physically
dissolved in 100 ml plasmatic HzO. No significant changes of pH, pCO z, Na",
K+, Cl', Ca z+, NaHC0 3 have been observed during 5-10 min .
2) Ozone passes continuously into solution and reacts with biomolecules. After the
mixing period, the ozone is practical1y exhausted and only some oxygen remains
in the gas phase.
3) During the ozonization process, the antioxidant reservoir decreases into the range
of 20-30%. TAS values vary among normal individuals and may be lower than
norm al in patients. At least one TAS test should be performed before
ozonetherapy. If it is below 1.3 mM, an oral supplement of antioxidants in the
morning before breakfast should be administered for 7-10 days before therapy ,
118 CHAPTER 13

when the TAS value (see Chapters 22 and 24) ought to be checked again .
Experimental evidence has shown that the TAS value returns to preozonization
levels by the time of reinfusion. Antiox idants should neither be added to the
blood sampIe nor administered IV before or after autohaemotherapy. They da
not serve any purpose because the antioxidant reservoir in plasma and interstitial
fluids is enormaus. It should be remembered that in the ozonized blood sampies
Hb0 2 is not oxidized to methaemoglobin, haemolysis is practically absent, the
PTG value is reduced and remains so (the PTG value does not recover, as does
TAS , because albumin oxidized-SH groups cannot be reduced) and TBARS are
increased 2-5 fold in relation to the ozone dose . As blood is reinfused, TBARS
mix with the blood pool but peroxidation is blocked and TBARS in the donor's
blood do not change from the preinfusion value.
4) The ozonetherapist does not need to carry out the outlined laboratory tests ,
except the TAS value. It is important, however, that he takes care concerning the
precision of 0 3 concentration and that he delivers the O 2-03 gas mixture in a I :I
ratio (i .e, 200 ml of blood, excluding the volume of citrate, + 200 ml of gas) and
then mixes the blood gently for at least 5 min .
5) The problem of the 0 3 concentration is critical because ozone is used as a real
"drug" and must be dosed according to the TAS value , the type and stage of the
disease, the patient's weight, the schedule, etc. (see following chapters for details) .
6) Experimental evidence gathered from hundreds of normal blood sampies has
indicated that the ozone therapeutic window ranges from 10 to 80 ug /ml (per ml
of blood). It is not possible to give an absolute value because each individual has
her/his own TAS that varies throughout the day , without mentioning the variable
capacity of intracellular antioxidants which is never assessed. Thus, an ozone
concentration of 10 ug/ml may be either effective or ineffective if the blood
sampIe has aTAS value of either 1.2 or 1.8 mM , respectively, because the latter
concentration may totally quench the ozone activity. In other words, the lowest
ozone concentration may be unable to generate a sufficient amount of H202 and
LOPs to trigger biological effects. In such a case, 0 3-AHT may elicit only a
placebo effect simply due to autotransfusion, oxygenation, the medical act and
other psychological effects. On the other hand , blood with a TAS value below
1.2 mM , exposed to an 0 3 concentration of 80 ug /ml , may reach a critical stage
and even present slight haemolysis and a minimal risk of toxicity. These two
extreme examples may never occur as they are at the opposite ends of the
therapeutic window and most cases will fall in between, as suggested in the
scheme reported in Figure 42 . If one thinks about it, one can sense the
uncertainties intrinsic to ozonization of blood and this must be stated c1early.
Although we must make an effort to identify the optimal dose, this is difficult
because an effector molecule, Iike H202, is influenced by the c1assical "all or
none law", by the efficiency ofbiochemical pathways, by the variability ofblood
composition in different pathologies and by the uncertain pharmacodynamics of
a number of LOPs. It is needless to say that the dynamism and complexity of the
ozonization process require that the procedure be performed in a standard
fashion if reliable and reproducible results are desired.
 How DOES OZONE ACT? 119

7) Skeptica1 physicians like to say that 03-AHT is a panacea for all diseases and
indeed it looks like that. However, it is easy to dismiss this superficial defmition
by considering the heterogeneity of blood components and their widely different
functions. Although some overlapping of the ozone concentration seems
unavoidable, we have experimental evidence that different cells are activated by
different ozone concentrations. Therefore , the aim is to identify a range of ozone
concentrations suitable for treating either vascu1ar (10-40 ug/ml) or infectious
(20-80 ug/rnl) or autoimmune (the range remains uncertain) diseases (for details
see Chapter 24). Thus the request for fixed "recipes" shows that the
ozonetherapist has not understood the subtlety ofthe ozonization process .

100

90
maximum
80 - .- "- "- .- "_ .. _ .- .- .- _ ._.. _ ._ ._.. _ .. _ .

70

60
Active
50

40

30

20 Threshold

10 .· l lnaclive
o -L.. --'--_--'-_-l-_-..l-._----'-_.....-lL-_.L-_...l...---=-- _

o 10 o 10 o
Minutes

Figure 42. A schematic representation of'the "therapeutic window" ot ozone in blood.

Concentrations below 15 ug/ml are practical/y neutralized by the antioxidant system and may
aet as a placebo. The threshold level varies between 15 and 25 ug/ml depending on the
individual TAS. Active doses range between 25 and 80-90 ug/ml. Above 90 ug/ml, an
incipient haemolysis warns about toxicity

8) In any case, bearing in rnind that the therapeutic "shock" always remains abrief
oxidative stress, in order to induce an adaptation, or ozone tolerance, it appears
rational to adopt the old strategy of "start low, go slow", i.e. in a patient with
hind limb ischaemia the initial ozone concentration of 20 ug/rnl can be
120 CHAPTER 13

progressively raised in 2-3 weeks to a maximum of 40 ug /ml . This sehedule is

likely to be useful within the TAS normal range.
9) Finally , how can we be sure that ozone eoneentrations above 80 ug /rnl are not
useful in cancer and in autoimmune diseases? On the basis of several e-mail
letters, Oxylist eommunications, etc., I have been informed that naturopaths
eontinue to make direet IV infus ion of O 2-0 3 and use huge amounts of ozone,
particularIy in the United States and Canada. This situation troubles me beeause
it can lead to further discrediting of ozonetherapy. The available experimental
evidence indicates that ozone concentrations above 80 ug /ml eause a marked
inerease ofhaemolysis and ofTBARS . We know that LOPs are toxie (Chapter 9)
and in spite of dilution , degradation, ete., the detoxifying system may be
overwhelmed, with toxie eonsequenees. I wish that 4-HNE eould bind
specifieally to neoplastic eells and clones of cytotoxie T eells and kill them but
this may not be the ease. Therefore, until we obtain more data, we should refrain
from using a dose of ozone higher than 80 ug/rnl.
 CHAPTER 14

WHAT HAPPENS IN THE INTRACELLULAR

ENVIRONMENT AFTER BLOOD OZONIZATION?

It has just been deseribed that ozone, after dissolv ing in plasma, generates H202
and LOPs . The eoneentration of H202 in the plasma results from adynamie
equilibrium between its synthesis and diffusion into intraeellular water due to the
eoneentration gradient formed between plasma and eytoplasm. Onee inside the cell
at a eoncentration above threshold levels, H 202 can switch on bioehemical
pathways, but it is simultaneously reduced by the potent antioxidant system
(Chapter 12). Most of the LOPs can interaet with cell receptors, membrane
cytoplasmic and even nuclear components. Owing to the heterogeneity and
potential toxicity of LOPs , the extent and relevance of their activities is currently
beyond our grasp , a situation that compels cautious action . On the other hand, the
ozone dose is calibrated agains t: a) the antioxidant capacity of blood, b) the
enormous cellular surface area (about 70 m2 for erythrocytes in 100 rnl of blood) ,
c) the plasma and cellular fluid and d) the capae ity of metabolie breakdown. It is
intriguing to think that this phase of "oxidative" stress ex vivo subsides in about
five minutes and it is indispensable for generation ofthe therapeutic "shock" to the
organism once the ozonized blood returns to the donor 's blood circulation.
How do the blood components behave under this stress? Erythrocytes,
leukocytes and platelets will be examined separately . Last but not least, it is
important to investigate if and how the blood container, i.e. the vascular system,
reacts to the ozone-acti vated blood .

I . ERYTHROCYTES
These cells are very suitable for the examination of any toxic and biologieal effects
of ozone and represent an ideal marker. It is unfortunate that past studies
(Goldstein and Balchum, 1967; Freeman et al., 1979; Freeman and Mudd, 1981;
van der Zee et al., 1987; Fukunaga et al., 1999) carried out on washed and
resuspended erythrocytes in physiological saline have concluded that direct
ozonization of erythrocytic membranes invariably causes: deerease in membrane
fluidity , hence diminished deformability and filterability, and formation of
hydrophilie eentres (peroxide and carbonyl groups) in the hydrophobie regions of
phospholipids, with eventual breakdown. These molecular modifieations have dire
functional consequences, such as disruption of ion transport meehanisms (Kourie,
1998) and consequently cell lysis with leakage of Hb and enzymes into the plasma .
Moreover, enzymatic functions may be impaired by loss of the normal lipid-lipid

121
122 CHAPTER 14

and lipid-protein interactions and by the fonnation of 4-HNE-protein adducts .

However, these results have no physiological significance because, in the presence
of plasma, erythrocytes are protected and do not show these alterations. This
critical point should be borne in mind by ozone toxicologists when they test cells
in culture not adequately supplied with antioxidants (Leist et al., 1996).
Moreover, ozone studies are always controversial and enthusiastic
proponents of ozonetherapy have claimed quite different findings that are
almost too good to be true: Rokitansky's group (1981) , Coppola et al. (1992) and
later Verrazzo et al. (1995) reported that after treating blood with 0 3 ex vivo or
after collecting blood sampies after 03-AHT perfonned in patients with peripheral
occlusive arterial disease (POAD), a slight peroxidation of the erythrocytic
membrane induces favourable consequences such as increased fluidity of the
membrane with enhanced defonnability and filterability . Moreover, an increased
negative charge of the membrane will be accompanied by a lower
erythrosedimentation rate which, in conjunction with reduced plasma viscosity
(due to reduced fibrinogen levels) , would explain an overall improvement of
rheologie parameters of ozonized blood in these patients.
On the other hand, how can we reconcile these claims with a mass of
experimental data showing that ozonization of blood, particularly using low-
medium ozone concentrations (10-40 ug /ml), does not involve any membrane
peroxidation (Galleano and Puntarulo, 1995; Caglayan and Bayer, 1995; Mudd et
al., 1997; Shinriki et al., 1998). These results are in line with older data showing
that, in spite of susceptibility of erythrocytes to lipid peroxidation, they are weil
protected by the antioxidant defence mechanism (Clemens and Waller, 1987;
Machlin and Bendich, 1987). Dur experimental data also imply a lack of
membrane peroxidation in ozonized blood, but it is necessary to verify if very
slight peroxidation does indeed improve haemorheologic characteristics.
The following is a summary of experimental observations during blood
ozonization :

• Levels of methaemoglobin remain practically normal . Shinriki et al. (1998)

reported a variation from 1.91 ± 0.11 to 2.21 ± 0.09 %. Actually, we have
always measured lower levels, i.e. about I% in comparison to Hb and we have
never visualized Heinz' bodies.
• The haematocrit value does not change, implying no modification of the
erythrocytic volume due to osmotic swelling or erythrocytic lysis .
• At high levels of ozonization, there is a minimal loss of K ' , which rapidly
returns to normal (Shinriki et al., 1998).
• There is negligible haemolysis, from the basic value of about 0.5 up to 1.2% if
the anticoagulant is citrate (the usual citrate, phosphate, dextrose , CPD) and up
to 1.8'Yo if it is heparin. Shinriki et al. (1998) reported lower values than ours :
from 0 up to 0.27%. However, as expected, washed erythrocytes yield a 10%,
value (Freeman et al., 1979) and even higher values (Fig . 43) when blood is
ozonized with excessive ozone doses (up to 200 ug/ml), The enhanced
haemolysis of heparinized blood is probably favoured by a concomitant Ca 2"
 WHAT HAPPENS DURING BLOOD OZONIZAnON? 123

influx. These data clearly establish that the ozone concentrat ion should not
exceed 80 ug/ml .
25

20

~
~
·a
b
15

."e
::c"
10

0 50 100 150 200

O J (,ugIml)

Figure 43. Kinetics 0/ haemolysis in relation to ozone concentrations (ug/mt pe r ml 0/

blood). Blood offive donors was treated with CPD (0) or with 30 Ulml heparin (~ . Mean
±SD

• Osmotic fragility of Ca 2+-chelated blood is not evident even when an ozone

concentration of 200 ug/rnl is used (Fig. 44), and interestingly it is moderately
changed (displacement of the haemolysis curve to the left) even in washed
erythrocytes (Freeman et al., 1979).

1 -.-
...-
120 ControI
0,

I - 2 5 ~ml
100
.. 5O~ml

I
7 5 ~ml

i
100 IJ9ImI
80 150lJ9Iml
200 lJ9lml

60-1
~ 40 I
20 '1
I
o i o ~ o

I
i
0.9 0.8 0.7 0.5 0.4 0,4 0.3 0.2
NOCI (',{,)

Figure 44. Osmoticfragility 0/ a normal blood sampie exposed to ozone concentrations

fro m 25 to 200 ug/m l. The TAS ofthis sampie was as high as 1.98 mM. which may explain
the lack ofhaemolysis
124 CHAPTER 14

• No damage to erythrocytie enzymes, such as Na/K-ATPase, acetylcholinesterase,

SOD, GSH-Px, GSSGR and catalase, has been noted in blood exposed to
concentrations as high as 100 ug/ml (Cross et a1., 1992a; Shinriki et a1., 1998),
confirming that the antioxidant system adequately protects enzymes and Hb.
Aldehydes forming adducts with these proteins would otherwise have denatured
the enzymes .

Blood ozonization implies the generation of an H202 gradient but its increase
in the cytoplasm is transient and kept under control by AH- , GSH, catalase and
GSHPx . In the erythrocyte, the GSH concentration is very high (- 2 mM) and the
GSH /GSSG ratio is about 50. The GSH tumover has a half-life of 70-100 hours
because GSSG is either extruded as such or is rapidly reduced to GSH by the
GSSGR. During ozonization, the intra-erythrocytic GSH level decreases by no
more than 20% because contemporaneously there is a new synthesis of GSH and,
more readily , reduction of GSSG at the expense of NADPH which serves as an
electron donor (Chapter 12). The decrease of NADPH/NADP ", or in other words
the increased level ofNADP+, switches on the hexose monophosphate pathway, in
which G-6PD is the rate-limiting enzyme. This leads to generation of NADPH and
ATP (Fig. 35). It is perhaps useful to remember that erythrocytes lack
mitochondria for the production of high-energy compounds and rely on the
anaerobic glycolytie (Embden-Meyerhoff) pathway and the just-mentioned aerobic
hexose monophosphate shunt. Normally, 90% of the glucose entering the
erythrocyte is metabolized by the former pathway and about 10% by the latter. The
glycolytie pathway yields three products: I) NADH, which is a crucial cofactor in
the methaemoglobin (MHb) reductase reaction regulating the constant conversion
of MHb (oxidized Hb) into oxyhaemoglobin (Hb0 2) , particularly during oxidative
stress; 2) adenosine triphosphate (ATP), the high-energy phosphate nucleotide that
powers the cell pumps; 3) the intermediate metabolite 2,3-diphosphoglycerate
(2,3-DPG), which regulates the globin chain interaction and thus modulates
oxygenation-deoxygenation of Hb, i.e. the release of O2 to tissues .
Of the eleven enzymes in the glycolytic pathway, the main regulators of the
rate of glycolysis are hexokinase (HK) , pyruvate kinase (PK), glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) and particularly phosphofructokinase (PFK)
(Magnani et al., 1988).
Does ozonetherapy exert any effect on glycolysis? It seems so. We found
increased ATP levels (from 1389 to 1968 11M) in patients with age-related macular
degeneration (ARMD) (atrophie form) after a therapeutic cycle (13-14 sessions) of
03-AHT (Fig . 45) (Diadori and Bocci, Chapter 24). Moreover, Viebahn (l999,b)
reported the same effect in athletes and elderly patients after rectal insufflation of
O 2-03. These results are surprising and the mechanisms of enzymatic activation or
induction have not yet been studied . Again in ARMD patients, Micheli et a1.
(2000) showed an increased (from 0.9227 ± 0.011 to 0.9456 ± 0.0039 , not
significant) energy charge (E.C.) as defined by Daniel Atkinson:

E.C. = (ATP + Y2 ADP) / (ATP + ADP + AMP)

 WHATHAPPENS DURING BLOOD OZONIZATION? 125

In this fonnula, the values can range from 0 (all nucleotides are present as
AMP) to I (all nucleotides are present as ATP) . In particular, PFK and PK are
strongly inhibited by a high ATP level, as shown in the scheme reported in Figure
46. Thus ATP-generating pathways are inhibited by a high E.C., while ATP-
utilizing pathways are stimulated by a high E.C. Increased glycolysis likely
renders erythrocytes more effective and less susceptible to premature lysis.
Figure 45 shows that the ATP increase occurs only after prolonged therapy:
indeed there was no change in ATP levels at the middle ofthe OrAHT cyc1e (6-7
sessions). We intend to investigate if the increase becomes more marked with
further 0 3-AHT sessions.
ATROPHIe EXUDATIVE
1,00 -
- °rOl ~ 200 -
0 ° 2 l.
U e,
w 0,95 ~ 100 -

Q,90 °1 ° -0 ,
1 n = 13

z
~
1:
..=,
200
1500 "- 150
"-
~ ~
100
1000 50
e,
0,8
~ 0,6 ° 8 n -l l
p s 0,05
J:
"- 0,4
0
-c 0,2
Z

700

0
<
l
1,0 "- 500
Z
~
J:
0 300
<
Z
0,5

10O
0,0

°
PRE POST PRE 8 13
p :; 0,025

Figure 45. Modifications ofATP, EC levels, NADPHINADP and NADHINAD ratios

measured in erythrocytes ofpatients affected by ARMD, atrophie and exudativeform,
before and after J3-14 OrAHT treatments with O2 only 01' with OrO) (left side) (Miehe/i et
al.. 2000). In eomparison (right siele). an increase oferythrocytic ATP levels has been
observed in young athletes by Jakl (above) und in elderly people (middle and below) after
reetal tnsufflation of ozonefor 8 and 13 weeks (Viebahn, 1999)
126 CHAPTER 14

ATP NADPH
AMP NADP

---PFK
G6PD
PK

ATP NADPH
AMP NADP
Figure 46. Mechanisms ofpositive and negativefeedback o(erythrocytic enzymes after
transient exposure of blood to ozone. High levels ofA TP inhibit phosphofiuctokinase
(PFK). while low levels activate pyruvate kinase (PK) . At the same time . lowered levels of
NADPH activate glucose-ti-Psdehydrogenase (G6PD)

In other words, is it possibIe that ozonetherapy can lead to the fonnation of

erythrocytes with increased capacity to synthesize ATP? And if this is true, how
does it happen? Our working hypothesis is that erythrocytes ozonized ex vivo are
not really modified but that reinfusion of some LOPs present in the ozonized bIood
sIowIy triggers a phenomenon of adaptation in the bone-rnarrow. In trace amounts,
LOPs may activate differentiation at the erythropoietic level, favouring the
formation of new erythrocytes with improved biochemical characteristics, which
could provisionally be considered as "supergifted erythrocytes". If this idea is
correct, every day during ozonetherapy the bone-marrow will release cohorts of
new erythrocytes capable of satisfying the increased biosynthetic and energetic
requirements imposed by the therapy . This hypothesis might not be so farfetched,
since it agrees weil with previous experimental results (Chow and TappeI, 1973;
Buckley et al., 1975; Freeman et al., 1979; Chow and Kaneko, 1979; Hemandez et
al., 1995); moreover, the cessation of ozonetherapy in ARMD patients does not
abruptly end the clinical improvement, which instead persists for 3-4 months
probably in relation to the life-span of the supergifted erythrocytes present in the
circulation. This problem will be further discussed in Chapter 22.
2,3-DPG is a highly anionic organic phosphate present in erythrocytes at a
concentration of4-5 mM at an atrnospheric pressure (AP) of760 mmHg . In 1967,
Reinhold and Ruth Benesch discovered that 2,3-DPG binds to Hb and has a
remarkable effect on its affinity for O 2. Without it, Hb0 2 is unable to unload O 2 in
tissue capiIlaries. On average, its presence, at p02 gradient of 98-40 mmHg at sea
level, allows Hb0 2 to release 4-5 ml O 2 per 100 mJ blood .
 WHAT HAPPENS DURING BLOOD OZONIZATION? 127

As mentioned previously, 2,3-DPG is formed along the glyeolytie pathway

when 1-3 diphosphoglyeerate is shifted towards the formation of 2,3-DPG by 2,3-
diphosphoglyeerate mutase (2,3-DPGM) :

1,3 diphosphoglyeerate _Z.3.PPGM ~ 2,3-DPG _z 3·PPGP~ 3-phophoglyeerate

where 2,3-DPGP is a phosphatase -allowing the transformation to 3-

phosphoglyeerate.
It is weil known that, by staying 2-3 weeks in high mountains (where the AP is
around 400 mmHg), we undergo an adaptation to low pOz (from 98 down to 50-40
mmHg). We ean survive beeause the bone marrow produees more erythroeytes
(polyeyternia) and beeause the inereased level of2,3-DPG bound to Hb allows the
release of Oz to hypoxie tissue even if the alveolar p02 value has fallen to 40
mmHg.
Physiologieal studies have shown that the reaetions:

Hb40 g --~) Hb406 + O2

Hb406 ) Hb404 + O2
Hb 404 ) Hb 402 + O2

Hb402 ) Hb4 + Oz

depend on the Oz tension in the tissues. Figure 47 shows that the dissociation eurve
of HbOz shifts to the right (p50 value inereases) as blood passes from pulmonary
(P) to systemie (S) eapillaries and even further ifthe 2,3-DPG level inereases . The
same shift oeeurs ifthe blood pH deereases (from pH 7.4 to 7.2, aeidosis) or the
pCO z value inereases (from about 40 to 60 mmHg, the Bohr effeet) or the body
temperature rises from 37°C to 39°C.
20

~
~
---
'5
, ,," 75

I ,,
i
,, ."
8 10 --- --i' 50 ~
~
....
::+r
:,
,
E
:'
•
,t·..··..····..················..
,, ::
15

~
,, .:'
20 40 60 10 .00 120 _
Po,mmH.

Figure 47. The dissociation curve ofRb0] shifts regularly from P (pulmonary capillaries)
to S (systemic capillaries). The increase oI2,3·DPG in the erythrocytes shifts the curve to
the right (broken Une). The presence ofCO (HhCO) and a decrease oI2,3-DPG shifls the
curve to the left
128 CHAPTER 14

Does ozonetherapy increase 2,3-DPG? Current results are controversial :

Rokitansky et al. (1981) and Rokitansky (1982) c1aimed a significant increase but
their results are difficuIt to evaluate. The study by Mattassi et al. (1987) is far more
meaningful : positive changes, although not statistically significant, were observed
in POAD patients treated with 03-AHT for several weeks . However, it appeared
that only patients who had a low baseline level of 2,3-DPG showed a marked
increase with therapy. We have confirmed this observation in our ARMD patients.
In normal subjects and diabetes type 11 patients, Coppola et al. (1995) found no
variation in blood ozonized ex vivo (in the range 6.5-78 ug/rnl) , Yet this negative
resuIt was conceivable because 2,3-DPG cannot change in vitro in a short time.
Therefore, the observed increase of p50 was likely due simply to the Bohr effect.
Figure 48 compares our data for ARMD patients treated with 13-14 sessions of
03-AHT with the results presented by Viebahn in young athletes and elderly
patients treated for weeks with rectal insufflation of O2-0.1 ' We could not
demonstrate any increase; thus Viebahn's results are surprising but they may have
been due to the lan ger cycle oftherapy (13 weeks) than ours (6.5-7 weeks).
,.
2,3 DrG

110
100

_ _ 0, - 0,

:;}1 -o.~

~ 1
,.
2,3 DrG

1 Atroph ie 150

~ 100
C 1
::l
~· __·_-··_·-l-- --_.. .~ 50

0
exudaöv.

PU
I
INTtRH POST
,.
2,3 DPG

150

100

50

0 J lJ
0 - 10
P <:0.05

Figure 48. Modifications oferythrocytic 2,3-DPG in patients with ARMD (leftside) before
(PRE), during (interim) and after (POST) J3-14 0rAHT treatments with either O2 01' with 0 ;-03
(see Figure 45) (Micheliet al.. 2000). In comparison (light side), a modest increase oJ
erythrocytic2.3-DPG levels has heen observed in young athletes by Jakl (ahove) and in elderly
people (middleand below) after rectal insufflation o(ozonefor 8 and J3 weeks (Viehahn, 1999)
 WHAT HAPPENS DURING BLOOD OZONIZAnON? 129

Thus , at this stage we cannot make a definitive conclusion. In the near future,
we will ascertain whether the 2,3-DPG level remains stationary or increases by
treating POAD patients with extracorporeal circulation of blood against Or03
(EBOO), which is a more intense therapy than the ordinary 03-AHT. Clarification
of this problem is important because areal increase of 2,3-DPG levels markedly
shifts the Hb0 2 dissociation curve to the right (p50 value increases) and the
peripheral venous blood p02 level may decrease to 15-20 mmHg with a 20-25%
increase of O 2 delivery to ischaemic tissue (Fig. 47) . The enhanced oxygenation,
possibly associated with microrelease of ATP from erythrocytes and with
vasodilatation (see endothelial cells for NO· release), can explain the beneficial
effect of ozonetherapy in ischaernic diseases. In 1956, Wenning suggested that the
polycythemia achieved after reinfusion of blood exposed to UV could be an
important factor, but we have observed neither an increased number of
erythrocytes nor an increase of the mean corpuscular Hb (27-31 pg).
A more theoretical problem to solve is why and how ozonetherapy should
increase the 2,3-DPG level in the first place? At first glance, oxygenation should
inhibit it. However, ischaemic tissues may try to release mediators attempting to
correct local hypoxia. A regulatory protein , hypoxia-inducible factor (Wang and
Semenza, 1996, Gassmann and Wenger, 1997), has been identified and it remains
to be seen whether factors released upon blood ozonization enhance the enzymatic
activity of 2,3-DPGM or upregulate its synthesis and/or inhibit 2,3-DPGP. In this
context, we should also check for any change of the erythropoietin level. It is now
feasible to use molecular biology methods to examine any change of expression of
proteins during erythrogenesis in order to explore the possibility of a continuous
output of"supergiftederythrocytes" during ozonetherapy. This study is within our
reach : by using appropriate density gradients, we can separate old (heavier)
erythrocytes from young (lighter) ones released during ozonetherapy and slowly
accumulating in the blood pool. If the hypothesis is correct, the young "supergifted
erythrocytes" may show subtle differences in the composition of the membrane, in
the phospholipid/cholesterol ratio, in the type and frequency of membrane
glycoproteins and they may display enhanced metabolie characteristics.
Examination of the life-span of ozonized erythrocytes has received minimal
attention. This is surprising because even slight peroxidative damage to the
membrane is bound to be recognized by the RES, with uptake of the impaired
erythrocytes. Numerous studies have shown that genetically comprornised human
erythrocytes, or those treated with heavy metals , xenobiotics or heat, have a far
shorter life-span than the normal 120 days . In 1996, on the basis of my project, Dr.
C. Kontorschikova and Dr. S. Peretyagin of the Central Health Institute at Niznhy
Novgorod (Russia) received funding from the Soros Foundation. By labelling
normal human erythrocytes with Technetium 99, we examined the fate of
untreated, Oj-treated and O 2-03 (40 ug /mlj-treated erythrocytes in normal
volunteers. The scheme reported in Figure 49 aims to clarify that, when ozonized
blood is reinfused into the donor, erythrocytes will rnix within the vascular system
and, if normal, will continue to circulate according to their age. In four months,
human erythrocytes undergo an average of 160,000 deoxygenation cycles, which
occur at random in more or less pervious sites . Every day, some 2xlO ll
 130 CHAPTER 14

erythrocytes, equivalent to the cells contained in about 40 ml of blood of anormal

man, die and are removed from the circulation. About three decades ago, we
showed that even a minimal loss of sialic acid from the membrane glycoproteins
(due to neuraminidase) markedly reduced the cell 's life time (Bocci , 1968; 198Ia).
Spleen, liver and bone marrow are the organs that will rapidly take up and
catabolize the damaged erythrocytes. The scheme also shows other sites that will
be discussed in the next sections.

02/03 Primary
BLOOD TUMOURS and
METASTASIS

Figure 49. After reinfusion , the ozonized hlood is distributed throughout the whole
organism. After OrAHT, the volume of'plasma (about J30 ml), slightly depleted of
antioxidants, returns to normal after mixing with 3 L plasma and 9-JJ L interstitialfluid.
Oxidized albumin is taken IIp by RES and cataholized. Erythrocytes, if not damaged.
continue to circulate while leukocytes migrate through post-capillary venules into various
organs and may return to the blood pool via lymph . The reinfused LOPs undergo dilution
but will deliver their message to the whole body. After ozonization. platelets will likely
release rheir hormonal contents into the blood and will disappear.

Experimental results obtained after measuring the hepatic and splenie uptake of
Tc 99-labeled erythrocytes (either oxygenated or ozonized), 10, 30 and 60 min after
reinfusion into the donor, show only an initial (10min) small uptake, which
suggests negligible cell damage (Fig . 50). Preliminary experiments in another two
subjects showed a similar trend . Subsequent technical difficulties prevented us
from completing the study by measuring the half-li fe of differently ozonized Cr 51_
 WHAT HAPPENS DURING BLOOD OZONIZATION? 131

erythrocytes. We hope that someone In a Western country will complete this

crucial study .

ClAUD1

60 Tc (liver).imp/nun
Tc (lJIlcen), imp/min
Tc (blood), unp/l1Iin

10

IGO'"

60 Tc (livec).unp/min
Tc (FPleen). unp/min
Tc (blood). imp/min
40

20

Figure 50. Erythrocytes were labelIed with Tc~~ and either exposed to oxygen (ox) 01' to
ozone (oz: 40 ug/ml) . Blood sampies were then reinfused into two respective donors and
Tc99 was detected by scintigraphy in blood. liver and spleen after 10.30, 60 and 90 (only
ox) min. Hepatic and splenie uptake Q! ozonized erythrocytes were only slightly higher than
those Q! oxygenated erythrocytes

In summary, according to several criteria, careful ozonization of erythrocytes

within the therapeutic window (most frequently 10-40 ug/ml) does not appear to
cause any damage . In spite of some controversy among different authors, ozone
132 CHAPTER 14

does not seem to even reach the membrane and induce peroxidation. There is good
evidence that oxidative damage is prevented by the powerful antioxidant system
and the activation of the pentose phosphate pathway certainly plays a role in that.
An increase of 2,3-DPG levels during ozonetherapy remains controversial and
must be verified with a more prolonged and reliable method than rectal
insufflation of O 2- ° 3' The activation of glycolysis and an increase of the E.C. have
led to the suggestion that a continuous flow of messengers present in ozonized
blood reaches the bone marrow and, by upregulating gene expression, may
improve the biochemical characteristics of differentiating erythroblasts.
Adaptation to the controlled oxidative stress may allow the generation of
"supergifted erythrocytes", which slowly increase in the circulation and may be
responsible for the therapeutic "shock", i.e. a revival of functional activities able to
explain the beneficial effect observed in patients with ischaemic diseases.

2 . LEUKOCYTES AND THE IMMUNE SYSTEM

In comparison to erythrocytes, leukocytes are a heterogeneous cell population

composed of lymphocytes (20-25%), monocytes (about 5%) and three types of
granulocytes, of which neutrophils are about 70-72%. I mentioned in Chapter 2
that, by mere chance, I came to investigate whether ozone could induce the
production of IFN because I remembered that other oxidizing agents were active
(AntoneIli et al., 1985 ; Dianzani et al., 1985) . As shown in Table 9, ozone is only
one (actually a minor one) of the several types of cytokine inducers. However, at
that time I thought that, in spite of its intrinsic toxicity, it would be worthwhile
investigating ozone. During the last decade, we have shown that ozone can induce
the production in blood of IFNy (Bocci and Paulesu, 1990), IFNß, IL-2, IL-6, IL-8,
TNF-a, TGF-ß and GM-CSF (Paulesu et al., 1991; Bocci et al., 1993a,b, 1994b,
1998b,c; Larini et al., 2001). Later, while trying to explain the deleterious effect of
ozone on the pulmonary system, other scientists confirmed that ozone can indu ce
the production of cytokines, adhesion molecules and proinflammatory eicosanoids
in leukocytes present in the bronchoalveolar lavage fluid (BALF) and in epithelial
cells ofthe respiratory mucosa (Beck et al., 1994; Arsalane et al., 1995; Takahashi
et al., 1995; Haddad et al., 1996; Jaspers et al., 1997; Cho et al., 2001).

Table 9. The variety ofinducers activating the production 0/cy tokines in BMC.
Mitogens Antigens Antibodies Proteinases Interleukins Oxidhing er:
Agents lonophores
PHA Virus Anti-CD3 Trypsin IL-Iß Periodate A23187
ConA Endotoxins Anti-CD4 Bromelain IL-2 HzO z
PWM Muramyl Anti-CD28 Thrombin IL-12 Galactose-
peptides oxidase
SEB Tumour TNF-a OZONE
proteins
Phorbol Polynucleotides
esther
 WHAT HAPPENS DURING BLOOD OZONIZAnON? 133

In 1988-90, owing to OUf inexperience, we had great difficulties in studying

this problem: our initial research on isolated BMC suspended in typical cell culture
media showed great sensitivity ofthese cells to ozone, and concentrations above 2-
3 ug/ml inhibited IFN production, confirming Becker et al.'s (1989) observation.
Our results became more interesting when we began to ozonize blood directiy and
we were able to show the release of cytokines in the plasma during the following
4-8 hours of incubation (Fig . 51).
6O ~-----------------,

50

40

f
g30
~
20

10

0
·2 0 2 4 6 8
Hours

Figure 51. Kinetics ofIL-6 production measured in hlood sampIes ofthree donors after
ozonization (concentration : 59 ug/ml per ml ofblood) and incubation at 37 °C in air-Cü,
for IIp to 8 hr. The increase oJ IL-6 was significantly (p-d), 05) higher than in control
sampIes after 8 hr

These initial studies shed light on several aspects, such as the protective effect
ofblood antioxidants, the dissimilar production of different cytokines (Fig . 52) and
the progressive inhibitory activity of increasing ozone concentrations, particularly
above 80 ug/ml. However, they had some limitations because whole blood can be
incubated only for a limited time and, most irnportantly, we could not decide
which cell type produced the cytokines. Moreover, in investigating the induction
of IL-8 in 13 sampies of blood donors, we noticed again, as was weil known for
IFN production, that there are high, medium and low responders (Bocci et al.,
I998b). Actually, in this case we went a step further because we could detect an
approximate relationship between the production of IL-8 and the TAS values.
Figure 53 shows that there are two high responders (no. 7 and no. 8) and two nil
responders (no. 3 and no. 12). Interestingly, the latter donors had very high TAS
values (1.90 and 1.98 mM) whereas the high responders had average values (1.45
and 1.56 mM). Most likely, genetic and other factors are also at play but this
observation needs to be investigated further by checking the values of PTG
oxidation and TBARS . These results suggest that a very high TAS can quench
most of the ozone dose and they emphasize that the ordinary empirical
performance of 03-AHT without knowing the basic TAS of the patient can easily
134 CHAPTER 14

lead to placebo treatment. It is distressing that most ozonetherapists do not

attribute any importance to this observation.

20 5
c::J TNf cl
_ 'FNy
~ I L- 2
4
15 -

...e 3
!
~
.E
~
10 ·

- 2
.f
~

5 w
r I :'";-':
Ilj~,~
,~, ~
~
0
~ ..~
, :.;s; ,_ , - ~
.~~-- - 0
0. 0 , 36 0 . 70

Figure 52. Production ofthree cytokines after treatment ofthree hlood samplesfrom
normal donors with either oxygen or ozone (36 and 70 ug/ml) and incubation .for 9 hr. The
asterisks indicate a significant difference (p<O.05) in comparison to oxygen

500

400

300

200

100

10 11 12 13
_ 0,
-400 § C, 4O.,pl
m O,to.......'
300

200

100

Figure 53. Effect of 1 min exposure ofeither 0 1 0 1' 0 3 (40 and 80 ug/ml) on the production
0/1L-8 after 8 hr incubation of 13 hlood samples. Average va lues are reported in the lower
panel ofter subtraction 0/ control values.
'"Significant difference (p<O.01) compared with samp les treated with O 2• The variable
production of1L-8 among donors is noteworthy. particularly the lack ofproduction 01'
donors 110. 3 and 12 likely due to a high TAS level
 WHAT HAPPENS DURING BLOOD OZONIZAnON? 135

During the last year, we decided to isolate from normal blood donors either
BMC or granulocytes in order to investigate their viability and production of
cytokines after ozonization . This is important because we do not know their
relative contribution and, as an example (Fig. 54), BMC produce mainly TNF-a.,
IFNy and small amounts of IL-2 (Larini et al., 2001). On the other hand
granulocytes release other cytokines (Larini et al., in preparation) .

40
38 h 62 h 86 h

~
}
20
...~
...J

0
' - -_ _- I : i:L.J....L"-'"=.L:>.L:.l_ .L..l hL

1&Xl

} 1000

r
Z
l!l eco

1500
ZBI Z I

E 1000
}
~
~ 500
I-

g
§•
r "'!ll<: lil ~I:! ~ 1
§•
r'" SI I<: fi1 s g~

Figure 54. Pattern o(cytokine production by BMC suspended in human serum before
ozonization. Samples were not exposed (control) 01' exposed to either O2 alone 01' Os-O, at
increasing concentrations (ug/ml). PHA indicates values after mitogen addition. Diagrams
represent the values ot one donor (H) who was a high responder

On the basis of these experimental results, we can draw some preliminary

conclusions :
136 CHAPTER 14

a) the ozonization process of blood or of a BMC serum suspension can lead to

strong modification of cell viability, ranging from modest proliferation to cell
death .
b) A comparison between blood and BMC suspensions shows that the production
of cytokines depends on the ozone dose and the cell types .
c) With some approximation, the production of cytokines is inversely
proportional to the ozone concentration and blood appears to be more ozone-
resistant than serum. With the benefit of hindsight, we should have expected
these results as we now know that blood but not serum rapidly reconstitutes the
original TAS values (Chapter 13).
d) Once again we reach the conclusion that too little ozone may be ineffective and
too much can be toxic, and it is difficult to envisage the perfect ozone
concentration for each patient because the production of cytokines is regulated
by a number of factors .
e) Nonetheless, these results at least partly support the hypothesis that
ozonetherapy has bactericidal and antiviral activities (Bocci, 1992a). It remains
questionable whether this is due to enhanced phagocytosis and/or
hypergammaglobulinemia, as was proposed by Washuttl and Viebahn (1986)
and Washuttl et aI. (1988). In any case, these immunological modi fications
depend upon cytokine activity.
f) Our data do not contradict studies showing the release of proinflammatory
cytokines (Becker et al., 1989; Beck et al., 1994; Arsalane et al., 1995; Haddad et
aI., 1995; Torres et al., 1997; Bayram et al., 2001 ; Cho et aI., 2001) and
eicosanoids (van Hoof et al., 1997) by bronchoalveolar cells. This is based on the
knowledge that antioxidant defences in RTLFs are easily overwhelmed during
exposure to ozone, with the formation of ROS, LOPs and consequent epithelial
damage (Kelly et al., 1995; Chen and Qu, 1997; Frampton et al., 1999).

In contrast, Iymphocytes have high levels of GSH and, although they have less
catalase than erythrocytes, they are equipped with GSH-Px and GSSGR.
Professional phagocytes (neutrophil granulocytes, monocytes and macrophages)
can also defend themselves, up to a point, against O2 . ' , H z0 2 and HOCI released
during phagocytosis. In the last decade, there has been great progress in
understanding the role of antioxidants and redox regulation of gene transcription
(Oe Forge et al., 1992; Anderson et al., 1994; Schenk et al., 1996; Sen and Packer,
1996; Flohe et al., 1997; Suzuki et al., 1997; Ginn -Pease and Whisler, 1998; Hack
et al., 1998, Arrigo, 1999; Tatla et al., 1999; Allen and Tresini, 2000) . The whole
story started in 1986 when Sen and Baltimore identified a transcription factor
called NF-KB (i.e, nuclear factor bound to an enhancer element in the K light
chain gene) present in the cytoplasm in association with inhibitory proteins, IKBs
(c, ß, y, etc .). NF-KB has subsequently been found in different cells ; it is
composed of several proteins (the Rei farnily) , of which a p65 (rel A) subunit and
a p50 subunit bind to specific sequences in the promoter regions of several cellular
and viral target genes, including transcription factors, viruses, cell adhesion
molecules, cytokines, immunoreceptors, haematopoietic growth factors and acute
 WHAT HAPPENS DURING BLOOD OZONIZATION? 137

phase proteins (Baeuerle and Henkel, 1994). This means that activated cells can
synthesize a variety of proteins, including IFNs, IL-I, 2, 6, TNFa., chemokines
such as IL-8, class land II proteins belonging to the major histocompatibility
complex (MHC), proteins ofthe HIV, orosomucoid and C-reactive protein.
A variety of inducers, such as growth factors, cytokines, mitogens, UV, ozone
and H2 0 2 , cause translocation of the heterodimer p65-p50 to the nucleus . This
happens when specific kinases (IKK-1/2) phosphorylate IKBa. and cause its
detachment from the inactive complex NF-KB-IKB, allowing rapid migration of
the heterodimer to the nucleus (Verma and Stevenson, 1997). IKBa. acts as a brake
in the quiescent cell and, after phosphorylation, enters the ubiquitin-proteasome
pathway where it undergoes rapid degradation (DeMartino and Slaughter, 1999;
Komitzer and Ciechanover, 2000) . Figure 55 depicts a scheme c1arifying the
sequences leading to NF-KB activation with consequent protein synthesis.
OZONE

l
"x
IlIUlO1001lllll1llllUllllI
ROS

Ca++ - : I
PLASMA "e"ORANE

~ t
New protein
.
Translation
mRNA
Ca++
T- H202--+-~
TK
AlP j.PO:f

PPase

."". - - % ~XIDANTS
ADP
IKB --+-IKB.P0:f
, . . . . . . . - - p50 p65
1
NUCLEUS ~ Proteasome
Transcription

~
Gene induction \

Figure 55.A schematic view ofsignal transduction in lymphoeytes due to oxidattve stress.
The nuclear transcriptionfactor NF-KB is a heterodimer composed oftwo subunits (p50
and p65). In resting T lymphocytes, it exists in an inactiveform complexed witb the
inhibitor IKB . Ozone decomposes in plasma and generates ROS. These may aet on lectins
situated in the plasma membrane, possibly opening Ca + + channels, and/or by aetivating
pro tein kinases. H10 1 activates a tyrosine kinase which phosphorylates IKB and causes its
detachmentfrom the inactive complex. While IKB -P01 is being degraded in the proteasome,
the heterodimer moves promptlyfrom the cytosol into the nucleus, where it regulates gene
expression. Activation 0/ a phosphatase (PPase) 01' an excess ofintracytoplasmic
antioxidants (GSH. NAC, CAT. thioredoxin. a-lipoic acid, ete.) inhibits the process
138 CHAPTER 14

The observation that NAC coul d suppress the activity of NF-KB was made in
1990 by Staal et al. and direct evidence of the activating role of 50-100 11M of
H 202 in Jurkat T cells was reported the following year (Schreck et al., 1991). In
Chapter 13, it was shown that ozone dissolved in plasma causes lipid peroxidat ion,
hence production of LOPs and H 202 • The sudden increase of H 202 concentration
allows its passive diffus ion through the cell membrane from plasma: "202
becomes the ozone messenger able to activate the specific kinäse (IKK-l/2)
which, by phosphorylating IKB, allows the migration of the transcription
heterodimer into the nucleus. The critical issue is that H 202 must reach a
concentration able to activate the kinase without be ing instantaneously reduced by
either GSH or the catalase-GSH-Px, thioredoxin system or be transformed into
OH". A c1ear demonstration of the dose -dependent activity of H202 is presented in
Figure 56, reported by Los et al. (1995).
50

'[
20 000 -
1-0- . l-HEI
___ - l.H E
40
s:
e 15 000-
::;-
.E 30 .'0~"
2- ~
~ 20 8 1000 0-
.s
CD
c
10 :;;
1 5000-

25 50 100 200
r=
a) HIOIIIIM) -I
0-
0 10 20 50 100 150 300
H10 1111M)

-4QOOO '

Ea. 30 000 -
s-
c:
0
:0

S 20 000 •
i
.s
11
c
:;; 10 000 -

1
b) 2 r= b)

0 10 20 50 100 ISO 300

H10 1 b IM)

Figure 56. H}O} induces the expression 0/ /L-2 in tissue culture. a) Maximum release of
/L-2 was achie ved wirh 100 I-JM H }O} (above left). b) Maximum expression of 1L-2 mRNA
appeared 12 hr after H}O} addit ion (below left). Thymidtne incorporation (right) is
influenced by 2-mercaptoethanol and catalase, below (Los et al., 1995) .
 WH.'!"T HAPPENS DURING BLOOD OZONIZAnON? 139

It can be seen in vitro that 100 J.1M ofH 2 0 2 achieve the maximal activity while
a double amount is partly inhibitory. The process is rapid and the maximal
expression of lL-2 mRNA is reached 12 hours after H 20 2 stimulation. Addition of
either catalase or 2-mercaptoethanol are partly inhibitory, as expected. Indeed
there are several antioxidants able to prevent or impair the generation of active
NF-KB (Schreck et al., 1992); conversely, the HIV-I TAT protein, being able to
suppress the expression of Mn-dependent SOD, acts as a strong inducer of NF-KB
(Chapter 24).
Although I could not go into details, the reader may have perceived the central
importance of NF-KB in the regulation of many immune and inflammatory
responses. Excellent and extensive reviews of this subject are available (Baeuerle
and Henkel, 1994; Barnes and Karin, 1997; May and Gosh, 1998).
We have been very interested in this factor because ozone induces the
formation of H2 0 2 , which acts as an intracellular messenger and has recently
acquired importance in animal and plant cells (Haddad et a1., 1996; Remick and
Villarete, 1996; Josse et al., 1998; Chua et al., 1998; Valen et al., 1998, 1999; Dei
Bello et al., 1999; Korzets et al., 1999; Pellinen et al., 1999; Chen et al., 2000a;
Desikan et al., 2000; Lee and Schacter, 2000 ; Kim et al., 2001). Obviously the
type of response depends on the H2 0 2 levels and these may act as either "Iife or
death signals". The ozonetherapist must be aware of the dilemma that either
too low or too high ozone doses ean be either ineffeetive or toxie, respeetively.
The dark side of excessive NF-KB activation has become evident in toxic/septic
shock, in acute respiratory distress syndrome (ARDS) and in chronic inflarnmatory
diseases such as multiple sclerosis, Crohn's disease and rheumatoid arthritis .
While the ozone dose can be carefully checked and adjusted to the disease and
patient, it is far more difficult to handle rather unspecific drugs that dampen the
transcription factor activities . Inhibitors such as glucocorticoids, gold salts, aspirin
and sodium salicylate are effective but displaya variety of side effects , and thus
selective inhibitors of IKB kinases are needed . IL-lO is an anti-inflammatory
cytokine that appears promising since it inhibits the action ofNF-KB (Wang et al.,
1995; Schreiber et al., 1995). Currently available antioxidants (Chapter 12) are
relatively useful and it remains to be seen whether prolonged ozonetherapy can
paradoxically reverse chronic inflammation by inducing an adaptation to chronic
oxidative stress . .
In order to clarify whether exposing leukocytes to ozone can lead to either
immune stimulation or suppression, we have addressed the following problems :

• How important is the maintenance of the plasmatic Ca 2+ level during blood

ozonization?
• Is there any advantage in ozonizing a large number of isolated leukocytes
rather than whole blood?
• Within the "therapeutic window" is there a range of either immunostimulating
or immunosuppressive 0 3 concentrations?
• On the experimental basis that ozone can act as a modest cytokine inducer,
does reinfusion of ozonized blood modify the plasma cytokine level in vivo?
140 CHAPTER 14

• Is OJ-AHT effective and is there an optimal schedule?

• Does the induction of asp and of adaptation to chronic oxidat ive stress have
an immunomodulatory effect?
• Can we select tests suitable for evaluating modifications of the immune status
during ozonetherapy?

Abrief discussion ofthese problems folIows :

2.1. How Important is the Maintenance ofthe Plasmatic Cal + Level during Blood
Ozonization?

In addition to protein-bound Ca, the physiological Ca H concentration is about 1

mM while is about 10,000 times lower inside the cell (- 0.1 11M). To prevent
blood coagulation, in most cases we use either sodium citrate (3.8% solution 10 ml
+ 90 m1 blood), which chelates Ca2+ and makes plasma Ca 2 >-free, or saline diluted
heparin (up to 30 IV per ml of blood) which preserves plasma Ca 2 + levels . When
BMC are stimulated with antigens, mitogens, Ca 2+ ionophores or ozone, the cell
Ca 2+ gradient decreases because of opening of intracellular and plasma membrane
Ca 2+ channels associated with the release of sequestrated Ca2 + from the
endoplasmic reticulum (Grafton and Thwaite, 2001) . During ozonization, the
formation of RaS may be responsible for the activation of phospholipase C, which
hydrolyses the plasma membrane phosphatidyl inositol 4,5-biphosphate (PIP 2 ) and
unleashes two messengers: inositol 1,4,5-trisphosphate (IP 3), which opens Ca 2 1
channels, and diacylglycerol (DAG), which activates protein kinase C. This
process, in conjunction with other kinases (IKK-1/2), seems to greatly enhance the
synthesis and release of several cytokines (Bocci et al., 1993a) . We examined this
finding by increasing the blood Ca 2+ concentration to 5, 10 and 25 mM ; we found
that the increased cytokine release was accompanied by significant haemolysis,
particularly after ozonization with 40-80 ug /ml OJ per ml of blood. Occasionally,
we also noted the worrisome presence of minute clots retained in the blood filter
used during reinfusion. This gave us the hint that , in spite of heparin ,
supraphysiological Ca 2 + level plus ozone may stimulate platelet aggregation and
coagulation (for details , see Platelets, Haemostasis and Growth Factor Release,
this chapter). Before this observation in 1993, we considered that the addition of 5
mM Ca (five times higher than the physiologicallevel) to heparinized blood before
ozonization might improve the efficacy of OrAHT in patients with chronic
bacterial and viral infections and possibly metastatic cancer. Although we know
that this procedure has been used for years in some private clinics in the New York
area, we have not been provided with clinical data assuring that it is risk-free and
beneficial. In conclusion, since chronic hepatitis patients may have altered
24
prothrombin time , the use of heparin (even without the add ition of Ca ) could be
hazardous if not properly monitored, whereas citrate may be less efficacious but
innocuous.
 WHAT HAPPENS DURING BLOOD OZONIZATION? 141

2.2Is There any Advantage in Ozonizing a Large Number ofIsolated Leukocytes

Rather than Whole Blood?
This idea may sound Iike a sterile laboratory exercise but it arose from the study
by Rosenberg et al. (1994) on Iymphokine-activated killer cells (LAK are
composed of natural killer, NK cells and T cells as weil) incubated with IL-2. It is
possible to isolate up to 1'10 10 leukocytes by leukapheresis, resuspend them in
plasma , expose them briefly to O 2-0 3 and reinfuse them. However, in a
preliminary experiment, we found that we first have to select the adequate 0 3
concentration (15-20 ug 0 3 per ml) because, unlike blood, leukocytes are very
sensitive to oxidation and higher ozone concentrations cause cell damage. This
represents a serious drawback because, during reinfusion, impaired leukocytes
may stick in the lungs and cause respiratory distress .
As if it were necessary, we leamt once more how critical is the equilibrium
between the antioxidant system, cells and ozone concentration. Wehave not
pursued this line because of the complexity of the procedure, the risk of
contarnination, the uncertainty of the outcome and the cost. Instead, we have been
developing the system of extracorporeal circulation of blood against O 2-0 3 , which
represents a far simpler method (Chapter 17).

2.3 Within the "Therapeutlc Window " ts There a Range ofeither

Immunostimulating 01' Immunosuppressive 0 3 Concentrations?
This question is important because ozonetherapists, without the support of
experimental and clinical data and sometime pressed by the patient's request,
venture into the perilous practice of performing OrAHT in either chronic
infectious diseases or autoimmune diseases (rheumatoid arthritis, multiple
sclerosis, systemic lupus erythematosus, SLE, etc.) without knowing which, if any,
is the optimal 0 3 concentration range .
In the ozonetherapy field, particularly in Germany and Italy, the dominant idea
is that small blood volumes and low 0 3 concentrations (10-30 ug/ml) are
stimulatory while large (probably 200 ml ?) volumes and fairly high 0 3
concentrations (40-50 u g/rnl) are suppressive. The idea is a vague recollection of
the bell-shape curve typical of antigen -antibody binding, but it is not backed by
any serious experimental results. We tested four 0 3 concentrations: 25, 50, 75 and
100 ug/rnl in blood sampies of normal donors . After 9 hours incubation, we
measured the cytokine concentration of proinflammatory (IL-I ß, IL-2, IL-12, IFNy
and TNFa) and inhibitory cytokines (IL-1O and TGFßI) : if the idea was correct,
we would have detected a preponderance of either proinflammatory or inhibitory
cytokines at low-medium (25, 50 ug/ml) or high (75, 100 ug/ml) 0 3
concentrations, respectively. The result was disappointing and not worth
publishing : within that time frame, IL-12 was not induced and, irrespective of the
0 3 concentration, the production of all cytokines increased up to 50 ug/rnl and
decreased at 75, particularly at 100 ug /rnl, likely owing to excessive oxidation and
cell inhibition. With hindsight, the problem appears more complex than we
envisaged and remains worthy of investigation. Rather than examining blood of
142 CHAPTER 14

normal donors, a more rational approach may be to examine the cytokine response
during relapse of either autoimmune diseases or allergie diseases . From an
immunological point ofview, these pathologies have an opposite polarity.
Figure 57 shows a simplified scheme regarding the interaction between 0 3 and
cytokine production by leukocytes of healthy blood donors . However, this situation
does not apply to the above-mentioned pathologies because after the pioneering
work ofMossman and Sad (1996), it has been shown that CD4+Iymphocytes (helper
T cells) undergo a profound shift towards either the Th l-phenotype (generally
producing IL-l , IL-2, IL-18, IFNy, TNFo.) or the Thl-phenotype (produc ing IL-3,
IL-4, IL-5, IL-I0 and TGFß I).
ERYrnR OCYTE

...
..
Clon,l e.pan,lon
01T.lymphotyln

Stemtell

Haematopolnl.

Figure 57. An overall view ofinteractions among OrROS and immune cells which.
after cytokine induction. home into various lymphoid microenvironments andfurther
release cytokines, thus enhancing humoral and cell-mediated immunity

A schematic representation is shown in Figure 58 and extensive analyses have

been published (Clerici and Shearer, 1993, 1994; Maggi et al., 1994; Kay, 2001;
Swain, 2001) . However, as a further complication, many CD4 ' T cells often cannot
be grouped into either a Th I or Th2 subset (Th3?) and they exhibit a heterogeneous
pattern of cytokines . Nevertheless , pathological immune responses at least partly
 WHAT HAPPENS DURING BLOOD OZONIZATlON? 143

support the pattern of cytokine production linked with the Th I or Th2 predorninant
immune state. Th I-type responses are associated with inflammation and defense
reactions, including cytolytic reactions, while Th2-type responses are antibody-
mediated immunity. It must be kept in mind that the interaction between the two
types of responses is reciprocal and thus Th I-type cytokines are inhibitory to Th2-
type responses and vice versa . As an example, IL-4 can inhibit IL-12 production,
while IL-4, IL-lO and IL-13 antagonise the macrophage-activating properties of
IFNy and IL-2.

Mycobacteria INDUCERS HIV

HSP Allergens

ThO
.-
Th 1 Th2
IL-1, IL-2, IL-12 IL-4, IL-5, IL-6
IL-15,IL-18 IL-10, IL·13
IFNy, TNF a TGFß
Cell-mediated Immunity
Inhibition

Autoimmune dlseases, HIV infection,

Leprosy, TBC , Pregnancy,
Diabetes Allergie diseases,
SLE , Scleroderma

Figure 58. A schematic representation ofthe immunological equilibrium between CD4+ T

lymphocytes with a 1711 or Th2 phenotype. Theformer release proinflammatory cytokines
while the laue r release immunosuppressive cytokines. There is a reciprocal inhibition and it
would be interesting to examine ifozon etherapy can re-equilibrate dysimmunity

This scenario necessitates a careful review of the use of ozone in these

diseases: the current idea may turn out to be detrimental because we are ozonizing
pathological blood with a prevalence of CD4~ lymphocytes of either Th l-type or
Th2-type. As an example, in allergic diseases (pattern: Th2 » Th l ), ozonization
of blood with low-medium 0 3 concentrations (20-40 ug/ml) may lead to further
stimulation, hence proliferation, of Th2-type lymphocytes and to increased
production ofIL-3, IL-4, IL-5, IL-lO, IgG, IgE eosinophilia, etc ., and consequently
to further inhibition of the production of IFNy, IL-2, IL-12, possibly aggravating
the disease. 1fthis reasoning is correct, we should ozonize blood with an inhibitory
dose able to suppress Th2-type Iymphocytes and their cytokine production.
144 CHAPTER 14

In rheumatoid arthritis and multiple sclerosis (pattern : Th I » Th2),

ozonization of blood with low-medium 0 3 concentrations (20-40 ug/ml) may
upregulate cytokines produced by Th I cells and accelerate the progression of the
disease, while inhibitory 0 3 doses may downregulate these cells, leading to a
quiescent phase . In practice, any strict categorization often defies our best
intention and, rather than trying to switch a Thl profile to a Th2 and vice versa ,
the aim is to rebalance the immune response so that it may lead to stabilization or,
ideally , resolution ofthe disease.
At this point , we must attempt to establish a guideline . The ozonetherapist,
who is only interested in being inforrned about the optimal ozone concentration
and getting on with the treatment, may be annoyed but, regretfully, I am aware of
only a few unreliable anecdotes. Certainly, even empirical but trustworthy results
would have been helpful . Unfortunately, most ozonetherapists neither possess a
reliable generator nor precisely check the ozone concentration and the blood/gas
volume ratio . Moreover, there is extreme confusion about the blood volume and
the system for ozonization : some ozonetherapists use small glass bottles and only
50-100 ml of blood, others use 500 ml glass bottles and collect between 100 and
250 ml of blood, while some even use the hyperbarie system, for which we have
no laboratory data. Others insist on using PVC bags of different volumes in spite
of the prohibition by the Italian Ministry of Health (see appendix) . Ouring the last
two years, I have tried to little avail to correct this anarchical situation that hinders
any progress .
Hopefully, a few readers will be interested in the following reasoning, which
highlights the complexity ofthe problem and tries to provide some cautious guidance.
Lets us first consider the crucial parameters:

I . the target is represented by C04 Iymphocytes present mainly as either Th I or

I

Th2 phenotypes. Although it may not be completely true, a fair assumption is

that some of these cells are somewhat sustaining the ongoing disease and a
possible approach is to suppress the secretion of Thl-type cytokines (and
cytolytic activity) or Th2-type cytokines (and antibody formation) in
autoimmune diseases or allergie diseases, respectively.
2. The volume ofblood appears critical for three reasons :
a) the number of present and active Iymphocytes during the ozonization
process , because they will be directly affected (via H20 2 and very short
half-life ROS) ;
b) the volume of plasma, because it contains all the substrates undergoing
direct peroxidation that will generate long half-life LOPs . These
compounds (OH-HNE, MOA, possibly acrolein, etc.) act immediatelyon
Iymphocytes and will also bind to circulating Iymphocytes (and other cells)
after blood reinfusion into the donor;
c) the ozone concentration (ug/ml per ml ofblood), which can be divided into:
- low (10-30 ug/ml)
- medium (30-50 ug/ml)
- high (50-80 ug/ml) ,
 WHAT HAPPENS DURING BLOOD OZONIZATION? 145

Depending on the capacity of the plasma antioxidant system, the fonnation of

ROS and LOPs, although not proportional to the 0 3 concentration, increases with
the 0 3 dose. The consequence is that amounts of these compounds, which are
supposed to act as cytotoxic drugs, depend on the volume of plasma and 0 3 dose.
Therefore , a low 0 3 dose may hardly affect the Iymphocytes present in the
blood during ozonization and, owing to minimal LOP fonnation, also may not
affect circulating cells. Indeed, in diseases with pattern Th2 » Thl, it willlikely
stimulate Th2-type Iymphocytes and possibly worsen the disease. Conversely, a
high 0 3 dose may prevalently inhibit, directly or indirectIy, Th2-type Iymphocytes
(via reinfused LOPs), thus dampening the disease. While it would be naive to think
that LOPs will selectively inhibit Th2-type Iymphocytes, they might preferentially
bind and inhibit these cells because they are in an activated state. Needless to say,
the same reasoning can be used for diseases with a pattern Thl »Th2.
It must be emphasized that this is only a working hypothesis and much remains
to be learned before making definitive recommendations. Moreover, as will be
extensively discussed in Chapter 22, we strongly advise the ozonetherapist to
apply the "up-dosing" system . In other words, in order to induce ozone tolerance,
the "start low, go slow" strategy appears most reasonable. The following is a
schematic example of a possible schedule for ozonetherapists perfonning 03-AHT
in 500 ml glass bottles :

Time Treatment Blood Ozone Total ozone

(weeks) Number volume (mI) concentration Dose (mg)
(ug/ml)
I 150 50 7.5
Ist
2 200 50 10.0
3 200 60 12.0
2nd
4 225 60 13.5
5 225 70 15.75
3rd
6 225 80 18.0
7 225 80 18.0
4th
8 225 80 18.0

and so on for at least 16 treatments (8 weeks), unless serious side effects appear.
Assuming that the patient has a high TAS and the disease symptoms tend to
ease, it may be reasonable to increase the ozone concentration to 90-100 ug/ml.
This may enhance the upregulation of oxidative stress proteins, strengthen the
adaptation to stress and further inhibit the clone of cytotoxic Iymphocytes.
Obviously, this possibility has many mighty "ifs" to be dispelled during the trial.

2.4. On the Experimental Basis that Ozone Can Act as a Modest Cytokine Inducer,
Does Reinfusion ofOzonized Blood Mod!fj: the Plasma Cytokine Level in Vivo?
146 CHAPTER 14

The immediate answer based on experimental results is NO! As a striking contrast,

either a sublethaI dose of LPS (4.0 ng/Kg as an IV bolus injection) or septic shock
causes a marked sequential increase of cytokines in plasma (TNFa, IL-6, G-CSF,
etc.) and considerable side effects, similar but worse than flu-like syndrome
(chilis, fever, headache, malaise, hypotension, etc.), which can be relieved only
partially by cyclooxygenase inhibitors (Mitchie et al., 1988; Machensen et al.,
1991; Bocci, 1988a). Since 1980, we underlined the fundamental difference
between the physiological IFN (cytokine) response and the emergcncy, or
inflammatory-toxic, response (Bocci, 1981c, 1988b, 1990b, 1992c).

GA LT INCUCERS

Wald.y . ~s ring Proloin.

Leetins
SUbmucosal lymphoeytes Bacterle
Virus
Lymph nodes Endotoxln.
Drugs
P.y. ~s plaques Ozone

LympraliCS

',: Cr---:.-....;..-r-- LIVER

. Portal vain
+ Kup"er 11 No's cells

( Venous circulation ( )

Figure 59.A sehematte view of'cytokine induction in the GALT by a number oflnducers.
among which ozone. The GALT is an important component able to interact with other parts
ofthe immune system

Cytokines are always produced after interactions between an array of inducers

(toxins, bacteria, LPS, virus mitogens, lectins, antigens , drugs , allergens, ozone ,
etc., presented in Table 9) and immune cells . However, the substantial difference
 WHAT HAPPENS DURING BLOOD OZONIZATION? 147

is that the physiological response, occurring continuously throughout life, is a

miniaturized release of cytokines in several microenvironrnents, such as the gut-
associated lymphoid system (Galt etc.), coming in contact with a trace amount of
inducers (see scheme in Fig. 59). In this case, by virtue oftheir migratory capacity
among the original microenvironment, lymphatic system, blood pool and then
lymphoid tissues (Westermann and Bode, 1999), the relatively small number of
stimulated immune cells can release small amounts of cytokines and prime other
cells, thus maintaining the immune system in an alert state, usually without any
imbalance between activation and suppression. The released cytokines are mostly
bound by neighbouring cells by a paracrine mechanism and barely emerge in the
plasma pool. Indeed in physiological conditions, only trace levels can be detected,
because of dilution, tissue binding and rapid catabolism via the kidneys, liver and
so on. Our studies on the pharmacokinetic properties of interferons have become a
paradigmatic model in the cytokine field (Bocci, 1981b, 1987b, 1990a, 1991a,b,
1992c, 1993a) and have indicated that cytokines are not meant to be circulatory
proteins like cIassical hormones. Their high turnover, due to a very efficient
catabolic system, preserves body homeostasis and prevents toxicity.
In contrast, the emergency response is characterized by a colossal release of
cytokines due to toxic stimuli inundating the blood pool. Plasma levels of
cytokines become so high that the catabolic system is overwhelmed, partly
because of multi-organ dysfunction, which leads to death in 40-50% of patients
with toxic shock syndrome . This excessive response represents the dark side of
cytokine activities and only a weil orchestrated administration of antibiotics,
anticytokines and human activated protein C may occasionally be life-saving .
Coming back to question no. 4, the answer is that classical 03-AHT and,
interestingly, the far more intensive treatment of extracorporeal blood circulation
against O 2-0 3 can be assimilated to the physiological response; neither treatment
causes side effects and most patients report a sense of weU-being after the
therapy. A likely explanation is the mild induction of only a small percentage of
immune ceIls, which after blood reinfusion horne in several organs (Fig. 49, 57 and
59); at best, they may reinforce the basic physiological response which, particularly in
ageing and in immunosuppressive states, may become subdued.
When, several years ago, we conducted the experiment on myself and a
collaborator we were disappointed by the results. 250 ml of heparinized (+5 mM
CeCl, to achieve maximal stimulation) blood was ozonized with 13.5 mg ozone
(54 ug/ml x 250 ml gas) and promptIy reinfused into each donor at 8.00 am. A
small blood sampie was incubated for 8 hours and, in comparison to the control,
we could distinctly detect a few IU of IFNß, IFNy, IL-2 and sorne IL-I ß, IL-6,
TNFa. and GM-CSF (see Table I in Bocci et al., 1994a). We were hoping that the
production of cytokines in vivo would be enhanced and that we could detect a
small increase in the peripheral plasma . Blood sampies were withdrawn I, 2, 4, 6,
8 and 12 hours after reinfusion. At the same time, we rneasured the body
temperature and compared it with the previously determined circadian values. We
did not detect any rnodification but at least we feit very weil the next day! The
interpretation was that, upon reinfusion, the activated BMC horne in various
 148 CHAPTER 14

lymphoid and parenchymal microenvironments and release small amounts of

cytokines that are bound by either bystander or migrating BMC. Therefore, unless
a far larger number of cells is involved, or they are superinduced, cytokine
spillover into the circulation is unlikely or, if it occurs to a small extent , dilution
and rapid cytokine turnover prevent the increase . The lack of any side effects is an
advantage but is the treatment effective?

2.5. Is DrAHT Effective and is There an Optimal Schedule?

This question would have remained unanswered if it had not been shown (Towbin
et al., 1992) that, after binding to BMC, IFN upregulates a number of genes, one of
which allows synthesis of the Mx protein. Besides having GTPase activity, this
protein of about 70 Kd is expressed during a flu-virus infection and blocks
transcription ofthe viral genome . While IFN has a half-life of a few hours after SC
administration (Bocci , 1990a), an interesting facet of the Mx protein is that it has a
half-life of 24-36 hours and it can be detected in circulating BMC even 2 days
after the total disappearance of IFN frorn plasma. It is intuitive that the Mx protein ,
like other specific enzymes induced by IFN, can be used as a biomarker to reveal
that IFN, albeit short-lived, had been present.

JOO

250

200

~ ISO

'00

50

o ?L...2'-
4 -4J-8-7.J-2-9.J-18-2.J..4-:'::48-=':72~9,8~24-:--'48:-:72~9,~6""':2':-4--:48~7~2--:9'!::,8--:2-::4-4-::8-::72:-:!9t·8

HOURS

Figure 60. Kinetics of Mt protein levels measured in 21 blood samp ies collected el'elY day
from one healthy volunteer. The six arrows indicate the time ofsix DrAHT performed using
300 ml ofblood (heparin) exposed to 70 mg/ml 0 3 (total dose 21 mg). There is a
progressive but variable increase ofMx pro tein levels. likely due to the release ofIFN in
lymphoid microenvironments

On the hypothetical assumption that reinfusion of ozonized blood can induce

IFN, we tested whether the Mx protein could be detected in circulating BMC after
OrAHT. Figure 60 shows that the Mx protein could be detected every time after
03-AHT in a healthy volunteer, with a peak usually 48 hours after reinfusion.
Although the Mx protein disappeared by 96 hours , it could be regularly re-
induced; in fact, it seems that the expression was gradually potentiated. The result
 WHAT HAPPENS DURING BLOOD OZONIZATION? 149

is interesting and suggests that the "biochemical memory" of the cell is fairly
short; however, two treatments per week may be sufficient to maintain an anti viral
state. In contrast, both Neopterin and 132 microglobulin (132M) plasma levels (data
not shown) remained within the normal range and 132M levels remained unvaried,
even during repeated autohaemotherapy. Plasma levels of acute phase proteins,
namely orosomucoid, C reactive protein and haptoglobin, remained constant 24,
48 and 96 hours after autohaemotherapy. This excludes the possibility that liver
synthesis was influenced by IL-6 . We plan to repeat this experiment with the
method of extracorporeal blood circulation, involving ozonization of the whole
blood volume.
In conclusion, the use of this amplification marker suggests that the reinfused
ozonized blood cells release IFN in vivo but, as discussed in Chapter 24 , the
efficacy of 0 3-AHT in chronic HCV infection remains controversial. It remains to
be seen whether more intensive treatment or a different schedule can improve the
outcome. A typical and frequently posed question is: how many autotransfusions
are needed to achieve a valid reactivation of the immune system? It is almost
impossible to provide an answer because each chronic HCV patient presents
variable parameters. However, the number of BMC producing IFNa/ß must be
substantial. On the whole, the immune system is a fairly large organ comprising
about 10 12 cells dispersed in the spleen, lymph nodes, Iymph pool, thymus, bone
marrow and lymphoid tissue associated with the gut (GALT), bronchi (BALT),
skin (SALT), etc . About 1% of these cells die every day and are replaced by virgin
cells (Westermann and Pabst, 1992) . Our approximate calculation estimated that
ozonization of 300 ml of blood with an ozone dose of 21 mg (70 ug/ml x 300 ml
gas) may stimulate - 6 x 10R cells, i.e. only 0.06% ofthe total cell number. After
50 treatments (twice weekly for 6 months), we may have activated - 3 x 10 10 cells,
equivalent to a mere 3% ofthe cell pool (Bocci, 1998c) . However, when activated
T cells horne in lymphoid microenvironments, they produce cytokines and express
co-stimulatory molecules. By inducing proliferation of bystander Iymphocytes,
these substances may amplify this process several fold, probably yielding 10 11
cells which is considered a desirable dose to treat human tumours by mononuclear
cell adoptive immunotherapy (Rosenberg et al., 1987 ; Lee and Klein, 1994) . It
would be interesting to investigate the distribution and fate of Indium'{'vlabelled
BMC and neutrophils after ozonization and to check whether they horne in the
lungs or throughout the body. This methodology has been used for LAK and TIL
cells (Rosenberg et al., 1987) .

2.6. Does the Induction o/OSP and ofAdaptation to Chronic Oxidative Stress
have an 1111111uno111odulat01Y Effect?
This is the most fascinating topic because ozone, the dreadful toxic gas, may
paradoxically become an important drug to induce adaptation to chronic oxidative
stress. There is a wealth of experimental data reported since the 1970s showing
that both animals and plants can develop resistance by upregulating the expression
of a number of proteins, generically denominated chaperones.
150 CHAPTER 14

When a Iiving being undergoes a stressful situation, such as ischaemia,

hypoxia, hyperoxia, heat, haemolysis or ozone, it has only two options : adaptation
or apoptosis, i.e. Iife or death. It is an exciting possibility that by carefully "up-
dosing" ozonetherapy we may be able to correct a chronic imbalance between
excessive oxidants and depressed antioxidants. If this is proved to be true , our
"calculated and brief oxidative stress" will merit the term therapeutic "shock",
symbolizing the fact that even stress can be therapeutic. So as not to repeat myself,
this problem will be discussed in more detail in Chapter 22.

2.7. Can We Select Tests Suitablefor Evaluating Modiftcations ofthe Immune

Status during Ozonetherapy?
On the assumption that one day we will be able to conduct randomized c1inical
trials with the appropriate control (0 2 only), it will be necessary to analyse (before,
at mid-cycle, at the end, and 3 months thereafter) several immunological
parameters in the hope of understanding if ozonetherapy is able to rnodify and
possibly improve immunological activities. Some ofthese tests (listed in Table 10)
may prove useful in evaluating the effect of ozonetherapy in acute and chronic
infections, allergie and autoimmune diseases and metastatic cancer.

Table 10. Possible parameters to test the activation ofthe immune system
before, eil/ring and after 0J-AHT.

• Evaluation ofthe number and subclasses oflymphocytes.

• Activation ofNK cells towards unspecific targets and autologous
tumour cells.
• Degree of activation of eytotoxic activity by T-cell Iymphocytes,
macrophages, PMN leukocytes.
• Enhancement of ADCC .
• Antibody production and specificity.
• Evaluation of the proliferation index of BMC (PHA-induced
versus spontaneous).
• Evaluation of the release of cytokines in eulture (spontaneous or
mitogen-induced).
• Plasma levels of cytokines.
• Evaluation of cytokine levels to assess a possible imbalance
between Th 1- and Th2- lymphocytes.
• Tests for evaluating immunosuppression.
• Levels of suitable biomarkers.
• Concurrent opportunistic infections.
• Remission rates and recurrence-free intervals.
• Relevance of cachexia.
• Psychosomatic state ofthe patient.
 WHAT HAPPENS DURING BLOOD OZONIZATION? 151

Therefore, a great deal of experimental and clinical work remains to be done.

Yet it remains doubtful that this will ever be accomplished unless official medicine
or the Department of Complementary Medicine (NIH, Bethesda, USA) takes this
approach to heart .

3. PLATELETS, HAEMOST ASIS AND GROWTH FACTOR RELEASE

Human platelets or thrombocytes are small (2-4 u), anucleate cells present in
blood (130,000-400,00011l1), which derive from the segmentation of bone marrow
megakaryocytes . Their life-span in the circulation is 7-10 days and, in
physiological conditions, old platelets (smaller and denser than young platelets)
are removed by the RES present in the spleen, liver and bone marrow. However, a
small amount of platelets is used continuously to "repair" any discontinuity of the
enormous endothelial surface and this process is markedly heightened by
pathological processes (injury, inflammation, atherosclerosis, immunological
reactions, etc.). Their life-span is conditioned by two facts: I) they have only
residual ribosomes and a little RNA to accomplish minimal protein synthesis, and
2) they are extremely sensitive and reactive to any modification of the vascuIar
ecosystem. Platelets contain several organelles (Harrison et al., 1990) filled with a
number of compounds promptly released into the microenvironment during
platelet aggregation (Table 11); the latter process involves the progressive
accumulation of platelets followed by the release of their components, leading to
further aggregation and blood coagulation. Thus platelets interact with each other
and with all the components of the vascular ecosystem by virtue of a wealth of
membrane receptors. Typical inducers of platelet aggregation in vitro and in vivo
are: damaged endothelium, ADP, thrombin, collagen, adrenaline, noradrenaline,
serotonin (5-hydroxytryptamine), platelet activating factor (PAF: I-O-alkyl-2-
acetyl-Sn-glycero-3-phosphocholine), Ca z+ , arachidonic acid derivatives such as
prostaglandins (PGE z, PGFzu and thromboxane, TxA2) and ROS. Among the
coagulation factors, the active ones are: Factor Va and Factor Xa after the
interaction with platelet factor 3 (PF3) . Typical inhibitors of platelet aggregation
are: PGDz produced by platelets, and prostacyclin (PGIz) produced by endothelial
cells, which, by activating adenylate cyclase, increases the intraplatelet level of
cAMP. POIl is far less potent and is rapidly degraded. NO· is also a powerful
inhibitor (see Endothelial cells and the vascular system).
It may be useful to mention the most common pharmacological inhibitors
such as acetylsalicylic acid (aspirin) , indomethacin , ticlopidine, suIoctidil,
dipyridamole, etc. On the assumption that the TxA2/PGI z balance is a key factor in
haemostasis and thrombosis, we are always facing the clinical dilemma to
selectively block aggregation without affecting PGIz synthesis.
From this brief outline, it is evident that, during blood ozonization,
platelets may react by aggregating and releasing their factors and we must be
aware of possible consequences. When we performed OrAHT with heparinized
blood (30 UI/ml blood without ci+addition) at 0 3 concentrations of 70-80 ug/ml
152 CHAPTER 14

Table 11. Main constituents 0/platelet organelies and their activity.

Constituents Activitv
ADP Aggregation
ATP
Dense bodies: CA 2+ Pro-aggregation
Serotonin Aggregation
Platelet factor 4 (PF4) Heparin-neutralizing
protein
Thrombomodulin Inhibition of thrombin
activity
ß thromboglobulin (ß- TBG) Antiheparin
Thrombospondin (TSP -1)
Fibronectin Adhesive glycoprotein
von Willebrand factor (vWF)
PDGF
TGFßl
u-granules: Hepatocyte growth factor (HGF) Growth factors for
Basic fibroblast growth factor fibroblasts, endothel ial
(bFGF) keratinocytes and museIe
Vascular endothelial growth factors cells
(VEGF-A, VEGF-C)
Epidermal growth factor (EGF)
Cl inhibitor Inhibitor
Fibrinogen Procoagulant
Factor VIII antigen Enhancer of platelet
adhesion
U2macroglobulin Antiproteases
U2antiplasmin
Lysosomes : Acid hydrolases
Proteinases
Cathepsin

per mJ blood, we occasionally noticed the presence of miniclots trapped in the

blood filter after reinfusion. This compelled us to investigate the cause of it. Rather
than thinking about prothrombin activation, an obvious hypothesis was that, during
ozonization and in the presence of physiological Ca 2 + (about 1 mM), platelets can
aggregate and possibly trigger coagulation, even in the presence of heparin. This
hypothesis was mainly based on previous observations (Maresca et al., 1992;
Iuliano et al., 1991, 1997) that ROS can induce platelet activation . Several
mechanisms could be at work, even simultaneously :

a) formation of H202, which if not sufficiently quenched by antioxidants could

diffuse into the platelets (Iuliano et al., 1991).
 WHAT HAPPENS DURING BLOOD OZONIZAnON? 153

b) H202 or other ROS may activate phospholipase C, which acts on P1P2 to

generate IP3 and DAG. The former compound causes the release of Ca2+ from
intracellular stores, which (possibly associated with an extracellular Ca2+
influx) causes the release of ADP and platelet aggregation .
c) HZ0 2 may activate phospholipase Az, cyclooxygenase II and thromboxane
synthetase, allowing the release of arachidonic acid and formation of PGE2,
PGF2u and TxA2 with consequent irreversible aggregation.

0 3-AHT has always been performed using blood anticoagulated with citrate, and
we and others have never observed any tendency to blood clotting. On the other
hand, by preserving the physiological Ca2+ level, the use of heparin may not be
sufficient to avoid minimal coagulation, a risk that cannot be underrated. Thus, using
biochemical and morphological criteria, we investigated the behaviour of platelets in
the presence of either citrate or heparin. Results of the evaluation of both ACD and
heparinized platelet-rich plasma (PRP) from five healthy donors are briefly
summarised, as details can be found in the original paper (Bocci et al., 1999a).
Figure 61 clearly shows that ozonization of both ACD- and heparin-treated
PRP induces an almost linear increase ofTBARS, which (as mentioned in Chapter
10) have been used as a practical ozone marker. Both plasma PTG and, to a lesser
extent, TAS also decrease progressively during ozonization carried out within the
usual therapeutic window used in medical practice (20-80 ug/ml per m1 of PRP).
As expected on the basis of the high capacity of platelets to restore their GSH
content after oxidation (Bosia et al., 1989), the intracellular GSH content does not
appear to be modified, even after exposing PRP samples to 0 3 concentrations as
high as 80 ug/ml (lower panel of Fig. 61). It is remarkable that under similar
experimental conditions, a GSH decrease of about 20% has been measured in
erythrocytes , although even these cells recover their normal GSH content soon
thereafter (Bocci et al., 1993b).
The surprise came when we monitored platelet aggregation:
control and 02-treated sarnples did not show spontaneous aggregation . However,
when heparinized PRP was pretreated for 30 seconds with three concentrations of
0 3 (20, 40 and 80 ug/rnl per ml PRP), there was a striking dose-effect relationship
(Fig. 62, right panel) : a mean spontaneous aggregation of either 20 ± 6% or 68 ±
14%) occurs immediately with an 0 3 concentration of 40 or 80 ug/ml, respectively.
However, the aggregation appears reversible (at least in part) and the addition of
ADP 4 min afterward induces the second wave. The phenomenon is quite
reproducible and it could easily be missed if the exposure to 80 ug/ml 0 3 lasts
longer than 30 sec. In contrast, PRP, lacking extracellular Ca2r, shows negligible
aggregation (left panel). The platelet aggregation can be observed
macroscopically , but Figure 63 illustrates, by both scanning electron microscopy
(SEM) and transmission electron microscopy (TEM), how the platelets are
clumped together. At present , the biochernical andrnolecular events leading to
aggregation remain unclear. However, extracellular Caz+ certainly has a crucial
role, since aggregation does not occur after its chelation. Another important factor
is the transient formation ofH 202 in the plasma, which, as was hypothesized, may
 Figure 61. Effect 0/30 sec exposure to either O2 or 0 3 (20, 400/1(180 ug/ml) per 1111 0/
human platelet-rich-plasma (PRP) on lipid peroxidation (TBARS), total antioxidant status
(TAS). protein thiol groups (PTG) and platelet GSH content . PRP was anticoagulated witti
either ACD (Cl) or heparin (~ .
 WHAT HAPPENS DURING BLOOD OZONIZATION? 155

cause a Ca 2 + influx or activate enzymes in the cell membrane. Other studies (Dei
Principe et a\., 1985; Iuliano et a\., 1991, 1997) have shown that H202 can trigger
aggregat ion of platelets even at nanomolar concentrations, when ozonization of
PRP yields concentrations up to 40 J..1M (Vala cchi and Bocci, 1999).
ACD Heparin
100 c.m...

80
0~
c: 60
."III0"
~
Cl
40
Cl
-c 20
..
0

0 2 4 6 8 10 0 2 4 6 8 10

t Time (mln) t
Figu re 62. Rep resentati ve p atterns ofplatelet aggregation induced by p rogressively
increas ing 0 3 concentrat ions (20. 40 and 80 ug/ml p er ml PRP) . 0 3 causes imm ediate and
dose -depende nt aggrega tion only in heparinized PRP (right panel). Aggregation profiles 0/
PRP in ACD are reported in the left pa nel. After 4 min, ADP inducesfull aggregation
(arrow)

The problem ofplatelet sensitivity to ozone has been tackled by Matsuno et a\.,
who presented a poster at the 13th Ozone World Congress (IOA, Kyoto 1997). Yet
they examined only PRP anticoagulated with citrate or washed plateiet suspension.
Moreover, the experimental approaches cannot be compared because those authors
did not study the direct effect of ozone on aggregation. Using 0 3 concentration in
the range 9-71 ug/ml, they observed that pretreatrnent of PRP with ozone actually
inhibited, in a dose-dependent manner, ADP-, collagen- and thrombin-induced
platelet aggregat ion. Matsuno et al. suggested that the inhibition was due to
reduced express ion of activation antigens, CD62P and particularly CD63,
accompanied by a reduced appearance of cytosolic free Ca 2+. We cannot confirm
their data because our measurements of CPD-PRP (Fig . 62, left panel) do not show
any inhibition when ADP was added 4-5 min after ozon ization . Thus , at present it
appears that platelets in heparin ized blood are sensitive to ozonization and tend to
aggregate, while Ca2 +-chelated platelets are either insensitive (Bocci et al., 1999)
or may become resistant to ozone (Matsuno et a\., 1997). Further studies are
needed to clarify this uncertainl y but, clearly, blood ozonization would seem
superfluous if we want only to inhibit platelet aggregation; this is better
156 CHAPTER 14

accomplished with drugs. Although no side effects appeared in the few patients for
whom we noted miniclots retained in the filter , we have nonetheless to be cautious
and avoid the use of heparin if we intend to use an 0 3 concentration above 40
ug/ml. Jt would seem that the simple use of citrate eliminates any risk and platelets
remain either insensitive or resistant to ozone. However, this simplification does
not answer the question of whether heparin is more or less beneficial than citrate .
It was mentioned that platelets contain a number of growth factors : Platelet-
derived growth factor (PDGF), TGFß 1, Yascular endothelial growth factors
(YEGFs), etc. (see arecent minireview by Browder et al, 2000), that may be
responsible (at least in part) for accelerating the healing of torpid ulcers observed
in hind limb ischaemia patients. If this is true, a slight activation of platelets ex
vivo by heparin-ozone may be more proficient than ACD .

Figure 63. Scanning electron microseopie (SEM, left panel) and transmission electron
microscopic (TEM. right panel) examinations o(human PRPs in heparin. Platelets exposed
to 0; (ahove) appear normal, while after exposure to ozone (concentration 80 pg/lIIl) they
form visible aggregates (below) . In contrast, PRP in ACD do not undergo aggregation afte r
exposure to ozone.For the total sequence see Bocci et a!. (/999a)
 WHAT HAPPENS DURING B LOOD OZONIZAnON? 157

In order to shed light on this possibility, we investigated whethe r the use of

heparin or citrate added to five different PRP sampies (from heait hy donors)
before ozonization would differently affect the release ofplatelet factors (Valacchi
and Bocci, 1999). As usual , several markers were controlled in the PRP sampies to
make sure that ozone acted correctly. The PTG values decreased in appr oximate
relation to the 0 3 concentration while the TBARS values increased several fold
(Fig.64).
3 c::::J ACe
_ Heparin

-
,I, ,..J: ,r.

o -- :--
.
- '- '-'-

0,4 +
. . +

~ 0,3

~ 0,2 -

0,1 -
... _-
0 ,0

0,6
.. '--

2'
.= 0 4
~ ,
s 0,2
~

0,0 --'------'----.I....:::!--...I.-
20 40 80

Figure 64. Effect 0130 sec exposure ofeither Oz 01' Or03 (20, 40 and 80 ug/ml p er ml
ofp lasma) on total antioxidant status (TAS). protein thiol group (PTG) and thiobarbituric
acid reactive substances (TBARS) ofthe same human PRP samples col!ected either in
heparin or in ACD. Statisti cal significance is indicated by (*) for intergroup analysis and
(+)./i}r intragroup analysis

Moreover, we observed that oxidation of PTG was far higher in the heparinized
sampies than in the Ca 2+-chelated sampIes , suggesting that physiological Ca 2+
levels favour ROS activity. Indeed, intragroup analysis showed a significant
difference at medium (40 ug/ml) and high (80 ug/ml) 0 3 concentration. After a 30-
sec ozonization in sterile silicone-coated syringes , PRP samples were dispensed
158 CHAPTER 14

into tissue culture plates and incubated at 37°C in air-C0 2 for I, 2 and 4 hours.
Control sampies were either untreated or mixed with an equal volume of O 2 • After
incubation, sampies were centrifuged and the supematant platelet-free plasma was
used to measure human PDGF-AB, TGFß 1 (after activation of the latent form),
IL-8 and the stable compound thromboxane B2 (TxB 2, derived from TxA2) as
typical platelet markers. Results obtained from five donors were expressed as the
mean ± standard deviation (Fig . 65) .

18000 c::J ACD

_ Heparin
15000
12000
1h 2h
+
. 4h

9000
• +
6000
3000
O...L~LULlJLLa.l""'--.I LaJ...IIL.LlLU --LlILLA..l..ILJLA..La.....J

9000

6000 TGFIlI

3000
E
~ 0 ..L..J~.A.l..a..LA..L ~w...u.a..La.L.A.-.l...A..l..a..LA..L-..w~.

::: ~ .I
800~
400
20~
IL-8

L-.- '---
JillJ • '. •

..
•• iIi ,.

4500

3000 TBX2 +
+

1500

__--l...ILJLA..La~LUII___LlLUILLA..l L.a-l
Cant,. 0, Cant,. 0,
O...L1A~LULlJLL

Cont,. O. 20 40 10 20 40 10 20 40 10

Figure 65. Release offactorsfrom human platelets during I. 2 and 4 hr incubation. The
same PRP samples collected either in heparin or ACD were not exposed (control). 01'
exposed 10 O2 alone or to OrO.! at concentrations 0/20. 40 and 80 pglml/or 30 sec before
incubation. Statistical significance is indicated hy (*)/01' intergroup analysis and (+)/01'
intragroup analysis
 WHAT HAPPENS DURING BLOOD OZONIZATlON? 159

Figure 65 shows the striking and significant difference between the heparinized
and Ca 2 +-chelated PRP sampIes in the release of POGF AB, TGF131 and IL-8. For
the first two cytok ines the difference is c1ear at all times, while for IL-8 it becomes
evident only after 4 hours of incubat ion. As far as the release of TXB2 is
concerned, heparin does not appear to prevail over citrate .
Thus experimental results have proved the hypothesis that activation of
platelets by heparin -ozone (even at a concentration of 40 ug/ml) significantly
favours the release of at least two important healing factors : POGF and TGFß I .
These findings should not be underestimated because, after reinfusion of ozonized
blood, the release occurs in vivo ; in patients with chronic limb ischaemia, this may
permit healing of necrotic ulcers. Mustoe et al. (1987) speculated on the
physiological role of TGFI3, both in wound healing and in the formation of scar
tissue , and Beck et al. (1993) showed that IV administration of TGF131 in rats
enhanced defective healing . This presaged important clinical application has not
yet materialized (Shah et al., 1999).
There are three isoforms ofTGF-ß (131, 132 and 133). The prototype (TGF-ßI),
identified as a growth factor for transformed cells, was first purified from human
platelets (Assoian et aL, 1983). They display pleiotropic activities (Sporn and
Roberts, 1993; Lawrence, 1996) but they are most active in promoting deposition
of collagen and matrix (Schrnid et aL, 1993). Slavin (1996) wrote that "if any
particular cytokine deserves to be described as a wound hormone it is TGF-
13". As yet, there is no consensus on how much TGF-ß I is present in normal
human plasma (Grainger et al., 2000).
There is no doubt that human blood and platelets release TGF -ß after ozone
treatment (Bocci et aL, 1994b, 1999a) and, most interestingly, Murphy-Ullrich and
Poczatek (2000) have identified mechanisms for the activation of latent (inactive)
TGF-13 by thrombospondin-l. Thus platelets contain all the ingredients to make
TGF-131 pharmacologically active.
If the healing of a skin wound is a complex process requiring the participation
of several growth factors and cytokines acting together or sequentially (Moulin,
1995; Martin, 1997; Browder et aL, 2000), the healing of a necrotic infected ulcer
in hind limb ischaemia is a paramount enterprise that often ends with amputation.
OJ-AHT can often avoid that and we must obviously ask what other compounds
are generated in the ozonized blood to explain the incredible improvement that
Rokitansky et al. (1981), Werkmeister (1995) and ourselves have observed
(Chapter 24) in several hind limb ischaemia patients (lII-IV stages) with
apparently incurable lesions?
Table 11 reports other critical factors released by platelets, such as VEGF
(Mohle et al., 1997). Furthermore, TGF-ß, angiopoietins and HGF have a potent
ability to induce angiogenesis (Roberts et aL, 1986; Yang and Moses, 1990;
Browder et aL, 2000) which , in association with vasodilatation and enhanced
oxygen delivery , is critical for reducing ischaemia .
Figure 65 shows the more marked release of POGF AB in the presence of
heparin in comparison to citrate; it also shows that there is no advantage in
doubling the ozone concentration (from 40 to 80 ug/ml). PDGF is one of the
160 CHAPTER 14

growth factor signals at the wound site (Ross, 1987) and besides being
chemotactic, it enhances fibroblast proliferation and extracellular matrix
production (Pierce et al., 1995).
The fairly late release of IL-8 has been interpreted as due to the time lag
necessary for its synthesis. It is known that induction of IL-8 by 0), while it is
promoted by a temporary rise of HzOz (Bocci , 1996d, 1998b; Jaspers et al., 1997)
in cytoplasmic water via the activation of NFKB, is inhibited by ROS scavengers
(DeForge et al., 1992). As this chemokine is capable of initiating the chemotactic
gradient that draws leukocytes from the circulation into tissues, it may have the
additional role of favouring phagocytosis of bacteria and necrotic tissue present in
torpid ulcers. Finally, release of TXB2 appears to be a drawback but we cannot
draw a conclusion unless we carry out determinations of other eicosanoids, such as
PGE2 and prostacyclin, that induce vasodilatation and inhibit aggregation.
These results suggest that ozonized blood may transport a wealth of growth
factors . Thus it is reasonable that we make a comparative evaluation of the
effectiveness of O)-AHT in patients with chronic limb ischaemia treated with
either citrated or heparinized blood exposed to low-medium ozone concentrations
(from 20 up to 40 ug/ml) . While these mild concentrations have never yielded
miniclots, they appear to generate pharmacologically relevant doses of growth
factors and may represent the optimal treatment. Low molecular weight heparins
have not proved to be advantageous but, as soon as hirudin (a specific inhibitor of
thrombin extracted from the salivary gland of the leech) becomes available, it will
be interesting to test it; unlike heparin, it does not appear to cause any negative
potentiation of platelet response.
If the reader has browsed Chapter 2, he/she has realized that ozonetherapy is
very unpopular and scorned by orthodox scientists. The paper on the release of
factors from ozonized human platelets (Valacchi and Bocci, 1999) was submitted
to the journal "Platelets" in 1998 and, onee aga in, it may be interesting to read the
cornments of a senior referee, who advised the editor to reject it:
"Thanks for sending me the manuscript from Valacchi and Bocci to review . I had
very mixed feelings on reading this article . On the one hand I know that you want to
keep Platelets as a very "open " journal where a wide range of contributions are
acceptable. On the other hand I found that although this article contains some solid
results, on the whole it has a very naive approach to the subject that it treats in a
preliminary fashion . I assume that the basic reason for treating autologous blood with
ozone is because it is recuperated during operations and has to be steri lized before
transfusion although this is never mentioned. The criteria for optimal levels of ozone
during such treatment are not discussed either. It is weil known that reacti ve oxygen
species are harmful to the body in general and to the cardiovascular system in
particular, hence the protective effects of vitamin E and the polyphenols from red
wine and other sources . The authors suppose however that the ozonized blood might
have positive effects on the patient's circulation, without providing any evidence that
this is effecti vely the case, and propose that this might be an effect of increased
release of factors such as PDGF and TGFßI. Indeed they go on to show that ozone
treatment of blood does increase release of these two components . However , as they
themselves note, this is accompan ied by a high degree of platelet aggregation.
lt seems to me that this can be possibly a very dangerous approach to treatment of
chronic limb ischemia by transfusing already partly, or even mostly, activated
platelets could contribute to a worsening of the condit ion. If the presence of platelet -
 WHAT HAPPENS DURING BLOOD OZONIZAnON? 161

derived growth factors might alleviate chronic limb ischemia this could be tested (first
of all in animal models) by administering the growth factors in the absence of
activated platelets to see if it really has positive effects. If this is a current practice to
use autologous transfusion with ozonated blood the incidence of vascular problems
could be compared with that in surgery accompanied by conventional transfusions . At
present 1would advise rejection ofthis manuscript."

This is another typical response by an anonymous referee who, firstly, did not
carefully read the methodology and wrongly assumed that blood has to be
sterilized. Secondly, as usual, he repeated the old stories that ROS are always
harmful to the body and polyphenols from red wine are protective. Finally, he
pontificated by advising the use of animal models when we know already that
there is absolutely no risk at low-rnedium 0 3 concentrations and that those
experiments would be uninformative.
Thus we should not get discouraged by this continued criticism and we must
hope to carry out controlled clinical trials that may dissipate doubts even in the
most skeptical referees.
Finally I must mention an important detail : the application of ozonized water
and oil in torpid ulcers does not only exert disinfectant activity but induces the
local release ofthe mentioned growth factors. This explain why the combination of
03 -AHT with topical therapy is so effective.

4 . ENDOTHELIAL CELLS AND THE VASCULAR SYSTEM

"These pipes and these conve yances 0/011" blood... ..
Shakespeare (1564-1616), Coriolanus, V i

Endothelial cells (Ecs) have long been considered a simple lining of blood vessels
with the unique property of being a non-thrombogenic substrate for blood. The
first breakthrough occurred in 1973, when laffe et al. learned to cuItivate human
endothelial cells (HUVECs) obtained from umbilical veins. The second was in
1980 when Furchgott and Zawadski described the endothelial-derived relaxing
factor (EDRF).
Today, we know that the vascular system is lined by about 10 13 cells covering
an enormous surface of about 400 m 2 . It is a real organ weighing about 900 g, with
innumerable paracrine-endocrine activities, the last one being to facilitate the
growth of metastasis.
In the last three decades, great progress has been made in understanding the
elose relationships among endothelium, platelets, leukocytes, procoagulant and
ant icoagulant factors during haemostasis, inflammatory and immunological
reactions. In spite of this new knowledge, thrombosis or haemorrhage remains the
primary cause of death in Western countries. Moreover, ischaemic diseases of the
hind limbs, heart, brain and kidneys take the heaviest socioeconomic toll and lead
to a very poor quality of life for several millions of people worldwide. A
discussion of the several factors involved in vascular deterioration is beyond the
scope of this book. The main purpose of this section is to discuss how
ozonetherapy may contribute to attenuate this devilish situation.
162 CHAPTER 14

There is a wealth of experimental and clinical studies suggesting that the

endothelium regulates vascular function through numerous autocrine-paracrine
interactions. Endothelial cells and smooth muscle cells are coupled to each other
and they sense the microenvironmental signals that are continuously at play,
generated by mechanical forces such as flow and pressure, by vasoaetive
honnones and by many inflammatory mediators including cytokines. The main
vasodilator and vasoconstrictor constituents are Iisted in Table 12.

Table 12. Factors exerting their activity on vascular tone.

Some ofthem are produced by endothelia! cells.

Relaxing Factors (EDRF) Con!l'acting Factors (EDCF)

Nitric oxide (NO) Endothelin I (ET -1)
Carbon monoxide (CO) Angiotensin II (Ag II)
Prostacyclin (P0I 2) Thromboxane A 2 (TxA 2)
Endothelium-derived Anion superoxide (0 2 . ' )
hyperpolarizing factor (EDHF)
Histamine Contraction factor I (EDCF -1)
Bradykinin Epinephrine
Adenosine Vasopressin
Acetylcholine Prostagiandin H 2 (PGH 2)
Serotonin Leukotriene B4 (L TB 4 )
Substance P Acidosis
Hydroxyeicosatetraenoic acid
(HETE)
Alkalosis

It is not surprising that the endothelium, representing a sensory interface

between the blood and the vessel, has a number of receptors able to pass on
infonnation leading to the synthesis and release of factors that continuously
modulate vascular tone. In pathological conditions, the acute or chronic disruption
of the physiological balance triggers the release of vasoconstrictor, procoagulant,
antifibrinolytic and chemo-attractant compounds, RaS and mitogenic hormones,
and progressively leads to platelet aggregation, leukocyte adhesion, coagulation,
thrombosis and infaretion. For space limitations, I can only eite a few authoritative
reviews on this topic (Ware and Heistad, 1993 ; Levin, 1995; Katusie, 1996 ;
Ferrara and Davis-Smyth, 1997; Shattil and Ginsberg, 1997 ; Thorin and Shreeve,
1998; Cines et al., 1998 ; Yancopoulos et al., 2000; Lum and Roebuck, 2001).
It is gratifying that at least Lum and Roebuck (2001) in their review on
"Oxidant stress and endothelial cell dysfunction" write that "at low levels, RaS
can function as signalling molecules participating as intermediates in regulation of
fundamental cell activities such as cell growth and cell adaptation responses, .. ."
and that " an increased amount of RaS constitutes a serious pathophysiologicaJ
factor for a wide variety of vascular-bed disorders", These comments support our
thesis that ajudicious and controlIed use of ozone can be useful and atoxie.
 WHAT HAPPENS DURING BLOOD OZONIZATION? ]63

Empirical observations (Chapter 24) have shown that 03-AHT markedly

improves the symptoms of chronic limb ischaemia in atherosclerotic and diabetic
patients, leading to necrosis of extremities. Conventional management, carried out
with infusion of prostanoids, vasodilators, antiaggregants and supporting physical
therapy, delays amputation. The reinfusion of autologous blood briefly exposed to
OZ-03' on its own, is more beneficial and frequently precludes amputation.
Unfortunately, the obstinacy and ostracism of orthodox angiologists have so far
prevented controlled and randomized clinical trials : the lack of means and suitable
clinics hinder us from moving in this direction.
As an alternative, we decided to investigate whether ozonized human plasma
elicits any effect on HUVECs in culture (Fig . 66) , which might help us to
understand the supposed therapeutic effect. lt is emphasized that we carried out the
study with fresh human ozonized serum (it would have been very impractical to
layer blood on HUVECs in culture) with medium and high 0 3 concentrations (40
and 80 ug/ml), simulating the timing and steps followed during 03-AHT.
Immediately after ozonization, the control , Oz-treated and Oz-Ortreated serum
sampIes were layered onto HUVECs for only 20 min, aperiod oftime corresponding
to blood reinfusion . Full details can be found in Valacchi and Bocci (2000) .

Figure 66. Human endothelial cells (from umbilical cord) in culture .

Figure 67 shows that ozonization of serum carried out in standard conditions

(plasma/gas volume, I :] ratio) induces a significant reduction of PTG levels while
TBARS and I-Iz0z levels increase. The new finding is that 40 or 80 ug/m! 0 3
164 CHAPTER 14

yields 19 ± 3.8 or 40 ± 2 I-lM of H 20 2, respectively, which reassuringly is within

the physiological range . Control sampIes do not show any change. Interestingly,
after being in contact with ozonized serum, HUVECs significantly increase NO·
production after 24 and 48 hours in an 0 3 dose-dependent fashion . We were
surprised that NO· levels remain unchanged during the first 6 hours of incubation,
as ifthe constitutive endothelial NO· synthase (eNOS) did not modify its activity.

4 _._- - - - -- - - - - - - - - - - ---.
_ 24 hours
CJ 48 hours
3

:E 2
::1.
B
.-z
'E

-
0
[=:J PTG 5 50
350 TBARS
****
300 HZ02 ij1' 4@ 40 :::r:
~lf
~~~ ~ '"0
:E 250 **
l'f 3C1:l '"
301=
1=
.&
.,
::1. 3::
200 3::
0
t j ** fit 2 20
150 ~!\ j i~~
100 .1"J 1"1
:,~
1f.4 10
rJi. ~.
50 in
'.~
~,

0 ~~ 0 0
Control 02 0 2-0 3 0 2-03
40 80

Figure 67. Effect 0/ different concentrations o[ozone on the production o[nitrite by

HUVECs, 24 and 48 hr alter addition otozonized human serum (top panel) . Effect 0/ either
oxygen 01' ozone on PTG , TBARS and H202 levels in the serum before addition to HUVECs.
The data are presented as the M :!:SD ol6 different experiments

Production of NO· is markedly enhanced by the addition of L-arginine (20 I-lM)

and is potentiated by 0 3 , while it is always inhibited in the presence of the NO·
inhibitor (L-NAME) or Nomega-nitro-L-arginine methyl ester (Fig . 68) .
 WHAT HAPPENS DURING BLOOD OZONIZATION? 165

4
_ Untreated
c:::J + L-Arg 20 ~M
+ L-NAM E 20 mM
..
.I ..
~
3 .... ~
.. cl-
:::E
::1.
2
I~
~
'5
Z

o '--
0 2-03 0 2-0 3
Control 02
40 80

Figure 68. Production ofnitrite by HUVECs. measured after 24 hr incubation. after

addition ofnormal human serum either oxygenated 01' ozonized (at 40 and 80 pg/m/).
Effects ofaddition ofl-arginine and L-NAME. The data are presented as the M :t SD of6
different experiments

Having observed that 0 3 induces H20 2 production (Pryor, 1994; Bocci et al.,
1998c and Fig. 67), we checked if H202 , on its own, is able to influence NO·
release.

Figure 69. Production 01 NO by HUVECs , measured after 24 hr incubation, after

addition ofthree concentrations ofH 20 ]. Effects ofthe addition ofLrarginine and L-NAME.
The data are presented as the M :t SD qf 6 different experiments
166 CHAPTER 14

Figure 69 shows that there is an almost linear relationship between NO· releas e
and the H 20 2 dose (20 , 40 and 100 I-lM) added to HUVECs. To be sure , addition of
L-arginine reinforces NO· production while L-NAME inhibits it.
2h

I
.5
"0
oS
o
]

:LI ~.~ ~ _I_I _.1

...
Untreated
_ 0 .0 110 o·.cJ so 0 -40 10 Ozcoe J,l;.'ml 0 40 100 40 11(1 0 40 10
Arginine
I.-NAME
" ·NAME

-.- I ;
2h

.mt
_ . _' -:""I . ~ .•!or._ .......... _ ..r... Ie<"',

4h

6h

Unneated - - - -
Ozone Jlglml - 0 .&0 .0 0 010 SO 0 -4 0 Rn
Ar&ininc:
l-NAME

Figure 70. Kinetics of release ofendothelin-I , E-selectin and lL-8/i'0111 HUVECI' after
addition ofhuman serum, after either oxygenation or ozonization. Effect ofthe addition o{
l-arginine and L-NAME. The data are presented as the M :f: SD of 6 different expe riments
 WHAT HAPPENS DURING BLOOD OZONIZATION? 167

As discussed earlier (this Chapter, Leukocytes and the Immune System), H 2 0 2

is now considered a crucial trigger of NF-KB. Thus it was obvious to examine
whether the stimulation of OJ-H2 0 2 on HUVECs induces the release of endothelial
proteins such as endothelin I (ET-I), the adhesion molecule E-selectin and the
chemokine IL-8 after 2 ,4, 6 and 24 hours incubation. Ozone at both
concentrations (40 and 80 ug/ml) significantly enhances the release of IL-8 after 4,
6 and 24 hours (Fig. 70), confirming the ability of ozone to induce IL-8 production
in blood (Bocci et al., 1998b) and respiratory epithelial cells (Jaspers et al., 1997).
Release of E-selectin or ET-I are not shown here because they were either
depressed or practically unmodified, suggesting that endothelium did not
upregulate their synthesis in our experimental conditions.
The finding that ozonized blood can enhance NO· production is promising,
particularly if it occurs in atherosclerotic vessels where it could counteract the
excessive local release of O,", which acts as a potent vasoconstrictor (Table 12). A
few basic concepts regarding NO· have been described in Chapter 9.

In physiological conditions, the endothelium produces minute amounts of 1-10

/!M NO· and 1 nM O 2. - (Wink et al., 1996). Moreover, as a result ofconsumption
by erythrocytes, in which the iron (Il) haeme of Hb acts an avid scavenger of NO·,
the intravascular half-life of NO· is approximately 2 msec while the extravascular
half-life has been estimated to range from 0.09 to > 2 sec (Thomas et al., 2001).
On this basis , the release and consumption of NO· appear to be so localized that
any enhanced release of NO· by ozone will be practically meaningless. In fact,
reinfusion of ozonized blood may yield a maximal effect on the vessels between
the venous access and the vast pulmonary bed . Therefore, how can NO· improve
the circulation and cellular oxygenation in remote ischaemic sites such as the
limbs or the retina? Only very diluted ozonized blood reaches these areas ;
certainly H 20 2 has been reduced long before and only traces of LOPs may still be
able to activate the endothelium. However, thanks to recent findings, more
convincing explanations can be found: the first regards the formation of far more
stable products than NO·, namely S-nitrosothiols (RSNO) occurring naturally after
the combination ofNO· with GSH (GS-NO) or cysteine (Cys-NO) or albumin (S-
nitrosoalbumin) (Butler et al., 1995; Kashiba et al., 1999; Gaston, 1999; Hogg,
2000 ; Al-Sadoni and Ferro, 2000).
SNO
SNO
HOOCyJ~~~'-/COOH Hl'+ ~CO' 2
NH2 0

GSNO Cys-NO
These NO-donor drugs act as slow-release compounds with half-lives of 5-50
min. and may allow prolonged vasodilatation even at distant sites. In our
experiments, we have also detected a small amount ofRSNO.
168 CHAPTER 14

The second explanation, provided by Stamler's group, has even farther

reaching implications and has been reported in two landmark papers (Jia et al.,
1996; Pawlosky et al., 2001) : after entering erythrocytes, NO· returns to the vessel
wall as an active RSNO molecule. A good deal of NO· is first salvaged by cysteine
residue ß93 forming S-nitroso haemoglobin and then transferred to the abundant
anion-exchange protein (AE I) present in the erythrocytic membrane. This system
appears not only to relax and increase blood flow in vessels of ischaemic tissues
but also to provide more oxygen where it is really needed (Kosaka, 1999) . This
advantage is lost when, during haemolysis or infusion of Hb-based blood
substitutes, haeme proteins are cell-free and scavenge NO· (Patel, 2000).
Vasoregulation is a complex process, dynamically regulated by several factors :
besides the heterogeneity of endothelial cells (Thorin and Shreeve, 1998) among
and within tissues (continuous, discontinuous and fenestrated capillaries), blood
pressure and blood flow are continuously controlled by the chemical compounds
listed in Table 12. Table 13 summarizes the activity of the most important one s:
NO·, prostacyclin and endothel in-l.

Table J3. Multiplefunctions o(the main endothelium-derived relaxing

and contractingfactors.

Nitrogen monoxide Prostacyclin Endothelin-l

(NO) (PGld (Er-I)
Vascular tone Relaxation Relaxation Contraction
Vascular Increase No effect Increase
permeability
Activity on Inhibits platelet, Inhibits or retards Enhances
platelets and leukocyte adhesion platelet activation, leukocytic
leukocytes and aggregation adhesion and adhesion and
aggregation aggregation
Angiogenesis and Inhibits the Cytostatic Stimulates the
vasal remodelling proliferation of proliferation of
vascular muscle vascular smooth
cells muscle cells

Several investigations have shown that, by relaxing vascular smooth muscle

cells, NO· participates in the control of peripheral vascular tone and contributes to
blood pressure control: inhibition of NO synthase in vivo by analogues of L-
arginine results in a striking increase of arterial blood pressure (Rees et al., 1989),
and mice lacking the eNOS gene are hypertensive (Huang et al., 1995) . On the
other hand, transgenic mice overexpressing the eNOS gene are hypotensive
(Ohashi et al., 1998). The endotheliurn-derived hyperpolarizing factor (EDHF)
cooperates with NO· and , by activating ATP-sensitive K + channels and /or smooth
muscle Na +-K+ ATPase, promotes vascular relaxation (Garland et al., 1995) .
Interestingly, Matoba et al. (2000) have indicated that EDHF may correspond to
HzOz, at least in small mesenteric arteries ofthe mouse.
 WHAT HAPPENS DURING BLOOD OZONIZAnON? 169

PGh is a rapidly synthesized eicosanoid which, acting in a paracrine fashion,

relaxes smooth muscle cells and causes vasodilatation. lt powerfully antagonizes
PAF activities. The action of PGh involves activation of adenylate cyclase and
formation ofcyclic AMP .
ET-I is a peptide of 21 amino acids, synthesized within minutes by endothelial
cells and released mainly toward the smooth-muscle side where it acts as the most
potent vasoconstrictor so far identified. lt is 100 times more potent than
norepinephrine, which potentiates the activity of ET-I . The plasma half-life is 4-7
min. and it is cleared by the lungs during the first passage (for details see Levin,
1995). In our study, ozone slightly increased the synthesis ofET-I, which was not
inhibited by addition of L-NAME. In pathological conditions, auto-regulatory
mechanisms are impaired and many other factors (Table 12) perturb the vascular
tone and favour inflarnmatory and fibroproliferative responses. Some of these
conditions will be considered in Chapter 24 .
An important aspect that, for trivial technical reasons, we could not evaluate
was if ozone, and then enhanced release of Nt)", was accompanied by a change of
VEGF expression. This topic needs to be discussed for three reasons: firstly, in
addition to the classical VEGF (Ferrara and Davis-Smyth, 1997), there has been a
recent explosion of newly discovered vascular growth factors or angiopoietins :
five members ofthe VEGF family , four members ofthe angiopoietin family and at
least one member of the ephrin family (reviewed by Yancopoulos et al., 2000).
These findings necessitate are-evaluation of therapeutic efforts, at least in
ischaemic vasculopathies, because delivery of a single agent may weil be either
ineffective or allow the formation of abnormal and leaky vessels.
Secondly, preliminary studies (Isner et al., 1996; Laitinen et al., 1998) with
adenovirus-mediated gene transfer of VEGF to the limb artery of patients with
ischaemic limb have generated unrealistic expectations; in fact, the process of
vascular formation is not as simple as was naively thought. Although we may be
on the verge of a revolutionary treatment beneficial to patients with ischaemic
diseases, there are many problems to overcome before vascular gene transfer
becomes an effective and practical reality (Laharn et al., 200 I) . The death of a few
patients has recently highlighted serious adverse events and , last but not least, we
must remember that the placebo effect in patients with this type of end-stage
disease is particularly significant. This gives me the opportunity to remind the
reader that this very problem occurs in ozonetherapy, where it has been totally
disregarded.
Thirdly, endothelial cells express and secrete VEGF, particularly during
hypoxia (Namiki et al., 1995). Chua et al. (1998) have demonstrated that H20 2 acts
on rat heart endothelial cells to induce VEGF mRNA in a dose- and time-
dependent manner. In their study, effective concentrations of H20 2 were 0.5 and I
mM, which are 12.5-25 times higher than the one measured in our experiments (40
~M), after addition of serum ozonized with 80 ug/ml 0 3 • Lactic dehydrogenase
(LDH) released from endothelial cells was less than 2% of the total , indicating no
cytotoxic effects. Thus , it would be interesting to ascertain if HUVECs synthesize
and release VEGF as weil as other angiopoietins after abrief contact with
170 CHAPTER 14

ozonized serum . It is also possible that the release of VEGF may be accentuated
during hypoxia, as occurs in ischaemic tissues . If this was true, ozonetherapy
would provide another useful factor, although it may not be as effective as
localized gene transfer owing to dilution .
A final remark concems the phenomenon of adaptation to chronie oxidative
stress (Chapter 22) and to the induction of stress proteins, particularly haeme
oxygenase (HO 1 or HSP32). This enzyme allows the formation of an antioxidant
(bilirubin) and CO (Verma et al., 1993; Otterbein et al., 1999; Snyder and
Baranano, 2001), a vasodilator that, like NO·, increases the level of cGMP via the
reaction catalyzed by guanylate cyelase.
It is truly remarkable that two gaseous moleeules thought to be toxic until
1987 have now become crucial physiological and pharmacological molecules.
I cannot envisage any mysterious chemical pathway able to synthesize labile
0 3, but I remain with the idea that ozone , when properly used, can reactivate a
series of biochemical processes gone astray . Unfortunately, as discussed in the
previous ehapter, besides the role of H20 2 , we have only a vague idea when,
where and how the array of LOPs genera ted during ozonization aet after blood
reinfusion. The ozonized plasma , disdainfully designated a "hodgepodge of
ozonized products" by an authoritative scientist, may offer the elue to solve the
puzzle of ozonetherapy.

Now I would ask the reader to meditate for a few minutes .

This chapter dealing with blood cells may appear boring and useless but I have
purposefully dwelt on describing some biological processes and the complex ,
almost infinite interaetions among erythrocytes, leukocytes, platelets , endothelial
cells and plasma components. In physiological conditions, these dynamie
relationships appear reasonably equilibrated but, if a pathological event intervenes,
many other actors , such as ROS, cytokines, adhesion factors and activated
enzymes, enter the scene and often lead to a chaotic situation. Orthodox medicine
strives to understand what is going wrong and is often able to restore normal
function . Yet it does not always succeed because if the therapeutic act is based on
a reduetionist approach, it may be unable to correet the complexity of some
diseases . It was and continues to be naive to expeet to eure ischaemie diseases by
the single gene transfer of VEGF or to eure cancer simply by reinfusing cells
eloned with the IL-2 gene. It will be equally naive, and even dishonest , to promise
to eure these disease with ozonetherapy. However, this approach should not be
neglected beeause it is simple , inexpensive, minimally invasive, without side
effeets and, by simultaneously activating several mechanisms in different cells,
may lead to an integrated and beneficial response.
 CHAPTER 15

ARE BLOOD LIPOPROTEINS OXIDIZED

AFTER OZONIZATION?

Chronic oxidative stress causes or accompanies many cardiovascular pathologies.

The concept that oxidized low-density lipoproteins (OxLOLs) recognized by
macrophage scavenger receptors (SRA, C036 and SR-PSOX) are internalized, thus
leading to lipid deposition and foam cell formation, is widely accepted as an early
step in atherosclerotic lesions (Steinberg, 1997; Shimaoka et al., 2000). It has been
clearly demonstrated (Graham, 1998 ; Bruckdorfer, 1998) that native (unoxidized)
LOLs do not cause formation of foam cells in macrophages in vitro and it is very
unlikely that LOLs are oxidized in the circulation, owing to the high capacity of
plasma antioxidants.
A reasonable sequence of events has been delineated by several authors (Fuster et.
al., 1992; Ross , 1993; Witztum, 1994; Berliner and Heinecke, 1996; Heinecke, 1999):

I ) the entry of a circulating monocyte into the intima, probably due to endothelial
ageing or a subtle inflammation.
2) inside the intima, native LOLs undergo oxidation by various agents (ROS, ONOO',
transition metals, lipoxygenase, myeloperoxidase) and are scavenged by the
monocyte which is activated to a macrophage, thus triggering a vicious circle.
3) The macrophage releases growth factors , which enhance the proliferation of
smooth muscle cells , and proinflammatory cytokines and chemokines, which in
turn upregulate the expression of adhesion receptors on endothelial cells.
4) Atherogenesis occurs progressively with macrophages transformed into foam
cells, endothelial thickening, platelet and leukocyte adhesion, plaque formation
with possible detachment and thrombus formation. As is weil known, some
factors that delay atherosclerosis and cardiovascular pathology are : NO·, low
LOL content «100 mg/dl ), high HOL content ( ~6 5 mg/dl), 10w plasmatic
cholesterol «190 mg/dl) and triglycerides «170 mg /dl) levels, normal glycemic
levels «100 mg/dl), high levels ofantioxidants (particularly vitamin E), etc .

Ozonization of plasma implies lipid peroxidation and possibly oxidation of

LOLs. Thus we must ask: Is there any connection between ozonetherapy and
atherogenesis? Can ozonetherapy worsen the situation?
Surprisingly, the answer to this question is that it may have a beneficial effect.
Common sense would suggest that ozonetherapy is a foolish approach, but ozone
seems to display unexpected effects. Cross et al. (1992b) investigated the oxidative
act ion of ozone on plasma proteins but did not detect any electrophoretic

171
172 CHAPTER 15

modification of lipoproteins. Albumin undergoes heavy oxidation and is considered

a "sacrificial" molecule, possibly preventing lipoprotein damage . This might explain
why the apoproteins exposed at the surface of the LDL particle evade oxidation.
However, these results are not altogether reassuring because methionine 358 present
in proteinase <X-I inhibitor is oxidized with inactivation (Evans and Pryor, 1994) and
it is difficult to envisage how either PUFAs or -SH groups present in lipoproteins
would not be oxidized, unless particularly inaccessible. On this basis, I believe that
the problem should be carefully re-examined using ozone concentrations up to 80
ug /rnl per 011 ofplasma .
My point of view is not in line with clinical results reported for vasculopathic
patients after 0rAHT. Hemandez et al. (1995) c1aimed that repeated treatments of
OrAHT lead to a lowering of plasma levels of LDLs and cholesterol, suggesting
that ozonetherapy has anti-atherosclerotic and hypolipemic effects. Mattassi et al.
(1987) and Bisetti et al. (1988) even noted an increased HDL level. In our studies,
the first with numerous elderly patients with ARMD (about 13 0 3-AHT treatments)
and the second with a dozen vasculopathic patients treated with 0 3 during
extravascular blood circulation, we observed neither a decrease of LDL and
cholesterol nor an increase of HDL. It is hoped that our trial in progress will c1arify
this controversy.
Moreover, it may be relevant to examine whether LDL oxidation in blood ex
vivo depends on the ozone concentration, i.e. it may be negligible at low-medium
levels (20-40 ug /ml per 011 of blood) and significant at medium-high levels (40-80
ug/ml) . If this is so, what is the fate of OxLDLs? It can be speculated that , during
reinfusion, these LDLs are recognized by the RES , rapidly taken up and degraded
without entering the endothelial intima. If this catabolic pathway is effective, it may
be beneficial, although there are better ways to reduce the levels of lipoproteins and
macroglobulins in plasma. By differentially labelling native LDLs and ozonized
LDLs, it will be possible to test the hypothesis in the rabbit and ascertain their half-
Iives and catabolic sites.
 CHAPTER 16

HOW IS OZONE ADMINISTERED ?

During the last five decades, several methods have been devised to perform
ozonetherapy. Surprisingly, except for the inhalation route (prevented by tracheo-
bronchial-pulmonary toxicity), many parenteral and topical routes have been used to
administer ozone without side effects or minimal discomfort (Table 14).

Table J4. Routes ot ozone administration.

Parenteral Topicalor Locoregional

Intravenous (IV) Nasal]
Intra-arterial (lA)* Tubalt
Intramuscular (IM) Auricular
Subcutaneous (SC) Oralj
Intraperitoneal (Ipe) Vaginal
Intrapleural (IPL) Urethral and intrabladder
Intra-articular (Iat)
a) Periarticular Rectal
b) Myofascial.
Intradisc (ID) Cutaneous
Intraforaminal (IF)
Intralesional (ILes)**

* /t is no longer usedfor limb ischaemia. Hepatic metastasis

could be embolized via the hepatic artery.
**/ntratumouralor via an intra-abscess fistula
t To be performed during 30-60 sec apnoea
Ozone is an unstable gas, present at most at 5% of gas mixture (0 2-03) , and must
be used extempore. The gas mixture, with a variable positive pressure (depending on
0 3 concentration), can be collected with a calibrated syringe (g1ass is ideal but
impractical and has been substituted with disposable, silicon-coated, polyethylene
syringes) or, if a continuous flow of gas is needed, by inserting a connection to the
exit valve . Rubber tubing cannot be used because ozone disintegrates the rubber;
however, silicone tubing is ideal.
Although ozone is a potent disinfectant, medical oxygen, pipe fittings, O-rings
and taps are not necessarily sterile. Once the gas has been collected, with rare
exceptions (rectal insufflation), it must be filtered before application. Since February

173
174 CHAPTER 16

1998, ozonetherapists should be eomplying with this regulation to avoid possible,

albeit very rare, infeetions. We are eurrently using an antibaeterial, ozone-resistant
hydrophobie filter (porosity 0.2 11 , with a female Luerloek at the inlet and a male
Luerloek at the outlet).

I . ADMINISTRATION VIA PARENTERAL ROUTES.

1.1. Can the Gas Mixture Be Directly Iniected Via the IV Route ?
No. Today It is Prohibited!

The idea to injeet the O 2-0 3 gas mixture direetly via the IV route was proposed by
Payr in 1935. Although extremely dangerous, it was used at first with good eommon
sense by very slow injeetion (in about 5 min) via the eubital vein of no more than 20
ml gas with ozone eoneentrations between 3 and 57 ug/ml (more frequently 20-33
ug /rnl) . The very slow injeetion favours the formation of a train of bubble gas,
where ozone (more soluble than oxygen) dissolves and reaets quiekly with blood
while oxygen (more than 95% of the gas mixture) reaches the right ventricle and
then the pulmonary artery . Oxygen solubility at 37 (Je is only about 0.23 ml per 100
ml of plasmatic water and therefore venous plasma cannot solubilize the excess
oxygen, leading inevitably to formation of agas embol us.
Unfortunately, it appears that some naturalist praetitioners and quacks without
any medical qualification perform this practice in Canada, Mexieo, Jamaica, Kenya,
etc . and actuaJly teach this technique in underdeveloped countries where there is no
medical control (see Oxylist in Intemet). On the basis of Sartori's protocol, they
proclaim excellent therapeutie achievements in HIV patients and it seems that
Sartori and Yuan have even published a book in eight chapters entitled: "Ozone. The
etemal purifier of the earth and c1eanser of all living beings". To the best of my
knowledge, it seems that up to 500 ml of gas with an ozone concentration of 70
ug /ml (about 2 ml/min for a total ozone dose of 35 mg) are directly injected IV, with
the idea that ozone, once dissolved in the plasma, inactivates HIV present in the
circulatory system, just as ozone is used to purify water flowing in an aqueduct. In
Sartori 's protocol, the gas infusion is intercalated with the infusion of "minerals"
and "vitarnins". It must be added that ozonetherapy was only one part of these eight
magical treatments, the other seven including a homeopathic AIDS treatment,
microcurrent therapy, vitamin-mineral and immunostimulating herbai treatments ,
mental reconditioning program, etc. Although patients reported acute thoracic pain,
cough, Iipothymia (possibly due to pulmonary and even cerebral embolization),
shivers and fever, the gas infusion continued relentlessly because these side effects
were considered positive reactions, showing that the body was "getting rid of toxins
and viruses" . In September 1992, it was c1aimed that more than 300 HIV patients,
after two weeks of treatment at a cost of about 8,000 US dollars, seroconverted to
HIV negative and were cured! These results were further supported by the
seroconversion noted in 30 horses affected by equine infective anaemia (EIA). Why
have these wonderful results not been submitted for objective scientific scrutiny? In
contrast, some years ago, two young American men came to ask my opinion and
 How IS OZONE AOMINISTEREO? 175

told me that the two-week treatment had been a nightrnare without any
improvement.
It is not surprising that the US Food and Drug. Administration and the Medieal
Establishment has beeome dead against the use of ozone. Health Authorities of all
eountries should devote great attention to bloeking this abuse and preventing
desperate patients from being enticed to undergo this sort of praetiee, exploited and
perhaps even killed . The number of deaths is uneertain but, in Italy, negligenee and
ineompetenee have resulted in at least one well doeumented death (1997) . At
autopsy , air was even found in the suprahepatie veins! However, it must be said that
sinee 1984, the praetice ofIV infusion of gas has been prohibited in a few European
eountries, even though the law is not always respeeted. This deeree followed the
exhaustive study of ozonetherapy by Jacobs (1982), which showed the surprising
lack of side effects but also clearly pointed out that four previous deaths were due to
lung embolization after IV gas injeetion. The practice of hyperbaric
autohaemotherapy, still performed in Germany and Austria, is also dangerous, as
shown by some deaths ; moreover, we have no data about the chemical effects of
ozone under pressure . Nitrogen (N 2) or air (78% N 2) must never be present during
ozone formation, because N0 2 compounds are highly toxie and nitrogen, per se, is
poorly soluble and far more likely to eause emboli.
Is there any rationale bebind direct IV gas injeetion? As mentioned, about 500
ml of gas mixture are injeeted in 4 hours (-2 ml per min) with a total ozone dose of
35 mg (70 ug /rnl X 500 ml = 35,000 ug) . Anormal 70 Kg human has about 5 L of
blood whieh, at rest, circulate entirely in one minute . This means that a total blood
volume of 1,000 L circulates in 4 hours . The plasma volume is about 3 L but it
continuously exchanges eomponents (and antioxidants) with 10-12 L of
extravascular liquid . This means that 35 mg ozone will dissolve and react with a
plasma volume that far exceeds the theoretical 3 litres .
Therefore, the final ozone concentration may range between 0.3 and less than 3.0
ug /rnl, which is equivalent to a placebo effect. This is so because of the enormous
capacity ofregenerating antioxidants (Chapter 12); thus the ozone concentration is
unable to reach a virucidal concentration in the plasma. Seeondly, the bulk of
infective viruses and proviruses is intracellular and, ironically, remains weil
protected by the intracellular antioxidant system . A slightly higher ozone
concentration may be achieved in the cubital-subclavian vein during gas infusion but
this possibility depends upon the variable venous blood flow . This is undefined and
if it has a very low rate , the gas may even cause haemolysis with unpredictable
consequences. Similar difficulties occur during the infusion of a solution of HZ02
(Chapter 34). The proponents of the IV method emphasize the therapeutic role of
oxygenation. However, considering the risk of embolization, this becomes
irrelevant. Needless to say, oxygen therapy can be performed efficaciously by
breathing hurnidified oxygen for a couple of hours at horne or under pressure in a
hyperbaric chamber for one hour according to a standard proeedure (Kindwall,
1993; Tibbles and Edelsberg, 1996).
A final consideration is that the awful side effects of direct IV gas injection are
not due to ozone but rather to oxygen embolization.
 176 CHAPTER 16

1.2. Direct Gas Administration Via the lntra-Arterial Route

This route was first used by Wolff (1974), who injected 10-20 ml of gas
(concentration of 0 3: about 30 ug /rnl) into the femoral artery of patients with
advanced Iimb ischaemia. At first, it causes a transient ischaemia, probably owing to
a local nervous reflex with some discomfort, followed after a few minutes by
vasodilatation and hyperaemia (release of NO?) . Because of the small gas volume
and the gas fragrnentation into the Iimb capillary bed, IA administration does not
involve a risk of embolization. Yet, according also to Mattassi (1985) , it has no
advantage over c1assical 0 3-AHT or even rectal insufflation of gas. Therefore, it is
no longer used , also because repeated arterial punctures should be avoided.
Regarding the current practice of therapeutic embolization (with a variety of
compounds) for hepatic metastasis, a possibility that may be considered is the slow
intra-arterial injection of 15-20 ml ofgas (concentration of Oj : about 30 ug /ml) , The
risk is practically absent because the gas will be dispersed into the sinusoidal and
tumour capillaries, possibly with direct ozone ,cytotoxicity on neoplastic cells . In
comparison to chemotherapeutic agents, ozone does not cause unpleasant side
effects. I would be glad to help anyone interested in testing this approach.

1.3. Other Parenteral Routes

Before the advent of 03-AHT, ozone was habitually injected by either IM or SC
routes for the treatment of chronic viral hepatitis and vasculopathies. Volumes of up
to 200 ml of gas were subdivided in different sites (03 concentrations of 10-15
ug /ml). Even with the utmost care, when a large volume of gas is injected, there is
always the risk of pulmonary embolization. The death of a young woman being
treated for a trivial lipodystrophy with SC injections of O2-0 3 occurred in ltaly in
March 1998 . Apparently, as much as 600 ml had been injected into various areas of
SC tissue of the lower part of the body .
IM and SC injections can produce acute pain (albeit lasting only a few minutes)
if the gas volume exceeds 7-8 rnl and the 0 3 concentration is near or above 20
ug/ml. In the last few years, it has become fashionable to inject ozone at trigger
points in the paravertebral muscles in patients with low-back pain . This sort of
"chemical acupuncture" has a therapeutic effect in about 70%, of patients (Chapter
24) . Most ozonetherapists also perform SC gas injection for Iipodystrophy or IV
injections for sclerotizing venous ectases. It is unfortunate that so much importance
has been given to ozone applications in cosmetic treatments. However, I was
reminded at a meeting that this activity proeures some 80% ofthe "bread and butter"
of ozonetherapists.
How oxygen and ozone are distributed in the tissue, how they act and what
biological mechanisms they elicit will be discussed in Chapters 24 and 27 .

J.4. Intraperitoneal and Intrapleural Administration

In peritonitis and pleural empyema, Russian physicians (probably lacking wide-
spectrum antibiotics) wash out purulent material with ozonized water: 100-300 ml of
 How IS OZONE ADMINISTERED? 177

gas (0 ) concentration from 50 down to 10 ug /ml depending on the gravity of the

infection) are insufflated into the cavities (Bulinin et al., 1995 ; Kasumjan et al.,
1995; Kudravcev et a1., 1995) . Ozone dissolves quickly and reacts with exudates
while oxygen is slowly reabsorbed. This treatment apparently does not damage the
peritoneum or the pleura. We performed experiments in normal rabbits and we
insufflated up to 300 ml of O 2-0 ) into the peritoneal cavity (0) concentration: 20
ug /rnl) without noting either animal discomfort or any damage to the peritoneal
lining at autopsy after 24 and 48 hours.
In view of the disappointing results with chemotherapeutics, peritoneal and
pleural carcinomatosis could be treated with daily insufflation of OrO). 200-500 ml
gas could easily be insufflated without risk of embolization because oxygen is
reabsorbed slowly. Treatment could start with an ozone concentration of 5 ug/ml,
which could be upgraded slowly to 30 ug/ml as the reactivity of the patient was
evaluated. Ozone is likely more cytotoxic to neoplastic cells than
chemotherapeutics, it does not allow resistance, it does not cause side effects typical
of chemotherapy and it costs almost nothing. The additional advantage of
oxygenation cannot be neglected. A permanent polyethylene cannula can easily be
inserted permanently in the cavities for daily administration. It is shameful that two
"farnous" Italian oncologists, specialized in this topic, have not even bothered to
respond to my proposa1. I am indignant at their disdainful behaviour, mostly because
I feel they are not being fair with the patients.
Another possibility, applicable to infected patients undergoing peritoneal dialysis
(p .d.), is to treat chronic viral hepatitis by insufflating O 2-0) intraperitoneally.
Obviously the preferred routes for treating hepatitis are : 0rAHT, EBOO and rectal
insufflation possibly associated with ingestion of ozonized water. Yet p.d. patients
have an intraperitoneal silicone catheter already implanted. With a suitable ozone
generator at horne , autotherapy could easily be performed between p.d. sessions and
perhaps the incidence of occasional peritonitis could be further reduced. Insufflated
vo lumes could be 200-300 ml starting with an 0 3 concentrations of 5 ug/ml and
slowly increasing it to 10 ug /ml (to allow for tolerance).

1.5. Intra-Articular , Intradisc, Intraforaminal Administrations

These topics will be discussed in Chapter 24 (section: Orthopaedic diseases) .

1.6. Intralesional Administration

The application of ozonized water and gas will also be evaluated in Chapter 24
(section: Infectious diseases and cancer) .
178 CHAPTER 16

2. BASIC CONCEPTS REGARDING TOPICAL APPLICATIONS

A Swiss dentist, Dr . E. A. Fisch, was the first to insufflate ozone into a tooth cavity
complicated by gangrenous pulpite. Subsequently, either ozone or ozonized water
were used to treat nasal, tubal and oral (gingival, mucosal and tonsillary) affections
by means of suitable metal or silicone catheters. 5-20 ml of gas (the initial ozone
concentration is about 20 ug /rnl and is scaled down as the infection recedes) can be
cautiously insufflated while the patient is in apnoea. The patient must be instructed
to take a deep breath and remain apnoeic for 30-60 seconds while the insufflation is
performed. He can then expire deeply to avoid breathing in ozone. The treatment can
be repeated a few times.
Ozone treatment of chronic vaginal (bacterial, fungal, protozoan) infections that
are resistant to conventional treatments are particularly successful and one can adapt
the most suitable seheme for each patient. After inserting about 10 cm of a
polyethylene catheter (lubricated with olive oil) , we can start to wash the vaginal
cavity with ozonized water if the secretion is abundant and purulent. Then, we can
insufflate 30-50 ml of gas (03 concentration no higher than 40 ug/ml) for a few
minutes, keeping the ostium closed, followed by the insertion of ozonized oil , either
directly or in the form of a vaginal suppository to be reapplied before going to bed at
night. It should be remembered that the ozone concentration must be progressively
scaled down to about 5 ug/rnl to avoid any damage to the regenerating epithelial
mucosa, normally protected by the physiological mucoprotein layer.
A similar strategy can be used to treat urethral and bladder infections. Siow and
careful insufflation of 50-100 ml gas (03 concentration between 5 and 15 ug /ml) is
occasionally painful and can be substituted with small volumes of ozonized water
every other day .
Cutaneous applications of either ozonized water or oil, as weil as gas , involve an
array of pathological situations, such as accidental and war trauma, bums and all
sorts of infections. These will be dealt with specifically in Chapter 24 but some
basic concepts can be outlined here . The gas mixture can be used whenever possible
but we must be careful to avoid contamination of the room, even if an efficient
exhaust fan is operative. A monitor sensing up to 0.1 ppm 0 3 , with an alarm system,
must be tumed on all the time.
The skin to be treated must be sealed hermetically with various devices, e.g. a
perspex bell or a teflon cup, with an inlet to deli ver the gas and an outlet connected
to the suction pump and the ozone destructor. With the rigid cup , a slight vacuum
(localised hypobaric pressure) can be achieved; according to Werkmeister (1995), it
favours local vasodilatation and may enhance healing. If adynamie exposure is not
feasible, particularly in the case of large wounds, the static system can be achieved
with a large polyethylene bag sealed with a wide adhesive tape (not too tight to
cause venous stasis).
All ofthese devices must contain sterile water. The internal environment must be
saturated with water vapour so that the ozone can dissolve in it and react with the
organic material of the wound . Usually 15-20 min exposure sufftces and the
remaining gas can be aspirated with the pump.
 How IS OZONE ADMINISTERED? 179

If the use of gas is risky, ozonized water can be freely used to clean the wound
and a wet compress can be applied for about 20 min in the moming and aftemoon,
followed by application of ozonized olive oil throughout the night.
Both ozone gas and ozonized water are excellent disinfectants (probably only
inferior to iodine, whieh is aetually too harsh) and eause a marked deerease of
bacterial, viral and fungal loads . Most infectious agents, either Gram negative
tSerratia marcescens, Escherichia coli, Pseudomonas aeruginosa, Klebsiella
pneumoniae), Gram positive (Staphylococcus aureus, Staphylococcus epidermidis,
Streptococcus pyogenes, Enterococcus faecalisi or myeetes (Candida albicans) do
not resist ozone during a 20 min exposure. In a careful study (Polignano et al.,
2000) , ozonized water and iodine were found to be equally effective, far more than
hydrogen peroxide and K permanganate. Only Streptococcus pyogenes needed a
contact period longer than 5 min before irreversible inhibition. The treatment is weil
tolerated, painless, does not have noxious effeets and the healing time is far shorter
than with any eonventional treatment. The latter point is important and is likely due
to a number of concomitant factors, such as vasodilatation, enhanced oxygenation,
normalisation of tissue pH, reabsorption of oedema. These proeesses of metabolie
aetivation are diffieult to docurnent scientifieally and step by step, but beeome
evident and very gratifying when one can follow the positive day-by-day progress of
the ulcers. Obviously, the primary point is the disinfeetant action, whieh initially
requires a rather high ozone eoneentration and then a progressive deerease as the
tissue begins to regenerate. This aspeet has been sehematieally represented by a
diagram (Martin, 1997), where the three stages of wound healing are also reported
(Figure 71). Number I indieates the inflammation stage, whieh is more or less
rapidly reduced (depending on the gravity and ehronieity of the baeterial infeetion)
after ozone application. Number II indicates the intermediate stage, when tissue
granulation oeeurs. The final phase III includes sear tissue remodelling and may take
a long time, partieularly in e1derly and/or diabetie patients.
It must be emphasized that successful and fairly rapid healing of a necrotic
ulcer in arteriopathic patients (probably the worst of all wounds) can be
achieved only by combining the parenteral treatment (OJ-AHT, EROO) with
the appropriate and necessarily tedious local application of ozonized water and
oil. Onee the patient notiees the improvement, eomplianee beeomes perfect.
Personal experienee has weil taught me about the validity of the eombination of
these therapies, whieh seem to act synergistieally. I had the fortune to follow several
patients at our hospital, and the results were so striking that today I feel it would be
unjustified and unethieal to perform a trial eomparing a) the exclusive loeal
treatment, b) the exclusive parenteral treatment, e) both treatments and d) both
treatments using only oxygen.
Table 15 surnmarizes the known and still hypothetical biological effeets of the
eombined treatment.
180 CHAPTER 16

Tahle J5. A summar y ofbiological effects induced in blood and tissue compo nents
by combining O;-AHT with the local application ofozone on necroti c ulcers
in patients with limh ischa emia.

Or AH T

Plasma Erythrocytes Platelets

Fibrinogenemia ~ Glycolysis t PDGFt
Cholesterol ~ ATPt TGF-ß I t
C-reactive protein ? 2-3-DPG j.; Fibronectin t
Oj-availabiliry t
Erythrosedimentation ..j,
Arterial p02 t
Venous p02..j,

Leuko cytes Endothelial Cells

PGE 2 t NO·t
Cytokines t VEGF (?)r

TOPICAL APPLICATION OF 0 3

Fibrobla sts and Keratinocytes

Hyaluronic acid t
Sulphate dermatan t
Fibronectin t
Collagen IIIII t
KGFt
BFGFt
EGFt
t = increase ; ..j, = decrease ; ~ = stable ; ? = unknown
 How IS OZONE ADMINISTERED? 181

I do hope that some angiologists, most likely unaware of the advantages of the
ozonetherapeutic treatment, will abandon the laborious and hardly useful orthodox
treatment and try ozonetherapy. For the sake of the patients, let us hope that they
cease to be sceptical and have the courage to try ozone.

80 ·

~
2: 80
c
.9
T§
s
0
c
40

8 20
Ö

lIJ
Ql
lIJ
ro 11 111
.c
e,

6 12 15 18 21 24 27 30

Days

Figure 71. The three phases ofwound healing. In thefirst (I) phase. inflammation prevails,
with the presence 0/ neutrophils, macrophages. mastocytes, platelets. bacteria and toxins .
Application ot ozone inhibits the infection and promotes the second (Il) phase, lasting about
nm weeks. During this phase, the constant application ot ozone at progressively lower
concentrations not only prevents a sup erinfection but stimulates cell proliferation, the
sy nthes is offibronectin. collagen !lI/I. hyaluronic acid and chondroitin sulphate.
Macrophages are still present but there is an active proliferationoffibroblasts and
keratinocytes. The restitutio ad integrum, i.e. comp lete reconstruction ofthe wound, occurs
during the last (IlI) phase. However, excessive release 0/ TGFßl may stimulate excessive
fibrogenesis with cheloidformation. The above diagram shows the approximate ozone
concentrations that must be progressively lowered to avoid inhibition ofhealing.
 182 CHAPTER 16

3. MAJOR AND MINOR 0 3 AUTOHAEMOTHERAPY (AHT)

These terms indieate a proeedure by whieh a volume of blood is drawn, exposed to

O 2-0 3 for a few minutes and reinfused either IV (major AHT) or IM (minor AHT )
into the donor. "Major" and "rninor" are only meant to indieate a different volume
of blood: 50-250 ml for the former and 5-10 ml for the latter. The original idea to
expose blood ex vivo to agas mixture was proposed by Wehrli and Steinbarth
(1954), who published the method of irradiating blood with UV light in the presenee
of pure oxygen. This proeedure, ealled HOT (Hamatogene oxidations therapiei, is
no longer used because it was uneertain with regard to the real and effeetive
eoncentration of ozone during irradiation of oxygen and was eumbersome and risky
beeause the quartz ampulla had to be c1eaned and sterilized after eaeh treatment.
Indeed, in a few eases, there was cross-infection with HCV due to imperfeet
sterilization (Gabriel et al., 1996) . In the 1960s, reliable medieal generators beeame
available and Hans Wolffproposed that blood be exposed direetly to ozone, with the
advantage of knowing its exaet eoneentration. In 1974, he reported that he had used
this method many times without any problems.
Unfortunately, modifieations were subsequently introdueed that possibly
worsened the proeedure; for example, the use of only one tube to eolleet and
reinfuse blood (involving the risk of embolization) and even worse, sinee 1991 in
Italy, the substitution of neutral glass bottles with plastie bags beeause they are
ehe aper and easier to dispose of. These bags are made of 50-55 %. polyvinyl chloride
(PVC) mixed with a number of additives (among whieh about 43 % of phthalates)
for the remaining 45-50% (Whysner et al., 1996) . The latter eompounds make the
PVC elastie and flexible but traees of phthalates are released into blood,
representing minimal and permissible eontamination. They are eommonly used
world-wide for storage of blood to be reinfused into the donor during or after a
surgical operation. I must eonfess that sinee 1993, we have used them (uneritieally)
to earry out experimental work on myself and eollaborators and sinee July 1995 in
ARMD patients undergoing a c1inieal trial at our Uni versity. By 1997, I beeame
aware that patients undergoing dialysis displayed high levels of phthalates in their
plasma due to large volumes of dialysing fluids stored in plastie containers. This
problem has been brought to medieal attention sinee 1970 (Jager and Rubin 1970a,b ;
Valeri et al., 1973; Lewis et al., 1977; Lawrenee 1978; Thomas et al., 1978;
Callahan et al., 1982; Di Vineenzo et al., 1985; Labow et al., 1986), but so far the
eeonomieal and praetieal advantage of plastie over glass has left the problem in
limba, even though dialysis " patients suffer from many ailments and whether
phthalates might worsen them remains uneertain.
My first enquiry with the Italian Ministry of HeaIth established very c1early that
plastie bags are authorised only for the temporary storage of blood or other fluids,
and that ozone must never be added. To make a long story short, we examined the
effeet of O 2-0 3 in routinely used plastie bags eontaining sterile saline, aeeording to
the European pharmaeopoeia and using the same volume ratio and O, eoncentrations
up to 70 ug/rnl per ml saline.
As reported in Tables 28-30 ofthe appendix, the results were worrisome :
 How 15 OZONE AOMINISTEREO? 183

a) not only did phthalates inerease several fold but mieroparticles (2,5, 10,20 and
25 /1) ofplastie material eould be measured in suspension . Their value inereased
from 3.3 to 10.7 times the minimum aeeepted level.
b) Table 30 also shows that the proliferation index ofhuman BMC ineubated with a
eomparable dilution (after reinfusion) of ozonized saline was inhibited up to
27.2%, resembling the immunosuppression typieal of dialysis patients .
e) during the elinieal trial in ARMD, a few patients have presented allergie
manifestations (Chapter 21) possibly due to the plastie material or phthalates
aeting as a hapten.

In May 1999, we sent the results of our investigation to the Department of

Toxicology of the Italian Ministry of Health, asking for their verifieation and for a
deeision as to whether plastic bags eould be used for ozonized autotransfusion . After
a few months, we reeeived a laeonie reply that there was no need of further
investigation because plastic bags should be used only to contain blood without
any further addition, let alone that of ozone . It took another few months but, on
Deeember 17, 1999, the Italian Ministry of Health sent a memorandum to all
regional Health authorities c1early stating that autotransfusion bags ean be used only
to store blood and not for Oj-autohaemotherapy. In the meantime, we updated and
optimized the old method with absolutely ozone-resistant and atoxie materials,
including a baeterial filter and suitable tubing with filter to eorreetly reinfuse blood
without any risk of clotting or air embolization (see Appendix). At this point, I
believed that the ordeal had eome to an end. However, I was wrong beeause even
after two years some ozonetherapists unlawfully eontinue to use plastie bags
beeause, being less eostly, they inerease their profit. Moreover, they calumniate me
by c1aiming that phthalates are not dangerous and that I have invented the whole
story to ruin their business. At several meetings, I have invited ozonetherapists to
make their own test and see with their own eyes how opaleseent the saline becomes
after being ozonized in the bag (due to released and suspended plastie particles).
Someone has gone as far as saying that I have added ozone to the bag before the
solution to purposefully damage the plastic or that, in any case, there is no need to
worry beeause blood can protect the plastic! Fortunately, in civilized eountries
(Germany, Austria, UK, USA, Cuba, ete.), all deeent ozonetherapists use glass bottles.
Obviously I remain very concemed about the patients who unknowingly are
transfused with blood that is heavily eontaminated , not so mueh with phthalates but
with mieroplastic particles that are eertainly taken up by the RES in the spleen, liver
and bone marrow and that, with time, may produee a caneerogenic stimulus. When I
pointed out this risk at the Verona Congress (March 1999), a very eynical comment
from an ozonetherapist was "to wait and see" . The whole story has distressed me
tremendously because I have realized my inability to straighten out the emblematic
method of ozonetherapy . One serious problem that has prevented real progress has
been the lack of a standard method; the one deseribed in the Appendix was an
attempt to obtain a general consensus. Obviously the method must be simple and
work perfectly, must be atoxic, must be flexible in the sense that one can use a blood
volume from 100 to 225 ml (depending on the patient), must accommodate a
184 CHAPTER 16

corresponding gas volume that has to react slowly with blood (for at least 5 min) and
must be absolutely safe .
How can we compare anecdotal results (already questionable) if everyone
disagrees about the blood and gas volume, ozone concentration (and its reliability),
exposure time , etc.? What is most disheartening about this chaos is that behind it
there are comrnercial interests (plastic or glass, small or large glass bottles, very
cheap system, etc .), mental reservations, lack of basic knowledge and plain
stupidity. One ltalian ozonetherapist has boasted of performing the whole OrAHT
procedure in 6 min when the correct time is 30-40 min !
Besides these depressing remarks, two modifications need to be c1arified: the
first regards the technique of exposing blood to O 2-0 3 . One system proposed to
circulate blood (collected in the usual glass bottle) by means of a peristaltic pump
through hollow capillary fibres against O2-0 3 to ensure ozonization. Needless to say ,
this system was expensive, unnecessarily complex and without any advantage. lt
was a blunder resulting in a comrnercial faHure. Another system allows the delivery
of gas in "micro-bubbles" and claims that full blood ozonization is achieved in a few
seconds. We tested it and we repeatedly found considerable blood foaming , with
greater haemolysis than normal and with a finallow p02 (about 90 mmHg).
By comparison, Wolffs classical method (Appendix) is simple, inexpensive and
very effective provided the gas is insufflated correctly into the glass bottle kept in
the horizontal position with slow manual blood mixing. Kinetic measurements of
both oxygenation and ozonization reach the plateau (p02 - 500 mmHg) only after 5
min of gentle mixing. Foaming and haemolysis are minimized.
The second issue that remains to be settled scientifically is the volume of blood
to be treated in each session. Obviously the volume of blood should not be imposed
by any comrnercial bottle or bag preparation. It must be flexible and the volume of
blood withdrawn must be in approximate relation to the patient's body weight, sex
and blood volume. To avoid any risk of lipothymia, no more than 250 ml blood
should be withdrawn. Thus a 500 ml glass bottle appears to be suitable. The problem
is that some ozonetherapists, particularly in Germany, believe that 50 ml, or at most
100 ml, are optimal. There is absolutely no experimental support for this contention
but the fanciful idea is that just "a little volume of ozonized blood ean trigger the
biological effects with the speed of fire driven by the wind through the dry
undershrubs" . This reasoning is bewildering and disagrees with the ordinary
biochemical and pharmacological concepts expressed in Chapters 9, 13 and 14. If
we aeeept the idea that ozone generates erucial messengers (0 202, LOPs,
metabelle intermediates, autaeoids, etc.) that are subjeet to dilution, eatabolism
and exeretion but ean express pharmaeologieal efTeets, we have to eonsider that
minimal stimulation may eorrespond only to a placebo effect (Chapter 25) . Our
contention is supported by the finding that, in a few terminal cases (stage IV,
Fontaine) of hind limb ischaemia, a dramatic improvement was observed
immediately after the first treatment performed with ESOO (ozonization of about
5 L blood in one hour). The problem of blood volume, appropriate 0 3
concentrations, the schedule and the induction of ozone tolerance will be discussed
from different angles in the next few chapters. Our standard approach has been to
 How IS OZONE AOMINISTEREO? 185

perform 2 (or at most 3) treatments weekly, using 225 ml ofblood each time , for 13-
15 sessions. This schedule is practical and appears effective in responsive patients.
Has classical 03-AHT any other disadvantage? The limitation ofblood volume can
be laboriously overcome by performing up to three classical 03-AHT in a row (about
750 ml blood ozonized and reinfused in two hours), as I have tested on myself.
Unless the ozonetherapist owns a reliable portable generator, dorniciliary treatment
cannot be perfonned. Nevertheless, superficiality and malpractice are endless: one
famous German ozonetherapist boasted of perforrning several 03-AHT every morning
by first loading small glass botdes at his clinic and then going around town to the
patients' hornes for the treatments. When I mentioned the fact that, firstly, the tap is
somewhat sensitive to ozone and, secondly, that the ozone concentration decreases
rapidly and a precise time cannot be calculated, he looked at me with commiseration
and replied that I was giving too much importance to scientific details and that it was
better to do a treatment (placebo I would say!) than nothing.
A correct reinfusion of 250 ml (225 ml blood + 25 ml Na citrate 3.8%) takes
about 20 min and then we must carefully check the haemostasis and avoid haematic
extravasation which may compromise the continuation of the therapy. Great care
must be exercised to maintain the venous access in the best condition, particularly in
women . Risk of infections (HIV and HCV) among patients and ozonetherapists must
be prevented; we fully agree with Daschner (1998) and Webster et al. (2000) that
some mistakes, e.g. repeatedly using a contarninated syringe, are inadrnissible.
Finally, if several AHTs are performed simultaneously, all glass bottles must
have the patient's name to prevent mistakes during reinfusion, with possible
dramatic consequences.
Fairly often, I have been asked: in AIDS patients or in cachectic, immunosuppressed
and anaemic patients, can we perform an ozonized allogeneic blood transfusion?
Obviously it would be exceptional to find homozygous twins (syngeneic
transfusion). To my knowledge, this has not been done and we can briefly exarnine
this thomy subject. Human leukocyte antigen (HLA)-identical donors must be
excluded to avoid the risk of a GVHD or graft versus host disease (Anderson and
Weinstein, 1990; Ludewig et al., 2000) . Donor leukocytes, partly like the receiver
(HLA haplotype-related donors), have been used in metastatic cancer and could be
effective in AIDS as weIl. However, after a long debate (at least in the UK) about
the advantages and disadvantages of the blood (allogeneic) transfusion effect, it was
dec ided for several good reasons that, since November 1999, all blood components
must be subjected to a leukocyte depletion (LD) step (Williamson, 2000). For our
specific problem, this does more good than hann. I would suggest that both
leukocytes and platelets should be removed before ozonization and reinfusion into
the patient. With these precautions, also the problem of TRAU (transfusion-related
acute lung injury) (Popovsky et al., 1992) is unlikely to develop; it must be said,
however, that it has never been observed during 03-AHT. We can conclude then that
the ozonized allogeneic (LD) blood transfusion may help critical patients provided it
is done with great caution. If venous access is not available, the c1assical 03-AHT
cannot be accomplished, but now we have four options: a) cannulation of a central
vein, with inherent drawbacks, b) quasi-total body exposure to O 2-03 (Chapter 18),
c) rectal insufflation of gas (Chapter 19), and d) IV infusion of ozonized solutions. It
186 CHAPTER 16

is in fact possible to slowly inject solutions into small veins , but is there an ideal
solution to substitute blood? Medical personnel working in infectious disease wards
are somewhat reluctant to deal continuously with infected blood and needles, and
they often ask me to develop a blood substitute. This is not a trivial request and
requires a serious analysis ofhow we can solve this problem (Chapter 34) .

3.1. Minor 0 3 Autohaemotherapy

In the 1950s, when I was a medical student, we did not have immunomodulators and
we used to do IM injection of either autologous freshly drawn blood or sterile milk .
Thus the pract ice of "minor" autohaemotherapy is quite old and even these days
continues to be used without ozone (Olwin et al., 1997). Wolfprobably had the idea
of ozonizing blood in the hope of activating it. The technical procedure is very
empirical and I will describe our procedure: firstly, we collect the blood (5 ml) in a
10 ml syringe, and secondly, via a two-way stopcock we add an equal volume of
filtered O 2-03 • The blood, vigorously mixed with the gas, develops abundant
foarning and certainly in this case all the ozone dose reacts in one minute . After
disinfecting the buttock skin and checking to have not penetrated avessei , we inject
the blood and foam in one site, usually without causing pain . As far as 1 know, a
negative aspect is that everyone claims to have his own method.
What is the rationale of this sort of unspecific proteintherapy coupled with Or
0 3? There are no experimental data that , with appropriate funding, could easily be
obtained frorn laboratory animals. Thus we can only speculate. There is no doubt
that blood infiltrates the muscle tissue and undergoes rapid coagulation due to
platelet activation, etc. This happens already in the syringe, if we delay IM injection .
A number of biological processes, such as fibrinolysis, serum reabsorption via
Iymphatic vessels and mild sterile inflammatory reactions, likely take place , as
indicated by the symptoms (slight swelling and pain at the injection site) reported by
the majority of patients during the next few days . Chemotactic compounds released
at the site may favour the local infiltration of monocytes and neutrophils, which
degrade haemolyzed erythrocytes, and proteinaceous compounds. If they become
activated, they may release cytokines either in loco or along the Iymphatic system ,
upregulating the physiological cytokine response (see Chapter 14, Leukocytes and
the immune system) . Thus it would be very interesting to evaluate some
immunological parameters and ascertain if there is a concomitant induction of
haeme-oxygenase I (HOI) and some heat shock proteins (Tamura et al., 1997) that
may enhance immune reactivity.
These hypothetical possibilities are not farfetched and , if demonstrated, would
justify and support the practice of minor 03-AHT, which is simple, atoxic ,
inexpensive and easy to perform. However, we have no data from controlled clinical
trials, which ought to have been carried out already, at least with an O 2 control. So
far there are only anecdotal data without controls in patients with herpes land B,
and herpes zoster (Mattassi, 1985; Konrad, 1995). At the IOA meeting (London,
September 200 I), Konrad reported further data regarding post-herpetic neuralgia. A
similar approach has been publicized by the commercial firm "Vasogen Inc.", whieh
claims great advantages by using a particular formulation in which blood is treated
 How IS OZONE ADMINISTERED? 187

with ozone, heat and UV light ; this closely resembles the methodology published by
Garber et al. (1991) that proved use1ess in AIDS patients. The "Vasogen" approach
seems to have yielded good results in patients with Raynaud's disease (Cooke et al.,
1997). I do not share their enthusiasm because we know almost nothing about the
effects of ozone alone and it becomes even more difficult to understand the
contributing role of heat and UV irradiation. Most likely, the association of all of
these factors leads to total blood denaturation and to the hypothetical formation of
an autovaccine.
In this connection, I feel that we should pursue the idea of a possible
autovaccination by heavily ozonizing small volumes (3-5 ml) of plasma with ozone
at high concentration (up to 100 ug/ml), having previously shown that they
inactivate the virus in plasma. Although this seems a naive idea, oxidation of viral
components may represent an effective immune stimulant in several chronic viral
diseases, from herpes to HIV and HCV . Instead ofplasma, why not use the infected
blood containing intracellular virus es as wel\? lt mayaIso have an adjuvant activity,
but I am concemed about the potential development of an autoimmune reaction
against oxidised cellular proteins. For this reason, if I were able to perform a trial, I
would prefer the local use of a safe immunoadjuvant like GM-CSF added to the
ozonized plasma. It remains to be ascertained if an autovaccine would be more
effective after a single IM or SC injection, or rather after several small (0.1-0 .2 ml)
intra-epidermal injections, in which immunologically active Langerhans cells (as
antigen-presenting cells, APC) are concentrated.
There is no record of significant side effects due to rninor OJ-AHT. However, I
recently refereed an excellent paper by Webster et al. (2000), who reported that
care1ess operators performing minor AHT (without ozone) in a naturopathic clinic in
London infected over 70 patients with HCV simply by diluting blood (was it
necessary?) with saline collected from a contaminated bottle. This episode iIIustrates
once again how trusting patients can run into danger.
It is almost needless to say that one can use a double or tripIe dose of ozone by
adding 2 or 3 volumes of gas (at an ozone concentration of 100 ug/ml) to I volurne
of plasma. According to the evidence that multi-drug-resistant Mycobacterium
tubercolosis (MDR-MT) bacteria already kill about 2 million people each year,
there is an urgent need of a new vaccine. MDR-MT has a lipid coat and it is
probably inactivated by ozone so that it would be interesting to evaluate the degree
of inactivation and immunogenicity after ozonization. If it works it will help to
reduce TB infection in India and Africa .
 CHAPTER 17

EXTRACORPOREAL BLOOD CIRCULATION

VERSUS O2-03 (EBOO)

"Est quadamprodire tenus. si non datur ultra ..

Horace (65-8 B.C.), Epist., I, 1,32
(At least we have done a first step)

During the 1980s, I became acquainted with several methods to activate immune cells
in cancer patients during extracorporeal circulation of blood . In Chapter 14, section
Leukocytes and the Immune System, an approximate calculation indicated that to
activate a sizable proportion of immune cells, we should perform at least 50 03-AHT
in a six month period. In 1992, after we had shown that 0 3 can act as a mild inducer of
cytokines, I became very keen on examining whether intensive blood ozonization
could help terminal cancer patients , thus overcoming the disadvantage of classical 0 3-
AHT. This could be realized by a dialysis-like system, substituting the dialysis liquid
with a continuous flow of Or03. It seems that, around that time, other people in
various countries had a similar idea. However, regretfully without any scientific and
medical background, they lured and exploited desperate patients . I remember distinctly
the winter morning when I was passing by the Siena Polyclinie and I suddenly decided
to go and talk with the Director of the Nephrology and Dialysis Unit, Prof. Nicola di
Paolo. Unlike many other distinguished clinicians, he let me talk and immediately
grasped the meaning and the possible implieations, with an enthusiastic mood that I
eould never have even dreamt of. It has taken almost a decade of laboratory,
preclinieal and preliminary clinical work before optimizing the method and I can reeall
many ups and downs. In the end, we shall see if it was a good or irrelevant idea. Yet,
whatever the outeome, I owe Nieola much gratitude, as without him nothing would
have been aecomplished.
The following is a sehematie aeeount of our results, detailed elsewhere (Bocei et
al., 1999b, 2001e ; Di Paolo et al., 2000):

I ) after several phases, the final EBOO system is shown in Figure 72. It eonsists of a
precise ozone generator, fed by therapeutie oxygen on line, able to deliver a
constant flow of the gas mixture (- 99% O2- - 1% 0 3) for hours. Wehave assessed
biochemical parameters and toxieity using 0 3 concentrations from 3 to 80 ug/ml,
but now we routinely use 4 ug/ml throughout the session. The 0 3 eoneentration is
continuously monitored by photometry and visualized in real time. We periodically
check the photometry by iodometrie titration.

189
190 CHAPTER 17

t
6

Figure 72. A schematic view ofthe simplified EBOO apparatus. J) Oxygen supply 2) Ozone
generator with photometer 3) Roller blood pump 4) Hollow-flbre oxygenator-ozonizer 5) Two
air traps with bloodfilters in series 6) Blood pressure monitor 7) Silica gel trap 8) Ozone
destructor

2) All materials used in the system are the best available and ozone resistant (teflon,
silicone tubing, etc .).
3) The oxygenator is a crucial part, representing (so to speak) the lungs ofthe system .
In the course of the years, we have tested some 20 types of dialysis filters (I believe
that quacks use them) made of various materials (cuprophane, cellulose acetate,
polysulphone, etc.) but only one ofthem allowed blood to be reasonably ozonized.
Moreover, they activate the complement and leukocytes (Rousseau et al., 2000) . In
this case, the blood circulated inside the hollow fibres and the ventilating gas
flowed outside the fibres in the opposite direction. Naturally, all dialysis filters
produced more or less ultrafiltrate which could be compensated for, but the major
risk was the release of potentially toxic materials because dialysis exchangers are
not supposed to be ozone-resistant. During the last three years , we have
experimented with typical oxygenators, currently used during cardiovascular
surgery. After testing various types, we selected the hollow polypropylene fibres
enclosed in a polycarbonate housing and a suitable potting . We studied the
problem in the lab, firstly using saline , then pig blood and then heparinized human
blood. With the oxygenator, the blood flows outside the hollow fibres while the gas
flows inside . Figure 73 shows two representative sets of data obtained by
 EXTRACORPOREAL BLOOD OZONIZAnON 191

eomparing the oxygenator that (C) does not work (left panel) and the one (E) that
works satisfaetorily (right panel) (Bocci et al., 2001c).

Figure 73. The diagrams show the modification 0/ several parameters such as p02.
TBARS. PTG and venous pressure (VP) before the gas exchanger throughout the EROO
session. The basic sampie was collected by ventilating the gas exchanger with oxygen only.
Then, blood sampies were collected before (Pre = venous blood, white column) and after
(Post = arterial blood, grey column) the gas exchanger at 10. 30. 45 and 60 min during Or
0 3 exposure (0 3 concentration : -t ug/ml) , The comparison between exchangers C and E
clearly shows the inefficiency oftheformer, particularly the small trans/er ot ozone and the
progressive increase of VP. indicating clogging ofthe exchanger. Numbers on the abscissa
indicate the time 0/pe/fusion.

Blood sarnples were eolleeted before and after the oxygenator, before returning into
the patient who represents the blood reservoir. Several parameters were followed from
the start (time 0), with O 2 alone and then after 10, 30, 45 and 60 min during OrO)
exposure . Only the most critical data are presented, i.e. p02, TBARS (peroxidation
marker), PTG (marker showing oxidation of protein-SH) and hydrostatic pressure
(mmHg), continuously monitored before the oxygenator (for details see Bocci et al.,
2001e). It ean be noted that oxygenator C functions poorly even in the first 10 min:
although it allows a good oxygenation, the increase of peroxidation is negligible,
indicating a minimal transfer of ozone. The worst result is the progressive increase of
hydrostatie pressure, which after 30'-45' indieates irreversible clogging of the
oxygenator. Indeed a transitory saline washing is ineffective. In contrast, oxygenator E
allows such a high exchange of oxygen-ozone, particularly during the initial 10min,
192 CHAPTER 17

that we must keep the ozone concentration at the rmrurnum level (3-4 ug/rnl).
Subsequently, peroxidation levels and PTG values progressively decline but indicate
sufficient transfer of ozone . Importantly, venous pressure remains stable, suggesting
good exchanger viability after one hour of perfusion . Blood flow is kept at 75-90 ml
per min, so that about 5 L blood are exposed in one hour. Thereafter, the gas flow is
stopped and 250 ml of saline are added to the circuit so that blood loss is minimal. A
elose inspection of the oxygenator highlights the presence of a very thin, irregular
coating in most ofthe fibres, but the lack ofminiclots. Needless to say, the oxygenator
is used only once and each session is perfonned with a new sterile set. There is no
need of a heat exchanger, thus reducing the priming volume , and the oxygenator in
current use has a surface of 1.7 m 2.
4) Venovenous circulation: blood, nonnally drawn frorn a large vein of one arm, is
directed toward the oxygenator by a standard roller pump and returned to the vein
of the contralateral arm with the interposition of two blood filters and air-traps .
Two are virtually unnecessary but eliminate any risk of embolization. Standard
arterial-venous fistula needle sets (usually G 17) are used and great care is taken to
maintain the venous access in good condition .
5) Five minutes before beginning the extracorporeal circulation, we slowly inject a 10
ml bolus of 10,000 IU Na-heparin diluted with saline, and a subsequent slow
delivery of a diluted solution has proved unnecessary . In the preclinical study, we
used a standard Na citrate solution (ACD) that was continuously mixed with blood
before the oxygenator: chelation of Ca 2+, in the presence of ozone, minimized
platelet activation , but subsequent recalcification with calcium gluconate and often
correction of acidosis by NaHC03 infusion was necessary . Even the use of heparin
may present the shortcoming of thrombocytopenia (Chong, 1995) but, on the
whole, with monitoring of platelet counts and use of a biocompatible oxygenator, it
appears to be less problematic.
6) Although there is no ultrafiltration , the outflow gas still passes though a trap of
silica gel to remove any trace of humidity and protect the thermostatically
controlled ozone destructor.
7) An ozone-sensing monitor with audio alann is always turned on in the EBOO
room.

Why did the usual oxygenators give us so much trouble? They have been used
regularly in cardiovascular surgery and although the polypropylene surface is not
biocompatible and elicits an immune response (Acseil and Riley, 1993), it allows
stable and efficient oxygenation. Conversely, owing to the critical fact that we were
using agas mixture with ozone, the oxygenators in our hands had a useful Iife of less
than 10min. The explanation has come from a collateral study on the effect of ozone
on platelet-rich plasma either with citrate or with heparin (Bocci et al., I999a). With
the latter anticoagulant (Chapter 14, section Platelets, Haemostasis and Growth Factor
Release), we showed that ozone almost instantaneously activates platelets, causes their
aggregation and the subsequent chain of events leading to coagulation. The same
reactions occur in a few minutes on the external surface of the fibres between the
flowing blood and the gas. As ozone solubilizes in the blood, it immediately causes
platelet adhesion and the progressive fonnation of a coating composed of platelets,
 EXTRACORPOREAL BLOOD OZONIZAnON 193

fibrin and trapped erythrocytes. At' first, oxygenation is not much affected, but ozone
movement toward the flowing blood is totally impeded because the gas remains
blocked and reacts at the coating level. Eventually the oxygenator is clogged with
coagulated blood, weil indicated by the progressive increase ofvenous pressure and no
exit of oxygenated blood. Luckily this problem has now been practically resolved by
the preparation ofbiocompatible oxygenators.
From a selection of polypropylene fibres either heparin-coated (which as expected
did not solve our problem), paraffin-coated, Trillium-coated or albumin- coated, we
are now using the last type .
About four years ago, we performed several experiments in sheep using ACD and
we obtained much information about the priming solution, volume of blood flow per
min, volume and concentration of the O 2-03 mixture flowing counter-current with
respect to blood and the time necessary for perfusion in vivo. The parameters showed
that an 0 3 concentration as low as I0 ug/ml was biochemically effective in terms of
P02' TBARS, T AS, PTG, GSH and GSSG changes measured in the post-oxygenator
blood. This meant that the gas exchange and 0 3 reactivity were rapid and capable of
inducing biological effects. The sheep showed no adverse effects even after 50 min of
extracorporeal circulation at high 0 3 concentrations (20 to 40 ug/ml), but the
exchanger became less effective (low pOz values) due to progressive clogging with
cells (Bocci et al., 1999b).

Figure 74. After the preclinical study, the author was the.first volunteer to prove that
extracorporeal blood circulation against OrO] was atoxic. P: Roller blood pump ; S: Hollow-
fibre oxygenutor-ozonizer; G: Ozone generator.

Thereafter, with a new oxygenator, I convinced my friend Nicola to perform a

couple of EBOO sessions on myself, which this time proved very encouraging. The
first EBOO was carried out in 1993 using the dialysis-type technique, but we realized
that it was unsuitable (Fig. 74) . Later on, after obtaining permission from the
194 CHAPTER 17

University Ethical Committee and informed consent frorn patients, we started a pilot
study in one patient with Madelung disease, a few serious cases of hind limb
ischaemia, ischaernic cardiopathy and one case ofnecrotizing fascitis (Chapter 24).
A prelirninary report (Oi Paolo et al., 2000) on seven patients included 98 EBOO
treatments . The patients were treated twice a week for a total of 14 sessions using
heparin . Outing and after treatment , body temperature, breathing and blood pressure
were unchanged. The patients did not note any subjective feeling of any kind but five
of them reported a sense of wellness and euphoria after the first few treatments. Three
patients reported improved visual acuity which, based on OUT ARMO study, is not
surprising. All chemical, biochernical and enzymatic parameters measured in the blood
before, at the end ofthe entire cycle and after 4 months remained unmodified .

0 .5 De
eI
il
Wilco.on a,b.c,d.o,f,g,h,i,l,m.n,o.p P<O05 no
p
0.4

0.3
~
Ul

~ 0.2

0.1

0.5 Wilcoxon a,b,c ,d.e,r,g,h,I.I.m.n,o P<0 .05

f. i
a d g f I 9
b h I m 1
0.4

0.3
:!!
:J.

f:2
CI. 0 .2

0,1

0
basal 02 03 51J9/rrl 03 1 O~g/m 03 151J9/ml
C preaft!tar • posl-fifter

Figure 75. Pre- and Post-gas exchanger TBARS and PTG plasma levels during thefirst
EROO treatment. A three-fold increase ofozone concentration does increase the oxidative
effect, but it is not necessaryfor the efficacy and rapidly leads to clogging ofthe exchanger
 EXTRACORPOREAL BLOOD OZONIZAnON 195

The scientists that eritieise the use of ozonetherapy may be interested in observing
the ehanges in TBARS and PTG values measured in plasma during the EBOO (Fig .
75) and throughout it (Fig. 76).
0.5

04

:2 03
""
U)

~
~ 0.2

0.1
n=7 n=4 n=4

0.5

04

0.3
:::;;
::I.
o
l-
o,
0 .2

0 .1
n=7 n=4 n=4

0+-_'--_-'-_-.-_.1..-_......1-_--,----_-'--_-'-_
basal 1st treatment basal7lh treatrrent bual14th trealment

Figure 76. TBARS and PTG basal levels (M i:SD) he(ore the Ist. 7th and 14th EBOO
treatment. It is reassuring that these values, indicative ot'oxidative stress, remain practically
the sante throughout the study.

During this pilot trial, we were still searehing für the optimal 0 3 eoneentration and
it ean noted that 15 ug/ml gave the highest inerease ofTBARS and deerease ofPTG.
In spite of this dosage, the values of both TB ARS and PTG remained markedly stable
throughout the whole cycle (Fig. 76). These data indicate that ozonetherapy does not
irreversibly inerease lipid peroxidation and that the dilution, eatabolism and exeretion
of LPOs are effeetive and unaltered . Moreover, these seven patients (and myself as
weil) have shown no side effeets. Thus the dogmatie sentenee that "ozone is toxie any
way you deal with it" does not appear to be true, and indeed this is not surprising to a
biologist. It suffiees to eonsider the enorrnous potentiality of the antioxidant system
and the eapacity to renew itself all the time. Even though seeptical seientists may not
196 CHAPTER 17

believe this result , they ought to seriously ponder on it. This group of patients was
heterogeneous, but all of them showed c!inical improvement. In fact, the Madelung
case, two peripheral vasculopathy patients (one with cholesterol embolism) and one
subject with severe coronary disease showed very great improvement that lasted
several months . To maintain the improvement, the treatment has to be resumed after 3-
6 months and repeated twice a month . The study has continued with the albumin-
coated oxygenator and by lune 200 I the number of patients had grown to 21. Thus we
were able to draw some conc!usions:

a) the extracorporeal circulation ofblood against O 2-03 has become a reality;

b) all the technical and methodological aspects have been resolved satisfactorily;
c) owing to the improved efficiency of the oxygenator, up to 5 L of blood can be
exposed to very low 0 3 concentrations (3-4 ug/ml), Ta enhance ozone tolerance
the first and second EBOOs last only 30 and 45 min, respectively ;
d) as occurs in the pulmonary circulation, the great efficiency of the hollow fibres
allows total gas exchange in one minute ;
e) both oxygenation and ozonization remain effective without any increase of venous
pressure ;
f) in arteriopathic patients (grade III and IV) subjective and objective c!inical
improvements have often been noted after the first treatment. Orthodox treatments
usually do not provide such improvement;
g) neither metabolic derangement nor changes in blood chemistry nor any toxic effect
has been observed during or months after the cycle;
h) it is necessary to prove objectively the c!inical data and support them with
laboratory data evaluating: I) adaptation to chronic oxidative stress, by measuring
levels of antioxidant enzymes, 2) various oxidative stress proteins, particularly
HSP32 or HOl, 3) 2,3-0PG values, 4) hormonal levels (CRH, ACTH , cortisol,
OHEA, endorphins, etc.) able to explain the feeling of wellness and disappearance
of pain, 5) any modification of LOL and HOL, cholesterol and fibrinogen levels ,
and 6) the immune status;
i) the possible disadvantages must be taken into due consideration : I) the cast of the
disposable oxygenator, inc!uding all ancillary materials, 2) the cost of a qualified
specialist technician, 3) the potential deterioration of venous access, 4) the
occasional need of inserting a catheter into a central vein to continue the cycle,
with the related risk ofinfection (this recently occurred in two patients, who had to
stop the treatment). The last problem may be reduced by using improved catheters
impregnated with antibacterial substances (Wenzel and Edmond , 1999).

At this stage, we feel campelied to vigorously ascertain the therapeutic benefits of

EBOO, particularly in a few areas :

a) critical, inoperable ischaemic limbs (stage III and IV, Fontaine) when amputation
remains the only option. Medical treatments (iIoprost infusion, pentoxyphylline,
electrical spinal-cord stimulation, anticoagulants, platelet anti-aggregation, anti-
atherosc!erotic drugs, etc.) help but are rarely successful (Bergquist, 1999). The
 EXTRACORPOREAL BLOOD OZONIZATION 197

surgical procedure of distal "venous arterialisation appears promising (Taylor et al.,

1999) and on a conceptual basis should be more useful than EBOO . Therefore, a
comparison ofthese approaches appears useful for patients;
b) severe cardiac angiostenosis, previously operated on with no success;
c) acute cerebral ischaemia, to be treated with EBOO as soon as possible to
reoxygenate the hypoischaemic (penumbra) and infarctuated areas, thus limiting
neuronal death and favouring a more rapid recovery;
d) chronic HCV hepatitis in patients who are IFN-resistant or IFN-intolerant or
because they refuse orthodox therapy ;
e) inoperable metastatic cancer, to check if EBOO can stabilize the disease;
f) severe lipodystrophies.

This is an ambitious program that we will tackle in the next three to four years .
Hopefully it will finally c1arify the validity and cost-benefit not only of EBOO but of
all ozonetherapy.
 CHAPTER 18

QUASI-TOTAL BODY EXPOSURE TO O2-03 (BOEX)

V. Bocci and E. Borrelli

Some six years ago, we raised the possibility of exposing the body (excluding the
head and neck to avoid pulmonary toxicity) in an ozone-resistant container (even a
very large polyethylene bag could be used) for patients who refused rectal
insufflation and for those who had no pervious venous access for OrAHT or EBOO
(Bocci 1996c,d). However several problems must be evaluated:

1) Is ozone as toxic for the skin as it is for the respiratory mucosa (Menzel,1984
Lippman, 1989; Devlin et al., 1991; Kelly et al., 1995; Chen and Qu, 1997)? In
common with ozone, chronie UV irradiation of the skin generates ROS, which
after life-long exposure can result in skin changes such as wrinkles, pigmented
spots and possibly cancer. Interestingly, Maeda et al. (1991) showed that hairless
mice initially enhance defence mechanisms, but these deteriorate later with
prolonged irradiation. Further studies have shown that both ozone treatment and
UV-irradiation of epidermallayers of murine and human skin cause peroxidation
and depletion of vitamins C and E (Thiele et al., 1997a,b; Podda et al., 1998;
Fuchs and Kern, 1998). It has also been shown that these oxidizing agents, hence
ROS, activate NFKB and activator protein-l (AP-I), but that LA, NAC, Trx and
Selenium can inhibit the activation to a large extent and induce adaptive
protection, such as over-expression of MnSOD and GSHPx as a response to
oxidative damage (Haas et al., 1998; Saliou et al., 1999; Meewes et al., 2001;
Didier et al., 2001). Arecent book entitled "Oxidants and Antioxidants in
Cutaneous Biology", edited by 1. Thiele and P. Elsner (2001), reports a wealth of
information. It is clear that the skin has a multiform antioxidant defence system,
far more potent than that present in RTLF, and that it cannot be overwhelmed
provided the attack by ozone or UV irradiation is not too harsh. These fmdings
lend support to the empirical observation that during topical ozonetherapy of
necrotic ulcers, we have never noticed any damage to normal skin. Moreover,
during balneotherapy with slightly ozonized water, no local or generalized
untoward effects have been reported.
2) Are there anatomical-physiological reasons for the relative tolerance of skin to
ozone? Yes, if one examines the scheme of Figure 77 showing the structure of
skin, with the epidermis, the derma and the disposition of the vascular system.
The most external layer is the stratum corneum, i.e. the end product of
keratinocyte function, which is a compressed and tough layer. This "dead layer"
is more or less overlain by a very dynamic film, containing some proteins and

199
200 CHAPTER 18

water, due to the secretion of the eccrine glands. 1t is partly responsible for
thennoregulation, since it allows cooling of the skin surface (-580 cal/g) as the
water changes frorn liquid to vapour. Moreover, the layer of lipids, produced by
sebaceous glands, consists of unusual oily material, partly modified by the
resident microflora (Nicolaides, 1974); in our opinion , this represents the first
line of defence against ozone and UV rays . Progressing towards the dennis, there
are the stratum granulosum, the stratum Malpighi and the proliferating basal cell
layer. The dennis and the subcutaneous tissue contain a very flexible vascular
system with a heat-exchanger, represented by capillaries and mainly by the
venous plexus associated with the opening of arteriovenous shunts. 1t is able to
accommodate up to 30% of the cardiac output so that heat transfer through the
skin can increase up to 8 fold from astate of total vasoconstriction to extreme
vasodilatation.

Figure 77. A sehematie view 0/ the skin and cutaneous circulation. Numbers indicate:
J)The stratum corneum overlain hy a superficial hydrolipid film, in whieh ozone dissolves and
generates RaS and LOPs. 2) Malpighi 's layer. 3) The basal eelllayer and basement
membrane. 4) The dermis , with a sebaceous gland and a sweat gland. 5) The arterial and
venous vasculature with arteriovenous anastomosis. 6) The subcutaneousfatty tissue

3) A crucial question is: when the skin is exposed to ozone , does this gas penetrate
all the cell layers to reach the dermis and enter the capillaries? Advertising
would have one believe that ozone reaches the blood circulation and has a
 QUASI-ToTAL BODY EXPOSURE TO O 2-03 201

c1eansing effect, with the elimination of viruses and toxins. Yet this claim is not
correct and has only commercial purposes. O2 and CO 2 do indeed move freely
through cell membranes . However, owing to its dipolar moment (Chapter 4) and
high solubility , ozone dissolves in the superficial water film and reacts
immediately with PUFAs of the sebum, generating H202 and an array of peroxyl
radicals, 4-HNE , on the whole denominated LOPs . Therefore , it is more than
likely that ozone does not even reach the phospholipids of the outer corneocytes,
a conclusion already advanced by Pryor in 1992 for the pulmonary air-tissue
boundary . However, the generated ROS and LOPs can be partly absorbed and
pass through the epidermis , derma and capillary wall to enter into the blood
stream. Obviously H202 and other ROS have a very short half-life and will be
quickly reduced ; indeed it has been c1early reported that several antioxidants
(vitamins E and C, etc.) are readily oxidized (Thiele et al., 1997a,b; Podda et al.,
1998; Fuchs and Kern, 1998).
4) The obvious corollary that comes to rnind is: does skin vasodilatation enhance
the transfer of O2, CO 2, ROS and LOPs? It probably does and we will examine
some experimental results . The "thermal stress" that is easily induced with sauna
bathing (Finnish bath) increases cutaneous capillary perfusion, which may
greatly increase the "perspiratio sensibilis" through activation of sweat glands
and mayaIso favour absorption of ROS and LOPs produced during an "ozonized
sauna" . Around 1995, we were informed that beauty centres in Italy had used
sauna bathing with a trace of ozone for a decade, but this had remained only in
the realm of cosmetic treatment of lipodystrophy and obesity. Moreover, on
October 10, 1997, we received a letter from Canada stating that steam sauna
combined with ozone had come into widespread use and "weil over 2,000 people
had been treated with uniformly excellent results" . Apparently some terminal
cancer patients had been cured!!! Needless to say, no scientific reports had been
published . At that time, we were still developing the EBOO system, but we
thought that the ozonized sauna might be another therapeutic option with the
advantage of non-invasiveness, particularly important in patients with
deteriorated venous access .

Dr. Borrelli (Institute of Thoraeie and Cardiovascular Surgery, University of

Siena) found an excellent place to perform our study : athermal resort in the middle
ofthe Dolomite Alps (Raphael Clinic at Roncegno, Trento) . We were lucky to have
the enthusiastic collaboration of seven middle-aged physicians who acted as
volunteers . In Italy, it is almost impossible to find healthy human volunteers and in
any case we had no money to pay them! The aim of our programme was to evaluate
the following aspects :

a) possible variations of arterial and venous P02, pC0 2, pH, exarnined before (pre),
immediately after (end) and then 0.5, 1.0 and 24 hours after aperiod in a sauna
cabin in the presence of either O2-0.1 (May 1998) or only O2 (control, September
1998). Unfortunately , only venous p02 values were obtained because our
colleagues objected to the arterial blood collection .
202 CHAPTER 18

b) Modifications of body mass , oral temperature, diastolic and systolic blood

pressure and ECG pattern.
c) One important question was to examine any possible variations of peroxidative
markers in plasma during and after treatment. In other words, we wanted to ascertain
whether a 20 min exposure to ozone of almost the entire cutaneous surface could
induce an oxidative stress and, if so, if this would be tolerable and lead to a
therapeutic benefit. All details can be read in the original paper (Bocci et al., 1999c).

The cabin was made of laminated plastic and, after subtraction of the body volume,
had an internal residual volume of about 440 L. The flow of gas through the cabin
(either a mixture of about 97 % O 2 and 3%. 0 3 or pure medical O 2) was I Llmin. The
0 3 concentration was assessed in real time with a portable photometer. Any internal
increase of barometrie pressure in the cabin was prevented by an external silicone
tubing connected to an 0 3 destructor. The maximum 0 3 concentration was reached
at the end of the session and was estimated to be no higher than 0.90 ug/rnl, i.e.
many times lower than the minimal 0 3 concentration used during local treatment of
torpid ulcers for the same period (Werkmeister, 1995) . Steam was generated in the
cabin by a thermostatically controlled heater set at 90 Co and turned on 10 min
before the subject ente red the cabin. Two towels and one polyethylene sheet were
wrapped around the subject's neck. Although the doors were tightl y closed by
means of Oj-resistant gaskets, they were further insulated with the pol yethylene
sheet and towels to avoid any leakage of 0 3 into the room. An improved, better
insulated cabin is now being tested. The session lasted 20 min , during which the
maximum temperature inside the cabin reached 46-50 Co. Just before the doors were
opened, the gas flow was interrupted and the internal gas was rap idly aspirated via
the outlet to prevent any breathing of 0 3 by the subject and the assistant.
Determination of several variables was performed before, immediately after, and
then 0.5 , 1.0,24 hours after the session. Body (oral) temperature was also measured
in the middle of session. Standard 12-lead e1ectrocardiograms were recorded before
and after the session. Body mass was assessed with an electronic balance with an
error of ± 50 g. Blood gas analysis was performed with an IL-1620 blood gas
analyser (Instrumentation Laboratory, Lexington, MA,USA). Systolic and diastolie
arterial blood pressures were measured with a standard cuff sphygmomanometer.
As mentioned before, biochemieal determinations in fresh plasma sampies
(namely TAS, PTG, TBARS, free Hb, levels of hepatic enzymes and creatinine)
were measured according to standard methods. Haemocytometric determinations
were performed with an automated haematology analyser. Cytokines (IL-8 , TGFß 1
and myeloperoxidase) were quantified with commercial ELISA kits . Latent TGFß 1
was determined after acid activation. The results are expressed as the mean ± SD
and the level of statistical significance (indicated with an asterisk) was set at p<O.05.
On the whole, the results were more interesting than we had expected:
Each volunteer was subjected to one 20-min exposure in the water vapour-
saturated cabin, in the presence of O 2-0 3 (black bars) or O 2 only (white bars ), i.e. he
served as his own controI. Modifications ofbody mass, oral temperature and systolic
and diastolic pressures are shown in Figure 78.
 QUASI-ToTAL BODY EXPOSURE TO O2-03 203

80 _ ozone
c::::J control

--~-
..c
Cl
75

.~ 70
>-
-g 65
m
60
6L..-
Q)
~
38
;:,
"§
E 36
Q)

~
34
120
~ f100
~ ~ 80
(J)~

60
a. 120
~

~ ~100
.!!!
o~
~ 80
60
pre end 0.5 1.0 24

Figure 78. Modification ofbody mass. oral temperature, diastolic and systolic blood pressure
(Diast BP and Syst BP, respectively) ofsix subjects before (pre). at the end (end), and 0.5, 1.0
and 24 h after aperiod in the sauna cabin in the presence 0/ either OrOs (black bars) 01' O2
only (control, white bars). Values represent the M :tSD. No significant intragroup 01'
intergroup differences were found.

There was a significant increase in body temperature, which reached a peak at

the end of the treatment and declined rapidly thereafter. The maximum oral
temperature ranged between 37.5 Co and 39.3 Co, There was a concomitant
reduction in body mass (200-600 g). Similarly, blood pressure decreased slightly,
but recovered within the next 30-60 min. Other data are summarised in Table 16.
The results for the venous partial pressure of O2 (PV02) and CO 2 (PvC0 2 ) are
ilIustrated in Figure 79.
204 CHAPTER 18

80 _ -
o ozone ..
control
. ..
60 -

40 -

20 -

C)
60-
.. ..
J:
E
E .. ..
'-'" 40 -
o
N

o
> 20-
o,

0-- -~ ~ ~- -
pre end 0.5 1.0 24

Figure 79. Modiflcation ofpartial pressure 0/0] in the venous blood (P,'o ]) and partial
pressure ofCO] in the venous blood (PvCO;) ofsix subje cts before (pre), at the end (end). and
0.5, 1.0 and 24 hr after aperiod in the sauna cabin in the presence 0/ either Or03 (black
bars) 0 1' 0] only (control, white bars) . Values represent the M :f:SD. Asterisks indicate
stat istical difference (p<O.05)/or the intragroup comparison

There was a significant increase of PV02 and decrease of PvC0 2 at the end of the
session and for I h after exposure to both O 2-03 and O 2 alone ; the increase in PV02
after exposure to O 2 alone was not significantiy higher than that after exposure to O 2-
0 3. Values for both erythrocytes and haematocrit increased immediately after the 20-
min exposure. They decreased thereafter, probably due to rehydration, and were
almost normal after 24 h (Table 16).
We noted an initial significant increase in leukocytes, followed by a decrease 1 h
after O 2-03 exposure (Fig . 80) .
Figure 81 shows the interesting results observed after O 2-03 exposure. TAS and
PTO decreased after both O 2-03 and O 2 exposure, but the PTO values were
significantly lower only after 0r03 exposure. Surprisingly, the baseline values of
both TAS and PTO were lower in mid -September (0 2 alone) than 3.5 months before
(0 2-03), in spite of the fact that the same subjects were studied. The difference may
have been due to seasonal variation. There was a concomitant, progressive and
significant increase of TBARS in the plasma after O 2-0 3 exposure, which receded
completely 24 h after the end ofthe exposure. In spite ofthis increase, it is important
to emphasize that no haemolysis was noted at any time.
 QUASI-ToTAL BODY EXPOSURE TO O2-03 205

. . Ozone
7,5 c=JControl

6'
o
oT'"" 5,0
o
U
~ 2,5

0,0
*
25

-
c»
E 20
~ 15 + +
"-'"

~ 10
5
o
pre end 0.5 1.0 24
Figure 80. Modification ofleukocyte (WBC) and interleukin 8 (IL-8) plasma levels ofsix
subjects before (pre), at the end (end). and 0.5. 1.0 and 24 hr after aperiod in the sauna
cabin in the presence 0/ either OrOj (black bars) 01' Oionly (control, white bars). Values
represent the M :1:SD. Asterisks indicate statistical difference (p<0.05)/or the intragroup
comparison . Crosses indicate statistical difference (p<O.05)/or the intergroup comparison .

We investigated whether the plasma levels of three representative markers

changed after the O2 -0 3 exposure . Levels of IL-8 significantly increased 30 min
after exposure (Fig . 80). Conversely, levels of MPO and TGF-ß I either did not
change or tended to decrease (Table 16).
Plasma levels of hepatic enzymes and creatinine remained within the normal
range. No abnormalities (except increased systolic pressure after exposure) were
noticeable from the tracings carried out before and after the session. All subjects
tolerated the exposure to both O2-0 3 and O2 without reporting either immediate or
subsequent adverse effects . Oral intake of water was allowed at the end of the sauna.
Four subjects enjoyed the sauna, but two reported that they would find it difficult to
tolerate aperiod longer than 20 min in the cabin .
206 CHAPTER 18
_ Ozone
c=J Conlrol
2,0 -
+ +
+
~ 1,5 -
.I
E
-10-
Cf)
,
~
r; 05 -
•
0,0 -'--

+
~0,4 -
..=!.
C9
r- -02
0 , -

0,0 -'--
*

~2 * +
Cf) * +
0::: +
~ 1

o
pre end 0.5 1.0 24

Figure 81. Modification oftotal antioxidant status (TAS). protein thiol groups (PTG) and
thiobarbituric acid reactive substan ces (TBARS) in the plasma ofsix subjects before (pre). at
the end (end). and 0.5. 1.0 and 24 hr after a period in the sauna cabin in the presence 0/
either OrOJ (black bars) or O2 only (control. white bars). Values represent the M i:SD.
Asterisks indicate statistical difference (p<0.05J/or the intragroup comparison. Crosses
indicate statistical differen ce (p<O.05)/or the intergroup comparison.

This study was intended to be an initial exploration in "terra incognita" and we

are weil aware of its pitfalls. If we had had c1inical and financial support, we would
have proceeded more rationally and not have mixed too many parameters at the
same time . First of all, we ought to have examined the problem without heat ,
working at room temperature in a simply humidified cabin (necessary to solubilize
the ozone).
 QUASI-ToTAL BODY EXPOSURE TO O 2-03 207

Table 16 A summary ofparameters measured before (PRE). at the end (END), and 0.5 h, 1.0 h
and 24 h after a period in the sauna cabin in the presence ofeither ara; (experimental) 01' O2
only (control) . Values are given as the mean (SD). Intragroup and intergroup comparisons
revealed no significant differences, (n.d. Not determined), RBC red blood cells, Ht
haematocrit. Pl.T'platelets, [HCO;] bicarbonate concentration, Sv02 venous oxygen
saturation, TGFß transforming growthfactor ßl. AST aspartate amino transferase, ALT
alanine amino transferase)

Parameters PRE END 0,5 h t.o» 24h

OrO; °rO; OrO; OrO; o-o,
RBC (xI0 6 rnnr') 4.94 (0.22) 5.06 (0.22) 4.71 (0.14) 4.62 (0.21) 4.87 (0.26)
HT(%) 45.37 (1.91) 46.5 (1.92) 43.27 (1.l9) 42.5 (1.82) 44.13 (2.91)
PLT (x103 rnm') 183.5(17) 207 (20) 182.5(19) 173.2(23) 191.5(26)
pH 7.33 (0.03) 7.43 (0.04) 7.38 (0.04) 7.39 (0.03) 7.38 (0.03)
[HC03] mmoll1 28.8 (0.98) 25.3 (1.24) 26.9 (2.32) 26.07 (1.34) 27.32 (1.02)
SV02 sat(%) 54.2(21.8) 88.3 (8.7) 76.3 (20.3) 86.5 (6.4) 62.4 (32.2)
TGFß (pg/ml) 54.5 (10.9) 32.8 (9.7) 32.7 (9.2) 30.5 (7.3) 45 .7 (8.7)
AST (U/I) 23.2 (3) 24 (2.5) 23.7 (6.3) 23.2 (4.7) 24 (2.9)
ALT (U /I) 19.8 (3.5) 21.3(4.6) 21.7 (3.9) 20.3 (3.8) 22.6 (4)
Creatinine (mg/dl) 1.03 (0.2) 1.17 (0.2) 0.97 (0.1) 0.92 (0.1) 1.02 (0.45)
Parameters
O2 O2 O2 O2 O2
RBC (xI0 6 mm') 4.66 (0.16) 4.73 (O.l) 4.54(0.1) 4.39 (0.1) 4.56 (0.2)
HT(%) 42.54 (2.02) 43.3 (1.8) 41.68 (1.8) 40.5 (1.82) 42.36 (1.58)
PLT (xI03 mm') 187.3 (16) 199.5(20) 162.8 (3.9) 169(11.7) 178.3 (12.3)
PH 7.3 (0.02) 7.4 (0.02) 7.38 (0.01) 7.37 (0.01) 7.36 (0.04)
[HC03] nunolll 27.68 (1.40) 24.03 (1.20) 25.30 (1.31) 26.70 (1.l8) 27.20 (1.60)
SV02 sat(%) 50.1 (19.3) 93.2 (2.8) 91.1 (4.3) 81.7 (12.6) 60.1 (18.25)
TGFß (pg/ml) n.d. n.d. n.d. n.d. n.d.
AST (U/I) 21(4) 22(2) 22 (4) 23(6) 24 (4.8)
ALT (U/I) 20(2) 22(3) 21(3) 20(4) 20.8 (4.5)
Creatinine (mg/dl) 1.01 (0.1) 1.08 (0.3) 1(0.2) 0.98 (0.1) 0.99 (0.38)

Then we could have examined the effect of the sauna alone, which in itself is
quite interesting. We enjoyed reading arecent review on the "Benefits and risks of
sauna bathing" (Hannuksela and ElIahham, 2001). Unlike the Turkish bath, the
sauna has a high temperature (80-100 °C at the level of the bather's face and 30°C
at floor level) and a relative humidity of about 20%. One good point of our study
was to control the same subjects with O 2 alone. In fact , Figure 81 surprisingly shows
that insufflation of O 2 alone is weil able to reduce plasma levels of T AS and PTG
and to increase peroxidation (TBARS levels) during the sauna period. The results
are even more surprising if we consider that the gas flow was only 1 Llmin and thus
the total volume of 20 L of O 2 was diluted in about 440 L of air contained in the
cabin. This suggests that the heating per se must overwhelm the effect of O 2 alone.
However, ozone must account for the significant linear increase of TBARS values
measured up to 40 rnin after the session.
208 CHAPTER 18

Naturally the project was evaluated and authorized by the Ethical Committee of
our University and all volunteers knew the problem and potential risks very weil .
My own data were not included for not increasing the variability of the age statistics.
Let us examine the risks : frrstly, ozone toxicity for the respiratory tract. There
must be neither contamination of environmental air with ozone nor any ozone
inhalation and we took precautions to avoid that. The cabin must be tightly c1osed,
the room must be weil ventilated, the gaseous contents of the cabin must be quickl y
aspirated before it is opened and a monitor sensing the ozone level must be switched on.
Secondly, ozone toxicity for the skin . Oepletion of antioxidants and the increase
of MOA in the outer epidermal layers are welI documented, but in our study the
final ozone concentration in the cabin could reach at most 0.9 ug/ml at the end ofthe
20 min session. The final ozone concentration increases slowly because we must
take into account the large dilution, a slight loss because the cabin remains at normal
pressure and the rapid ozone decay at about 40°C (about 18 min) . Thus the final
concentration is about 10 times lower than that used during the final topical
applications in skin ulcers or decubitus (Werkmeister, 1995). In conclusion, we did
not observe any acute or chronic toxicity.
Thirdly, systemic toxicity of ozone. We had no information about this but we
reasoned that ozone would decompose entirely on the cutaneous surface and only
some of the generated ROS and LOPs might be absorbed and enter the circulation.
The scheme shown in Figure 82 gives an idea of the site of action and fate of ozone
in the skin . However, we knew already that blood is quite resistant to ozone, and
body tissues and fluids have a great reservoir of antioxidant compounds, as weil as
the ability to regenerate them (Chapters 12 and 13). We envisaged that dilution,
metabolie breakdown and renal exeretion would minimize the inerea se, if any, of
TBARS in the plasma pool. Contrary to our expectation, there was a very signifieant
increase of TBARS, which eontinued long after the session , suggesting a slow
steady inflow prevailing over catabolism. It would be interesting to follow the
kinetics at 1.5-2-3-4 ho urs to localize the peak and the pattern of decrease. PTG
va lues showed a consistent decrease, while (reassuringly) TAS values declined only
slightly and temporarily. The induced oxidative stress had abrief lifetime and did
not cause haemolysis or any modification of important blood parameters (Table 16).
Hepatic enzymes and creatinine plasma levels remained unmodified. Plasma levels
of myeloperoxidase (MPO), a sensitive marker of the activity of neutrophils
(Weissman et al., 1980; Boxer and Smolen, 1988), did not change. At this stage , we
cannot say anything about toxicity after repeated BOEX, but it is most probably
harmless for the skin. However, subjects with moles at risk may protect them with a
crearn rich in vitamin E.
None of our volunteers, nor several patients, have reported acute or late side
effects. For experimental reasons, one of us (VB) has undergone four BOEX , at
different times, and he has experienced a feeling of great energy and euphoria for
the next couple of days. In fact, it would be pleasant to have the time to do it twice
weekly! A similar sense of welIness has been claimed by most of the patients
(mostly vasculopathic), some ofwhom were in very poor condition.
Is there an explanation for this good feeling and is it due to ozone or the sauna or
both? We can certainly say that 0 3-AHT and EBOO (rectal insuftlation is less
 QUASI-TOTAL BODY EXPOSURE TO O 2-03 209

effective) also give a sense of weil being, but in the case of BOEX the sauna itself
may contribute . For a long time, we have wanted to evaluate the hormonal changes
related to ozonetherapy and such a study would probably clarify this issue and
broaden our vision. We found that the short-tenn hormonal changes during and after
sauna bathing, particularly the increase of growth honnone and beta-endorphin, are
quite interesting (Hannuksela and Ellahham, 2001). It is intriguing that long-tenn
sauna bathing helps to lower blood pressure in hypertensive patients in spite of
transient activation of the renin-angiotensin-aldosterone system. As expected, these
changes are brief and reversible, and the same may occur for ozonetherapy. Whether
ozone potentiates the effects ofthe sauna remains to be seen.

Figure 82. A schematic view ofthefate ot ozone in the skin during BOEX Ozone
dissolves in the water-sebum film overlaying the outer layer ofthe stratum eorneum and
reacts immediately witb PUFAs . generating ROS (among whieh H 20JJ and LOPs . These
compounds ean he partly absorbed and pass througb the transcutaneous barrier, facilitated
by the intense vasodilatation indueed hy the sauna . Botk lymphatic and venous eapillaries ean
rapidly trans/er LOPs into the general circulation, thus inducing systemie effects . Numbers
indicate : J) The stratum corneum overlain by a superflcial hydrolipidfilm. in whiclt ozone
dissolves and generates RaS and LOPs . 2) Malpighi's layer.3) The basal eel/layer. 4) The
dertnis
210 CHAPTER 18

Fourthly, does ozone switch on dangerous oxidative stress? Although we noted a

remarkable systemic increase of peroxidation, it was transitory, since the levels
retumed to baseline after 24 hours . If the reader has gone through the previous
chapter, he likely realizes that we purposefully want to induce an acute oxidative
stress in patients, using either OJ-AHT or EBOO (and perhaps even rectal
insufflation). Probably he also realizes that this stress must be adeguate (otherwise it
is a placebo), calculated (i.e. neither below nor too much above threshold levels) and
transitory. This is important because we do not want to ovenide the antioxidant
defence system nor cause any toxicity. Let us make a very unscientific remark : the
central airn of ozonetherapy is to give a precise, atoxic shock to an organism
which for various reasons has gone astray; the hope is that repeated, timely
shocks will readjust several biological functions by means of many rnessengers
(ROS, LOPs and autacoids generated by ozone) delivered by circulating blood
to the whole body. We really wonder if this book will serve to revise the dogmatic
concept that "ozone is toxic any way you deal with it" and will prompt some
orthodox scientists and c1inicians to try it. One study we should do as soon as
possible is to evaluate and compare the pharmacokinetics of LOPs and TBARS
(even ifthey are several and heterogeneous) in single patients during :

a) a standard 0 3-AHT (as described in the Appendix)

b) an EBOO treatment (Chapter 17)
c) a BOEX
d) a standard rectal insufflation (Chapter 19)

By assessing several parameters and comparing them after each of these four
procedures, we could gain a fair idea of the magnitude of the biochemical
modifications and their therapeutic benefit.
Another important study is to evaluate which of these four procedures is most
effective in raising the adaptation to COS and, in so doing, yielding c1inical
improvement (Chapter 22) .
Fifthly, does BOEX to 02::llJ have some advantages? During the treatment, there
is a loss of 300-500 g of water due to intense perspiration, normal for sauna bath ing.
This loss of water is ridiculously advertised as greatly bencficial because the "body
gets rid of oxidised toxins" in this way! Transitory hyperoxygenation is also
considered relevant, but it would be absurd to increase pOz levels through the skin
when we could increase them far more simply by breathing humidified oxygen for
one hour. The transitory thermal stress (due to the sauna) associated with the acute
oxidative stress is possibly an advantage because it may enhance and accelerate the
adaptation to COS . It is weil known that moderate hyperthermia positively
modulates the immune system during infection and cancer, and we will discuss this
topic in Chapter 24 (cancer section). On the other hand , excessive hyperthermia
presents several risks (cardiovascular failure, etc .), induces a hypercatabolic state
and immune depression; hence it must be avoided. An initial leukocytosis, followed
by a modest leucopenia, was observed after exposure to OZ-03 , as weil as to Oz
atone, in our study and was probably due to a transient release of IL-8. This agrees
weil with our previous data (Bocci et al., 1998b) showing that [L-8 is a chemokine
 QUASI-ToTAL BODY EXPOSURE TO OrO] 211

that is released rapidly by leukocytes in blood that has been briefly exposed to O 2-
0 3. It may be useful in patients with infections, but it is necessary to explore this
finding further and look for other cytokines such as 11-2, 11-12, IFNy and GM-CSF.
The observed hyperthermia is more likely due to the sauna than to the pyrogenic
effect of some cytokines.
In spite of our necessarily approximate approach, we feel that our studies have
some merit because they were the first to evaluate scientifically new ideas which
have revived a stagnant field, restricted for three decades to 03-AHT and rectal
insufflation.
What might be the practical usefulness ofBOEX and does it have a future? Ifwe
listen to commercial advertising, which claims to eure cancer and AIDS, it will have
a bright future. Yet we do not believe that the future of ozonetherapy lies in the
claims of charlatans. However, we would like to compare the pros and cons of the
four current methods. If one uses the standard, optimised 03-AHT method (Chapter
16 and Appendix), one is able to slowly treat several ailments without any risk to the
patient, but one venous puncture is necessary. EBOO (Chapter 17) seems to be more
rapid and perhaps more effective (it is too early to be sure), but two venous
punctures are necessary and the technical application is more costly and complex.
Rectal insufflation is extremely easy to do (once instructed by the
ozonetherapist, the patient can do it at horne by hirnself), very cheap and practically
free of risk . Yet it is often objected to and the delivery of a precise dosage is always
uncertain , although it may be beneficial in certain pathologies (see Chapter 19).
BOEX has distinct advantages: it is simple to perform, fairly inexpensive, non-
invasive (no venous puncture) and does not involve the handling of potentially
infectious blood, a point highly appreciated by medical personnel. We have noted
some problems: the cabin must be technically improved and BOEX is best
performed in a well-organised c1inie or in athermal resort with an entrance room,
treatment room , adjacent room to allow a comfortable one-hour rest for the patient
and another room with a shower. Whether this approach will truly become useful
remains to be established by RCTs , but at this stage it seems to represent a
promising tool to rnodify the biologieal response in some pathological states:

• Chronic viral diseases (HBV, HCV, herpes I and II, HIV, HPV). It may be useful to
treat chronic fatigue syndrome (CFS), even though it is probably not a viral disease.
• Metastatic cancer, to avoid palliative chemotherapy, whieh is usually useless and
associated with a very poor quality of life. However, it could be tried as an
immunoadjuvant at earlier stages with polychemotherapy.
• Vasculopathies, particularly hind limb ischaemia due to atherosclerosis, Buerger
disease and diabetes. Necrotic ulcers and dystrophie lesions must be
simultaneously treated with topical therapy (Chapter 16). Patients with severe
coronary atherosclerosis, recent myocardial infarction or severe hypertension
may undergo BOEX, but without sauna bathing, starting with a lO-min period
and scaling up slowly . Patients with asthma and BPCO must be treated
cautiousl y.
• ARMD , particularly the atrophic form. Keeping the sauna at a low level.
212 CHAPTER 18

• Sclerodennia with Raynaud's phenomenon.

• Moderate bums, to prevent or reduce bacterial infections and enhance healing .
• Some muscular-tendinous lesions in athletes, to reduce muscle contraction and
alleviate pain .
• Skin disease, such as infections, psoriasis, perhaps atopic dennatitis and eczema .
• Advanced Iipodystrophies, such as Madelung disease . The lipodystrophy
occurring during HAART mayaIso be advantageously treated

Our provisional protocols involve a course of therapy every other day (three
times weekly) during the first and second weeks . We always insist on the "start low,
go slow" paradigm to allowing for adaptation to COS . The sauna temperature should
be gradually scaled up from 70 °C to no more than 90 °C, with periods from 10 min
to a maximum of 25 min.
Only time, careful observation and hard work can tell us how best to proceed.
 CHAPTER 19

RECTAL INSUFFLATION OF O 2-03 (RI)

As early as 1935, before the advent ofOJ-AHT, Payr and Aubourg (1936) suggested
to insufflate 02-0J into the colon-rectum. This approach has been widely adopted in
Europe, as weil as in Russia , Cuba and other countries, because of its
inexpensiveness and the lack of aversion to rectal medication. Even in several states
of the USA, where ozonetherapy has been prohibited (mostly because it is badly
used by quacks) , many HIV patients used to do their own auto-insufflation using a
smalI, often imprecise generator. In Califomia, Carpendale et al. (1993) were
allowed to carry out a study in AIDS patients with profuse diarrhoea due to
opportunistic Cryptosporidium infection; they obtained temporary improvement in
some of the patients. The main field of application is represented by rhagases, anal
and rectal abscesses with fistulae, proctitis , bacterial cholitis, Crohn's disease,
ulcerative cholitis and chronic viral (8 and C) hepatitis (Knock et al., 1987; Knock
and Klug, 1990). Even ischaemic diseases and dementias (Gomez Moraleda, 1995)
have been treated with RI, which was postulated to have a systemic effect.
Anecdotes reporting beneficial results are scientifically worthless and, besides the
Cuban study in patients with dementias , there is an urgent need of RCTs . A possible
systemic effect seems supported by recent studies in the rat (Leon et al., 1998;
Barber et aI., 1999; Peralta et aI., 1999, 2000), in which it was shown that IR for two
weeks induced adaptation to COS .
Rectal insufflation of 02-0J is an easy, inexpensive method of delivering ozone,
which is practically risk-free, apparently beneficial and without side effects . In spite
of the fact that hundreds of thousands of treatments are performed in patients every
year, it is not really known whether and how these gases affect some fundamental
physiological and biochemical parameters. It is unfortunate that this simple
treatment has been neglected, indeed scomed, by mainstream medicine . Thus there
are several questions to be addressed:

I) Is 02-0J absorbed by the intestinal mucosa?

2) Does RI have only local effects or systemic ones as weil?
3) Bearing in mind the rapid and intense toxic effects of ozone on the respiratory
mucosa, is it wise to expose the intestinal mucosa to ozone?

Only Knoch et aI. (1987) have examined the PV02 modifications after rectal
insufflation in the rabbit. They found that PV02 values in a mesocolonic vein, portal
vein and liver parenchyma increased by about 230, 121 and 127% of the basal
values, respectively, 8-20 min after rectal insufflation of 150 ml gas. The PV02
values retumed to baseline after 50 min.

213
214 CHAPTER 19

The result that oxygen is absorbed is not surprising because it is weil known that
several gases (C0 2, CH 4 , N 2, H2, O 2, H 2S) either ingested or produced by the
bacterial flora are partly absorbed and even exhaled with expired air. But what about
ozone? As we cannot measure a PV03 directly, we have to assess it indirectly by
measuring compounds generated by ozone. As explained in the previous chapter,
ozone is not passively absorbed Iike oxygen. In contrast to the respiratory mucosa,
overlain by a thin film of fluids (hence, almost unable to quench the harsh oxidant
activity of ozone), the gut mucosa is abundantly covered by the glycocalyx and a
thick coating of water containing mucoproteins and other secretion products with
marked antioxidant activity. Luminal contents mayaIso have a higher antioxidant
concentration than plasma (HaIliweIl et al., 2000). However this gel-mucous layer is
not uniformly dispensed and certainly there are more or less protected areas.
Residual faecal contents are also variably present and may interfere with gas
absorption. In order to c1arify this problem, we carried out experiments in two rabbit
models after insufflation of either O 2 alone or O 2-03 into the colorectal lumen. Two
approaches were used: firstly , conscious animals were slightly seda ted with 5 mg
diazepam and kept comfortably in arestrainer cage that allowed the collection of
small blood sampIes after cannulation of the marginal vein of the ear . Secondly,
owing to the need to simultaneously examine portal and peripheral (jugular vein)
blood after laparotomy, the animals were deeply anaesthetised for the experimental
period (up to 65 min) . AIthough the results cannot be translated into the human
situation owing to profound anatomical differences, they are informative about a
systemic effect. Details are reported in Bocci et al. (2000).
To minimize the experimental stress, we first evaluated the effect of colorectal
insufflation of O2-03 in non-anaesthetised rabbits, simply collecting blood sampies
from the marg inal vein ofthe ear. The results reported in Figures 83 and 84 show:

a) a slight increase of PV02 values already 20 min after gas insufflation, confirming
the rapid absorption of O 2 previously described by Knock et al. (1987) . PvC0 2
and pH values remain within the physiological range;
b) concomitantly, there is a constant increase (not statistically significant) of
TBARS va lues up to 60 min after gas insufflation, when they start to decline.
Conversely, the PTG values decrease and reach a minimum after 90 min . Both
parameters return to baseline values 24 hours thereafter.

In the second phase ofthe study, we evaluated the parameters by simultaneously

collecting blood sampIes from the portal and jugular veins . The latter vein gives
more precise information about peripheral plasma levels than the marginal vein,
where unpredictable arterial-venous shunts occur. As expected, PV02 values rise
quickly (10min) in the portal vein, are slightly higher than in the jugular vein and ,
after 40 min, slowly return to the pre-insufflation levels . pH and PvC0 2 values vary
within a normal range in anaesthetised rabbits (Fig . 85) . The results regarding a
possible effect of 0 3 on levels of peroxidation and thiol group oxidation are new and
interesting : there is a more rapid and substantial increase of TBARS in the portal
plasma than in the peripheral plasma (Fig . 86) . The peak is reached in both veins
within 40-50 min and then the levels decline. PTG values decrease progressively
 RECTAL INSUFFLATION OF O2-03 215

and reach a minimum at 50-65 min in both portal and jugular blood. Thus for both
parameters, the effects of ozone are more evident and rapid for portal blood but they
appear to die off about 40 min after gas insufflation. Control animals insufflated
with oxygen showed only negligible modifications of TBARS and PTG values
indicating that the experimental procedure was not responsible for the change
observed after O 2 -0 3 insufflation.

90
80

--1
Ci
"T"
70
E
Er.r 60 •.
0
50
?
40 J
30 r---
. - - .--.-----..- / ~--'-------i

I
T.-t-Y--t~
-.'1~ _...
"C) · 40 ~ 1

~ !i T
Er
0
o
N
35 -j
i
,i
-1
~ 30 ~

25

7,5

:r: 7,4
a.

7,3

7,2 ·~__r-..,.I7'
/<'~/----------
I
O' 20' 35' 60' .90' 120' 24h
Time

Figure 83. Modification ofpartial pressure 0/02 (PvO?J. CO 2 (PvCO?J and pH values 0/
venous blood withdrawnfrom the marginal ear vein ofsix rabbits before (0) and after
colorectal insufflation 0/150 ml gas mixture (Or03) with an ozone concentration of 20
ug/ml. Values represent the M :f: SD.
216 CHAPTER 19

0,6 -,---- - - -- - - -- - - -,

0,5

i 0,4
2>
~ 0,3
~
__ 0,2

0,1

0,0 _'--TUu.=-

0,7
0,6
~ 0,5
Cl 0,4
li: 0,3
0,2
0,1
0,0
0' 20' 35' 60 ' 90' 120' 24h
TIme

Figure 84. Modification ofthiobarbituric acid reactive substances (TBARS) and protein
thiol groups (PTG) in the plasma (marginal ear vein) ofsix rabbits before (0) and aft er
colorectal insufflation 0/ J50 ml gas mixture (02-0 J) with an ozone con centration 0[20
IJg11l11. Values represent the M r SD

~ ..
5~

.~ .0 '
.,

'

o~ ~5

~ ae

r
25 -J-- - - - - - -...-.- .- - ...- -
.0 ·
8·
~ 40

7,5 +-- - - -

--,----,.---- - ,'- -
o 10 25 40 so 05
Tkne (min)

Figure 85. Modi/kation ofpartial pressure 0[02 (P,.o2Y. CO 2 (Pl'C0 2) and pH values of
venous blood withdrawnfrom portal (0) andjugular (~ veins offive rabbits before (0) and
after colorectal insufflation 0/ J50 ml gas mixture (Or03) with an ozone concentration 0{20
ug/ml. Values represent the M rSD,
 REeTAL INSUFFLATION OF O2-03 217

In conclusion, it appears that RI can exert a local and a rapid systemic effect
due to absorption of compounds generated by the interaction of ozone with the
luminal contents. Figure 87 attempts to illustrate that ozone dissol ves rapidly in the
lumin al wate r, where it partly reacts with faecal material or/and is reduced by a
myriad of antio xidants. It cert ainly gen erates both ROS (some H 202 ) and LOPs by
reacting with residual, un absorbed PUF As. The former compounds, like oxygen,
pass through the muscularis mucosa (MM) and enter the circulation via lymphatic
and venous capillaries . This is interesting and would support the contention by
Mattassi et al. (persona l communicat ion) that the beneficial effect of RI in chronic
limb ischa emia is equ ivalent to 0 3-AHT. If this result can be confirmed, it will be
helpful for patients because they will be able to do automedication and avoid
repeated ven ous punctures. Furthermore, in Chapter 14, Figures 45 and 48 show that
prolonged RI in athletes and aged subjects caused an increase of both ATP and 2,3-
DPG in erythrocytes. These results are the more surprising because every
ozonetherapist knows how imprecise and uncertain can be the application of ozone
and the volume of gas ret ained in the gut.

0,7 .f.

j
_ Jugular • • .,..L
0,6 ~ c::J Portal r= ,.
!
~ 0,5 -i

~~ 0,41 T
,.I I ,·r
0,3 I
I
... 0,2
r-- II
0,1
0,0 _..
I
I
-- '-- ,~-
I
0,6 -
I,
0,5
T .*
~
.,:;,Q.4 .
...
l
(!:l
0,3
a..
0,2
0,1
0,0 I
0 10 25 40 50 .65
Time (min)

Figure 86. Mod ification of thiobarb ituric acid reactive substan ces (TBARS) and protein
thiol groups (PTG) in plasma withdrawnfrom the portal (emp ty boxes) andjugular (black
boxes) veins of five rabbits befor e (0) antialter colorectal insufflation of 150 ml gas mixture
(Or O.!) with an ozone concen tration o{ 20 ug/ml. Values represent the M ± SD. Statistical
signific ance (p <O.05) is indicated hy an asterisk
218 CHAPTER 19

Figure 87. A schematic vie w ofthe trans/ er ofthe 0 r0.! gas mixturefrom the colonic lumen
into the submucosa. Both gases dissolve in the luminal mucous lay er. but ozone reacts
immediately and decomposes into a number 0/ ROS and LOPs. These are absorbed with
water via venous and lymphatic capillaries in the submucosa below the muscula ris mucosac
(M M) .

This leads to the discussion of some technical details in terms of gas volume, 0 .1
concentration and schedule of administration.
RI should be done after defecation or after an enema, when the rectal ampulla is
empty. The patient must lay on one side and try to rela x; often he/she prefers to
personally insert the disposable, oil-lubricated polyethylene (rubber must never be
used ) catheter (30-40 cm long ). The insert ion is easy and it should not stimulate
peristaIsis. In this regard, the gas has to be introduced slowl y and in step s of 50- 100
ml every 1-2 min . If it is done quickly , the gas will be expelled at once. The gas can
be introduced via a) a manual two-wa y silicone pump connected to the gas just
collected in a polyeth ylene bag , or better with b) a 50 ml silicone-coated syring e,
clarnping the catheter each time after insufflation. We can obta in goo d compliance if
we start with 150 ml and slowly scale up to about 500 ml depending on the patient's
 RECTAL INSUFFLAnON OF O 2-03 219

tolerance. This volume can easily be retained for at least 20-30 min. Knock et al.
( 1987) insufflated up to 800 rnl in 1 min, but I cannot confirm this and it is likely
that the patient would rapidly expel most of the gas . Carpendale et al. insuffiated
from 700 to 1300 ml of gas (up to 30 mg ozone daily) in AIDS patients, hoping the
gas would diffuse into the whole colon. This was adesperate, almost useless
enterprise because Cryptosporidium contaminates the whole gastro-intestinal and
biliary tract.
The patient should be left to rest for at least 15 min after RI to avoid rapid gas
expulsion and to allow the reaction of ozone with the luminal contents.
The 0 3 concentration is important to induce local and generalized effects but
there is general consensus that it should not exceed 40 ug/ml, In my experience, this
concentration often elicits painful cramps, particularly in patients with ulcerous
cholitis or when the application is done after an enema, suggesting a dangerous
stimulation ofthe local gut reflexes. Ifthe overlaying mucus has been washed away,
this high concentration might cause direct damage to the enterocytes and we sho uld
not forget that ozone can be mutagenic (Chapter 20) . Thus I suggest to begin
treatments with 3-5 ug /ml and slowly scale up to 30 ug/ml if the patient tolerates it
weil. It has been written that in the case of haemorrhagic ulcerative cholitis, an
ozone concentration of 70-80 ug/ml should be used for haemostatic purposes, but
this could induce cytotoxic damage and is not advisable. Moreover, on the basis of
the concept of inducing ozone tolerance, it appears reas onable to reach the
concentration of 30 ug /ml in 2-3 weeks. Whether it is worthwhile reaching the
highest ozone concentration of 40 ug /ml will depend on the type of pathology,
patient tolerance and other information that can only be obtained by daily
observations during a weIl controlled clinical study. Treatment can be done daily or
every other day . Table 17 provides an example of a flexible schedule.

Table 17. A possible schedule ot ozone administration by RI.

Weeks DllYS Concentration Gas volume Total 0 3 Range

0 3 (J1g/ml) (ml) dose (mg)
I 3 100 0.3
3 5 150 0.75
5 8 200 1.6
Low-Medium
1 10 200 2.0
2 3 10 250 2.5
5 15 250 3.75
I 20 300 6.0
3 3 25 350 8.75
5 30 400 12.0
Medium-high
I 35 400 14.0
4 3 40 450 18.0
5 40 500 20 .0
 220 CHAPTER 19

If the patient responds positively to the therapy, it could be continued 2-3 times
per week , maintaining a high or medium 0 3 concentration.
I am not in favour of the IR approach because the effective ozone dose is never
known due to the faecal contents and other variables. Yet ladmit that it is the
simplest and most practical option to be adopted in poor countries. I must
emphasize, however, that the catheter and syringe must be disposed of after each
treatment to avoid cross-contamination.
If we can prove (by appropriate RCTs) that IR also has therapeutic activity in
vascular disease, chronic hepatitis and intestinal diseases, we will have to promote
RI, the Cinderella ofapproaches, to the rank of0 3-AHT and EBOO .
Sixty-six years after the introduction of RI and after millions of applications with
no cause for complaint, we can say that this approach, if properly performed, does
not seem to induce adverse local effects. It appears reasonable to think that a
judicious ozone dosage, the mucous layer, the antioxidant system and the adaptive
response of enterocytes are all responsible for the lack of toxicity. In Chapter 24, we
will briefly examine the pathogenesis of the diseases where RI is best employed, but
here it may be useful to speculate (unfortunately I have no experimental data) about
the local effects of ozone. These may be as folIows :

a) Biochemical effects. In the studies already cited (Leon et al., 1998; Barber et al.,
1999; Peralta et al., 1999, 2000), RI in rats upgraded the enzymatic antioxidant
response in liver and kidney but the enterocytes were not examined.
b) Bactericidal effects. The human colon-rectum contains up to 500 g of about 400
species of mostly anaerobic bacteria, and O2-03 may partly change the
environment for a short while. Except in particular conditions, like clindamycin-
associated enterocolitis (Schulz, 1986), bactericidal activity per se is probably
unimportant but may cause the release of LPSs and muramyl peptides. These
compounds are among the most potent cytokine inducers (Chapter 14, Table 9)
and in large amounts are responsible for toxic shock syndrome and likely death .
However, in physiological conditions, the daily absorption of traces of LPSs
bound to specific proteins and to lipoproteins is considered essenti al for
maintenance of the basic cytokine response and an alert immune system (Bocci,
1981b, 1988b, 1992c) . Particularly in the last paper, it was postulated that the
somewhat neglected gut flora has a crucial immunostimulatory role. This idea
remains valid today and it is possible that RI favours a slight increase of LPS
absorption with the consequence of enhanced activation of intrahepatic
lymphocytes, Ito 's and Kupffer's cells (O'Farrelly and Crispe, 1999), which may
change the evolution of chronic hepatitis.
c) Modification of the bacterial flora eguilibrium. Ow ing to the multiplicity of
bacterial species, this remains a complex area . However, the normal flora
contains Lactobacillus (Lb) acidophilus, Lb. bifldus, Lb. fermentum , Lb. casei,
Streptococcus faecalis , S. thermophilus, S. bulgaricus, Escherichia coli, Proteils
and a variety of enterocci. The bacteria and their products interact with each
other and with the enterocytes, goblet and enteroendocrine cells (producing a
myriad of hormones) and the GALT (Hooper and Gordon, 200 I) . On the other
hand, it is weil known that contaminated food, water and antibiotics can subvert
 RECTAL INSUFFLAnON OF OZ-Oj 221

this dynamic symbiosis by allowing the establishment of pathological bacteria

and fungi like Candida albicans, C. tropicalis, Torulopsis glabrata, etc. The
successive dysmicrobism usually has far-reaching deleterious consequences,
ranging from transient to chronic enterocolitis and to autoimmune reactions. We
shall examine (Chapter 24) several attempts to correct it in order to restore
normal homeostasis. Whether RI with a daily input of OrOj can re-equilibrate
the bacterial flora and lead to normal immunoreactivity remains to be
demonstrated (and explained), although anecdotal results suggest a beneficial
effect.
d) Effects on the GALT. The gastrointestinal compartrnent represents almost 40%
of the whole immune system. Besides the famous plaques described by Johann
Konrad Peyer (1653-1712), over a total intestinal surface ofsome 300 m Z, there
are about 1011 immunocytes per m Z or about one per 6-7 enterocytes .

Intra-epithelial immunocytes are mainly T Iymphocytes, either o-ß of thymic

origin or y-8 of local origin. The latter induce a Th-2 type response that is anti-
inflammatory and immunosuppressive, quite important to prevent excessive
stimulation due to alimentary, bacterial, viral and toxic antigens . Perdue (1999) has
emphasized that a continuous cross-talk between immunocytes and enterocytes may
maintain healthy homeostasis and prevent breakdown of the mucosal barrier and
inflammation . In spite of interesting hypotheses (Fiocchi, 1998, 1999; van Parijs and
Abbas, 1998; Okabe, 200 I), the etiology and pathogenesis of both ulcerative colitis
and Crohn's disease remain uncertain and it is difficult to identify the culprits that,
step by step, constitute the disease. Using the current paradigm of T-cell
homeostasis , ulcerative colitis seems compatible with a poorly polarized Th-2
response while Crohn' s disease is characterized by an excessive Th-I response. In
other words, any alteration of the balance between pro-inflammatory (IL-I, IL-2,
IFNy, TNFu) and anti-inflammatory cytokines (IL-IO, TGF-ß) appears critical, and
an excessive release of IL-4, which affects the enterocytes , also appears important in
ulcerative colitis (Perdue, 1999).
Another piece of the puzzle is represented by a more or less adequate synthesis
of Hereman's "protective vernix" , i.e. A-type immunoglobulins (Ig) produced by
plasma cells (B Iymphocytes) . IgAs have a critical role in neutralizing foreign
antigens and this may limit the onset of an autoimmune process . Once this starts, the
vicious circle is complicated by other cells, namely cytotoxic Iymphocytes,
monocytes, macrophages and granulocytes, and by the release of other inflammatory
compounds such as ROS, proteinases, eicosanoids and PAF.
During the last twenty years, official medicine has made a great effort to sort out
this intricate problem. Yet still today Crohn's disease remains a serious affliction.
Nevertheless, with the usual superficiality, a few ozonetherapists have claimed that
the trivial RI can eure Crohn 's disease. If this were true, no patient should miss this
opportunity and we ought to present a rational basis for using ozonetherapy. Table
17 shows a possible treatment scheme that could be adopted for RCTs, but will it be
useful? The local treatment could be combined with two weekly OrAHT. However,
to be honest, I am not certain that it will work because it is almost impossible to
222 CHAPTER 19

predict if ozone will be able to re-equilibrate the immune response and lead to
normal mucosal metabolism. There is only one thing that is certain today and that is
that official Medicine does not even mention ozonetherapy. Hanauer and
Dassopoulos (2001) have reviewed some 20 therapeutic approaches and ifwe do not
start to be serious, ozone will remain a quack remedy.
 CHAPTER20

THE POTENTIAL TOXICITY

AND MUT AGENICITY OF OZONE

One reason for the unpopularity of ozonetherapy in the medical field is that the
toxicity of ozone is considered equal to that of RaS. In fact, there are substantial
differences because ozonetherapy is occasional and can be controlled whereas
endogenous ROS formation goes on unperturbed throughout Iife (Farber et al.,
1990; Ames et al., 1993).
The topography of formation of RaS is also quite different: mitochondria, which
convert 95% of the inhaled oxygen to harmless H20 , are the main source of RaS
since at least 3% of oxygen is converted to O2. ' (Richter et al., 1995). Dismutation of
O 2. ' by SODs (Fridovich, 1995; Carlsson et al., 1995) is the source of H 202, whose
reduction may generate the fearsome, non-specific OH·. Halliwell (1994) estimated
that a 70 Kg human produces no less than 0.147 moles or 5 g/day of O,", whereas
one 03-AHT uses less than 20 mg of ozone, equivalent to less than 0.4% of the
minimum daily production of O,"!
The huge formation of endogenous RaS in mitochondria, deeply imrnersed in
the cell, explains the damage to mitochondrial DNA (Wiseman and Halliwell, 1996),
which is oxidized about 10 times more than nuclear DNA (Richter et al., 1988) and
remains persistently damaged (Yakes and Van Houten, 1997) .
Conversely, ozone acts frorn the outside on the plasma, which is a huge reservoir
of antioxidants. Nonetheless, the ozone dose added to blood must reach a threshold
level in order to generate sufficient H202', which passes from the plasma into the
cytoplasm where it triggers several biological effects . It must be very c1ear that for
ozone to act we have to induce a calculated, transitory, acute oxidative stress
that is rapidly corrected by the antioxidant system. Thus, there is no doubt
regarding the formation of peroxyl radicals, hydroxyaldehydes and perhaps traces of
OH· and HOCl in the plasma. What is important to note is that all the vital cell
compounds, such as enzymes, proteins, RNA and DNA (Van der Zee et al., 1987;
Stadtman and Oliver, 1991; Ames et al., 1993), are spared during the extracellular
ozone decomposition.
Particularly in the USA, ozonetherapy is regarded as a "barbaric" therapy
(Chapter 2) and unscrupulous ozonetherapists and quacks have done their best to
reinforce this concept. However, it is now time to c1arify this issue; without
prejudices, we must evaluate the merits and demerits and put an end to the
confusion between the constant oxidative stress due to oxygen and the
occasional acute stress due to ozone.

223
224 CHAPTER 20

Knowing the importance of oxidative DNA lesions in ageing and cancer, I am

not surprised when often asked : is ozone mutagenic? And does ozonetherapy
accelerate ageing? To my knowledge, Fetner (1958) was the first to study this
problem and to demonstrate (Fetner, 1962) that human KB cells exposed to ozone
showed chromatid breakages similar to those produced by x-rays. While this report
caused much concern, it must be said that experimental conditions were
unphysiological because "cells were washed once in a protein-free saline solution
to remove extraneous reducing materials. Ozone is very reactive, and small
amounts of reducing materials provide considerable protection". Subsequent
investigations were conducted in somewhat more realistic conditions : Gooch et al.
(1976) exposed human leukocytes to ozone in culture medium without antioxidants
and, as expected, observed a slight increase in the frequency of chromar id-type
aberrations. However, leukocytes from mice exposed to ozone inhalation did not
show any chromosome damage. At about the same time, there were two discordant
reports: one by Merz et al. (1975) showing an increase in chromatid aberrations and
the other by McKenzie et al. (1977) unable to demonstrate any damage in
Iymphoeytes colleeted from volunteers exposed to 0.4-0.5 ppm ozone for several
hours . In order to overcome this inconsistency, Guerrero et al. (1979) carried out a
careful study using the sensitive analysis of sister chromatid exchange (SCE); they
concluded that there was no difference in the SCE values of Iymphocytes collected
from students exposed to either 0.05 ppm ozone or air for 2 h. Once again, however,
WI-38 cells incubated with serum-free medium showed a linear dose -related
increase in SCEs per chromosome spread over an ozone concentration range from 0
to 1.0 ppm for ) h, implying that the lack of serum allowed chromosomal damage.
The study by Sarto and Viola (1980), although suggesting cytogenetic damage in
chronic ozone-exposed workers, must be regarded as ineonclusive because smoking
or other confounding factors were not taken into consideration. Subsequently,
Victorin (1992) extensively reviewed this topie and stated that "no cytogenetic
effects have been reported for bone marrow cells or spermatocytes and the few
experimental and epidemiological studies with human subjects do not allow a
conclusion on the cytogenetic effects of ozone in human Iymphocytes". Victorin
(1994) also reviewed the genotoxicity of NOx . The latest study by Diaz et al. (1995)
is important because it was specifically carried out in Iymphocytes of eight Retinitis
pigmentosa patients before and after 15 treatments of 03-AHT. The results showed
no signiftcant differences in SCE, micronuclei frequencies and proliferation index
values between control and ozone-treated Iymphocytes.
As far as induction of tumours is concemed, lung adenomas were induced in the
sensitive strain A/J but not in Swiss- Webster male mice after 4.5 months of
inhalation exposure to 0.8 ppm ozone (Last et al., )987). In mice, prolonged ozone
inhalation at very low concentrations seems to prevent or reduce multiple lung
tumours promoted by urethane, probably by inducing adaptation to COS (Schulz,
personal communication). Kozumbo et al. (J 996) showed that in cultured human
lung cells incubated without catalase, the formation of DNA single strand breaks
could be due to hydrogen peroxide and/or ozonized arachidonate. Finally, Witschi et
al. (1999) concluded that animal studies do not support the idea that ozone is a
pulmonary carcinogen.
 THE TOXICITY AND MUTAGENICITY OF OZONE 225

Trying to sum up this important topic, it appears that the lack of natural
antioxidants is critical in allowing mutagenic changes in cells exposed to ozone
in vitro for a length of time . After the removal of plasma, washing and
resuspension in physiological media without or with only a small amount of
antioxidants, erythrocytes and other cells become very sensitive to even very low
ozone concentrations, as demonstrated by intense haemolysis or apoptosis . Instead
of stigmatizing ozonetherapy as toxic, published papers (Goldstein and Bachum,
1967; Gooch et al., 1976; Freeman et al., 1979; Sato et al., 1999; Fukunaga et al.,
1999) ought to have pointed out the importance of antioxidants in preventing
damage. It has correctly been shown that genotoxicity induced by H202 or iron
overload can be checked if tissue culture media contain adequate amounts of
antioxidants (Leist et al., 1996; Matos et al., 2000).
Another blunder has been made by several cell biologists by keeping ce11
cultures under constant ozone exposure (Tarkington et al., 1994) at extremely low
levels, but for several hours or days. The conclusion that ozone is toxic even at
minimal levels is misleading : firstly, the level of antioxidants in tissue culture media
is far lower than in plasma and, more seriously , the authors have not taken into
account the cumulative ozone dose . Although I have already mentioned this point
(Chapters 4 and 8), it is appropriate to remind the reader that ozone solubility is very
high : according to Henry's law, every second, ozone solubilizes into water, reacts
and disappears, so that more ozone solubilizes and reacts, and so on. Although
minimal, all ofthese continuous reactions lead to increasing concentrations ofH20 2,
OHo, 4-HNE, etc., which go unquenched on account ofthe scarcity and consumption
of antioxidants and thus become toxic . Therefore, with time, even the lowest ozone
concentration becomes toxic.
In contrast, exposure of blood to O2-03 is performed with ozone concentrations
within the therapeutic window and is over after one min during EBOO and about 5
min during 0 3-AHT . However, if the ozonetherapist uses either ozone
concentrations above 80 ug/ml or ozonized saline, he makes another blunder. A
typical example is represented by the IV infusion of ozonized saline : Foksinski et al.
(1999) infused into POAD patients 500 ml of saline ozonized for I h, obviously
without worrying about the high content of newly formed HOCI; they recorded a
450% increase of 8-0HdG in the Iymphocyte DNA isolated from some of these
unlucky patients . In Chapter 10, it was mentioned that 8-0HdG is a marker
indicating the occurrence of DNA oxidation. Thus Foksinski 's result should
preclude the use of ozonized saline . An interesting, but not unexpected, result ofthis
study was that only 3 of 6 patients showed the appearance of this marker, suggesting
a possible genetic sensitivity to oxidative agents . Kleeberger et al. (1997) were the
first to show that a susceptible strain of mice presents a different ozone sensitivity
(see also Cho et al., 2001). Unfortunately, the state of the ozonetherapeutic art is still
too primordial to allow examination of the genetic pattern of antioxidant enzymes in
putative patients. Nevertheless, it is necessary to check TAS levels in plasma and
ascertain ifpatients have a G-6PD deficiency.
A reassuring fact is that after millions of 0rAHT sessions performed in
Germany, Austria, Switzerland and Italy, neither serious acute nor chronic side
effects, nor an increased cancer incidence has been reported (Chapter 21). Yet this
226 CHAPTER20

does not absolve us from improving our eontrols by monitoring oxidative stress and
lipid peroxidation in patients during and after ozonetherapy, e.g. by measuring F2-iP,
hydroperoxides andlor other parameters in plasma or urine. This is easier said than put
into praetiee, but I am hopeful that a speeifie and reliable assay for routine clinieal use
will soon beeome available. Furthermore , we must never lower our attention to the use
ofprecise ozone generators and ozone doses that are biologically aetive but atoxie. If
we work eorreet1y, perhaps in due time the scientifie eommunity will aeeept the
eoneept that ozonethe rapy is not eomparable to life-long endogenous ROS toxicity .
In eonc1usion, I eannot avoid saying that ozone is potentially toxic and
mutagenic (like all cytotoxic drugs!). Yet so rar, our experimental data and
c1inical evidence (albeit not entirely reliable) has not shown any risk.
 CHAPTER21

SIDE EFFECTS AND CONTRAINDICATIONS

OF OZONETHERAPY

Jacobs (1982) is the only one to have published an extensive study on the negative
effects of ozonetherapy. In spite of the famous "toxicity" of ozone, it appears that
the incidence is only 0.0007 %, one of the lowest in medicine. Four deaths due to
direct IV injection oithe gas were included in his data , but since 1982 other deaths
for a similar reason have occurred, of which at least two (1997 and 1998) in Italy.
However, due to the HIV epidem ic and improper use of ozone , it is likely that other
deaths have occurred but have not been reported. Thus Jacobs' data are valuable
only with regard to side effects such as nausea, headache, tiredness and the like.
The reader will have to trust the Italian experience: at the Verona Congress
(1999 ), Dr. Giuseppe Amato , who has always worked at the Hospital in Conegliano
(Veneto) and is a very scrupulous ozonetherapist, reported only rninor side effects
and no sequelae in a thousand patients treated with 03-AHT for several years. Our
experience at the Siena University Hospital is also significant: since 1995, we have
performed about 6000 0 3-AHT in ARMD patients and about 100 in patients with
fibromyositis , as wel1 as about 350 EBOO sessions, countless topical applications in
chronic ulcers of the limbs , and either direct (intradisc) or indirect (chemical
acupuncture with Or03 in the para vertebral muscles) O 2-03 applications in about 40
patients with backache.
Firstly, regarding side effects occurring during and after 0 3-AHT, we have to
distinguish about 5000 treatments performed between 1995 and June 2000,
unfortunately using PVC autotransfusion bags. These contained 63 m1 of CPD (up to
450 ml blood could be collected), but usual1y only 200-250 ml blood was withdrawn
to treat ARMD patients . In order to avoid any contamination, the excess of CPD
(about 30 ml) was not discarded and it was likely responsible for one of the fol1owing
side effects. Plastic autotransfusion bags have the foUowing disadvantages:

a) Venous puncture must be done with a venous fistula needle set (G 17) and
occasional1y some patients faint with fear. No case of lipothymia has been
observed, probably because, after blood collection during the ozonization
process, about 100 ml saline was infused via the same needle.
b) Some patients (almost always women) reported a tingling sensation in the lips
and tongue, most frequently towards the end ofthe reinfusion. This did not occur
with very slow infusion, nor with the new atoxic system (ACD wel1 calibrated to
the blood volume), nor with heparinized blood; hence this symptom has been
attributed to a transitory slight hypocalcaemia due to the excess of citrate.

227
228 CHAPTER21

c) During blood reinfusion, more frequently women (l 0-15%) have reported nausea,
a feeling of stornach bloating and a strange metallic taste in their mouth, which
could be due to Zn-stearate or Zn-2-ethyl hexanoate present as additives in PVc.
d) For about 1 day after the first 4-5 treatments, 20-30% of both male and female
patients reported feeling tired . Another 10-20% had no symptoms, while 50%
reported a feeling ofwellness. It must be noted that in all ofthese patients (60-80
years old), the 03-AHT were perfonned with a constant ozone concentration of
65-70 ug /ml per ml of blood , without scaling up the dosage . In retrospect, this
was amistake and particularly in aged patients we must begin with a low 0 3
dose (20 ug/ml) and slowly scale up to 40-50 ug /ml .
e) After 4-12 OrAHT sessions, four women patients (one with the history of an
episode of anaphylactic shock to a wasp-sting) had a sudden appearance of a
diffuse erythematous skin rash, with itching, nausea , hot flushes and slight
hypotension, at the end of a blood reinfusion . IV infusion of I g methyl-
prednisolone Na-succinate relieved the symptoms in about 2 h. Interestingly,
before undergoing ozonetherapy , one ofthese patients had participated as a control
and had received 12 02-AHT without any problem . These eases of definitive
intolerance were attributed to progressive sensitization to an immunogen due to
phthalates bound to lipoproteins or to other PVC-additive components.

From June 2000 until the present, we have been using the new atoxie system
(gIass, etc.), a precise volume of 3.8% Na Citrate to blood (l :9 v/v or 25-225 rnl)
and the slow scaling up ofthe ozone concentration (from 20 to 50 ug/ml) . All ofthe
above-mentioned side effects have disappeared, and no others have appeared.
Moreover, no allergic-Iike intolerance has been observed. Because the glass bottle is
under vacuum, blood is easily drawn with a smaller needle (G 19).

I. CAN OZONETHERAPY INTERFERE WITH CONVENTIONAL

TREATMENT?
Yes, at least in the case of hypertensive patients being treated with ACE inhibitors.
This has been reported by Mattassi's group (unpublished), who repeatedly observed
a sudden marked hypotension upon reinfusion of ozonized blood in these patients.
This effect may be due to the activation of the kallikrein-kininogen cascade , as
reported by Shiba et al. (1997) and Abe et al. (1998) . However, plasma bradykinin is
degraded within minutes and very slow infusion reduees this adverse effeet.
In Chapter 16, it was mentioned that it has become fashionable to injeet O 2-0 3
into the trigger points detectable in the paravertebral muscles of patients (see also
Chapter 24, the problem of backaehe). I defined this approach as "chemical
acupuncture" (Bocci, 1998a) and a Iikely explanation is that ozone acts locally on
nociceptors and evokes a rapid and effective (in about 2/3 of patients)
antinociceptive response through chemical mediators. While direct intrad isc
injection of02-0 3 (to degrade the proteoglycans ofthe hemiated disc) remains in the
hands of orthopaedists and neurosurgeons, some physicians decide overnight to
become ozonetherapists and , with the opportunistie encouragement of an ozone
 SIDE EFFECTS OF OZONETHERAPY 229

generator salesman, begin to practise the indirect method without knowing anything
about ozone. This situation has some risks : in May 200 I, one death in Naples was
due to this therapy. Immediately after IM injection, ozone dissolves locally in the
interstitial H 20 and generates several ROS: if the 0 3 concentration is around 20
ug /rnl and the gas volume exceeds 5 ml, a very acute pain may cause vagal
hypertone (inotropic and chronotropic negative effects) , which may culminate in
cardiac arrest. If the patient is lucky , he will recover or undergo only transitory
lipothymia (bradycardia, hypotension, profuse perspiration, transitory loss of
consciousness, etc.). Therefore, it is advisable to practise "chemical acupuncture" with
appropriate precautions (Chapters 24 and 36). If these are taken, the backache may
show remarkable improvement after 6-10 sessions, but the patient must be warned
about the aphorism "no pain, no gain" and that, with the appropriate technique , the
pain will be bearable and will last for only 5-10 min. In general, the improvement of
baekaehe outweighs the transitory therapeutic pain, so that the compliance is good.
With a proper injeetion, the risk of oxygen embolization is nil and only one case of
subcutaneous haematoma has been reported (Fabris et al., 2001).
The direet intradisc injeetion may present very slight side effects and rare
transitory cephalea. However, in the case of a herniated eervical disk in a young
athlete, Alexandre et al. (1999) reported that the patient presented abilateral
amaurosis fugax after the injection, which fortunately reversed after one day . This
serious eomplieation ean more likely be attributed to transitory ischaemia of the
vertebral arteries due to an erroneous position of the head during ozonetherapy than
to the ozone itself.
For all of these negative effects of ozonetherapy, the physician must be ready to
resolve the problem (Chapter 36). Instead, it seems that a prompt intervention was
tragically de1ayed in the three cases of death . In this regard, a specific training
course would be most useful.
A positive side effect already mentioned in Chapters 16-19 is that most patients,
particularly those that feel depressed and asthenie before 03-AHT, EBOO , BOEX or
RI, report a feeling of weil being and euphoria. Whether this is due to the "staging"
of the proeedure or to ozone or to oxygen, or to all these factors, remains unknown.
For a long time , I have wished to perform a kinetic study of the hormonal pattern
(CRH, ACTH, Cortisol , DHEA, GH, ß-endorphin, somatostatin plasma levels) after
these types of treatment. Needless to say, such a study must be performed with
appropriate eontrols and this, unfortunately, will imply the collection of many blood
sampies. It will be more difficult to evaluate whether there is also a eoncomitant
serotonin and/or dopamine upsurge .
Wehave no reason to be1ieve that ozonetherapy represents an inflammatory
stimulation. Yet to be sure, we must also assess the kinetics of positive acute phase
reaetants (APR) , namely C-reactive protein (CRP) , serum amyloid A (SAA), 0.1-
antitrypsin, al -acid glycoprotein (orosomucoid), haptoglobin, C3 and C4 , ß-2-
mieroglobulin (ß2-M). In contrast to other ozonetherapists, we have often noted a
slight increase of plasma fibrinogen and prothrombin, which we have referred to
improved hepatic metabolism.
230 CHAPTER21

A final unresolved question is the optimal time of day to perform the systemic
approaches . On the basis of circadian rhythms of crucial honnones, I believe that the late
aftemoon is the preferable period (Bocei, 1985b), but this is not neeessarily praetieal.

2. ARE THERE CONTRAINDICATIONS FOR OZONETHERAPY?

This is partieulariy important for systemic therapy and the burden of ozonetherapy
(not so irrelevant for BOEX with sauna) must be weighed against the clinieal
eondition ofthe patient. Moreover, the following situations preclude its use :

a) Patients with a signifieant defieit of G-6-PD (Chapter 14, Erythroeytes). The

problem of genetie suseeptibility to ozone is surely appropriate (MeDonnel1,
1991 ; Prows et al., 1997; Kleeberger et al., 1997) and besides individual TAS
levels, eaeh patient has a different enzymatie profile, different absorption and
metabolism of antioxidants and so on . However, we are still at a very
rudimentary stage with regard to the resolution of these problems.
b) Pregnaney, partieulariy the early phase, to exclude any mutagenic risk, although
it is unlikely.
e) Patients being treated with ACE inhibitors.
d) Abnormal situations with hyperthyroidism, thromboeytopenia and serious
eardio- vaseular instabi lity.
e) Allergy to ozone has been c1aimed, but what is it? I reckon that the
hypersensitivity of asthmatie patients to ozone has ereated some confusion.

3. DOES PROLONGED USE OF OZONETHERAPY GIVE RISE TO SEQUELAE

SUCH AS TUMOURS, DEGENERATIVE DISEASE, ETC.?

The question is theoretically appropriate because ozone induces ROS and these are
at least partly responsible for many ailments and ageing. This is the fourth time that
I propose that all national Health Authorities should oblige al1 ozonetherapists (who
ought to be physieians with appropriate specifie training) to keep a medieal register
in whieh to preeisely folIowand reeord all pathologieal events appearing in patients
during and after ozonetherapy.
The following form may be useful :

Sumame and Name .

Sex Age .
Address .
Type of employment .
Diagnosis , ..
Type of0 2-0 ) treatment ..
Period oftreatment: from to .
Clinieal evolution .
 SIDE EFFECTS OF OZONETHERAPY 231

Whenever possible, the patient should be followed during subsequent years and
it should be noted if the disease improves or persists or worsens, as weIl as the
possible appearance ofnew pathologies related to oxidative stress .
Great attention should be given to:
agranulocytosis, asthma, atherosclerosis, bone marrow dysplasia or atrophy,
cataract, degenerative diseases, emphysema, fibrosis (paravertebral muscles),
gastrointestinal diseases, hepatitis, hypertension, leukemia and other haematological
neoplasias, multiple sclerosis, neurodegenerative diseases (Parkinson, dementias),
renal sclerosis, rheurnatoid arthritis, scleroderma, skin carcinomas, SLE, solid
turnours, others .
 CHAPTER22

THE ADAPTATION
TO CHRONIC OXIDATIVE STRESS (COS)

" To express an opinlon is as easy as it is arduous 10 acquire th e necessary knowledge "

Juvenal (ca. 55 - ca. 130 AD.)

In 1962, Ritossa observed a pattern of Drosophila salivary gland ehromosome puffs

indueed in response to 30 min exposure to 37"C (from the normal temperature of
25"C). Tissieres et al. (1974) demonstrated an inereased expression of proteins with
moleeular weights (MW) of 26 and 70 KDa and these proteins were denominated
"heat stress proteins" (HSPs) . I would guess that Ritossa never imagined how
important it was to have shown the great potentiality and flexibility of the eell to
respond to environmental stimuli. Sinee then, a number of HSPs with different MW
(- 15-30, 32, 60, 70, 90, 100, 110 KOa) have been deseribed and reviewed (Burdon,
1986; Welch, 1992; Cioeea et al., 1993; Csermely et al., 1998; Kiang and Tsokos,
1998; van Eden et a1., 1998; Oe Maio, 1999; Jolly and Morimoto, 2000).
Any change of the external environment or internal "milieu" disturbs eell
homeostasis, but if the stress is tolerable, or graduated in intensity, the cell can
adapt to it and survive. If it is too violent, the cell programmes its own death, or
apoptosis (Jaeobson, 1996). The large array of stresses includes hyperthermia,
hyperoxia, hypoxia , isehaemia, excessive ROS and LOP production , heavy metals,
ethanol, hypoglycemia , pH modifications, viral, bacterial and parasitic infections,
antibiotics, malignaney, radiation, metabolie inhibitors, amino acid analogs and
most likely mental stress and hormonal derangement. Obviously, ozone has to be
included: HSP70 is expressed after ozone inhalation (Su and Gordon, 1997) and an
attenuation of ozone-induced inflammation has been recorded after repeated daily
exposure (Christian et a1., 1998).
In relation to the variety of stresses, the cell either upregulates or synthesizes
probably a hundred -or more new proteins like HSPs, glucose-regulated proteins
(GRPs) and oxidative shoek proteins (OSPs), which allow the cell to resist against
new and even more intensive stresses. As has been observed in the cytokine field,
also in this ease there is an apparent redundancy, with the final aim of establishing
"stress toleranee" and insuring cell surviva1. The teleological significanee of the
HSPs can be deduced from the following observations:

a) They are universally present from bacteria to fungi to plants to mammals. The
anti-ozone responses in plants (Kangasjarvi et a1. , 1994; Benes et a1., 1995;
Schmieden and Wild, 1995; Robinson and Rowland, 1996; Ranieri et a1., 1996;
Sharma et a1., 1996; Maccarrone et a1. , 1997; Pell et a1., 1997; Sharma and

233
234 CHAPTER22

Davis, 1997; Bilodeau and Chevrier, 1998; Desikan et a1., 2000; Ranieri et al.,
2000) are fascinating and strongly suggest that humans can do the same.
b) The HSPs from organisms of the animal kingdom are highly conserved in regard
to their structure and functional activities.
c) The cell's ability to promptly and continuously respond to a moderate stress
condition.
d) At least some HSPs help to maintain protein folding and translocation of
polypeptides across membranes. That is why they have been indicated as
molecular chaperones (Hightower, 1980; Ellis and Van der Vies, 1991).
e) The role of HSPs varies widely from the induction of thermotolerance to
protection against ROS by upregulating the synthesis of antioxidant enzymes.
Moreover, they regulate inflammatory and autoimmune diseases, antigen
presentation, recognition of malignant cells and even inhibition of tumour cell
death . In this case, they do not display a useful activity because the over-
expression of HSP70 inhibits apoptosis, thus favouring proliferation of breast
tumour cells (Nylandsted et al., 2000) . Some ozonetherapists may find this story
boring and thus I must explain where it might lead .

1. THE PARADOX OF OZONE AS A DRUG AND THE CONCEPT

OF THE MULTIV ARIED THERAPEUTIC RESPONSE TO COS
In the "Nature of Disease", Paracelsus (1493-1541) wrote that "the body possesses
the high art of wrecking but also restoring health". Although he was not aware of the
existence of the oxidative stress response, his intuition (made in the Renaissance)
appears valid still today .
I believe that the future of ozonetherapy, if there is one, rests on the pedestal of
the OSP, but it will be necessary to demonstrate how it can be obtained, its
relevance and amplitude. The concept is old and it has been named in different ways
only because it has been observed in different pathological conditions: Murry et al.
(1986) pioneered the concept of "ischaemic preconditioning" for the heart, which
after undergoing abrief, non-lethal period of ischaemia can become resistant to
infarction from a sub sequent ischaemic insult. Once the mechanism of action is
c1arified, pharmacological preconditioning will be applied in the clinic and will
improve surv ival (Cohen and Downey, 1996; Nakano et al., 2000) .
"Hormesis", the term used by Goldman (1996), means "the beneficial effect of a
low level exposure to an agent that is harmful at high levels" , e.g. very low doses of
radiation induce an adaptive response to a high dose in human Iymphocytes (Olivieri
et al., 1984; Wolff, 1996). Calabrese and Baldwin (200 I) have presented numerous
examples of stimulatory responses following stimuli below the toxicological threshold .
"Oxidative preconditioning" has been achieved by warm ischaemia or
hyperthermia (Kume et al., 1996; Yamamoto et al., 2000), transitory Iimb ischaemia
(Sun et al., 1999), 03-AHT (Bocci, 1996a,c) and RI of ozone (Leon et al., 1998;
Barber et al., 1999; Peralta et al., 1999, 2000) . However, when ozone is used, the
term "ozone tolerance" or "adaptation to COS" seems more appropriate because it
 THE ADAPTATION TO THE OXIDATIVE Sl:RESS 235

specifies the inducing agent. We face a real paradox ; since ozone , the "toxic gas",
can be tumed into a useful drug able to readjust an otherwise irreversible state of
chronic oxidative stress. There are several pathologies, such as neurodegenerative
diseases, chronic viral infections (HIV and HCV) and autoimmune diseases, in
which a vicious imbalance between oxidants and antioxidants becomes firmly
established, leading more or less rapidly to death .
How can modem medicine correct this? In Chapter 24, we shall see that there are
several therapeutic possibilities, but in most cases they are not entirely successful.
While they can limit the damage, they are unable to modify the involution. What can
ozone do? It cannot remove the primary causes of these diseases, but it may reverse
the chronic oxidative stress (Fig . 88).

HIV infection,
ageing,
1
OZONETHERAPY
autoimmune and

I
neurodegenerative
disorders

Figure 88. The normal and pathological redox balance. The scheme suggests that, by
upregulating the expression ofantioxidant enzymes, ozonetherapy mayfavour normalization
0/ the impaired redox balance
In Chapters 11 -13, the ozone treatment has been envisaged as a transitory and
calculated oxidative stress resulting in a sort of therapeutic "shock" for the ailing
organism . Ozone realizes this shock because generates a number of messengers
that can reach all cells in the organism. It is not easy to imagine how it works, but
236 CHAPTER22

we must have some hypotheses and ascertain if they are correct. First of all, it is
necessary to distinguish local from parenteral treatments. Among the latter, OJ-AHT
and EBOO are reasonably precise, and perhaps HzOz, but especially LOPs with a
long half-life, are the most important putative agents. BOEX and RI are somewhat
imprecise approaches, but nonetheless likely to put LOPs, generated on the
cutaneous and mucosal surface, into the circulation. Thus, during and immediately
after one of these treatments, cells throughout the body will suddenly receive a
pulse of LOPs and newly generated autacoids. As discussed in Chapters 12 and
13, these compounds are heterogeneous and undergo dilution and metabolism
(Vasiliou et al., 2000) . Over a certain level they are cytotoxic, while below I 11M
they can act as physiological messengers after binding to cell receptors. Liu et al.
(2000) recently reminded us that 4-HNE is a key mediator (but not the only one!) of
oxidative stress-induced cell death; thus it would seem crazy to increase plasma
levels of LOPs. A possible idea is that cells undergoing intracellular COS (of
endogenous origin due to a virus or a mitochondrial dysfunction or inactivation of
antioxidant enzymes) are PARALYSED and unable to reverse it,
One possible way to interrupt this anergy might be an adequate and atoxic
stimulation ofthe cell membrane receptors via a few LOP molecules. Ifthe cell is still
able to transduce the message to the nucleus, via phosphorylation of protein kinases
and the like, it may represent the alarm signal able to reactivate gene expression,
leading to the synthesis of HSPs and antioxidant enzymes. While a too high LOP
concentration will detinitively kill the cell, a very low and gradual stimulation
may favour a re-equilibration of the oxidant-antioxidant balance, as shown in the
lower part ofFigure 88. Ifthe idea is correct, this strategy prescribes that ozonetherapy
should start at low concentrations just above the threshold level , which is in line with
the old concept "start low, go slow", Experiments in laboratory animals (Leon et al.,
1998; Barber et al., 1999; Peralta et al., 1999, 2000) treated daily with RI of ozone (I
mg per kg body weight) have shown that the adaptation to COS , with consequent
resistance to prolonged ischaemia or toxic compounds, can be achieved in two weeks
(10 treatments). In a healthy volunteer (Fig . 89), as weil as in HIV patients, we found
that it took from 2 to 4 weeks (5 to 9 O]-AHT; twice weekly) to detect an increased
plasma level of SOD and a concomitant decrease of the TBARS level.
Which proteins and enzymes are important in correcting the COS ? This problem
has been investigated in the last 15 years and it has been shown that hyperoxia and
ROS can induce increased levels of SODs, GSH-Pxs, GSSGR and catalase (Heng et
al., 1987; Rahman et al., 1991; Shull et al., 1991; Doroshow, 1995; Hernandez et al.
1995; Bocci, 1996a ; Tacchini et al., 1996; Sagara et al. 1998; Wang et al., 1998;
Barber et al., 1999; Chen et al., 2000b; Csonka et al., 2000), encouraging us to
evaluate the effects of ozonetherapy.
The practical application of EBOO in a gradual form should allow us to clarify
the problem. Indeed, we plan to systemically investigate the levels of antioxidant
enzymes, G6PD (Puskas et al., 2000) and some HSPs inducible by H 20 2 and ozone
(Jornot et al., 1991 ; Cardile et al., 1995; Kiang and Tsokos, 1998), before, during
and after EBOO. We are particularly interested in analysing the pattern of HO-I (or
HSP-32) because EBOO is likely to release traces of haeme and its breakdown
generates beneficial molecules, such as CO and bilirubin (Abraham et al., 1996), as
 THEADAPTATION TO THE OXIDATIVE STRESS 237

weil as free Fe 2 + whieh, if not promptly ehelated, may aet as a pro-oxidant (Dong et
al., 2000; Ryter and Tyrrell, 2000 ; Snyder and Baranano, 2001) . On the whole, HO-
l is beeoming a most interesting enzyme (Galbraith, 1999), involved in proteeting
the skin (Reeve and Tyrrell, 1999), in avoiding aeute haeme toxicity and iron
overload (Nath et al., 2000) , in suppressing endothelial eell apoptosis (Brouard et
al., 2000) , in rejeetion of mouse to rat eardiae transplants (Sato et al., 200 I) and in
proteeting heart, liver and lung against isehaemialreperfusion and hyperoxia injury
(Csonka et al., 1999; Amersi et al., 1999; Otterbein, 1999).

Adaptallve
1,4
phase
E
1,2 ~
\
1,0

-<
Q 0.8
:E
:E
:=. 0,6 0-_
- '"":9'"
./
0,4 ./
./

,..- . /
0,2 o:

0,0 L-L-_--'-_ _L-_--'-_ _L-_--'-_----' 0,0

f
25
°t 5 10 15 20 30

Mln DllYS
t t t t
Figure 89. An AMRD patient 's response to a single (left side) 01' tntermittent (right side)
infusion of 0 3-AHT (300 g blood treated with an ozone dose of 21 mg per session). MDA ,
malonyldialdehyde (0) and Mn-SOD (U/ml plasma. 0) are reported on the ordinate. Arrows
indicate the time of blood reinfusion.

Thus there is already supporting evidenee that the adaptation to COS ean be
realized with ozonetherapy. This seems the most effieacious possibility but ean the
same result be obtained with passive administration of antioxidants?

2. IS AN ANTIOXIDANT SUPPLEMENTATrON NECESSARY?

This is a reeurrent and fashionable theme, diseussed ad nauseam by vitaminologists
to promote their produets and by eharlatans, who may intoxicate patients with
megadoses of selenium, zine, iron and vitamins A, C and E.
Reeently, I was asked why the initial improvement of a CFS patient treated with
ozonetherapy was followed by a relapse after taking a megasupplementation of
antioxidants and seleniurn , indeed so mueh as to reaeh abnormally high plasma
levels. Obviously we eannot draw any conclusion from one patient, but we have
observed that high TAS plasma levels inhibit the release of IL-g, probably beeause
the ozone dose was totally quenehed (Chapter 14, Figure 53) .
238 CHAPTER 22

Authoritative scientists have often posed the question as to whether

supplementation with antioxidants (Antioxidant therapy, AT) reduces oxidative
damage in humans. The conclusion is that an equilibrated dose may be essential during
growth and useful in oxidati ve stress-related conditions, but there is little evidence that
it can be a defmitive remedy (Hennekens et al., 1994; Zino et al., 1997; Halli well,
I999b ; McCall and Frei, 1999; Pryor , 2000 ; Polidori et al., 200 I) . An excessi ve
amount may modulate the synthesis of HSPs and actually reduce the synthesis of HO-
I (Peng et al., 2000). Ifwe wish to tackle this problem realistically, we must consider:

a) the uncertainty ofintestinal absorption ;

b) the individual variability ofmetabolism and excretion;
c) the variable and often reduced uptake ofantioxidants by the cell ;
d) the possible reduced synthesis ofGSH (HIV infection) ;
e) the potential toxicity of excessive doses;
f) the inability of antioxidants to stimulate the synthesis of antioxidant enzymes;
g) ifnot, to inhibit this process.

Thus the problem of antioxidant supplementation cannot be neglected, but we

must use a correct and equilibrated amount, i.e. neither too little nor too much . "Est
modus in rebus! " (Horace, Serm. 1.1.1 06) . I repeat that , before starting
ozonetherapy, we should at least determine the TAS of each patient. If this is not
possible and if the patient is in a critical condition (cachexia, anorexia, great pain,
etc .), I feel it necessary to give a daily well-balanced and reliable supplementation of
antioxidants one week before ozonetherapy, calibrated at a low level. Moreover, in
the case of ademanding approach, such as EBOO perforrned in critical patients, we
can start with short treatment periods (20 min only, followed by 30, 40 , 50 and
finally 60 min , corresponding to the l ", 2nd , 3rd , 41h and 51h treatm ent , respecti vely)
and we prescribe the following oral supplementation:

0.5 g of vitamin C (morning ). As stated at pag. 85, this dose saturates the body
(Levine et al., 1996);
0.6 g of NAC (either morning or evening), see pag. 104 (Bridgeman et al., 1991;
Hack et al. , 1998);
an approved multivitamin complex (RD doses) including vitamin E and se1enium;
a rich dietary intake of fresh fruit and vegetables.

This AT regimen can be maintained throughout the therapy and it will allow us
to progressively increase the ozone dose without risk .
It may be useful to outline our standard treatment schedules:

I) 0 3-AHT, low-medium 0 3 concentrations (20-40 ug /ml) (225 011 blood + 25 011

3.8% Na citrate + 225 ml O 2-03; blood/gas ratio I : I). Two treatments weekly (M
and Th or Tu and F).
I st week : 20 ug/ml , total ozone dose per treatment 4 .5 mg.
2nd week: 25 ug /rnl, total ozone dose per treatment 5.63 mg .
 THE ADAPTATION TO THE OXIDATIVE STRESS 239

3rd week : 30 ug/ml, total ozone dose per treatment 6.75 mg .

4th week: 40 ug/ml, total ozone dose per treatment 9.00 mg.
2) 03-AHT, medium-high 0 3 concentrations (50-80 ug /ml). A schematic example
is given in Chapter 14.

If patients tolerate the treatment weIl, it can be continued for 2, 3 or 4 months

depending on the disease and the selected end-points. If patients have no side effects
and have shown therapeutic results (e.g. significant improvement of visual acuity in
ARMD; normalization oftransaminases and viral load reduction in chronic hepatitis;
marked improvement of circulation in chronic limb ischaemia with disappearance of
pain, healing ofulcers, etc.), the treatment can be continued for a further month.
After a rest period of one month, even if the improvement remains stable , it
seems worthwhile to begin a maintenance therapy of one treatment every two
weeks. From the outset, the patient must be informed that ozonetherapy can at best
only improve the outcome.
A possible scheme for RI has been reported in Chapter 19.
For BOEX and EBOO, a similar gradual approach to allow adaptation is advised,
and I must warn that these schemes are based on theoretical and anecdotal results.
One of the purposes of this book is to present the basic, yet still approximate,
knowledge of ozone activities in cells and body fluids, in the hope that the
ozonetherapist will become able on his/her own to modify the ozone doses and
schedules for different diseases and patients.
Therefore, Table 18 is intended to outline our present views. Only ifwe carry out
RCTs will we be able to optimize the treatments.

Table 18. Tentative guidelines regarding ozone concentrations

within the therapeutic window (20-80 ug/ml) to be used in different pathologies with the
classical 0rAHT (blood/gas ratio 1:1). twice weekly. Ozone concentrations are slowly
upgraded, no more than 5 ug/ml at a time, to achieve the adaptation to COS in 2-3 weeks.

PROPOSED 0 3 CONCENTRATIONS
initiaI final
Infectious diseases 25 70
Vascular diseases 20 40
Degenerative diseases 20 40
Respiratory diseases 20 40
Autoimmune diseases 50 80
Metastatic tumours 25 70

From examination of the table, two facts emerge: firstly, the idea "more is
better" is not always appropriate for ozone and its concentration must be calibrated
in relation to the effector and target cells; secondly, the need of serious
experimentation with appropriate controls to genera te valid c1inical data.
A RCT is ademanding enterprise (see next chapter) that will require a fairly
large number of patients, who will undergo either AHT with O 2-0 3 or only O2 . For
comparative purposes, a third arm involving AHT with the addition of an inert gas
240 CHAPTER 22

would certainly be useful, but it is not strictly necessary. A daily physiological

antioxidant supplementation should be administered to both groups of patients.
The results can be compared to a similar group of patients treated with the best
orthodox medical treatment.
 CHAPTER23

HOW TO EVALUATE OZONETHERAPY?

RCTs are indispensable to prove or disprove the validity of ozonetherapy. Yet,

because ozonetherapy is mostly in the hands of private physicians, very few studies
have been done . There are several problems and the first is that a complementary
treatment is always associated with opposite trends : most of the OPPONENTS are
physicians practising orthodox medicine who are skeptical despite knowing nothing
about ozone . This is the worst situation and I believe Einstein used to say that "it is
easier to split the atom than to remove a skeptic principle". On the other hand, some
physicians, quite correctly, are against charlatans, who entice desperate patients to do
ozonetherapy regardless ofthe physical, emotional and financial expense it may entail.
Some of the PROPONENTS, who seriously practice ozonetherapy and are in
good faith, perceive discrimination when their proposals are rejected or, even worse,
when regulatory agencies investigate them . Moreover, lack of funding leads to a
standstill. However, we cannot allow this any Ionger and we must decide beyond
any doubt if ozonetherapy is really effective and tolerable.
Depending on financial possibilities and the willingness of a University clinic or
a hospital unit to cooperate, proponents of a study could use two strategies:

1) preparation of a "best case series". This will not pronounce the final word but
will encourage further research. Interestingly, the National Cancer Institute has
clar ified that the Best Case Series programme was founded in 1991 to apply the
principles of evidence-based medicine to therapies not yet rigorously tested
(Vanchieri, 2000) . Patients included in a "best case series" MUST meet the
following criteria:
a) The diagnosis and stage of the disease MUST be clearly documented and all
pathological fmdings MUST be available for review.
b) Surnmaries ofpatients' previous therapies and responses MUST be provided.
c) A detailed description of patients' treatment with the complementary
approach MUST be available.
d) The treatment administered MUST be defined in detail so that other
clinicians can independently reproduce it.
e) NO OTHER THERAPY should be concurrently administered that could
account for the clinical response .
f) Valid supporting documentation (laboratory, radiological and clinical data)
MUST clearly show a positive response.
g) Depending on the entity of the clinical response a "sufficient" number of
patients SHOULD BE EVALUATED. Anecdotal experience of a few cases
is NOT relevant.

241
242 CHAPTER23

2) Conduction of areal RCT is the preferred approach if we want to interpret data

correctJy and reduce stubbom skepticism. Yet, there are plenty of problems:
a) Patients who accept ozonetherapy after being thoroughly informed are
usually reluctant to enter a randomized trial . Vanchieri (2000) reported the
case in which only 3 out of 200 patients accepted randomization. However,
we must explain that this is an unavoidable problem in the evaluation of new
therapies, conventional or not, and must be overcome (Jenkins et al., 1999).
Moreover, the control patient has the right to be properly treated at a later
date, free of cost. If patients cannot be recruited, a non-randomized,
prospective design is better than nothing. On the other hand, if it can be
clearly proved that ozonetherapy is absolutely beneficial, particularly in
critical patients, the control (0 2 only) becomes unethical, also on the basis of
the last Helsinki revision (2000).
b) Physicians practising complementary medicine , believing in good faith that
their treatment is effective, feel that randomization is unethical. Most of them
do not care about the possibility of a "placebo effect", indeed they actually
welcome it. This attitude is wrong and prevents acceptance of a complementary
approach. It must be explained that only results obtained with a posit ive, well-
conducted randomized trial are convincing and can be published by a peer-
reviewed journal, possibly stimulating eonfirmatory studies .
e) There are several requirements that must be fulfilled :
Permission obtained by the loeal Ethical Committee on the basis of a
preeise and well-defined protoeol including the analysis of all possible
variables. The definition of evaluation criteria, appropriate selection of
endpoints and possible toxic effects is very important.
Patient enrolment, clarifying the scope of the trial and the relevance of a
control group.
If possible, it is desirable to have a "blind" study, although it can be
shifted at a later stage .
Random alloeation of patients to the active or control groups. Groups
must be balanced for age, sex, stage of disease , etc.
Signed, informed consent by all participants.
Statistical advice about the analysis before starting and after the study .
d) Last but not least, there is the problem of funding a clinical trial. This is a
costly enterprise and, in the field of ozonetherapy, it is practieally impossible
to find finaneial support. Manufacturers of ozone generators are eertainly not
giant pharmaceutical industries. Nevertheless, they are myopie, since they
wish to seil their instruments but show little interest in supporting studies that
might prove the usefulness of ozonetherapy.
 CHAPTER24

OZONETHERAPY IN VARIOUS PATHOLOGIES

"Natura inflrmttatts humana e tardiora sunt remedia quam mala "

Tacitus (-54- 120 A.D.), The Life ofGnaeus Julius Agricola, Chapter 3
(From the nature ofhuman frailty, remedies work more slowly than illnesses)

What Tacitus asserted remains true even today with modem medicine, although he
could not know that some diseases (atherosclerosis, cancer) take years to set in.
The title of the 14th Annual Euro Meeting (March 5-8, 2002, Basel) is "The
Patient is Waiting", meaning that "At the start of the 21'1 century only a relatively
small proportion of all diseases can be adequately treated or even cured and only a
small proportion of all patients have access to medicinal products at affordable
prices" . Although this seems a pretty harsh judgement, it is true that for some
diseases we do not yet have a rational drug treatment, that diseases are often poorly
treated , and that too many patients in poor countries do not receive any benefit or
even worse are the prey of quacks . However, we can forget neither the value of
antibiotics and vaccines nor the long effort to fulfil Paul Erhlich's dream of the
magic bullet, which , in spite of enormous expenses, has so far yielded meagre
results and remains amirage.
I often complain that basic and clinical research on ozonetherapy has been too
little and too slow . Yet, therapeutic progress in cancer therapy has also not been as
fast and positive as had been predicted. Three decades have already passed since
President Richard Nixon dec1ared war on cancer and , although knowledge about
tumorigenesis has shown an incredible expansion, the mortality rate has barely
decreased. I very much hope that the new generation of drugs , like the specific
inhibitor 2-phenylamino pyrimidine (STI-57 1), which precisely targets the AbI
tyrosine kinase in chronic myeloid leukemia cells , lives up to expectations and does
not disappo int us as several potential miracle eures have done (Gorre et a1., 2001).
Indeed there always appears to be unforeseen toxicity observed during prolonged
treatment, as occurred for the HAART (Hruz et al., 2001 ; Fellay et a1. , 2001) which
in 1996 seemed to have resolved the problem of HIV disease.
All the hype during the last decade about research on gene therapy of tumours
and more recently therapeutic angiogenesis (Isner et al., 1996; Patterson and Runge,
2000; Simons, 2001), once again obtained at huge cost, has indeed generated new
knowledge and allowed authors to publish interesting papers in the best medical
journals. Yet, all ofthis has , so far, yielded only minimal practical results and even a
few deaths .
Recent developments in autoimmune diseases have highlighted potentially useful
new therapies with an anti-TNFa monoclonal IgG 1 antibody and a protein made of
two chains of p75 TNF receptor monomer fused to the Fe domain of IgG 1 (Hanauer

243
244 CHAPTER24

and Oassopoulos, 2001). However, it remains uneertain whether they are simply
able to alleviate symptoms for a while or, more importantly, to modify the course of
these diseases.
If I believed aneedotal results of ozonetherapy, obtained with minimal resourees
and manpower, I would say that perhaps ozonetherapy is not as bad or obsolete as
orthodox medicine depiets it. Yet rather than resting on false laureIs, I would incite
ozonetherapists to work hard and seriously; the sooner we clarify the validity of
ozonetherapy the better it will be for everyone.
As will be discussed in this chapter, the medical applications of ozonetherapy are
innumerable (Table 19) and this fact exposes the approach to medical derision. Is it
possible that ozone acts as a panacea or the ill-famed Theriaca? Aecording to
tradition, Andromachus (who was Nero's quack) invented Theriaea, a very complex
mixture able to neutralize poisoning and eure every illness! Indeed, he wrote a poem
in 175 verses (Oe Theriaca) to describe and praise it.

Table 19. Ozonetherapy is used in thefollowing medical spectalities.

Angiology Gerontology Oncology

Cardiology Gynaeeology Orthopaedies
Cosmetology Hepatology Pneumology
Oentistry Infectivology Rheumatology
Oermatology Intensive therapy Surgery
Gastroenterology Neurology Urology

The answer is a decisive NO and only a superficial observer or a sareastie

skeptic would say YES : In reality, ozonetherapy seems to exert beneficial effeets
in so many, and somewhat unrelated, pathologies beeause ozone aets at abasie
level on several blood eomponents with different funetions. Moreover, the
generated ROS and LOPs not only have the most powerful disinfeetant aetivity
but ean aet either loeally or systemieally in praetieally all eells of an organism.
The most important ROS are listed in Chapter 9 (Table 6), while only the most
signifieant LOPs (we know very little about them as ozone effeetor molecules) are
mentioned in Chapters 9 and 10. Target cells vary from bacteria, fungi , parasites to
blood and all eells ofthe body.
The reason why I decided to continue working in this controversial field is that I
believe, in contrast to the erroneous (that is only my opinion!) axiom that "ozone is
toxie any way you deal with it", that oxygen-ozone, if properly used, can act
contemporaneously as a disinfectant, an oxygen donor, an immunomodulator, an
inducer of antioxidants (this is a real paradoxl) a metabolie enhancer and perhaps as
an activator of resident stern cells . It is difficult to figure out how beneficial effects
can develop during ozonetherapy, but Figure 90 attempts to give a visual image of
how this can happen. The centre of action is always the plasma, where ozone
dissolves and generates all the effector moleeules that interaet with erythrocytes
(ER), BMC, granulocytes (GRAN), platelets (PLAT) and the Endothelium. All of
 OZONETHERAPY IN V ARIOUS PA THOLOGIES 245

these cells will be more or less affected depending on the ozone concentration
and the ozonization modality, which in any case must be absolutely risk-free.

Figure 90. The multivaried biological response of the organism to ozonized blood can he
envisaged hy considering that ozonized hlood cells and compounds interact with a number of
organ s. Some of these represent real targets (liver in chronic hepatitis, vascular systemfor
vasculopathies) , while other argan s are prohahly involved in restoring normal homeostasis

This aspect is important to understand the consequent effects that will be

directed to twelve different organs or functional sectors. The plasma and the
extracellular fluid are the media interconnecting the ozone generated compounds,
cell released moleeules (cytokines, growth factor, autacoids, PAF, metabolites, etc.)
and a multitude of target cells . Therefore, the ozonetherapeutic stimulus
simultaneously triggers many functional activities, thus allowing a multivaried
therapeutic response, but also prornotes a calculated, transitory oxidative stress
which, unlike chronic endogenous oxidative stress, has the value of a
therapeutic "shock". As an exaggerated example, somewhat like the dreaded
246 CHAPTER24

e1ectroshock that we used to inf1ict on schizophrenic patients when I was a medical

student! In our case , the difference is substantial as we do not harm the patient and
yet we induce one of the most important body responses, i.e. upregulation of the
antioxidant system which is far more than the simple adaptation to COS (Fig . 88) . If
this is true, and we can prove or disprove it experimentally, ozone may stimulate and
correct the natural resources gone astray, in accordance with the old saw "Medicus
curat, natura sanat" (The physician treats, nature heals).
There is a profound difference between modern orthodox medicine and
ozonetherapy. On the basis ofbiochemical, physiological and pha rmacological data,
the former tries to develop a specific drug able to correct the cause of the disease,
and if this succeeds the result is wonderful. Unfortunately this does not always
happen or the success is not complete because a disease generally causes many
dysfunctions and the use of a reductionist approach is inherently disadvantageous.
Although ozonetherapy has limited specificity, it has the considerable advantage that
"the hodgepodge of ozonized products" can mobilize natural resources that
eventually may resolve the overall problem .
With this, I am not saying that ozonetherapy is preferable; my deep feeling is
that, in any circumstance, we must first offer the patient the best treatment that
orthodox medicine provides. Only ifthis does not work, or has serious side effects,
or the patient does not comply with it, can we offer the option of ozonetherapy if it
is applicable to the disease . I would also take this opportunity to reject the term
"alternative" because ozonetherapy, as empirical as it is today, cannot be antithetical
but only complementary. This is the correct term. It would certainly be a wonderful
achievement if, one day, official medicine would state that "for this disease.
ozonetherapy is the therapy (4' choice" , This would mean that ozonetherapy has
gained respect but, I guess that plenty of water will pass under the bridge before that
day arrives.
In spite ofimportant advances, conventional medicine is still unable to provide a
definitive improvement in some pathologies. Thus it is reasonable and ethically
correct to take advantage of ozonetherapy when the best orthodox treatment has
failed. As typical examples, I will report treatment of a) suppurative infections
refractory to all antibiotics, b) III and IV grade hind-limb ischaemia facing
amputation, and c) chronic hepatitis patients either resistant to or intolerant of IFNa.
Occasionally, I may only touch upon the etiopathogenesis of a disease; the reader
can fare better by consulting Harrison's "Principles of Internal Medicine",
Nevertheless, the ozonetherapist must know all the pros and cons of all
conventional therapeutic modalities before suggesting the use of ozonetherapy. If they
have been unsuccessful or are unavailable, ozonetherapy can then be considered.

l. INFECTIOUS DJSEASES (BACTERIAL, VIRAL, FUNGAL, PARASITIC)

It is intuitive that ozone can have an important therapeutic role in various types of
infections because it generates ROS (0 2 OH', HzOz, NO' and HOCI), also produced
0
' ,

by granulocytes and macrophages during an infectious process (Badwey and

Kamowsky, 1980; Chanock et al., 1994; Anderson et al., 1997; Saran et al., 1999;
 OZONETHERAPY IN VARIOUS PA THOLOGIES 247

Titheradge, 1999; Babior , 2000) . Moreover, neutrophils have a wealth of antimierobial

proteins in their granules and release eytokines and autaeoids whieh, by exerting a
variety of effeets , eause tissue damage as weil (Witko-Sarsat et al., 2000) .
We observe that , owing to diffuse antibiotic-resistant baeteria, rieh eountries
continue to use often useless, expensive antibiotics, while poor countries, by sheer
necessity, use ozone whieh is quite aetive and so far has hardly induced resistance.
Ozone is profitably employed either as agas (0 2 + 0 3), which must be weil
contained in an ozone-resistant bag saturated with water vapour, or as ozonized
saline (to be used only topieally) , or better as ozonized bidistilled water, or as
ozonized oils for the treatment of war wounds, anaerobie infections, trophic ulcers
and bums (Miroshin and Kontorshikova, 1995). Abscesses, anal fissures, deeubitus
(bed sores), fistulae, fungal diseases, furunculosis, gingivitis, inveterate
oste omyelitis, peritonitis, sinusitis, stomatitis, vulvovaginitis and wound healing
disturbances have been shown to improve rapidly beeause ozonized solutions
display a c1eansing effeet and act as a powerful disinfectant, whieh kills even
antibiotic-resistant or anaerobic baeteria. On the whole, ozonized solutions eontrol
the bleeding, improve the metabolism and reduee the infection (Payr, 1935;
Aubourg, 1940; Rokitansky, 1982; Werkmeister, 1995; Shaschova et al., 1995;
Filippi and Kirschner, 1995; Wasser, 1995; Bulinin et al., 1995; Kudravcev et al.,
1995; Kasumjan et a1. , 1995; Steinhart et al., 1999).
In poor countries (I repeat), by sheer necessity, physieians have had to devise all
sorts of ways to employ the gas, or more easily the ozonized water, to avoid
environmental contamination. In Western countries, we still need to create the
mental attitude to profitably use ozone. Yet , I am eonvinced that, once medieal
personnel realize the advantages, it will be put into general use, to the benefit of
patients. Moreover , with the eurrent inerease in medieal costs, ozonetherapy
deserves attention because it reduces hospital assistance and is extremely cheap.
Obviously we will need to explain how ozone works and show what ozone
concentrations are appropriate for the particular infeetion or lesion. The sehe me
reported in Figure 71 (Chapter 16) shows that a concentration of 80 ug/ml (as gas)
can be used only during the first phase, in which there is pus, bacteria and necrotie
tissue. The wound must be cleaned and exposed to the gas for only 10-15 min.
Bidistilled water ozonized with 80 ug/ml has an effective content of about 20 ug /ml
0 3 and is far more practieal for cleansing the wound and changing the moisted
compress throughout the day . Ozonized oil can be applied for the night. As the
infection regresses, ozone concentrations must be lowered to 2-5 ug /ml to avoid
cytotoxieity and to activate local metabolism, cell proliferation and synthesis of
cytokines (POGF, bFGF, TGFß I, EGF, KOF), so as to promote synthesis of the
intercellular matrix and the healing process (Beck et al., 1993; Pierce et al., 1995;
Sporn and Roberts, 1993; Schmid et al., 1993; Slavin, 1996; Martin, 1997). Topical
treatment is easy to perform because daily observation of the wound is a good guide;
however, it helps to know that time , patience and complianoe are good allies .
The problem is more complex in systemic infeetions (peritonitis, large abscesses,
pleural empyema), possibly complieated with toxie and septic shock. Removal of
purulent material and rapid washing with ozonized water is useful , particularly
combined with 0 3-AHT whieh can be earried out 1-3 times a day at low 0 3
248 CHAPTER 24

concentrations (20-30 ug /ml per ml ofblood) . OJ-AHT is intended to improve tissue

oxygenation and metabolism but not to increase production of pro-intlammatory
cytokines, which are already superinduced by toxins. It is also not intended to
sterilize blood: although bacteria and viruses suspended in water are sensitive to
ozone, they become fairly resistant in plasma beeause of the proteetion exerted by
endogenous antioxidants.
Direet IV injeetion of gas , similar to the sterilization of drinking water in a
aqueduet, is simply a mad idea and is proseribed.
OJ-AHT (2-3 sessions weekly) must eomplement topical treatment of uleers and
wounds beeause the synergism leads to more rapid healing.
I will briefly deseribe three eases that have eonvineed me that ozone has great
therapeutic aetivity in suppurative infeetions :
• The first ease was reported by Dr . Salvatore Mieeli, of the Hospital at Corleone
(Palermo), during the I st Ozonetherapy Meeting in Palermo (Mieeli, 1999). The
patient was a woman, 51 years old, born with a eleft spine and sueeessive
paraplegia of the hind limbs. A previous infected deeubitus probably eaused
osteomyelitis of the right coxo-femoral joint with a fistula releasing a foul-
smelling seeretion (Fig . 91 a,b; Fig. 92) . For about six weeks, she was treated
intensively with several wide-spectrum antibiotics to no avail ; in the meantime,
she developed septie fever (-39 Oe), progressive caehexia, lethargy and profound
debilitation. With her permission, ozonetherapy was carried by : 1) 0 3-AHT (30
ug /rnl per ml of blood) every day during the first week and then onee weekl y,
and, most importantly, 2) repeated direet insuftlation (via a polyethylene catheter
introduced as far as it could go) of 02 -0J at a concentration of 40 ug /ml every 5
min for 1 hour. After only about 12 hours, the fever receded and purulent
secret ion ceased. Loeal gas treatment was then repeated every day for one week
and then twiee weekly for three months, when total resolution of the
osteomyelitis was shown radiologieally (Fig . 93). The general condition of the
patient returned almost to normal shortl y after ozonetherapy, with out antibiotie
support. After six months (Fig . 94), the patient was perfeetly healthy.
• Another ease of osteomyelitis was referred to me but thi s eolleague had no
radiological records . It appeared in a multiple myeloma pat ient und er eytostatie
therapy at the level of implantation of a eoxo-femoral prosthesis. The physieian
also used the dual approach : 0 rAHT twice weekly, but laeking a fistula he
repeatedly infiltrated the area surrounding the lesion with 50 ml of gas at a
coneentration of 20 ug /ml thrice weekly. He elaimed to have resolved the
problem after 5 weeks. Osteomyelitis can easily become a ehronic infeetion and I
feel that the timely use of ozone should be weil kept in mind .
• The third ease was a 67 year-old woman in dialysis at our Hospital since 1977 .
The problem started with an initial deeubitus in the eoceygeal area . In spite of
conventional therapy, the infeetion spread and she presented an extensive
necrotizing faseiitis in both legs and at the site of primary deeubitus (Fig. 95 ).
Antibioties and con ventional loeal therapy carried out by a dermatologist were
ineffective and the patient worsened, with septie fever and a semieomatose state .
Ozonetherapy was carried out in the Nephrology Unit , using the comb ined
 OZONETHERAPY IN V ARIOUS PATHOLOGIES 249

treatment. In this case, it was the EBOO approach three times weekly in
conjunction with intensive local therapy consisting in the continuous change of
compresses soaked in freshly ozonized water during the day, substituted with
ozonized oil at night. The therapeutic activity of ozonized oil is simply
unbelievable. The patient returned practically to new after two months . I
personally followed this case and I was amazed at her rapid recovery (Di Paolo
et al., manuscript in preparation).

Figure 91. (June 14. 1999). a)Radiograph ofthe rightfemoral head and iliac bone: the aspect
of thefemoral neck indicates the presence of an osteomyelitic process. b) The external aspect
ofthefistula
250 CHAPTER 24

Figure 92. (June 14. 1999) Radiographie image 0/ the osteomyelitic process visualized after
injection ofradiopaque material via a catheter. The catheter was also used to insufflate ozone
into the purulent cavity

Figure 93. (September 25. 1999) After
topical ozone application and OJ-ART. the
radiograph shows a striking improvement
and a marked reduction ofthe abscess.
There are signs ofosteoblastic
hyperactivity
Figure 94. (April 11. 20(0) The
radiograph shows an intense
osteometaplasia with disappearance of
the abscess cavity and resolution of'the
osteomyelitis
 OZONETHERAPY IN V ARIOUS P ATHOLOGIES 251

Figure 95. The amazing results obtained in one patient with necrotizingfasciitis treated with
parenteral (EBOO) and topical (ozonized water and oil) treatments. Extensive necrotic
lesions were present between the buttocks , Oll the legs and heels. Before (left) and after (right)
the treatment
252 CHAPTER24

1.1. The Special Case 0/ Helicobacter Pylori (Hp)

Hp is a gram negative, microaerophilic bacterium that is acquired in childhood; it
infects the stomach of about 50-80% of children and remains for life (Rowland,
2000) . In about half of subjects, Hp may cause peptic ulcer disease, chronic gastritis
and possibly gastric adenocarcinoma and gastric B cell lymphoma. Hp thrives in the
acid environment of the stomach by activating its own cytoplasmic urease, which
converts urea into CO 2 and ammonia (NH) . The ammonia neutralizes the gastric
acid that otherwise would diffuse into the periplasm and kill the bacterium or inhibit
its colonisation. A protein denominated Ure I, a member of the amidoporin family,
has the crucial role of allowing up to a 300 -fold passage of urea into the cytoplasm
of Hp (Weeks et al., 2000). Tombola et al., (2001) have also shown that the
cytotoxin VacA increases the transepithelial flux ofurea.
There is evidence suggesting that Hp infeetion is associated with inflamed
gastric mucosal epithelium accompanied by a marked local enhancement of CD4'
Th I-type response. This may explain why even eradication of the infeetion does not
always improve the dyspeptic symptoms. However, the current therapeutie approach
aims at eradicating the infection, which is possible in about 80% of patients after
treatment with omeprazole (a protonic pump inhibitor) and a combination of two
antibiotics chosen among amoxicillin, clarithromycin and metronidazole (the latter
two seem most effective). However, there are already problems of bacterial
resistance, lirnited patient compliance, high costs and the Iikelihood of reinfection
about I month after discontinuing the treatment.
It appears to me that Hp colonisation is a prototypie case in which the use of
ozonetherapy would be ideal: the bacilli are localized in the deep portions of the
mucus gel layer and in between this layer and the apical surfaces of the gastric
epithelial cells. They do not invade the mucosa and are rarely situated between
adjacent epithelial cells. Thus , there is nothing better than ozone and the ROS
caseade to transitorily create a hostile environment to Hp, since it is sensiti ve to
ozone . I would Iike to propose a protocol investigating the effeet of ozonized water
and ozonized oil on the model already used in experiments on Cryptosporidiosis and
Giardiasis. It may be sufficient to ingest a teaspoon of ozonized oil in the moming
on an empty stomaeh, followed by two-three glasses (200-300 ml) of freshly
ozonized water (final 0) concentration about I0 ug/ml) I hour before breakfast. The
oil is a bit disgusting but the water is easily drinkable. I would suggest continuing
this treatment for four weeks and then repeating all the tests (Hahn et al., 2000),
possibly the non-invasive ones (Hp antigens in faeces, urea breath test , whole blood
antibody tests) during the exploratory phase . A possible substitute of ozonized oil is
to drink a glass of ozonized full cream milk rieh in PUF As. It may weil have a
similar effect and it is more palatable.
Is there any danger? No adverse effects have been reported in children with
Giardiasis. Moreover, ozonized water is promptly distributed over the large mucosal
surface and reacts instantaneously with mucoproteins, which at least partly
neutralize it. Hp bacilli are fully exposed to the oxidative stress and will be
eliminated. Obviously the enthusiastic patient should not exceed in drinking too
 OZONETHERA,PY IN VARIOUS PATHOLOGIES 253

much ozonized water to avoid the loss of cytoprotective mucus, with the risk of
insulting epithelial cells.
There is a practical disadvantage since the patient must take freshly prepared
ozonized water or perhaps milk from the hospital pharrnacy at least every other day
(for four weeks). Ozonized oil is quite stable in the refrigerator. I have no doubt that
an effective vaccine will eventually be developed; this mayaiso solve the problem
in poor countries where Hp infection is widespread.

1.2. Viral Infections (HIV-1 Infection)

Most lipid-enveloped viruses in aqueous media are sensitive to ozone because it
easily oxidizes glycoproteins and lipoproteins of the external envelope (Akey and
Walton, 1985; Shinriki et al., 1988; Vaughn et al., 1990; We11s et al., 1991;
Carpendale and Freeberg, 1991). However, the virucidal activity becomes less
certain when viruses are in biological .fluids or, even worse, when they are
intracellular (hepatocytes, epithelia, CD4+ lymphocytes, monocytes, glial and
neuronal cells, etc.), where the potent antioxidant system protects viral integrity.
This emphasizes once again the irrationality of direct IV injection of gas. Moreover,
by means of some viral components, e.g. HIV-1 trans-activator of transcription (Tat
protein), HIV and HCV are able to inhibit or downregulate the synthesis of
antioxidant enzymes such as SOD and GSH-P x. This induces an intracellular
chronic oxidative stress (increase of 0/', OHO), which favours viral replication and,
by accelerating cell death, enhances expansion of the disease (Ho, 1997). There are
unequivocal experimental data (Westendorp et al., 1995; Oe Maria et al., 1996;
Ranjbar and Holmes, 1996; Schwarz , 1996: Akaike et al., 1998; Larrea et al., 1998;
Romero et al., 1998; Rubartelli et al., 1998) that fully agree with the fact that an
excess ofNAC, GSH and cystamine suppresses in vitro HIV replication (Roederer et
al., 1990; Kalebic et al., 1991; Bergamini et al., 1994), while a GSH deficiency
impairs survival (Herzenberg et al., 1997). The increased release of extracellular Tat,
associated with circulating IFNa, also suppresses immune ce11 activation and inhibits
the production ofC-C chemokines , leading to immune co11apse (Zagury et al., 1998).
Since 1993, the mass media have misinforrned the public, boasting that
ozonetherapy could eure HIV infection . I was so perplexed about these rumours that
I wrote to Dr. A. Fauci and I appreciated his concern that ozone could promote
further oxidative stress in HIV patients (Chapter 2).
The spreading of false, sensational news is a typical but reprehensible propensity
of complementary medicine, particularly ozonetherapy, favoured by quacks to
exploit anguished patients and by some producers and salesmen of ozone generators.
In the period 1993-95, the epidemie was mounting, AZT monotherapy was hardly
useful and yet the wenderful results with ozone had not been published in scientific
journals, except for a pilot study of its safety and efficacy by Garber et al. (1991) .
Unfortunately, this work was very badly conceived: although it showed neither
efficacy nor toxicity, it could not support any valid conclusion and I am still puzzled
as to how it was accepted for publication. Can you imagine the scientific validity of
254 CHAPTER24

a test using 10 rnl ofblood (HIV infected) treated with an unknown 0 3 concentration
plus heat (?) plus irradiation with IV (?) and then reinjected IM.
In 1994, I feit as if I was between the devil and the deep blue sea : many patients
at the hospital refused AZT and other therapies and solicited me to perform
ozonetherapy. Garber's study was uninformative but news from Germany was
c1aiming exceIlent results and I wondered what was true. I must confess that even in
these days, when I receive news that ozone (direct IV injection or hyperbaric 0 3 !!)
works weIl, I get confused and I start to wonder if I am mistaken.
I had mixed feelings when I tried to evaluate the pros and cons (Bocci , I994a, b,
1996a) in order to elaborate a rational approach : I) By oxidizing the viral gp 120 or
gp41 , ozone may inactivate some free viruses in plasma, but the required
concentration is between 40 and 80 ug /rnl gas per ml of blood. 2) If it were true that
infected leukocytes have a decreased content of antioxidant enzymes, we might even
induce their death, but this could lead to further viral dissemination. In any case , the
ozonization of 250 mI blood (about 1/20 of the blood mass) would have only a
negligible impact on the total viral load. 3) It remained uncertain whether the free
inactivated viruses may either induce tolerance or may act as an endogenous
immunogen and/or as an activator of cell-rnediated immunity. While an increase of
antibodies is hardly helpful, the activation of cytotoxic T Iymphocytes (CTL) could
be . 4) By acting on BMC, ozone may stirnulate the production of irnmunoregulatory
cytokines; in 1994, we hoped that CD8 + T Iyrnphocytes present in long-term
survivors might either release the phantom cell antiviral factor (CAF; Walker and
Levy, 1989) or Th l -type cytokines, such as IFNy and IL-2, to block the shift
towards the production of Th2-type cytokines (IL-4, 5, 6, 10) (Clerici and Shearer,
1993, 1994). At that time, we did not know that CTL could release ß-chemokines
(MIP-Ia, MIP-Iß and Rantes) , which by binding to the second receptor (CCR5) on
CD4 T Iymphocytes (the first receptor is known as CD4) impede the infection of
4

ceIls by HIV-I (Cocchi et al., 1995; Feng et al., 1996; Alkhatib et al., 1996; Deng et
al., 1996; Zagury et al., 1998). On the other hand, release of GM -CSF and TNF-a
may have increased viral replication and accelerate the progression of the disease
(Pemo et al., 1989; Mellors et al., 1991). 5) It was difficult to predict if improved
oxygenation and activation of rnetabolism could have exerted a prevalently
beneficial or negative effect. 6) One great hope was the possibility of inducing the
adaptation to COS and I don 't think that the daily oral antioxidant supplement could
inhibit the process. 7) Activation of psychosomatic factor might have been helpful
but , at the same time, an increase of ACTH-cortisol release with a reduced DHEA
secretion may have enhanced imrnunosuppression (Clerici et al., 1994; Corley,
1995). 8) A pitfall was that in 1995 we were using PVC bags for autotransfusion,
which we now know may cause immunosuppression and PVC toxicity, to the
patients' disadvantage.
In July 1995, we began a trial on 10 patients (8 men, 2 wornen), aJl of them also
HCV positive. The patients had CD4 + T cell counts of about 260 cells /ul and an
average plasrna HIV-I RNA level of 138,000 copies per ml.
After a few sessions, the women refused to continue owing to emotional stress
and one man was very depressed and gave up because his girlfriend had left hirn.
 OZONETHERAPY IN V ARIOUS PATHOLOGIES 255

They were between 26 and 37 years old and, after talking with them often, I realised
how unhappy and strained they were. They were very grateful for what we were
trying to do and occasionally when I apologized for an imperfect venous puncture,
they were most kind and said: don 't worry , we have been so stupid to inject and
drug ourselves so many times , throwing away our lives , that you are always perfect.
Obviously I wished very much that the treatment would be beneficial, but in any
case their gratitude was so sincere that I rarely feit more rewarded.
The patients had never been treated, because they refused AZT and other
complementary therapies, and they signed an informed consent form for
ozonetherapy. The trial ended in February 1996 and three patients underwent as
many as 54 OJ-AHT, receiving an overall ozone dose of 1080 mg evenly distributed
in 16.2 I of blood. Although the study analysed a limited number of patients,
repeated measurements of relevant virological markers indicated that ozonetherapy
carried out with great care neither improves nor worsens the dynamics of HIV-1
replication. CD4 + lymphocytes slightly increased (p=0.066) from 272±99 to
341±133. Therapy was stopped in one patient after two months because the viral
load in plasma showed a marked increase. Plasma HIV-I DNA remained stable
(-57,000 copies/Iü" CD4) and HIV -I RNA levels also remained practically
unvaried, except in one case . Serum ß2-micro-globulin increased significantly,
possibly as a result of OJ-AHT -mediated immunological enhancement. Analysis of
the three long-term ozone-treated patients at week 24 confirmed sustained CD4
counts and a stable viral load . While in the lay press there have been many
undocumented claims that OJ-AHT is effective in HIV -I infection, we could not
document any substantial advantage, even though no patient reported side effects,
haematology parameters remained stable and some patients reported a feeling of
well-being and a decreased incidence of oral candidosis and herpes labialis. The full
report has been published (Bocci et al., 1998c). In any event, against the most
pessimistic predictions of distinguished scientists, ozonetherapy did not harm the
patients and it is possible that the adaptation to COS induced by ozonetherapy
countered the COS established by the virus . Indeed in two patients, we measured a
significant increase of erythrocytic SOD after 4 and 5 OJ-AHT (Bocci, 1996a).
Even in these days, I continue to ask myself if I was wrong in selecting the ozone
concentration (-68 ug /ml per ml blood), or the schedule, or the use of PVC bags or
what else? I also very much regret that I was unable to retrace these patients and see
how they fared, but the physicians in charge at the hospital did not bother to help me.
Needless to say, I have often been solicited to perform 03-AHT in the occasional
patient, but the ID unit at the Polyclinic is not willing to perform a study. One good
reason is that HAART (no venous punctures) can be done at horne and is usually
very effective. This therapy has been able to inhibit HIV -1 replication, resulting in
undetectable levels of free viruses in plasma in about two-thirds of patients for at
least 3 years, and thus has been a great success (Pomerantz, 1999; Gulick et al.,
2000 ; Montaner and Mellors, 200 I) because it has reduced morbidity and mortality.
Unfortunately, the initial hope to totally eradicate the virus has not come true
because the virus remains hidden in resting CD4 + T cells and in sanctuaries (Chun
and Fauci , 1999); as soon as HAART is stopped, plasma viremia becomes detectable
in about 3 weeks (Chun et al., 1999). The benefit of complementing the therapy
256 CHAPTER24

with SC admin istration of IL-2 (Levy et al., 1999; Davey et a\., 2000) or with the
promising option of "structured intermittent therapy" (Ruiz et a\., 2001) remains to
be assessed, but it is now eertain that eontinuous HAART is toxie (Hruz et a\., 2001 ;
Fellay et a\., 2001), diffieult to adhere to and very expensive, even for Amerieans
(Steinbrook, 2001) .
Does it make any sense today to think that ozonetherapy eould help HIV
patients? My answer is: yes and no! No, if we want to substitute HAART with
ozone . The former is in eontinuous evolution and there is great hope of having even
more potent and less toxie drugs, thus redueing treatment failures due to the
induetion of resistanee or poor eomplianee (Weller and Williams, 2001) . Despite the
news I reeeive from quaeks, I am eonvineed that ozone eannot match HAART in
removing HIV from the plasma, when we know that blocking viral replication is a
fundamental step . There is no need to eomment about the belief that HIV is not the
erucial eulprit.
However, ozonetherapy may be useful as a eomplementary therapy for the
following reasons:

a) Now, with the new option of BOEX (or at least RI), we have a praetieal ,
inexpensive and above all non-invasive approach (no venous puncture or risk of
infeetion) .
b) Using a gradual increase of ozone eoneentrations (from low to medium : 20-40
ug/rnl), we may aehieve:
• adaptation to COS, hence a re-equilibration of the eellular redox state , which
is a fundamental proeess for inhibition of HIV replication;
• eorrection of hyperlipidemia and peripheral lipodystrophy . With the EBOO
proeedure, we have already eorreeted two serious eases ofMadelung's disease;
• a eorreetion of the wasting syndrome instead of adm inistering reeombinant
GH and DHEA (Murphy and Longo, 2000) ;
• a feeling of euphoria, counteraeting asthenia and depression.

The same objeetives can be aehieved using EBOO or OJ-AHT (in very
preearious patients, even using allogeneic AHT with LD blood), but these
approaches are technically more eomplex , invasive and more expensive.
It is not yet elear what will be the most profieient strategy for intermittent
therapy, i.e. HAART either on a monthly or a seven-day-on-off sehedule, with
ozonetherapy performed during the periods ofHAART interruptions.
I would Iike to elose this section by offering my enthusiastic eollaboration to
anyone seriously interested in eondueting a eontrolled study . I cannot do it here
beeause I have neither funds nor support . Yet I would bet that official medicine will
disregard my offer and eontinue to test IL-2 and hormones because , eoneeptually,
the injection of drugs is preferred. Nonetheless, I insist in saying that if we want to
assess whether ozonetherapy has any value, we must eonduet appropriate studies in
eollaboration with expert infectivologists, virologists, pathologists and statistieians.
 OZONETHER..-\PY IN V ARIOUS PATHOLOGIES 257

1.3. Chronic Hepatitis (HBV, HCVand HDV)

It has been estimated that 300 million people in the world are chronically infected with
HCV and about 2 million live in Italy. Chronic hepatitis diseases are somewhat less
dramatic than HIV but are certainly very serious ailments from a socio-economic point
of view . In Italy about 80% of infected people have a chronic disease but, luckily
about 60% remain with a mild infection. On the other hand, 20% develop an
aggressive disease leading to cirrhosis and liver cancer with some 20,000 deaths
yearly. Current knowledge about the potential usefulness of ozonetherapy is
inconclusive. In the recent past, I failed to elicit the interest of five of the best Italian
hepatologists. Because of my inability to produce experimental evidence or asound
rationale, they all dedined my request to perform a RCT using OrAHT.
Is there any valid reason to justify the study?

1) The demonstration of a virucidal effect against HAV in vitro (Vaughn et al.,

1990) has little relevance in vivo because hepatitis viruses are more resistant to
ozone than HIV and they may be protected by the plasma antioxidant capacity.
2) The oxidation ofviral particle components during blood ozonization may generate an
inactivated and immunogenic vaccine, but this idea remains hypothetical.
3) The induction of cytokine synthesis (IFNß, IFNy, IL-4, IL-6, IL-8 and TNFa) in
ozonized blood incubated in vitro has been demonstrated (Bocci and Paulesu,
1990; Paulesu et al., 1991; Bocci et al., 1993a,b, 1994b, 1998b). Yet the amount
of cytokines released is small and the evidence that this happens in vivo upon
reinfusion of ozonized blood is limited to the demonstration that the Mx protein
is induced in leukocytes (Bocci et al., 1994a). It is unknown whether the
ozonization process leads to activation of CD4+ helper T cells, (CD8 +) CTL
responses (Cemy and Chisari, 1999; Lechner et al., 2000) and NK cells . We also
do not know whether infected hepatocytes increase the expression of MHC class
II on the membrane, which is an important co-signal for their identification and
subsequent clearance. In the past, it was claimed that ozonetherapy increases IgG
production (Washuttl et al., 1988), but there is no clear evidence of increased
antibody-dependent cellular cytotoxicity. The possible involvement of Kupffer
and Ito ceIls (O'Farrelly and Crispe, 1999) with infiltrating CD4+ T lymphocytes
and CTL in destroying infected hepatocytes remains another possibility.
Obviously, if the cellular immune response is weak because of insufficient
quantities of cytokines, or because the heterogeneous viral population evolves
rapidly and becomes IFN-insensitive, or the immune system becomes tolerant to
HCV antigens, the infection is not eradicated and becomes chronic (Bendinelli et
al., 1999; Cemy and Chisari, 1999; Farci et al., 2000) .
4) We always say that OrOJ therapy improves oxygenation and hepatic metabolism,
but this is more a cliche than a proven reality. Indirect, but rather tenuous, evidence
of this has been discussed in Chapter 19 (Rectal insufflation): after OrAHT or
EBOO, fibrinogen and prothrombin plasma levels tend to normalize.
5) It is weIl documented that hepatic viral infections induce a COS, to the
advantage of the virus, and the release of cytokines (TNFa and TGFß) enhances
the process (De Maria et al., 1996; Schwarz, 1996; Romero et al., 1998; Akaike
258 CHAPTER24

et al., 1998). In line with this ftnding is the fact that administration of NAC (plus
IFN) reduces the plasma level of transaminases (Larrea et al., 1998). The local
release of TGFß is doubly deleterious because it inhibits the synthesis of
MnSOD (while IL-I and IFNy increase it) and enhances hepatic fibrogenesis
(Poynard et al., 1997; Poli and Parola, 1997). It can be hypothesized that
prolonged ozonetherapy may be able to reverse the COS by upregulating
antioxidant enzymes in the liver. This would be an interesting and positive
result, but it remains to be demonstrated. It is also unknown whether
ozonetherapy can induce the release of growth-stimulating factors (mainly
hepatocyte growth factor, HGF, and TGFo.) and/or growth- inhibitory factors to
regulate liver homeostasis during infections, which implies continuous destruction
and regeneration ofliver tissue (Fausto et al., 1995; Ankoma-Sey, 1999).

1.3.1. 1s There Any Clinical Evidence That Ozonetherapy is Useful in Chronic

Hepatitis?
This is a sore point and it is shameful that neither in Germany nor in Italy have we
been able to produce serious documentation. In practice, we are still at the stage of
vague, insignificant oral communications by Dorstewitz, Konrad , Mattassi (1985)
and Knock et al. (1987) who reported "more than satisfactory results" (?) in patients
with chronic HBV infection.
The only diagrammatic example of treatment, reported by Viebahn ( 1999) as a
"diamond", is shown in Figure 96.
- - - Bill
.- - - - - - - GOT
---y-GT

~:r! ;1t'lJj!! !~ !j1 j! 1J

200

150

100 2 \

".,
I I

'J I r , \
50 I "
' , '\

25/08 24/09 15/10 29110 04/12 04/02 27/05 28/06

1980/81

Figure 96. In the ozonetherapy literature, there is only this diagram showing the improvement
0/ three markers in a patient with chronic HB V hepatitis. 21 DrAHT were p erform ed between
August 25. 1980 and June 1981. The bilirubin (BILl) level increased during theflrst month
and then declined to normal values (Viehahn. 1994) .
 OZONETHERAPY IN V ARIOUS PATHOLOGIES 259

Apparently after 21 0 3-AHT carried out discontinuously in about 9 months,

there was normalization of the levels of two hepatic enzymes and bilirubin.
Apparently two to five 0 3-AHT were already effeetive in reducing HCV RNA levels
in plasma (Yamamoto et al., 1996); these results are at variance with our data. The
0 3 eoneentrations used are uneertain, but a famous German ozonetherapist onee told
me that either 20 ug /ml or sometimes 100-150 (?) ug/ml produce "good results" and
"patients fee! mueh better". Any eomment appears superfluous. Regretfully, we also
have done very little. As I mentioned previously, my seminars in Rome , Florence,
etc. proved useless. Eventually, I was lueky enough that Dr. Giuseppe Amato, one of
the most re!iable ozonetherapists, who works at the hospital at Conegliano Veneto,
agreed to evaluate our protoeol in the period 1996-98. We planned to evaluate the
treatment of ehronie HCV patients with 0 3-AHT: 250 rnl blood eolleeted in ACD
using at first an 0 3 eoneentration of 40 ug/ml per ml blood (total dose : 10 mg) twiee
weekly for 5 months (about 40 treatments). One big problem at that time was that
we were using PVC bags and the protoeol was stopped around Fall 1998 when we
realized the risk of the release of plastic mieroparticles and phthalates. Of twe!ve
patients, only nine received the full treatment while the other three reeeived between
17 and 31 treatments. However, the results were very disappointing: none of the
nine patients showed any normalization of hepatie transaminases, while the viral
load was not measured. At least no side effeets were reeorded. It was then decided to
begin a study (June 1999) with new patients using the atoxie system (giass bottles)
and testing the 0 3 eoncentration of 70 ug/ml per rnl of blood (225 ml) eollected in
Na citrate (25 ml) to prevent any risk of plate!et aggregation (total 0 3 dose : 15.75
mg). Fifteen patients were treated but only 14 were evaluated beeause one woman
withdrew after the third treatment. The schedule followed was somewhat unusual :
three 0 3-AHT weekly for the first 3 weeks and then onee a month for a total of 20-
21 sessions. The sehedule had to be adjusted aecording to the hospital labour supply!
In addition to the initial sampie prior to therapy, 13 sampies were colleeted per
patient (about every month) to evaluate liver enzymes, namely serum aspartate
aminotransferase (SGOT), alanine aminotransferase (SGPT) and y-Glutamyl
transpeptidase (GGT), and the viral pattern (anti-HCV, HCV-RNA-PCR, anti-HBs,
anti-HBC, anti-Hbe, HbeAg, HbsAg) beeause four patients were also HBV positive.
Figure 97 shows that all three hepatie enzymes deereased progressive!y and were
eventually within the normal range (the statistical signifieanee between the
pretreatment values and the last values was p<O.OI). However, the viral tests
remained stable throughout the year . These results were reported at the I st
International Medical Ozone Society Meeting (IMOS, Siena November 2-4, 2000).
On the whole, the results were promising, but the ineompleteness of the study made
it impossible to draw any conclusion.
Firstly , the schedule was not optimal, because to start abruptly with the highest
concentration is unsound and it may compromise the induetion of ozone toleranee.
SecondIy, one treatment monthly is most likely insufficient; it should be performed
at least onee weekly and thus a more intensive schedule must be devised. One
problem for patients is that they must come to the hospital for a few hours; if we
require too frequent treatments, the compliance is not so good. Then there are
problems of not having enough personnel at the hospital. Cleary the best goodwill
260 CHAPTER24

does not overcome the problem of lack of funding to pay for laboratory exams and
medical personnel and we are now at astandstill.

-. , \
120 . , - - - - - - - - - - - - - - - - - - - - - - - ,

100 ···..··SGOT

.., \ - ·SGPT
80
.......... " --GGT
"-
-~,

..............,
- ----
60 "
.•......'-. -----
.'
40

20
~
.

-
.... ......_-~----
&
........
•
~

..
.
'*
.--~~

.. '*
:::-
"':
..

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sam pie number

Figure 97. The pattern oftransaminase plasma levels ofpatients with chronic Hel'
hepatitis treated with OrAHT throughout one year. Ordinate: lU/mi enzymes. All three
decreased levels are statistically significant (p<.O.01) (Amato et al.• 2000)

I must mention that, at the Siena congress, areport was presented by Luongo et
al. (2000) regarding 0 3-AHT in 82 HCV patients. They c1aimed to have monitored
and recorded the redox potential of cell membranes (?) with a non-invasive method
for each patient so that, on the basis of the ongoing measurements, they could
modify and optimize the 0 3 concentration. This was not specified but presumably
ranged from 0 to 60 ug/ml, according to the redox potential.
My comment was that the results sounded too good to be true, but the proof of
the pudding is in the eating. Several types of the redox potent ial apparatus appear
often in the field of complementary medicine; while their real validity remains
questionable, the fee of the "physician" goes up. Nonetheless, I have the duty to
summarize their data :
a) 30% of patients initially presented between 250,000 and 1,000,000 HCV
copies/ml.
b) 40% of patients initially presented between 1,000,000 and 5,000,000 HCV
copies/mI.
c) 20% of patients initially presented between 5,000,000 and 7,000,000 HCV
copies/ml.
d) 10% of patients initially presented between 7,000,000 and 10,200,000 HCV
copies /ml
The HCV-RNA was quantitatively assessed by the Reverse PCR method.
 OZONETH ERAPY IN V ARIO US PA THOLOGIES 261

The amazing results are that 99% of the 82 patients treated for 3 to 6 months
(schedule unknown) showed a more than 80% reduction of the viral load, with 9
patients achieving negativity between 4 and 16 weeks. Biochemical and histological
responses were not mentioned. Patients previously pretreated with IFN or diabetic
subjects were particularly resistant to the treatment and this is not surprising.
I very much hope that this group will publish these data in an international, peer-
reviewedjournal, because, iftrue, they will represent a significant advancement.
In conclusion, ozonetherapy may be useful in chronic HCV hepatitis, but firm
evidence ofthis is stilliacking.
Therefore, the refusal of orthodox hepatologists to test this approach is
comprehensible. It is more so because, since the 1980s, IFNs (particularly u type)
have proved to be useful, although not always resolutive. Indeed even after intensive
6-12 months therapy, up to 50% of patients may show a good clinical response, but
about 1/3 of them soon relapse. Particularly during the first month of therapy, most
patients report significant and well known side effects, which partially recede later
on. Moreover, elderly patients may show a worrisome depressive state (Bocci,
1988a; Spat-Schwalbe et al., 2000; Malaguarnera et al., 200 I; Musselman et al.,
2001 ). About 20% of patients do not tolerate IFN therapy, while about 70%
experience acceptable side effects and a lucky 10% have no problems. Patients with
extensive liver fibrosis or thrombocytopenia or anaemia have to be treated with great
caution.
Resistance to IFN is frequent in older patients, in those with a highly chronicized
disease or with high serum viral load or with HCV genotypes land 4 (while
genotypes 2 and 3 are favourable), in men more than women, in black patients, in
alcoholic or immunodeficient (HIV) patients, or in hepatitis complicated by
cryoglobulinemia, vasculitis, membranoproliferative glomerulonephritis, arthritis,
etc. (Johnson et al., 1994).
Hundreds of good studies (Dusheiko, 1995; Hoofnagle and Bisceglie, 1997) have
been performed, mostly using IFNu, and to make a long story short, IFN is now
considered the treatment of choice. The use of Peg IFNu-2a (with a very long half-
life, so that only one dose per week is required to maintain effective levels in the
blood) in combination with ribavirin (1 .0-1.2 g a day) for at least six months appears
effective in about 40% of patients, although half of them may continue to have
viremia once the treatment is stopped. (Heathcote et al., 2000; Zeuzem et al., 2000;
Manns et al., 2001). Ribavirin, an oral purine nucleoside analogue, is modestly
effective on its own, but it seems to develop asynergie effect with either IFNu alone
or Peg IFNu (Reichard et al., 1998; McHutchison et al., 1998; Poynard et al., 1998;
Andreone et al., 1999; Cummings et al., 200 I; Younossi et al., 200 I) . Ribavirin may
induce haemolytic anaemia, which occasionally is severe enough to require the
discontinuation oftreatment. Even though Peg IFNu-2a is a "retard" IFN, it induces
adverse effects similar to those with the unpegylated counterpart.
Chronic HBV infection affects more than 350 million people worldwide, Like
HCV infection, it carries the risk of developing cirrhosis and , once viral DNA has
been integrated into hepatocytes, liver cancer. The therapeutic aim is to stimulate a
valid immune response, which with time may lead to viral clearance and reduce liver
262 CHAPTER24

inflammation, necrosis and cancerogenesis in vivo . Orthodox medicine is now

providing new effective therapeutic strategies based on IFNa, whieh has anti viral
and immunomodulatory properties, and several nucleoside/nucleotide analogues,
name1y lami vudine, famciclovir, adefovir dipivoxil , etc ., which inhibit HBV
polymerase. Vaccines and anti sense oligonucleotides complete the armament, whieh
is promising particularly because it combines drugs with different mechanisms of
action (Boni et al., 1998; Dianzani, 1999; Pianko and McHutchison , 1999). The
optimal dose of IFN is somewhat contentious, but usually 8-10 megaunits three
times a week for 4-6 months e1icit a positive response in about 40% of patients, as
documented by lack ofviral replication and improvement of liver histology.
What about the remaining 60% of patients? Clearly the remarkable scientifie
progress in this area must be translated into improved clinical praetices for all
patients, particularly those with the associated delta agent who are at a high risk. In
reality some hepatitis patients seek complementary treatments : an interesting review
has evaluated the effects and toxicity of frequently used herbaI produets while
ozonetherapy was not even mentioned (Seeff et al., 2001)
This digression has two purposes : the first is to inform the ozonetherapist of the
state of the art, because she/he has the duty to inform the patient thoroughly
about IFN therapy. The second is to point out that orthodox rnedicine reeeives
plenty of funding from national agencies and multinational pharmaeeutical
industries, which are interested in developing drugs to recover their investment and
making a profit. In comparison, ozonetherapy is like an ant to an elephant: no
funding , no laboratories, no clinics and total disorganization; the gap is too big to be
closed. Moreover, there is another huge disad vantage: IFN therapy is intrinsieally
expensive, but the patient, once instructed, can do it casily at horne and visit the
hospital every three months for acheck up . In eontrast, except for the very empirieal
RI, OrAHT, EBOO and BOEX requ ire a da y hospital and expensive (for the
National Health Service) medical assistance. Because it is performed privately, the
patient must bear the financial burden out of his own pocket. Moreover, medical
personnel are hostile to both ozone and the handl ing of infected blood. Thus,
although it is likely that ozonetherapy represents a money-saving approach, we
cannot evaluate the cos tlbenefit ratio beeause the benefits have not yet been
demonstrated.
In spite of enormous difficulties, I still believe that we should clarify whether
ozonetherapy has some merits. This can only be done by RCTs , comparing OrO..
therapy against O 2 alone, because the relevance of spontaneous remissions must be
c1arified .
The most suitable and practical methods are I) OJ-AHT; 2) RI and 3) BOEX, but
we could start with the first one and then proceed depending on the results .
Among ehronic hepatitis diseases, we eould examine hepatiti s C with defined
HCV genotype, possibly without any previous treatment beeause of refusal of IFN.
Patients should be of both sexes, between 30 and 50 years old . Informed eonsent is
needed.
 OZONETHERAPY IN VARIOUS PATHOLOGIES 263

1.3.2. Proposal ofa Protocol

The most practical schedule seems twice a week (M and Th or Tu and F):
225 ml blood in 3.8% Na citrate (25 ml) plus 225 ml O2 alone or 02-0J.
Use of citrate instead of heparin may reduce ozone's effectiveness but avoids
possible complications due to dyscoagulation and potential formation of miniclots.

I SI week : 30 ug/ml for a total OJ dose of 6.75 mg per treatment,

2nd week: 40 ug/ml for a total OJ dose of9.00 mg per treatment,
3rd week: 50 ug/ml for a total OJ dose of 11.25 mg per treatment,
4lh week : 60 ug/ml for a total OJ dose of 13.50 mg per treatment,
t h week: 70 ug/ml for a total OJ dose of 15.75 mg per treatment,
to be continued for 24 weeks (48 sessions) unless a problem arises . Therapy may be
continued once a week during the second semester depending on the results. Patients
should take the usual daily . oral antioxidant supplement. Evaluation of therapeutic
effectiveness should consider the following end-points:

a) permanent serum HCV RNA clearance, tested with the most precise system .
Viral load should be assessed before treatment, after 3 and 6 months therapy and
then after a further 3 months .
b) normalization of hepatic biochemistry (SGOT , SGPT, GGT, bilirubin levels).
Test as in (a).
c) liver histological results, whenever possible before and 3 months after the 6-
month course . If liver biopsy is refused, a surrogate test to indirectly evaluate
liver fibrosis may be used. Moreover, in addition to all the routine biochemical
tests, TAS, TBARS and PTG should be measured every 3 months. Of particular
interest is the evaluation of cholesterol, LOL, HOL, albumin, fibrinogen,
prothrombin and CRP .

Patients with HIV, autoantibodies, autoimmune hepatitis, hypergammaglobulinemia,

haemochromatosis, liver metastasis, incipient cirrhosis, extrahepatic manifestation of
HCV infection should be excIuded.
Treatment is obviously cost-free and control patients have the right to be treated
with 02-0J after the first semester. This switch-over might actually be interesting to
clearly demonstrate the role of ozone. It would be very important to have the results
of this study and I would be glad to collaborate with anyone seriously interested in
conducting it. If the results are clearly negative, we should forget about
ozonetherapy. If they show that at least 50% of patients are good responders,
ozonetherapy could be useful in patients who do not tolerate IFN, in elderly patients
particularly sensitive to psychotic effects, in hepatitis C patients with normal serum
aminotransferase levels but with viremia (Hirsch and Wright, 2000), in patients after
liver transplantation and in patients who cannot afford the cost ofIFN.
As the current best conventional comb ination (Peg IFNa-2a with ribavirin) is not
entirely satisfactory, it could be supplemented with one OJ-AHT treatment per week,
which may reduce the severity of adverse effects and enhance immunoactivation.
264 CHAPTER24

Moreover, as I doubt that a brief course of ozonetherapy can reduce the viral
load (as observed with HIV infection), we could test a hybrid approach: firstly ,
knock down the viral load with a short (1-2 weeks) intensive treatment with IFNa
(Neumann et al., 1998) or IFN-ß (Ikeda et al., 2000) followed by 0 3-AHT according
to the schedule described above. ladmit that these may be hopeless speculations.
Yet, for many reasons, hepatitis C is a very heterogeneous and difficult disease, for
which ozonetherapy might be useful in complementing conventional therapies to
achieve a favourable outcome.
Finally, a sort of minor plasma autotherapy can be envisaged and utilized for
priming and activating natural immunity: it may be worth while to evaluate a
protocol based on repeated, weekly IM injections of 1-2 ml autologous plasma
heavily ozonized to produce a viral immunogenic vaccine.

1.4. Herpetic 1nfections and Herp es Zoster

Herpes simplex viruses (HSV-I and HSV -2) cause human infections involving
mucocutaneous surfaces, the CNS and possibly visceral organs in
immunosuppressed patients.
HSV-I is mostly responsible for causing oral-facial herpes, but it can spread to
give an herpetic eye infection that may lead to corneal blindness. HSV-2 is mostly
responsible for lesions on the genitalia, and it recurs petiodically. HSV infection of
the finger (herpetic whitlow) usually represents a complication of oral or genital
herpes.
Although these infections are usually limited, their frequent recurrence
compromises the patient's quality of life (Arvin and Prober, 1997). Current antiviral
chemotherapy is prevalently based on systemic (oral andJor IV) administration of
nucleoside analogues: aciclovir, famciclovir and valaciclovir. These drugs are not
always effective because of aciclovir-resistant strains .
Control of HSV infection may be achieved by a vaccine, which has been late in
coming and has showed effectiveness only in women not previously infected with
HSV-I (Stephenson, 2000) . A promising therapy for genital herpes is the local use
of a gel containing an immune response modifier called resiquimod, which is able to
stimulate antibody and cytokine production (Bishop et al., 2001) .
Herpetic cheratitis can be treated with ophthalmic IFNa or IFNß plus aciclovir.
Herpes zoster (HZ), or shingles, is a distressing disease affecting about 1% of the
over-60 population. It is caused by the varicella-zoster virus, which remains in a
quiescent state in the nerve root ganglia after recovery from chicken pox . The virus
may be reactivated during an immunosuppressive state. It causes a unilateral
dermatomal, vesicular rash associated with severe pain . The frequency of location
is: trigeminal (16%), thoracic (50'%), cervical (14%) and lumbar (12%)
dermatomers. If the disease goes untreated, the pain can last for months and can be
complicated by post-herpetic neuralgia (PHN). This complication is rare in young
middle-age patients (30-50 years) but is frequent in elderly patients. PHN should be
prevented by intensive therapy as early as possible. Unfortunately, the incidence of
this complication increases with age and with immune depression. It seems that
 OZONETHERAPY IN V ARIOUS PATHOLOGIES 265

microinfusion of anaesthetics via the peridural route, initiated no later than I week
from the appearance of the cutaneous exanthema, may reduce the incidence and
minimize the pain . By blocking the axonplasmatic transport, local anaesthetics can
prevent diffusion of the HZ virus to neurones in the spinal cord, thus reducing
neuronal death and the consequent allodynia and abnormal sensations. The anti-
epileptic, gabapentin, is widely used, but is not always effective. The sooner an
appropriate treatment is started, the better. Prophylaxis in patients over 60 and at
risk has been partially accomplished by the administration of specific zoster immune
globulin (ZIG) or by shingles vaccine (NIAID, Bethesda, USA, 1999). Antiviral
chemotherapy is based on aciclovir, valaciclovir or, probably even better,
famciclovir with or without prednisolone (Wood et aI., 1994, Whitley and Roizman,
200 I), but they have little effect on the healing of skin lesions or pain. The use of
corticosteroids is controversial: although they reduce inflammation, they inhibit
healing and enhance immunosuppression, which is exactly what favours the virus.
Administration of amitryptline (25 mg for 3 months) seems to reduce the pain
(Dworkin, 1999). Taking drugs continuously can reduce or suppress herpetic
infections, but it is expensive, can cause adverse effects and induce viral resistance.
This is what official medicine offers today, but it cannot necessarily satisfy all
patients. Although these diseases are not deadly, they are serious and we must foster
a sense of concern and the need ofbetter therapeutics.
As this book is aimed at critically evaluating ozonetherapy, I have the duty to
report the usual litany of wonderful results obtained with ozonetherapy. The
problem is that these results have been reported at best in an abstract form, are very
difficult to trace and analyse, and can be considered anecdotaI. It seems that
Mattassi (1981 , 1983) treated 20 patients, of which ll presented herpes simplex and
9 had HZ. I believe the pat ients were treated with 5 to 12 IV injections of 02-03!
After a few injections, all patients overcame the infectious episode and only a few
had a recurrence over several years. None of the patients had side effects. Mattassi
(1981) stated that results were incredibly rapid and that to be successful the therapy
should be started as soon as the lesion appears. Dr . J. Delgado, of the Center of
Medical and Surgical Research in Havana, treated 15 patients suffering from HZ
with daily IM infections of Or03 and topical applications of ozonized sunflower oil.
He noted a marked improvement after a few days and alI patients were cured after
two weeks, without showing any relapse. He concluded that "the low cost , the easy
ava ilability and simple application made ozonetherapy the treatment of choice". I
believe that Dr . H. Konrad works in Sao Paulo (Brazil); he has reported (1995,
2001) that 03-AHT was effective in both herpetic infections and was able to
minimize the complication of PHN evaluated in 55 patients.
I think it is important to report the experience of Dr . Giuseppe Amato, already
cited for the study performed in chronic HCV infection. I believe that bis work in
treating PHN patients is outstanding; it has been performed at the Hospital
"DeGironcoli" at Conegliano Veneto in Italy during the last decade. Although this is
an open study, it is praiseworthy and regards 180 patients (84 men and 96 women)
between 40 and 85 years of age :
266 CHAPTER24

• age 40-50 : 30 patients (16.7%);

• age 51-70: 60 patients (33.3%);
• age 71-80 : 54 patients (30%) ;
• age 81-85 : 36 patients (20%);

The location of HZ was as folIows :

• Ocular region: 18 patients.

• Head, neck and anns: 30 patients.
• Thorax : 30 patients.
• Lumbar region: 48 patients.
• Limbs : 54 patients.

Patients always arrived at the hospital with some delay when previous physicians
feit unable to deal with the intense pain of acute HZ infection. Evaluation of pain
was carried out with the visual analogue scale (VAS) . On the basis of previous
experience, Dr. Amato decided to abandon all conventional med ication and examine
ozonetherapy associated with the microinfusion of anaesthetics (usually 12 ml of
marcaine at 0.25% daily) mostly via the epidural route to block the sympathetic
system in relation to the dennatome presenting the cutaneous rash.
The concomitant use of two therapies or the lack of a control is usually open to
criticism, but in the case of PHN it had be done for ethical reasons in order to reduce
the pain.
Dr. Amato proceeded systematically to perfonn :

a) 03-AHT (150 ml of blood collected in Na citrate and a total ozone dose of 10.5
mg or 70 ug /ml) every day for 4 consecutive days and then every other day for 2
weeks (at least 10 treatments).
b) Local treatment using compresses moistened with ozonized water during the day .
The applicatlon of ozonized oil at night is effective and most irnportant.
c) Sympathicolysis ofthe stellate ganglion or other ganglia at various levels .

Owing to the fact that patients below 50 years rarely develop PHN, they
underwent only ozonetherapy. Pain disappeared after 2-3 days (i.e. after 2-3 0 3-
AHT) and the exanthema also improved very rapidly. Three patients (out of 30)
developed PHN after 2 months and they were promptly treated with anaesthetics.
However, in the subjects over 50 (150 patients), Dr. Amato believed it ethically
correct to practise both therapies on a prophylactic basis, because they are at areal
risk of developing PHN.
Anaesthetic treatment was perfonned daily for no more than 10 days at the level
ofthe stellate ganglion and for no more than 20 days in other locations .
. On average, pain disappeared after the first application:

• for about 12 hours in 36 patients.

 OZONETHERAPY IN VARIOUS PATHOLOGIES 267

• for about 18 hours in 114 patients.

• and for 24 hours in 30 patients.

After 3-4 days, the pain disappeared in about 90% of patients; although further
treatment seemed unnecessary, it continued for up to 20 days in order to prevent
PHN later on. All patients were followed for 2 to 5 years : of 99 patients older than
50 and treated as indicated above in the first week, only 12 developed mild PHN that
was successfully treated with both therapies . Of the remaining 51 patients treated
with a delay longer than one week, the percentage increased and was in relation to
the delay. In conclusion, it appears that the combination of ozonetherapy with
anaesthetic intervention is most effective in preventing PHN in patients older than 50.
In view of the difficulty of managing PHN, the results appear impressive. By
sheer necessity, they lack controls (0 2 only) and, in this regard, I must report another
surprising study. Olwin et al. (1997) found that minor AHT (10 ml of blood not
treated with O 2-03 or O 2) was effective in eliminating clinical sequelae in 8 of
12 (66%) patients with thoracic HZ, in 9 of9 (100%) patients with ophthalmic HZ
and in I with lurnbar-thigh HZ. They claimed (data not presented) that IFNa., IFNß
and IL-4 levels were increased in the patients within 24 hours after the IM blood
injection . They also mentioned that another 25 cases of herpes infections of various
types yielded favourable results , noting that the rate of success depends on early
intervention. A delay of 2-13 months between the first symptoms and treatment
yields negative results. As this report originates from reliable institutions (Rush
Presbyterian St. Luke's Medical Center and Life Seiences Department, IIT Research
Institute, Chicago, USA), the data ought to be true. If they are, they partly support
Amato's data; yet they totally refute the value of oxygen-ozone, Moreover, if they
are true, Health Authorities and official Medicine have the obligation to verify
them: irrespective of the skepticism toward ozone, it is outrageous that we
intoxicate HZ patients with expensive, but modestly effective, pills when a few
trivial injections of autologous blood into the patient's buttock could relieve awful
pain in 2 to 8 days. However , authoritative scientists and clinicians obviously do not
bother to believe, or to read, papers published in the Journal of Alternative and
Complementary Medicine and prefer to administer expensive drugs. This is even
more reproachable because simple AHT is an old medical practice (Maddox and
Back, 1935; Hardwick , 1940; Martindale and Capper, 1952); even I performed it in
1953 when I was an intern in Clinical Medicine!
If dermatologists and neurologists, as weil as general practitioners, ever read this
section, they might think that everything I have written is false and it would be
better to forget everything. I have no direct experience, but I fully trust Dr. Amato
and I am inclined to believe that, bearing in mind that orthodox medicine does not
offer much to highly distressed patients, we should attempt to prove or disprove the
validity of either simple AHT or 0 3-AHT. As soon as I fmish writing this book, I
will revive my proposal of a protocol evaluating herpetic infections and HZ, which
in 1999 was refused by the Professor of Dermatology . In spite of everything , I am
still optimistic that many physicians are not aware of ozonetherapy and that they
may be willing to try it. I am certainly ready to help anyone .
268 CHAPTER24

In Chapter 16, I extensively discussed the approaches of the so-called "rnajor"

OrAHT and "rninor" OJ-AHT . If venous access is lacking, we can use the option of
RI or BOEX . Minor AHT, without or with 0 2-0 J, is in my opinion an interesting
immunoenhancer approach and it is easy, simple, inexpensive and rapid to perform.
Starting with a low dose and gradually increasing it, we can ozonize 5 ml blood (30
ug /rnl and upward) followed by IM injection three times a week , and then slow
down as soon as the lesions are healed and the pain is gone . Local treatment is also
important and effective when combined with OrAHT. It can be performed easily by
applying and repeatedly changing a compress moistened with ozonized water (or
ozonized oil at night) . Vaginal or rectal suppositories of ozonized oil can be
employed in genital-anorectal herpes . We must try to start the treatment as soon as
cutaneous lesions and pain appear; the viral reactivation should be suppressed as
soon as possible because it reduces the PHN complication. It appears necessary to
alert all GPs to send HZ patients to the special PHN unit at the hospital as soon as
they make the diagnosis.

1.5. Papillomavirus Infections (HPV)

HPV infects the epithelium of skin or mucous membranes and may produce warts,
or benign and malignant neoplasias. Common warts (Verrucae vulgaris and plana )
may be present in children, while plantar warts (Verrucae plantaris) are painful and
fairly common in young adults . The incidence of venereal warts (Condyloma
acurninatum) has risen, particularly in women, and represents a common sexually
transmitted disease (Cannistra and Niloff, 1996). Viral genotypes 6 and 11 carry a
low risk and may cause modest dysplasia of the uterine cervical epithelium, known
as cervical intra-epithelial neoplasia (CIN I). Viral genotypes 16, 18, 31, 33 and 35
are more carcinogenic and can induce a CIN II or the more severe form, CIN rn
(Liaw et al., 1999). Laryngeal papillomas are typical of children and may produce
life-threatening airway obstruction. Anogenital warts (venereal warts) can reach
monstrous proportions and may be associated with cervical cancer.
Effective conventional therapies include cryosurgery, surgical excision and
ablation with a laser. Topical treatments with antimetabolites and podophyllum
preparations are scarcely resolutive because the virus is widespread in the basal cell
layer and persists if the immune system is unable to destroy infected cells . The use
ofboth IFNa and IFNß have been successful for laryngeal papillomatosis and partly
useful (30-40% response) in preventing venereal HPV recurrences even after
prolonged treatment (Friedman-Kien et al., 1988; Kirby et al., 1988; Weck et al.,
1988; Bocci et al., 1990). Both the cost and the adverse effects of IFNs reduce the
compliance. The fact that HPV infection is an important risk factor for carcinoma is
weil known and several HPV vaccines are undergoing trials. However, ozonetherapy
could be useful as a complementary therapy. To the best of my knowledge, there are
no data, but it may be worthwhile evaluating a protocol in the hope of eradicating
cervical-vaginal infections. Therapy should combine parenteral approa ches, such as
major or minor OrAHT, RI or BOEX , with local treatment. After the basic surgical
treatment, always important to remove the bulk of infected tissue, there are several
 OZONETHERAPY IN VARIOUS PATHOLOGIES 269

possibilities: one is intralesional injections ofsmall volurnes ofO r03 (from 10 to 20

ug /ml) . The infiltrating injections of gas must be done slowly and with great care,
possibly at the base of the wart; as reported for IFNß, they are pa inful and the
patient may get discouraged. Intravaginal insufflation of Or03 (concentration: 30-
50 ug /ml) for a few seconds is more acceptable, as noted during treatment of
bacterial and fungal vaginitis. Instillation of ozonized water (final 0 3 concentration
- 20 ug /rnl) for 5-10 min can be done at home and application of an ozonized oil
pessary every night is practical and certainly far less expensive than an IFNß gel.
The benefit of ozonetherapy remains to be ascertained, but there is no risk, no
side effects and certainly a low cost. The possibility of minimizing viral shedding,
thus reducing the potential of transmission to sexual partners is not a trivial
advantage.

1.6. The Common Cold

The well-known manifestations of the common cold, i.e . rhinorrhea, nasal
congestion, lachrymation and sneezing, are commonly experienced each year. Sore
throat, malaise and headache are also frequently present. Although the common cold
resolves without sequelae in 4 to 9 days, it is a very bothersome infection. Normal
individuals do not need particular treatment, but immunosuppressed patients are at
risk ofpulmonary infections and can be prophylactically treated with IFNa or IFNß.
Enormous funds have been spent in the hope that a few applications of IFNs
sprayed at the appearance of the first symptoms would abort the infection. As a
matter of fact, the applications are always too late : in order to establish the antiviral
state , the IFN should bind to the cell receptors a few hours before the viral invasion.
The IFN approach has been a financial blunder because the local adverse effects of
IFN are worse than the infection itself. I wish a small part of the money would be
spent on evaluating the effect of ozone! I would like to give an example of the
versatility of ozone, and I am not pretending to be too serious!
Ozone as agas is toxic for the nasal and respiratory mucosa and must not be
used . However, in our lab, during the last five years we have prepared a lot of
ozonized bidistilled water every day . It is ready after 5 min of bubbling ozone
(concentration 80 ug /rnl) in water. The final 0 3 concentration is about 20 ug/ml and
if it stored in a glass bottle with a tight teflon tap, it keeps for two days, even though
the ozone concentration progressively decreases. If anyone thinks he has caught a
cold, he can aspirate the ozonized water into the nostrils 3-4 times a day and can
take the bottle home for further use . Water passes into the rhinopharynx and is
eliminated, but it is not harmful if swallowed. It also helps to gargle the ozonized
water at the same time . Although nasal aspiration of ozonized water causes transient
irritation (10-15 sec) , it is unbelievable how rapidly the nasal congestion, sinusal
oedema and pharyngodynia disappear rapidly for 3-5 hours, after which it is
necessary to repeat the procedure. The infection resolves in 3-4 days, but it is far
more tolerable than if it went untreated. If I have time, I also perform 03-AHT on
myselfto reduce the feeling offatigue.
270 CHAPTER 24

This approach is quite empirical and it is difticult to produee praetieal

applications, so pharmaeeutieal eompanies have no reason to worry about it!

1.7. Fungal and Parasitic Infections

Most of these diseases are present in hot-humid countries and are seen less
frequently in Europe, either as opportunistic infections or after a trip to the tropies .
Among fungal infections, those that have been treated with ozone are
onychomycosis (tinea pedis or athlete's foot) and eandidiasis. Owing to the US
embargo against Cuba, scientists and physieians in Havana have used ozone , as
necessity is the mother of invention. At the XII IOA Congress (LiIle 1995), it was
reported that treatment of a sick nail with 1-2 drops of ozonized sunflower oil for
several days led to a complete eure in 69% of patients (the remaining 31'% showed
marked improvement). In contrast, in the group treated with tolnaftate solution twiee
a day, only 7% were eured, 25'% improved and there was no change in 68'%,
probably owing to drug resistance (Menendez et al., 1995) . Using ozonized olive oil
topically, we have achieved incredible results; in fact, we now believe that this
simple preparation is really effective because it is amply disinfectant and able to
stimulate the healing proeess. I predict that , as soon as prejudiees disappear and
physieians beeome aware of this fact and try ozonized oil with good results, it wiIl
become widely used worldwide with great patient satisfaction .
Candidiasis and any other fungal infection can obviously be treated with
systemic and topical antifungals, but I can guarantee that gas insufflation (whenever
possible), ozonized water and ozonized oil applied topically have equal , if not
superior, effectiveness.
Giardiasis is a parasitic infeetion eaused by the protozoan Giardia lamblia,
common in areas with poor sanitation and present even in the United States .
Cryptosporidiosis is also a diarrhoeal disease, caused by protozoa of the genus
Cryptosporidium . Good drugs Iike metronidazole are effective but have some side
effects. In Cuba, at first they used to drink ozonized water, at least four of five
glasses per day on an empty stornach for repeated periods of 10 days separated by a
I week interval. According to Sardina et al. (1991), up to 48% of patients became
asymptomatic after the second cycle . Ingestion of ozonized oil seems more
effective, but it is hard to swaIlow. An improved administration is represented by
capsules filled with ozonized oil. A 10-day cycle "cured" 79% of children, while the
remaining 21% showed a marked improvement of symptoms but still had cysts or
trophozoites in the faeces (Menendez et al., 1995) . No side effects were reported.
There is no need to report other studies because the therapeutic modality is the
same . However, it is certainly worth keeping this approach in mind for use in poor
countries of Africa, Asia and South America affected by several fungal and parasitic
diseases. Areas lacking electricity cannot produee ozone and ozonized water . Thus
the World Health Organization (WHO) ought to promote a standard and very
eeonomical production of ozonized oil (which keeps weil) and distribute it where
needed. I should try to promote this enterprise, although it may have little value
unless we ean reduce the rate of infection by improving sanitation in all directions.
 OZONETHERAPY IN VARIOUS PATHOLOGIES 271

Just a few .words about malaria, which remains another scourge of our time ,
exacting atoll of I million deaths each year . Unfortunately, both the mosquitoes and
the protozoan Plasmodium falciparum have become resistant , the former to
insecticides and the latter to drugs . Almost 20 years ago, Dockrell and Playfair
showed in mice that H202 is able to kill Plasmodium yoelii. At the XV 10A
Congress (London, September 10-15, 200 I), Viebahn-Hansler et al. reported that
parasite growth can be inhibited by ozone at a concentration of 80 ug/ml after
ozonization of a blood cell suspension. However, in contrast to the sarcastic opinion
of many scientists that ozone is a panacea, I doubt that ozonetherapy would ever be
useful because parasites are well protected by the plasma and cellular antioxidant
system , as weil as being hidden in the spleen and other sanctuaries. Moreover, 0 3-
AHT or EBOO are demanding approaches and would be difficult to organize in
tropical countries for the treatment of millions of people. I feel pessimistic about
wasting our meagre resources on diseases such as HIV and malaria for which the
administration of oral drugs or a long-sought vaccine appear rational and could be
more useful on a large scale.

2. AUTOIMMUNE DISEASES
Autoimmune diseases are areal thomy problem because serious diseases such as
rheumatoid arthritis (RA), Sjögren's syndrome, vaseulitis, multiple sc1erosis (MS) ,
systemic lupus erythematosus (SLE), Crohn's disease, systemic sc1erosis (SSc)
should have attracted our attention a long time ago, instead of treating unaesthetic,
albeit very lucrative, lipodystrophy. Since 1983, this has been the almost exclus ive
interest of Italian ozonetherapists, which explains why today ozonetherapy is
regarded so poorly. It is even worse that at the annual meetings, two or three
ozonetherapists claim , with great rhetoric, that they are able to obtain such
wonderful results with a few OrAHT (ozone concentrations and schedule are
always uncertain) so that invalid patients are able to rise frorn the wheelchair and go
bicycling or dancing . In obedience to holistic therapy and to the patient's advantage
(so they say), they invariably mix ozonetherapy with magnetotherapy, phytotherapy,
homeopathy, chelation therapy, etc., so that it becomes impossible to c1arify the role of
ozonetherapy . Assuming that this exerts areal effect, it would be important to confirm
the results with a proper RCT in order to understand what mechanisms have been
activated, how relevant is the placebo effect and how long the improvement lasts.
The aetiology of these diseases remains hypothetical, while the pathogenetic
mechanisms are fairly common, obviously with different locations and with a strong
prevalence in women. Different tissues (articular, gut mucosa, myelin, etc.) are
infiltrated by macrophages, neutrophils and CTL, responsible for an abnormal
release of ROS and proinflammatory cytokines (IL-I ß, IL-2, IL-8, IL-12, IL-15, IL-
18, TNFa, IFNy), while inhibitory cytokines (lL-IO, IL- I I, TGFß 1) are largely
suppressed (Kuruvilla et al., 1991; Brandes et al., 1991; Taga et al., 1993; Akdis et
al. , 1998; Letterio and Roberts , 1998; Mclnnes and Liew, 1998; Pizarro et al., 1999;
Perdue, 1999; Dinarello, 1999; Herrrnann et al., 2000) .
272 CHAPTER24

Activation of enzymes, such as phospholipase A2 (PLA 2) , metalloproteinases

(collagenases, elastases), cathepsins Band D, and plasminogen activators causes the
release of compounds leading to cell death and disintegration of the intercellular
matrix and/or myelin, thus perpetuating negative involution. Local release of
substance P, calcitonin grp (gene related peptide), bradikynin, leukotrienes, LTB4 (a
potent chemotactic and hyperpermeabilizing factor) , PGE 2 , PGD 2, PG12
(vasodilatators), TxA 2, and Frisoprostanes (both vasoconstrictors) wreak further
havoc and elicit oedema and pain (Cracowski et al., 2000). Interestingly, these
eicosanoids (2-series PGs and 4-series LTs) derive from AA (20:4n-6), while 5-
series LTs and 3-series PGs, deriving from 5, 8, 11, 14, 17 eicosapentaenoic acid
(EPA, 20:5n-3) and from 4,7, 10, 13, 16, 19 docosahexaenoic acid (DHA, 22:6n-3) ,
are far less phlogogenic but are practically absent (Purasiri et al., 1997). EPA and
DHA competitively inhibit the conversion of AA to PGs, thus exerting useful
inhibitory effects on inflammation and inappropriate immune responses (Calder,
1998). That is why a diet rich in n-3 PUF As has been advocated for the treatment of
various chronic inflammatory conditions typical of autoimmune diseases (Belluzzi
et al., 1996; Rose and Connolly, 1999).
Throughout the years, with the progressive understanding of pathogenetic
mechanisms, orthodox medicine has striven hard to offer the most effective therapy.
Yet it has succeeded only in part, owing to adverse effects and unforeseen
complications. Nonetheless, the ozonetherapist has the duty to present all the possible
options. For the sake of space, 1 can only give a few brief examples, but the reader can
find a wealth of details in Hanauer (1996) and Hanauer and Dassopoulos (2001) :

2.1. Current Therapies for Inflammatory Bowel Disease

• Sulfasalazine, oral and topical with slow release preparation.

• Corticosteroids, among which budesonide is a new compound with high mucosal
potency (enema formulation) and low systemic activity. I mention these two
compounds because they are specific inhibitors of NFKB (Chapter 14,
Leukocytes), which allows the synthesis of IL-113 and TNFu (Auphan et al.,
1995; Wahl et al., 1998).
• Antibiotics, such as metronidazole and ciprofloxacin, used alone or in combination .
• Immunomodulatory drugs: azathioprine, 6-mercaptopurine, methotrexate,
mycophenolate mofetil, cyclosporine, tacrolimus (FK 506), thalidomide. They
have different mechanisms of action , but substantially inhibit the production of
pro-inflammatory cytokines (lL-l, lI-2, lI-8, IL-12 , TNFu).
• Imunosuppressive cytokines to inhibit the Th I-type » Th2-type excessive
response. lI-IO and Il-Ll seem to suppress effector functions and Th I-type
cytokine production (Taga et al., 1993; Akdis and Blaser , 2001) . A few trials
have shown the safety and tolerance , but the ultimate efficacy remains unknown.
An interesting possibility, so far evaluated in mouse colitis, is the increased
release of IL-lO into the gut lumen by genetically engineered bacteria. IL-IO
may be absorbed via a paracellular route and may downregulate T cell activation
 OZONETHERAPY IN VARIOUS PATHOLOGIES 273

in the submucosa (Steidler et al., 2000) . TGFßl mayaiso be efficacious but has
not yet been tested . The usefulness of IFNa remains equivocal.
• Oral tolerance. If the responsible autoantigens ean be identified , their oral
administration eould induee an immune tolerance and represent a rational
treatment.
• Probiotics. In Chapter 19, the critical role of genetic susceptibility of the gut
mieroflora and its interaction with enterocytes and the MALT was discussed .
There are promising, yet unsubstantiated, results after administration of
laetobaeilli, bifidobacteria, Strepto coccus thermophilus, ete. This complementary
approach should not be disregarded, since it is non-toxie and may become more
useful by modifying the luminal environment by intermittent hydrocolon
therapy. A correet ecologieal environment can be restored by microflora
administered via enema .
• Dietetic support. As previously mentioned, a diet enriehed with n-3 PUFAs
present in fish oil generates (via cyclooxygenase and lipoxygenase) 3-series PGs
and 5-series LTs, which are anti-inflammatory and may re-equilibrate the Th l-
Th2 pattern (Hodgson, 1996). n-3 PUFAs can easily be taken in eapsules
(Belluzzi et al., 1996) or emulsionated with milk. Although this approach is
probably not sufficient to solve the problem, it is recommended and obviously
has to be continued for life.
• SC administration 0/ growth hormone for four months (Slonim et al., 2000) . The
optimal dose, schedule and duration of response remain to be defined .
• The last approach regards a biotechnological "jewel", i.e. the licensing in the
USA and Europe of infliximab, an antibody against TNFa (anti-TNFa). This
chimeric antibody is about 25% mouse and 75% human and is still
immunogenic. A humanised antibody, CDP571 (only 5% mouse and 95%
human) , is probably better, but is not yet available. RCTs involving the IV
infusion of these antibodies have shown clinical benefits in about 65% of
patients with severe Crohn's disease and fistulae. Four trials have examined a
total of 306 patients (Targan et al., 1997; Present et al., 1999) and a very
informative commentary has been published in which Bell and Kamm (2000)
conclude that "although treatment with anti-TNFa has improved the quality 0/
life for some patients with Crohn's disease, knowledge ofits proper clinical role
will come only with time". Another geneticallyengineered product is the soluble
TNFa receptors consisting of two identical chains of the extracellular human
p75 TNF reeeptor monomer fused to the Fe domain of human IgG I. This is
known as etanercept and is administered SC twice a week. These new products
are now limited by their cost, not only in Europe but also in the U.S.

2.2. Current Therapies o/RA

Beeause of similarities, I will now briefly report eurrent therapies of RA (Choy and
Panayi, 200 I), in whieh degenerat ion of cartilage and erosion ofjuxta-articular bone
seems mainly due to the presence ofTNFa (Feldmann and Maini, 2001).
274 CHAPTER 24

Orthodox therapy aims to relieve pain , reduce and possibly resolve inflammation
and enhance healing.

• Non-steroidal anti-lrflammatory drugs (NSAlD) . Besides the old aspirin , they

inc1ude ibuprofen, indomethacin, naproxen, sulindac, etc . They are all associated
with at least gastric irritation. The latest-generation cyclooxygenase I1 inhibitors
seem to be effective and have less adverse effects (Fitzgerald and Patrono,
2001) .
• Glucocorticoid therapy . Except occasionally prednisone to control symptoms,
this therapy is now proscribed.
• Immunosuppressive therapy. Azathioprine, cyc1ophosphamide, methotrexate at an
interrnittent low-dose may be useful, but attention must be given to adverse effects.
• Disease-modifying drugs , such as D-penicillamine, sulfasalazine, gold
compounds, are partly useful , but there is minimal evidence that they delay bone
erosion or allow real healing.
• The new drugs are the previously mentioned anti-TNFa antibodies and the
soluble TNFa R fusion protein etanercept. IV administered antibodies appear to
regulate the production of IL-6, IL-8, MCP-I and VEGF, reduce the influx of
inflammatory cells into joints and the blood levels of rnatrix metalloproteinases.
As has been observed in Crohn's disease, in a few RCTs, about two-thirds ofRA
patients have shown a c1ear beneficial response for up to 2 years (Elliott et al.,
1994; Maini et al., 1999). However, in RA, infliximab has been associated with
methotrexate. Similarly, the combination of etanercept and methotrexate seems
to provide greater c1inical benefit than methotrexate alone (Lovell et al., 2000;
Weinblatt et al., 2000) .

Lets us now consider the official therapeutic arsenal of Crohn's disease and
RA versus ozonetherapy. Because someone may think that I am biased against
official medicine, I invite everyone to read Sartor's commentary (2000) about new
approaches to Crohn's disease and the commentaries ofO'Dell (1999) and Pisetsky
(2000) about RA. Although all three authors agree that a new era of improved
treatment has arrived, they caution that a multitude of bad effects may appear. Sartor
(but also Bell and Kamm, 2000) is particularly critical. Possible problems include :
allergic reactions and serum-sickness-like reaction, induction of antibodies to
double-stranded DNA, occurrence of lymphoma (shown already in 6 patients) and
development of tubercolosis (Keane et al., 200 I). Use of infliximab costs about
12,000 pounds per year . About 33% of patients are unresponsive and some pat ients
are not suitable for immunological therapy. All previous therapies , as reviewed by
Sartor, are both scarcely effective and present serious adverse effects .
Regarding ozonetherapy, besides a few unbelievable anecdotes or scanty results
by Knock and Klug (1990) , there is only the study performed at Cuba's Institute of
Rheumatology in 1988 on 17 patients treated with IM injections of Os-O, (total dose
of ozone : 700 ug) for 8 weeks combined with NSAID . Apparently about 25% of
ozone-treated patients scored 25% better than controls (Menendez et al., 1989). It is
 OZONETHERAPY IN VARIOUS PATHOLOGIES 275

reasonable to conclude that we do not know today if ozonetherapy may be useful.

Nevertheless, in spite of a few charlatans' tales, we should not dismiss it a priori.
How and why could ozonetherapy be beneficial?
a) Using either 03-AHT or BOEX or even the more complex EBOO approach, we
may be able to enhance immunosuppression and increase the production of IL- 10,
IL-II, TGFß and perhaps IL-I Receptor antagonist (IL-I Ra). The immunological
investigation carried out in patients should aim at clarifying if ozonetherapy
induces anergy of the T cells that are cytotoxic. The RI approach, associated with
the parenteral ones, may be helpful in inducing immunosuppression in the gut (see
Chapter 19). RI mayaiso inhibit the bacterial flora that is partly responsible for
Crohn's disease . However, the possibility ofinducing immunosuppression has yet
to be tested, although a hypothetical rationale and a tentative protocol have been
proposed in Chapter 14 (Leukocytes, Point 3).
b) Generalized and localized induction of antioxidant enzymes, thus eliminating the
excess of ROS production. This is a reasonable proposition, which already has
experimental support and may be achieved by inducing the adaptation to COS.
c) Inhibition of the release of inflammatory enzymes, metalloproteinases etc ., with
a progressive decrease of plasma levels of PAF , LTB 4, PGE 2, TxA2 and
isoprostanes. The chronie inflammatory process can be slowly tumed off only if
we can, with 6 months oftherapy, implement points a) and b).
d) Fistulae and abscesses in Crohn's diseases can be dealt with by insufflation of
ozone or ozonized water and oil. An RCT could be performed in one or more
hospitals, but this is beyond our means and dreams at the moment. It will be
indispensable to have a control arm because the placebo effect may be relevant.

Who can pay the cost of endoscopic, radiological, histologieal, biochemical and
clinical exams? We are not backed by any pharmaceutical and/or biotechnological
firm because ozonetherapy does not produce profits. However, if ozonetherapy
proved to be useful, it would save money for the National Health Service. Yet, I
doubt that the Ministry of Health, biased and myopie as it has proved to be in the
past, will ever support this research.

2.3. Therapies ofMS

The same problems will apply to a RCT planned for MS. This is a tragic disease
because it very often disables young adults just when they are about to show their
merit. I worked enthusiastically on IFN research, but in the meantime 1 was longing
to evaluate if ozonetherapy would be useful for these patients. All physicians know
that MS is a T cell-mediated autoimmune disease that can either relapse (relapsing-
remitting MS) or be very aggressive (progressive MS) . Good reviews of the topic
are available (Rudick et al., 1997; Karp et al., 2000; Polman and Uitdehaag, 2000;
Wingerchuk et al., 2001) .
Orthodox medical therapy is based on a) corticosteroids, b) immunosuppressive
drugs, namely azathioprine, methotrexate, cyclophosphamide and cyc1osporine, and
more or less c) experimental biologicals, such as IV immune globulins, copolymer I
276 CHAPTER24

(COP) 01' glatiramer acetate (Duda et al., 2000; Kipnis et al., 2000; Neuhaus et a\.,
2000), which induces a shift from a Th -I to a Th-2 cytokine profile in COP-treated
patients. The present treatment of choice (for r-rMS) is IFNß-Ia (glycosylated) and
IFNß-Ib (a mutein). All of these drugs can cause immunosuppression to different
degrees, and particularly those indicated in a) and b) may cause serious adverse
effects. Despite their biochemical difference, both fonns of IFNß (approved by US
and European regulatory authorities) have a positive elinical effect, characterized by
a elear reduction of both the frequency and severity of exacerbations (Amason,
1993; Rudick et al., 1997; Polman and Uitdehaag, 2000). IFNßs are fairly weil
tolerated. Unfortunately, owing to striking phannacokinetic and pharmaeodynamic
differenees (Bocci, 1981b; 1987b; 1988a; 1990a; Bocci et al., 1988), IFNa-2a,
which eould be therapeutically useful, causes adverse events that negatively affect
the already poor quality of life of these patients (Nortvedt et al., 1999). IFNsß are
now in wide use, but problems such as the optimal dose and sehedule, the
appearance of neutralising antibodies (mostly to IFNß-I b) that may jeopardize
efficacy (AntoneIli et al., 1998), a possible relapse when stopping therapy and the
eonsiderable cost, provide a glimmer of hope that a serious RCT based on
ozonetherapy is still meaningful. In the ease of MS, nothing serious has been done
and my attempt to interest three neurologists was in vain beeause they were weil
sponsored by finns producing IFNß . Two ozonetherapists (one in Turin and another
in Milan) reported to me that they had achieved "good rcsults" treating MS patients
with OJ-AHT eombined with either magnetotherapy 01' ehelation therapy. No
comment!
Thus very \ittle hope remains and it also seems useless to speculate that in these
diseases, as weil as in others cited at the beginning of this section , it might have
been useful to combine 01' to alternate cyeles of IFNß 01' anti-TNFa antibodies with
ozonetherapy. An old adage says: "Be not afraid of going slowly, be afraid only of
standing still".
The problem of Raynaud's phenomenon (Block and Sequeira, 2001) in
selerodenna patients will be considered in the next section .

3. ISCHAEMIC DISEASES
(HlND-LIMB ISCHAEMIA, CEREBRAL AND HEART ISCHAEMIA,
VENOUS STASIS)

It appears logical to think that if blood briefly exposed to oxygen-ozone can exert a
benefit, this would be best noted in isehaemic tissues . Even a partial obstruction of
limb arteries due to atheroselerosis (Lusis, 2000) 01' diabetes 01' Buerger's disease
(thromboangiitis obliterans) leads to a progressive reduction of blood flow to the feet.
Lack ofperfusion leads to tissue ischaemia and possibly cell death. Any minor trauma ,
nonnally irrelevant, facilitates the fonnation of an ulcer, which will not heal beeause
oxygen , nutrients and soluble media tors involved in the repair process are laeking.
Aeute \imb ischaemia is frequently eaused by aeute thrombotie oeclusion of a
pre-existing stenosis 01' by an embolus; it requires immediate surgieal 01' medieal
 OZONETHERAPY IN VARIOUS PATHOLOGIES 277

attention . Chronic limb ischaemia becomes progressively evident. In Europe, we

distinguish four stages, as classified by Fontaine:

Stage J: Feeling of cold or nurnbness in the foot and toes. Skin temperature is
reduced. The foot is pale and frequently becomes cyanotic.
Stage JI: Paresthesia and hypoesthesia, firstly localized and successively diffused to
the whole foot. Hyporeflexia. This is the phase with incipient neurological defect.
Intermittent claudication. Pain may cease with rest.
Stage IJI: Pain at rest with noctumal exacerbation. Cyanosis becomes weil evident in
one or several toes , with an incipient trophic lesion or a frank uleer. (Rate of
amputation is - 15%).
Stage IV: Partial or total necrosis of one or several toes. Pain often becomes
unbearable (Rate of amputation is - 50%) .

The angiologist has several, precise non-invasive techniques to objectively

assess the severity of POAD. Extensive epidemiological studies have shown that
these patients have practically the same relative risk of death from cardiovascular
causes as do patients with a history of cerebrovascular or coronary disease. A useful
predictive value is given by the ankle-brachial index (ABI). The normal range of
values is 0.91-1.30, which decreases to 0.41-0.90 in mild to moderate POAD and to
below 0.40 in severe POAD. Patients with ABI below 0.40 are at high risk of a
cardiovascular event and present an annual mortality of about 25%. As I cannot go
into details, I suggest reading two excellent review articles (Dorrnandy and
Rutheford, 2000; Hiatt, 200 I). Each patient must be evaluated for any possible
revascularization and there are several operative procedures with a high success rate .
Lurnbar sympathectomy is no longer performed because it does not increase blood
flow to muscle. Spinal-cord stimulation also does not prevent amputation (Klomp et
al., 1999). At the extreme, Taylor et al. (1999) have shown that distal venous
arterialisation is a unique procedure with promising possibilities for salvage of
critically ischaemic, inoperable limbs (stage IV). Besides surgery, orthodox
medicine offers several therapeutic options, including useful supportive measures,
such as quitting smoking, proper diet, exercise (Davies, 2000), and pharrnacological
treatments as folIows :

1) Vasodilatators must be able to improve collateral blood flow and avoid

"stealing" blood away from underperfused muscle. Pentoxyfylline may enhance
oxygenation in ischaemic tissues by increasing blood flow to the
microcirculation. lt may improve blood rheology by decreasing blood viscosity
and enhance erythrocyte flexibility. However, arecent double-blind RCT
showed no significant difference in healing rates of pure venous uleers between
patients taking pentoxyfilline and those taking placebo (Dale et al., 1999). In
1999, the FDA approved cilostazol, an inhibitor of phosphodiesterase type 3,
which by increasing the concentration of cAMP causes vasodilatation and
reduces claudication. PGE 1 and a stable prostacyclin analogue (iloprost) have
been infused in patients with critical leg ischaemia. Both cilostazol and iloprost
278 CHAPTER24

improve POAD, but also cause frequent headaches, palpitations and dizziness
and should not be used with patients who also have heart failure.
2) Progression of atherosclerosis may be delayed by treatment of
hypercholesterolemia and platelet aggregation inhibitors (aspirin, ticlopidine,
clopidogrel), while thrombolytic intervention does not help POAD patients .
Propionyl levocamitine improves muscle metabolism and seems useful in
improving the quality of life, but certainly does not solve the central problem.
Needless to say, diabetic patients must be weil und er control (although it hardl y
helps), the homocysteine serurn concentration must be lowered and hypertension
must be treated with caution. The prognosis of POAD patients is dirn, with
progressive deterioration that limits their ability to perform daily activities.

In these circumstances, patients look for a treatment that may real1y improve
their condition. Thus oxygen-ozone therapy has attracted much attention. Whether it
deserves it and is really better than the multi form traditional therapy remains to be
seen, because most of the following data are questionable.
In the ozonetherapy field, the work by Rokitansky, who was president of the
Austrian Society ofOzonetherapy, is revered as the best. In 1981, he presented data
for 152 patients treated with OJ-AHT between 1974 and 1980.

Table 20. Results in POAD patients after ozonetherapy .

Fontaine stage:
II III IV
Patient number: 62 51 39
Very good
improvement: 87.1 % 70.6 % 53.8 %
Walking distance: > 1000 m > 800 m > 500 m
no pain at rest gangrene healed
Improved: 9.7 % 21.6 % 25.6 %
Walking distance: > 400-500 m > 300-400 m Amputation oftoes
occasional pain with healing of
stump
No improvement
or progression: 3.2 % 7.8% 20.6%

He examined 2,3-DPG levels in erythrocytes in 11 patients and reported a

marked increase in 9, no change in land a decrease in another (who had a very high
level before ozonetherapy). This study was without any contro\.
At a sub sequent meeting (1983), Rokitansky presented the following table (Table
21) : two groups of patients (232 versus 140), roughly similar in age and stage, were
treated with either O 2-0 3 administered by intra-arterial injection into the femoral
artery plus local treatment with gas, or conventional (C) vasodilatation therapy (a
sort of control ).
 OZONETHERA.PY IN VARIOUS PATHOLOGIES 279

Table 21. Comparison ofresults in two groups ofPOAD patients.

Stage Nutnber of Marked Modest Progression

p"tients improvement (%) improvement (%)
03 C 03 C 03 C 03 C
11 105 73 80 .0 43.8 11.4 19.2 8.5 37.0
III 72 46 70 .8 39 .1 19.4 17.4 9.7 43.5
IV 55 21 50 .9 28.6 21.8 19.0 27 .3 54 .0

The rate of amputations declined from 15 to 10% for stage III and from 50 to
27% for patients (Stage IV) treated with IA Ozone plus topical bagging.
This study is of no use today because IA injection of ozone has been abandoned
and the traditional treatment at that time is no match for the modern integrated
approach.
In 1987, Mattassi et al., working at the Vascular Surgical Unit at Garbagnate
Hospital near Milan, compared the old approach of IA injection of ozone (20 ml, 0 3
concentration: 40 ug/ml; 3 times per week or every day) to the c1assical 03-AHT
(200 ml blood in citrate, 0 3 concentration not specified, probably 30-40 ug/mI; one
session per week for a total of five sessions). They found that that OrAHT yielded
slightly better results than IA ozone (Table 22). They also reported (it is unclear if
they pooled the data) a significant increase of HDL-cholesterol and triglycerides.

Tahle 22. Comparison between IA ozone administration (IA03) and OrAHT in 101
patients (II stage). 53 patients (III stage) and 65 patients (IV stage).

Stage Optimal Result (%) Good Result (%) No Result (%)

/AOI DrAHT IA 0 3 DrAHT IA 0 3 DrAHT
11 16 27 59 49 25 24
III 7 32 67 36 26 32
IV 11 11 42 33 47 56

In 1988, Romero et al. perfonned a study on 60 patients at the National Institute

of Angiology and Vascular Surgery in Havana, comparing IA03, 03-AHT and
traditional (?) medical treatment. It is unclear if the IA administration of 0 3 was
better than O]-AHT, but ozonetherapy apparently produced a 20-35% improvement
over conventional therapy.
Three Polish studies (published in Polish) in 1990, 1991 and 1992 continued to
examine the effect of IA0 3 in POAD atherosclerotic and diabetic patients. On the
whole, the conclusion was that IAO} was "valuable and safe" and markedly
improved the intermittent c1audication distance measured before and after the
treatment. In one study, an increase of ABI was also shown. The measurement of
some biochemical parameters led the authors to say that IA03 caused "a significant
decrease in blood cholesterol levels and a marked reduction in blood and plasma
viscosity".
280 CHAPTER24

After Rokitansky et al. (1981) asserted that ozone increases 2,3-DPG, lowers
fibrinogen and plasma viscosity, and reduces plasma cholesterol, it has become a
regular ritual to confirm these data.
The aim of orthodox therapy is to improve blood rheology by changing the
lifestyle (no smoking, almost a vegetarian diet , exercise, etc.) and using several
drugs aimed at different targets . Yet, in spite of a great effort, the success has been
meagre . As discussed in Chapter 14 (Erythrocytes), it is hard to believe that ozone
can produce all these magie results , because during the last 6 years of c1inical work
(with 0 3-AHT and EBOO) we have not been able to confirm them. We have not
observed a decrease of cholesterol plasma levels, nor a change of LDL ; if anything,
the fibrinogen level tends to increase slightly. In our ARMD study (about 5,000 0 3-
AHT) , often with 13-14 sessions , we observed a slight increase of2,3-DPG only in a
few patients who had a very low level prior to therapy . This result was also obtained
by Mattassi (1985) before uso Romero et al. (1993) conducted a useful study ,
comparing the relative efficacy of ozone adrninistered to POAD patients by: a) IM
gas injection, b) OrAHT, c) RI, and d) standard conventional treatment. Irrespective
of the administration route, ozonetherapy markedly improved the ABI and reduced
c1audication. Thus the authors reached the conclusion that RI was as effective but far
easier to perform than 03-AHT. As I am not enthusiastic about RI, I was surprised
when Mattassi confirmed, at the Verona Congress (1999), that among IA, OJ-AHT
and RI, the last approach is as effective as the first two and concluded that we must
bear in mind this finding because the patient who owns a generator can perform a
self-administration at horne. Russian c1inicians working at the Ozonetherapy Centre
in Nizhni Novgorod reported at the XII 10A Congress in Lille (1995) that ozone
admin istered by various techniques to 132 elderly patients (IV and SC injections of
O 2-0 3, IV infusion of ozonized rheomacrodex solution and 3-4 0 3-AHT) is "highly
effective" and induces "significant improvement ofpatients' weil being".
Amato's observations (2000) on the effect of 03-AHT as a unique therapy for
angina abdominis (AA) cannot be overlooked. AA is arare, painful abdominal
syndrome that manifests itself after a meal , probably owing to a localized transitory
ischaemia of the gut. Surgical vascular correction normally solves the problern. but
in the three elderly patients studied by Amato it was not feasible . A cycle of 10 Or
AHT (150 ml of blood treated with 0 3: 20-40 ug/rnl per ml of blood) followed by
maintenance therapy (one treatment every month) resolved the problem very weil
and patients, no Ionger afraid to have a meal , showed a marked improvement
without any side effects. The oldest patient, a woman of 87 years, has undergone
this therapy since 1994!
Who says that ozone is toxic?
A final remark can be made regarding the extraordinary capacity of combining
03-AHT with topical therapy (either gas or, better, ozonized water and oil) to allow
healing of awful decubitus or neerotie ulcers in the limbs (also see Chapter 14,
Platelets, and this Chapter). It takes some time but they do heal. Figure 98, taken
from Werkmeister's work (1995), shows that extensive lesions after X irradiation
took up to 190 days with only loeal treatment, but I believe that association with
parenteral treatment would have shortened the resolution time. Regarding ulcers on
limbs, irrespective of the aetiology (atherosclerosis, Buerger's disease, diabetes,
 OZONETHERAPY IN V ARIOUS PATHOLOGIES 281

Raynaud's phenomenon), they do heal, even in the two exceptional cases described
by De Monte and van der Zee (2001).
160 r--------------------------,
140

_120
N

E 100
~
c:: 80 -
o
:3 60
..J
40

20
oL:::::::!;s;:::;:e:~ä3I;~~A--~--_..,._--~
, ,
o 50 100 150 200 250
Days

Figure 98. The diagram reports the time necessary /01' the healing 0/ decubitus ulcers in 8
patients after only topical hypobaric application o/OrOJ. according to Werkmeister (1995).
The ulcer q{ 153 cm' took about 190 days to heal. This time can be markedly shortened by
combining the parenteral (DrAHT) and topical (ozonized water and oil) treatments

In the first, a woman was initially treated with a percutaneous chemical (phenol)
lumbar sympathectomy, supplemented with a continuous infusion (0.5 ml/hour) of
bupivacaine 0.15% via an epidural catheter; this treatment only controlled the pain.
The second case was a man with painful bilateral leg ulcerations due to a vaseulitis.
A lumbar epidural catheter delivering 0.5 ml/hour of bupivacaine 0.20% and 0.125
mg/hour ofmorphine (3 mg/day) bare1y controlled the pain.and the ulcers worsened.
In both cases, healing was achieved by removing the catheters and perfonning 40
and 45 03-AHT, respectively, plus topical treatment with ozone.
Uleers from venous stasis have been treated and they also heal rapidly with the
combined treatment. However, phlebopathies have attracted less interest than
arteriopathies. If venous hypertension cannot be compensated by physiological
mechanisms, it leads to increased penneability at the level of the microcirculation,
Iymphatic hypertension, oedema and possibly torpid ulcers. I can report only one
investigation (Lo Prete, 2000) perfonned in patients with extended varicosity, which
examined subjective parameters (phlebalgia, feeling of orthostatic weight or pain,
fonnication and paresthesia), objective parameters (evening oedema, constant
oedema, haemosiderinic dermitis, fibrous hypodennitis, eczema, skin ulcerations)
and instrumental parameters (plethysmography, videocapillaroscopy and evaluation
of circumference at the calf and at the ankle-malleolus) , There were 15 patients (14
women and 1 man), from 20-60 years old, with marked varicosity complicated by
chronic venous insufficiency. Ozonetherapy was perfonned by SC and perivenous
injection ofup to 300 ml of gas (O~ + Oj) at an ozone concentration of 8 ug/ml in 60
282 CHAPTER24

sites (5 ml per site) . There were two treatments per week, repeated for 12 weeks
(total 24 sessions). The results are presented in the following tables:

Table 23a. Subjective parameters.

Before treatment Aper treatment

Phlebalgia 8(15) 53% 0(15) 0
Feeling of orthostatic weight 15 (15) 100% 4 (15) 26.6%.
Orthostatic pain 15(15) 100% I (15) 6.6'%
Formication 9 (15) 60% 0(15) 0
Paresthesia 7 (15) 40% o(15) 0

Table 23h. Objective parameters.

Before treatment After treatment

Evening oedema 15 (15) 100% 2 (15) 13.3%
Constant oedema 4 (15) 26.6'Yo 2 (15) 13.3%
Haemosiderinic dermitis 5(15) 33.3% 1 (15) 6.6%.
Fibrous hypodermitis 6 (15) 40% 4 (15) 26.6%.
Eczema 4 (15) 26.6% 0(15) 0
Skin ulcers 2 (15) 13.3% o(15) 0

Table 23c. Instrum ental parameter s.

Before treatment After treatm ent

Plethysmography - 18.0" 22"
Evaluation of circumference (cm) :
calf 34.3 33.1
ankle (malleolus) 25.2 24.5

There was a marked reduction of the peripheral venous stasis, likely due to
improved microcirculation. The SC and perivenous administration of gas caused
modest but transitory pain. No more than 5 ml per site ought to be injected. There are
no other adverse effects . Simultaneous topical treatment enhances the healing oftorpid
ulcers. The association with OrAHT may further improve the treatment.

Let us now try to reach some conclusions. It is unfortunate that the possibility of
improving tissue ischaemia with ozonetherapy has interested only a few vascular
surgeons. Conceptually, ozonetherapy is the warhorse of this approach and appears
to be very useful in skeletal muscle, myocardial and cerebraI ischaemia, because it
may : increase oxygen and glucose delivery by several mechanisms (Chapters 12 to
14), b) enhance angiogenesis via activation of resident stern cells, c) induce the
preconditioning phenomena by upregulating the expression of antioxidant enzymes
and HSPs, and d) trigger a neuro-humoral response to improve the quality of life .
This is exciting, but objectively we can conclude that none of the several studies is
 OZONETHERAPY IN V ARIOUS PA THOLOGIES 283

satisfactory because of inappropriate administration routes, unknown or uncertain 0 3

concentrations, too brief schedules and lack of areal contro\. The only available
RCT , in line with the rules of orthodox medicine, is that perforrned by Kraft et al.
(1998), who examined the effect of03-AHT on patients with mild hypertension. The
original abstract folIows:
"Autohemotherapy with ozone is widely applied by German therapists who prefer
complementary medicine, in eider patients with cardiovascular problems in order to
improve various symptoms and the quality of Iife. The strong oxidant ozone may further
increase the already high oxidative potential of these patients and thereby counteract
positive effects of antioxidants. In the present study, the effects of autohemotherapy
with ozone on cardiovascular risk factors were evaluated in 17 patients , with mild
hypertension. In a randomised, double-blind, placebo-controlled crossover-study, 24 h
ambulatory blood pressure and blood pressure load were significantly reduced after a
cycle of ten autohemotherapies with ozone, but not after a cycle with placebo (oxygen)
(p < 0.05). The initial blood pressure level was restored after the end of the therapy
within 4 months. Aggregation of thrombocytes was significant and the malondialdehyde
concentration was c1early increased, serum HDL concentration was reduced, and the
rate of side effects was higher during the cycle with ozone . The reduction of blood
press ure may be due to an increased oxygen supply of the vascular endothelium, but an
activation of the oxidative potential of polymorphonuclear leukocytes and/or
macrophages cannot be excluded . Considering the small reduction of blood pressure,
the potentially noxious oxidative mechanisms and the rather high expenses,
autohemotherapy with ozone cannot be recommended as a useful alternative to
pharmacological treatment ofpatients with mild hypertension."

Clearly Kraft et a\. are very critical and, in my opinion, they have verified the
validity of ozonetherapy on the wrong disease. Indeed mild hypertension is a life-
long ailment and there are optimal conventional remedies (Iow-sait diet, statins,
ACE-inhibitors, etc.) to keep it under control, so that the use of ozonetherapy is
unjustified. Moreover, the conclusions of these authors have been influenced by
prejudice and an incomplete knowledge of the potentials of ozonetherapy.
With regard to the standard set by official Medicine, we are practically back to
square one and if we really want to define the value of ozonetherapy in POAD
patients we must first define standard conditions and then perforrn serious RCTs .
The following guidelines are suggested:

I) among the four approaches, 03-AHT, RI, BOEX and EBOO, we can select the
first or the second because they are easy to perforrn and inexpensive. IA, IV or
SC injection of ozone as gas should be excluded. After a convenient wash-out
period, patient groups may switch over to compare 0rAHT and RI in the same
patient. For OrAHT, suitable ozone concentrations may range from an initial 20
ug/ml per ml of blood up to 40 ug /rnl at the beginning of the 3'd week. Two
treatments per week for a total of 16 treatments (8 weeks). In our experience a
shorter schedule is not valid. For RI, ozone concentrations may range frorn an
initial 5 J.lg up to 30 ug/rnl, increasing the gas volume progressively frorn 150 ml
to 600 ml in 2 weeks. Five treatments per week for 8 weeks. This schedule is not
practical for patients.
2) Groups of POAD patients belonging to stage 11, III and IV should be as
homogeneous as possible.
284 CHAPTER24

3) Objective assessment of the stage and severity of the disease should be

detennined by the best and most precise non-invasive techn iques before
treatment, after 8 weeks and again two months thereafter. Evaluation should be
perfonned by independent c1inicians.
4) Patients should be randomized into two groups: one receiving O 2-03 and one O 2
only, with the same technical modalities. A third group treated with the best
conventional treatment could be included for a comparison of the efficacy . If the
treatment is valid, the control group has the right to be switched over and receive
the ozonized treatment. These data may strengthen the results .
5) A specialized statistician should evaluate all the data .
6) The same protocol can be tentatively explored in patients with Raynaud's
phenomenon secondary to systemic sclerosis. A comparison can be made with an
historicalor a new control with infusion of iloprost, which has proved to be
effective for short-tenn palliation (Wigley et al., 1994; Block and Sequeira, 2001) .

Two RCTs, one dealing with POAD and another with terminal cardiopathic
patients, will evaluate the toxicity and efficacy of EBOO according to the optimized
methodology (reported in Chapter 17) at the Siena and Modena University clinics.
We hope to be able to report the results within 2003 .
Owing to the systemic nature of atherosclerosis, both the heart and CNS are at
high risk in POAD, and there is a rather high incidence of myocardial infarction or
ischaemic stroke and/or terminal stage lirnb ischaemia. That is why we are testing
the validity of EBOO in end-stage cardiopathic patients when either transplantation
or surgical revascularization is not feasible . Our preliminary study (Di Paolo et al.,
2000) on three patients yielded results that are encouraging but regarded as
anecdotal because angiocardiographic examination could not be repeated after the
treatment. This project is ongoing and we have already treated 15 patients
(manuscript in preparation). For the time being, there are two studies: the Russian
trial was carried out in 39 patients with advanced coronary atherosclerosis. They
underwent five daily infusions (for 20 days) of ozonized saline solution. I believe
that ozonization was carried out at a very low ozone concentration (perhaps 2-3
ug /rnl), so the levels of HOCI were not too high and thus not too caustic! I must say
that I am dead against this procedure. However, Zhulina et al. (1993) concluded that
the treatment was effective because angina attacks decreased from an average of 6 to
about 2 per day . There were no controls with either oxygenated saline or simple
saline, which may have shown a significant placebo effect.
The second study is one ofthe best reported in the field and I am glad to say that
the editor of "Free Radicals in Biology and Medicine", who strongly opposes ozone
in Medicine (Chapter 2), decided to publish this paper. Hemandez et al. (1995) quite
correctly performed 03-AHT, five days per week for a total of 15 treatments, in 22
cardiopathic patients. They found a significant decrease in plasma cholesterol and
LDL levels (we shall see ifwe can confinn this finding after EBOO) and an increase
of erythrocytic GSH-Px and G-6PD , which is in line with the phenomenon of
adaptation to COS paradoxically induced by ozone.
It is fair to mention that the great hope of modem medicine is to use gene
therapy to elicit therapeutic angiogenesis in patients with chronic myocardial
 OZONETHERAPY IN VARIOUS PATHOLOGIES 285

ischaemia (Patterson and Runge, 2000 ; Jackson et al., 2001) . However, while this
new approach matures, I do not see anything wrong in evaluating ozonetherapy.
In about 80% of patients, ischaemic stroke results from atherothrombotic or
thromboembolic processes. Stroke can strike relatively young persons at the peak of
their intellectual activity and, if not fatal, can be highly debilitating. Fortunately,
Handel, Pasteur and Roosevelt, to cite a few, were able to make great contributions
to music, science and politics in spite of suffering a stroke .
Modem medicine has developed prophylactic measures able to reduce the risk of
transient ischaemic attacks (TIAs) or of stroke in prone individuals by 20-30%
(Gubitz and Sandercock, 2000). Moreover, anti-atherosclerotic drugs and, if
necessary, carotid endarcterectomy appear beneficial. In case of an acute stroke,
therapy must begun within the shortest possible time (from 0.5 to 2-3 hours) to
reperfuse the ischaemic penumbra surrounding the core of a cerebral infarction .
Time deIays are predominantly in the pre-hospital phase and can be fatal. Hypoxia
induces a cascade of metabolic disorders, such as tissue acidosis, reduction of ATP
levels, Ca 2+ overload, activation of glutamate receptors, N-methyl-D-aspartate
(NMDA) channel opening and release ofproteinases, leading to neuronal death . The
interested reader will appreciate the complex sequence of events carefully analyzed
by Besson and Bogousslavsky (1995) , Back (1998), Small et al. (1999) and
Rosenberg (1999) . Since the 1990s, IV thrombolysis using recombinant tissue
plasminogen activator (Tpa), with due caution to avoid cerebral haemorrhage, has
been applied to reduce the time of reperfusion and neuronal damage . In Cuba, where
there is a lack of Tpa (owing to the embargo), many hospital emergency units have
ozone generators at hand and patients with stroke are luckily treated as soon as
possible with oxygen-ozone therapy . Thus, we again face the old dilemma, which
need not exist if we have been able to weigh the risks and advantages of the two
approaches.
To my knowledge, only a preliminary study has been reported by Wasser, a
German ozonetherapist, who has treated stroke patients privately, with all possible
inherent disadvantages. He reported at the XII IOA Congress (Lilie, 1995) that he had
treated several patients some time after they suffered an acute stroke. In spite of this
limitation, the use of 0 3-AHT every day seems to have improved the outcome, in the
sense that no patient died and they apparently recovered very rapidly. A1though the
study has obvious limitations, Dr. Wasser (1995b) has to be congratulated for his
adventurous enterprise, which deserves to be pursued. At my University, I have found
great disinterest; neurologists do not want to risk what they consider a reasonably valid
treatment (thrombolysis) for the uncertainty of ozonetherapy .
For what it is worth, my opinion is that a controlled study using either Tpa or
ozonetherapy, or a combination of the two, performed at the earliest possible time
after a stroke would be very informative. Indeed it would probably save lives or
reduce the disability.
On the basis of some experimental findings and on the uncertain validity of the cited
c1inical open studies, I believe that ozonetherapy could be useful in vascular diseases,
particularly in poor countries. However, it must be validated by RCTs in developed
countries. Regretfully, no support from Health Authorities or private organizations is
foreseeable and thus we have to continue the struggle and try to do our best.
286 CHAPTER24

4. RETINAL DEGENERATIVE DISORDERS

A. Diadori and V. Bocci

4.1. Retina: Anatomical Aspects

The retina, a thin transparent membrane lining the interior of the eye , is the
initial receptor for visual stimuli . Its outer face is in contact with Bruch's
membrane of the vascular choroid; its inner surface is in contact with the
vitreous body (Fig . 99) .

Sclera

Choroid

.'= ~~'t's memb rane

Pholo receplo rs

Relinal vessels

Figure 99. Image ofthe ocularfundus (left side) with the macula lutea at the centre.
Schematic drawing (right side) 0/ the several retinal layers, from the internal limiting
membrane to the sclera . RPE: retinal pigment ep ithelium

The neurosensorial retina is separated into 10 layers, with the photoreceptors

(rods and cones) located in the outer layer and the axons of the ganglion cells
(second-order neurons) collecting on the inner layer to form the optic nerve . At the
extreme outer layer of the retina, there is the retinal pigment ep ithelium (RPE), a
monolayer of cells resting on Bruchs membrane (which separates the RPE from
the choriocapillaris), ftmctionally connected to the photoreceptors. The RPE has
several important functions : it phagocytoses and processes the tips of the outer
segment of the photoreceptors, recycles vitamin A, transmits oxygen and other
nutrients from the choroid to the photoreceptors and outer retina, and differentiates
into macrophages and other types of cells in response to various stimuli .
The central area of the retina (c1inically defined as the area within the
temporal vascular arcade) is referred to as the macula; the highest visual acuity
(ability to see fine details) resides in the cone-rich centre of the macula, the
fovea. Retina! vesse!s are absent in the fovea, rendering the fovea critically
dependent on the choriocapillaris for its metabolic requirements.
 OZONETHERAPY IN V ARIOUS PATHOLOGIES 287

Several ophthalmologie disorders of the retina and the optie nerve, of

multifactorial aetiology, lead inevitably to peripheral and/or eentral loss of
vision by degeneration ofthe neurosensorial eells (0' Amico , 1994).
Reeent studies on the meehanisms mediating neuronal damage suggest that
apoptosis might be implieated in the death of neurosensorial eells in the retina
and optie nerve (Xu et al., 1996; Gregory and Bird, 1995; Lo et al., 1995; Adler
1996; Fraser et al., 1996). In partieular, advanees in the understanding of
eellular meehanisms of retinal ganglion eell and nerve axonal damage in
isehaemie injury suggest an isehaemie eomponent; seeondary effeets (seeondary
neuronal degeneration) are eaused by a toxie environment produeed by the
dying eells, resuIting in free radical produetion and further tissue damage .
There is an inereasing amount of experimental evidenee that neuronal
degeneration is mediated by numerous processes, including exeitatory amino
acid (espeeially glutamate) toxieity , bursts of reactive oxygen species (free-
radicaJ damage, oxidative stress), nitric oxide perturbation and Ca 2+-indueed
damage (intracellular calcium influx), whieh interact to provide a fmal common
pathway for eell vulnerability. By intervening in such processes, neuroprotective
agents have produced beneficial effeets in animal models of retinaI ganglion cell
damage and optic neuropathy.
These data open a novel path way toward really innovative pharmacologieal
research , aimed at protecting the retina and optie nerve from neurotoxic injury
(Steinberg, 1994; Orago et al., 1993; Nayak et al., 1993; La Vail et al., 1992).
In such an effort, ozonetherapy eould play a role, as a new, reliable,
eomplementary treatment for the management of several neurodegenerative
disorders . Indeed there is experimental evidenee that ozone ean have important
biological effeets (Coppola et al., 1992; Romero Valdes et al., 1993; Hernandez
et al., 1995; Bocci, 1996), such as:

• improved perfusion and eellular oxygenation(aetivationof erythrocytefimction)

• vasodilatation by release ofNO and CO;
• upregulation of the enzymatic antioxidant system;
• induetion and release of cytokines and growth factors.

These effects eould eooperate beneficially in an attempt to increase the

defence meehanisms of eells against isehaemie and neurotoxic injury, thus
preventing eell death or reducing tissue injury in reversibly damaged cells .
It has been speculated that several irreversible degenerative disorders of the
retina and optie nerve, for which no therapy has proven effective, could benefit
from ozonetherapy (Riva Sanseverino et al., 1990; Moraleda, 1995; Marmer and
Parker 1998; Diadori et al., 1996, 2000) :

• age-related maeular degeneration,

• degenerative myopia,
• retinal vascular disorders (such as diabetes) ,
• retinal inherited-degenerative disorders (such as retinitis pigmentosa),
288 CHAPTER 24

• ischaemic optic neuropathies,

• glaucoma.

We report our experience on the efficacy of OJ-AHT in the treatment of age-

related macular degeneration (ARMD). Other disorders are still under investigation.

4.2. Age-Related Macular Degeneration

ARMD is a progressive disabling bilateral condition affecting central vision . In
developed countries, it is the leading cause of irreversible loss of vision in
people over 50 years of age (Bressler et al. , 1988); its incidence increases with
age, varying from 1.6% in patients 50 to 60 years of age to 20-30 % of people
over the age of 65 (Framingham Eye Study, 1980; Pauleikoff and Koch, 1995).
Since this sector of the population is expected to increase during the next
century, the social and economic consequences of ARMD-related visual
impairment and blindness are destined to increase unless successful means of
prevention and treatment can be found . Therefore, ARMD is a public health
problem of severe and growing proportions (Evans and Wormald, 1996).

4.2.1. Clinieal Presentation

The most frequent signs of ARMD are:

I) drusen . These lesions are ophthalmoscopically visible as pale yellow spots

that may occur individually or in clusters throughout the macula. They
consist of an accumulation of amorphous material between the RPE and
Bruch's membrane, resulting in a microscopic elevation of the RPE .
Although their exact origin remains unknown, current theories favour the
accumulation of lipofuscin and other cellular debris derived from cells of the
RPE that are compromised by age or other factors.
2) disturban ee ur the RPE, which may appear disrupted into small areas of
hypo- and hyperpigmentation (pigmentary changes) or may become absent,
forming large areas of atrophy (areolar [geographi e) atrophy).
3) ehuroidal neovascularization. In response to as yet undefined stimuli ,
choroidal vessels proli ferate across Bruch's membrane under the RPE and, in
certain cases, continue their extension into the subretinal space .

According to recent studies, neovascularization could be stimulated and

mediated by VEGF , produced by the RPE cells und er hypoxic conditions
(Frank, 1997; Pournaras et al., 1997).
Copious leakage from these neovascular membranes may result in exudative
detachment of the RPE or haemorrhages, which may be confined to the area
under the RPE or may extend under the retina. The natural course of this process
is fibrotic evolution, with formation of a disciform sear.
Clinically, two forms or stages of ARMD are usually described :
 OZONETHERAPY IN V ARIOUS PATHOLOGIES 289

• "dry" or atrophie form (the most eommon, aeeounting for 80-95% of eases),
eharaeterized by disturbanee of the RPE with varying degrees of drusen
(hard, soft, mixed) and areolar (geographie) atrophy, in whieh loss of the
RPE is aeeompanied by fallout of photoreeeptors and ehorioeapillaris. The
visual deterioration is usually slow and gradual, and beeomes really
important only in 5-10% of eases, depending on the extent and loeation of
the area of atrophy.
• "Wet" or exudative-neovaseular form, eharaeterized by detaehment of the RPE
(serous or haemorrhagic), subretinal neovaseularization, and flbrovascular
disciform searring. It is less eommon (5-20% ofeases), but is associated with an
even worse visual prognosis (severe loss of eentral vision in 70-80% of eases).

The most frequent symptoms ofthese alterations ofthe maeula are:

deereased visual aeuity (loss of eentral vision, eolour vision, ability to see
fine details)
metamorphopsia (distortion ofthe shape ofobjeets in view)
paraeentral-central seotoma

Loss ofvision in ARMD is the result ofphotoreeeptor death, oeeurring when

RPE eells, with whieh they are assoeiated, deteriorate and die.
The loss of vision resulting from drusen and pigmentary ehanges (early
stages of the disease) is highly variable: most patients are asymptomatic or
experienee only a small visual loss or metamorphopsia . With the progressive
development of larger areas of atrophy of the RPE involving the fovea, visual
aeuity deereases abruptly and relative or absolute seotomas appear within the
eentral 10 degrees of the visual field.
Sudden substantial loss of eentral vision, over a larger area and often at an
earlier age, is generally the result of ehoroidal neovaseularization, with serous or
haemorrhagie detaehment ofthe RPE.
The natural elinieal eourse of ARMD is progressive and the final visual
aeuity is usually < 20/200 (Piguet et al., 1992; Sarks et al., 1988; Barondes et
al., 1990; Klein et al., 1993, 1997).

4.2.2. Pathophysiology and Risk Faetors

The eauses and faetors related to the onset and progression of ARMD remain
unknown, but it now appears likely that ARMD is a multifaetorial disease,
triggered by environmental faetors in those who are genetieally predisposed .
Although multiple possible riskfactors for ARMD have been identified, the
importanee of eaeh remains uncertain . Those thought to be important include:

ageing (> 55 years);

genetic predisposition [familial trait], (Silvestri et al., 1994; Silvestri, 1997;
Allikmets et al., 1997);
290 CHAPTER24

smoking (Vingerling et al., 1996);

exposure to sunlight [photo-oxidative stress], (Cruickshanks et al., 1993;
Darzins et al., 1997);
blue iris (Sandberg et al., 1994);
hyperopia (Broker et al., 1993);
systemic hypertension, cardiovascular disease, and vasculopathic trait
(Vingerling et al., 1995);
nutritional factors, such as a low zinc and antioxidants intake (Newsome et al.,
1988; Sperduto et al., 1990; Seddon et al., 1994; West et al., 1994; Chew, 1995).

The cause and pathogenesis of ARMD is unclear (Young, 1987), but an

ensemble of nutritional, degenerative, oxidative (phototoxic), haemodynamic
and genetic factors, appear responsible and are being actively investigated.
Three main theories can be distinguished:

• primary dysfunction of the RPE (genetic predisposition or environmental

exposure).
There is a consensus that the crucial lesion in ARMD involves the RPE cells.
Light damage, ageing and a variety of dietary deficiencies have been
invoked as possible primary causes of RPE failure in ARMD , with
secondary atrophy of the choroid.
There are data suggesting that the outer retina, in particular photoreceptors
and RPE cells, may be particularly susceptible to the oxidation process,
which leads to the formation of free radicals and highly reactive singlet
oxygen within the cells, causing cell death .
Animal studies suggest that exposure to intense sunlight or UV radiation
may cause changes in the RPE similar to those seen in ARMD . The high
metabolic rate of the outer retina and chronic exposure to light
(photochemical damage) may contribute to an underlying oxidative stress
(Organisciak et al., 1997; Rapp et al., 1997, Tate et al., 1995).
The retina contains a wide variety ofprotective mechanisms (e.g. antioxidants)
against such damage under normal conditions, but these natural defences may
decrease with age, or may be inadequate if environmental exposure to oxidants
increases, or if there is a genetic deficiency of such enzymes.
• Haemodynamic dysfunction in the choriocapillaris.
The vascular theory (Friedman, 1997) postulates that ARMD is caused by a
progressive decrease in the compliance of the sclera (a higher coefficient of
scleral rigidity was found in patients with ARMD) and choroidal vessels,
leading to an increase in the resistance of the choroid to the flow of blood .
This process initiates with the deposition of lipids in the sclera and Bruch's
membrane, and results in decreased choroidal perfusion, with reduced
supply of oxygen and glucose to the retina (ischaemic damage) , and higher
pressure in the choroidal vessels . According to this haemodynamic model,
the decompensation of the RPE is the result of its incapacity to transport
 OZONETHERAPY IN VARIOUS PATHOLOGIES 291

debris and metabolites against progressively unfavourable hydrostatic and

osmotic gradients.
• Primary dysfunction ofphotoreceptors (ABCR mutation).
ABCR mutation was found in patients with ARMD, especially in those
affected by the "dry" form. Since the ABCR protein is a transporter protein
found in the outer segment of rod cells, its mutation could cause degraded
material to accumulate, thus interfering with retinal cell function.

4.2.3. Prevention and Treatment

At present useful therapy to prevent the natural history of ARMD remains
controversial. On the basis of the role of oxidative stress in the pathogenesis of
the disease , the protective effect of several food supplements (minerals and
antioxidant vitamins) have been investigated, but conflicting results have
emerged (Newsome et al., 1988; Sperduto et al., 1990; Prashar et al., 1993;
Cohen et al., 1994; West et al., 1994; De La Paz et al., 1996; Larkin, 2001) .
There is no known treatment for the "atrophic" form of ARMD, which
accounts for most cases of ARMD .
Most potential therapies are addressed to the wet form of ARMD, with the aim
of reducing the neovascularization . However, these treatments do not improve
sight; they only help to slow visual deterioration, so they are unlikely to have an
important impact on the blindness caused by ARMD. Laser photocoagulation
(Macular photocoagulation group, 1986, 1991) is indicated only for selected
patients with well defined extrafoveal and juxtafoveal neovascular membranes,
but it is often followed by a decrease in visual acuity and it does not prevent the
long-term recurrence of neovascularization (Lambert et al., 1992; Thomas et al.,
1992). Photodynarnic therapy looks more promising, since it is more selective (it
does not affect the RPE, photoreceptors and choriocapillaris), but it is only
suitable for 10-15% ofpatients with wet ARMD and it is expensive.
Low-dose radiation therapy (teletherapy) has been proposed for the
treatment of subfoveal neovascular membran es that are unsuitable for laser
treatment, but its effectiveness has not been demonstrated.
Despite initial enthusiasm about their potential angiogenetic inhibitory
effects, antivasoproliferative substances, such as IFNo. (Fung, 1991; Ezekowitz
et al., 1992), have shown little benefit in the treatment of age-related subretinal
neovascularization. There are now far more prornising anti-angiogenetic
compounds under investigation (O'Reilly et al., 1994; Aiello et al., 1995;
Robinson et al., 1996).
Subretinal surgery, aiming to directly remove the offending neovascular
membrane, is very appealing, but not free from risks; moreover, in ARMD the
disturbances of the RPE make the process more complicated, and the results
may be disappointing.
292 CHAPTER24

4.3. ARMD: Personal Experience with Ozonetherapy

Owing to the lack of an effective therapy, it was decided to conduct a
preliminary investigation of the efficacy of 0 3-AHT in the dry form of ARMD;
this was carried out in the Department of Ophthalmology and Neurosurgery , in
cooperation with the Institute ofGeneral Physiology, University ofSiena.
0 3-AHT is a safe complementary approach widely used in the treatment of
ischaemic vascular disorders , such as peripheral chronic arterial occlusive disease.
It has been shown to activate erythrocytic metabolism and oxygen delivery to
hypoxic tissues and to upregulate the expression of antioxidant enzymes, possibly
leading to the correction of an endogenous oxidative stress (Bocci 1996a,c,d).
The purpose of our study was to check its c1inical efficacy in patients with
dry ARMD, compared with an age-matched control group (prospective RCT) .
The study population consisted of patients referred to the outpatient c1inic of
the Department of Ophthalmology and Neurosurgery of the University of Siena.
All patients signed an informed consent statement prior to inclusion in the study.
All patients were affected by the "dry" form of ARMD, documented by
fluorescein angiography, and had at least 10 ophthalmoscopically visible
macular drusen, characteristic pigmentary alterations, or geographic atrophy ;
one eye was considered for each patient, the functionally best one in the bilateral
forms . Subjects whose visual loss may have been secondary to media opacities
or any other ocular disease besides ARMD were excluded.
The following were carried out before the first treatment, after the last
treatment, and then every 3 months for up to I year : a fully corrected near and
distance visual acuity (V .A.) measurement; a complete biomicroscopic and
ophthalmoscopic examination, in which the refractive error, iris colour, lens
status (aphakia, pseudophakia or initial cataract), disc and macula morphology ,
and intra-ocular pressure values (measured by applanation tonometry) were
specifieally reeorded for eaeh eye.
The experimental group consisted of 50 "dry" ARMD patients, who
underwent 12-13 sessions of OrAHT within 6.5-7 .5 weeks.
Standardized 03 -AHT was carried out by exposing about 250 g blood in
eitrate-phosphate-dextrose (CPD) to agas mixture eomposed of about 96% O 2
and 4% 0 3, with a final 0 3 concentration of 40-70 ug /rnl gas per g of blood
(starting with 40 Ilg/m1 and gradually increasing by 5 ug/ml each session to
reach 70 ug/ml by the 7th treatment). After about 5 min of thorough mixing,
eaeh blood sampIe was reinfused into the donor. This procedure was repeated
twiee a week for 12-13 times .
As a "control" group, we treated 30 "dry" ARMD patients by exposing the
same amount ofblood to O 2 alone (the same number ofsessions twice a week).

Funetional ophthalmologie examination included:

Mean distance visual acuity (ETDRS charts).
Macula threshold test (Humphrey automated perimeter).
Self-assessment test of the quality ofvision (Carta et al. , 1998).
 OZONETHERAPY IN VARIOUS PATHOLOGIES 293

General examination included:

Blood pressure.
Haematochemical data (blood cell count, plasma proteins, plasma lipids,
coagulation and fibrinolysis tests) .

These data were recorded at the baseline time (before starting the therapy), at
the end of the last 03-AHT sessions, and then every 3 months for up to 1 year.
In all the follow-up examinations, the examiners were blind to the results of
each patient's initial examination.

4.4. Results
4.4.1. Ophthalmologie Results
Change in visual acuity from baseline at each examination was the primary
parameter used to verify the response, if any, to 03-AHT.
Mean distance visual acuity (ETDRS charts) was significantly improved in ARMD
patients (p<0.05), while no significant improvement was observed in the control
group . This data was confirmed by the results of the visual field (increased mean
sensitivity and foveal threshold in 54% oftreated patients and in 18% ofcontrols) and
the self-assessment test of quality of vision (improved quality of vision in 60% of the
treated patients and 23% of controls) .
In the treatment group, the improvement rema ined stable in the first quarter
and declined slightly in the next six months after the treatment (Fig . 100); at 12
months, up to 41.6% showed either a regression to their pre-treatment values
(33.3%) or had worse V.A. (8 .3%).
0.30

200
0,25
• • 175

ä 0,20
:;
'"
~'" 150 .

0,15 12S

100
0,10
Pr. Post 6 9 12 Pr. Post 6 9 12
Months

Figure 100. Modifications ofvisual acuity before, during and after (3, 6, 9 and 12 months)
one cycle 0/ /3-14 OrAHT sessions. The left diagram reports the actual change, expressed as
a percentage ofthe right diagram .
294 CHAPTER24

4.4.2. Laboratory and Biochemi cal Results

The laboratory resuIts reported in Table 24a show that OrAHT did not cause
significant modifications of critical parameters measured just before and at the end of
the treatment. The levels oftypicalliver enzymes were also unmodified.

Tahle 24a. Laboratory tests carried out in 34 ARAfD patients before and after 12-13
sessions ofOJ-AHT .

PRE FINAL
Blood cells:
RBC (M/~I) 4.58 ± 0.7 4.50 ± 0.7
HGB (g/dl) 14± 0.2 13.6 ± 0.2
HCT (%) 41.7 ± 0.6 40.6 ± 0.7
MCV (0) 91 ± 0.8 9 1.4 ± 0.8
MCH (pg) 30.5 ± 0.3 30.7 ± 0.3
MCHC (g/dl) 33.5 ± 0.1 33.5 ± 0. 1
PLT (K1~1) 232.2 ± 9.2 237.4 ± 9.8
WBC (KfI.,t1) 6.3 ± 0.3 6.4 ± 0.3
Coagulation tests:
Fibrinogen (rng/dl) 293 .6± 12.5 327.6 ± 14.7
F.VIIIvV (%) 151.6±12.8 153.6± 14.0
F l +2 (nM/I) 1.42±0. 14 1.15 ± 0.11
AT III (%) 100.9 ± 3.6 100.9 ± 2.6
PT(%) 96.2 ± 3.1 96.1 ± 1.8
a PTT (sec) 31.7 ± 0.7 30.3 ± 0.6
TT (sec) 19.4±0.7 19.6 ± 0.3
Fibrinolysis tests:
t-PA (ng/ml) 11.2 ± 0.8 10.4 ± 0.9
PAli (IU/ml) 11.2 ± 1.4 13.1±1.6
FM test (uz/ml) lO.1 ± 1.2 13.5 ± 3.0
FDP (ug/ml) 6.4 ± 0.8 7.6 ± 2.4
D dimer (ng/ml) 111.3 ± 5.5 114.5 ± 8.4
Lp (a) (rng/dl) 43 .8± 10.4 35.7 ± 8.2
Plate/et tests :
PF4 (lU/mi) 4.6 ± 0.8 3.8 ± 0.5
ß-TG (IU/ml) 27.1 ±2.0 29 .2 ± 2.9
Plasma proteins:
Proteinemia (g/dl) 6.8 ± 0.4 6.9 ± 0.5
Plasminogen (g/l) 0.12 ± 0.4 0.12 ± 0.6
Fibronectin (mg/dl) 43 .3 ± 1.5 45.4 ± 2.2
Plasma lipids:
HOL (mgf/dl) 60.2 ± 2.6 54.9 ± 2.8
Cholesterol (mg/dl) 285 .5 ± 8.9 278 .9 ± 8.5
Triglycerides (mg/dl) 119.9 ± 13.9 114.4± 10.5
 ÜZONETHERAPY IN VARIOUS PATHOLOGIES 295

Table 24b reports the data on TBARS, 2,3-DPG and SüD content measured in
the blood of the ARMD patients before and at the 5th, 9th and 13th Ü3-AHT
treatment. Unfortunately, these determinations could be reliably carried out in only
some of the patients. It appears that there is no increased peroxidation and 2,3-DPG
levels remained constant. SüD levels increased after the first five sessions and then
retumed to normal. The slight increase of G-6PD was also not significant.

Table 24b. Evaluation ofplasma TBARS, 2.3-DPG content, SOD and G-6PD activities before
(0), at the 5th (5), 9th (9) and 13th (13) OrAHT treatment in ARMD patients.

Parameters Treatment
() 5 9 13
TBARS 32 .6±8 .10 (15) 28.4±8.9 (15) 36.4±13.7 (16) 26 .1±6.4 (15)

2,3-DPG 1.95±O.06 (25) 2.07±O.09 (25) 1.95±O.10 (23) 1.86±O.08 (22)

SüD 1.51±0.20 (14) 1.83±O.22 (14) 1.37±O.Ol (16) 1.45±0.2 (13)

G-6PD 7.46±O.89 (9) 7.76±O.62 (9)

Values are expressed as mean ± S.E. No significant differences were observed . Number of
observations in brackets .

4.4.3. Side-effects and compliance

We did not observe any important side effects during or after the treatment; thus
we can state that, in our experience, the procedure was safe . Actually, most of
the patients reported an improvement of their general condition, particularly in
terms of increased strength, mental concentration and memory.
The on1y noticeable problem was that a few patients had poor venous access and
this often required more than one venipuncture.
Compliance to the treatment was excellent, even in the patients who showed no
improvement and it must be emphasised that most of the patients undertook two
long journeys week1y to receive the treatment. On the other hand, adherence to the
se1ected follow-up examination was not fully satisfactory.

4.4.4. Conclusions
At the present time, there is no effective medical therapy for dry ARMD: the use
of minerals and antioxidant vitamins, while harmless, may delay but does not
recover vision loss. Most ARMD patients, still physically and mentally active,
are very concemed about the lack of an effective treatment and although there
are now interesting avenues of research, it will take time before the results
appear on the pharmacist's shelf.
On this basis, ozonetherapy could be proposed to ARMD patients as a
reliable cornplementary therapy able to offer, in most patients, a significant
296 CHAPTER24

general improvement of the quality of vision, so that they can experience a

better lifestyle.

5. DERMATOLOGICAL DISEASES
In 1989, the Director of the Institute of Dermatology of Siena University
surprisingly asked me to help study the value of O)-AHT in psoriasis, because one
of his colleagues was privately performing this therapy and c1aiming great success.
Unfortunately, at that time I had no practical experience. Firstly , I noted that an
assistant 'collected ozone in a syringe and, without closing the tip,' walked a long
corridor before insufflating it into the blood bottle. Secondly, they assured me that
the ozone concentration must be very 10w (probably 2-5 ug /ml ) and it was not really
very important because blood would become "very red" in any case . The surprising
result was that after 7-10 treatments, one patient showed extraordinary
improvement, another was slightly better and three patients remained the same.
They tried to publish a paper but it was rejected because there were no controls.
Thus my first c1inical experience was very disconcerting. Since then , I have heard
several other anecdotes of splendid results and 11eave the judgement to the readers.
In spite of this puzzling story, it seems that Russian dermatologists and a
German ozonetherapist (Kief, 1993b) also succeeded in treating various forms of
eczema and atopic dermatitis (AD) , which, to my knowledge, is challenging and
frustrating to treat. I had been interested in this disease from an immunological point
of view : the hallmark of AD is a Th l/Th2 imbalance (Bohn and Bauer, 1997;
Campbell et al., 1999) with a reduced production of IFNy and an elevated release of
IL-4 and IL-5, which favours IgE production and eosinophilia, a typical disorder of
atopic diathesis (Beltrani, 1999; Leung, 1999) .
Prophylactic measures such as avoidance of irritants, allergic food (eggs, soy ,
peanuts, etc.), contact with house-dust mites or other aeroallergens, are helpful but
the mainstays of therapy have been and still are topical corticosteroids. More
recently, in severe forms , phototherapy, cyclosporin A, azathioprine and tacrol imus
ointment appear to be effective but with some side effects (Rudikoff and Lebwohl ,
1998; Hanifin and Tofte, 1999; Fleischer Jr., 1999).
Does ozonetherapy have any future in dermatology? If seriously performed, 0 )-
AHT may be useful and my "gut feeling" is that we should progressively try from
medium (40-50 ug/ml) to high ozone concentrations (80 ug /ml), as discussed in
Chapter 14 (Leukocytes), to readjust the Thl/Th2 balance. However, it should be
remembered that this is a proposal that may not be effective.
The BOEX procedure, combining systemic and cutaneous treatment, may be an
ideal approach. However, patients with these diseases are often very distressed and
understandably anxious to receive the most effective treatment immediately .
Therefore, they are not interested in our controversies and this rnakes it awkward to
recruit patients for a RCT . Ozonetherapy may yield some benefit at a slow pace and
patients will accept it only if, at least in the initial period, they are assisted with the
proven topical anti-inflammatory options.
 OZONETHERAPY IN VARIOUS PATHOLOGIES 297

6. PULMONARY DISEASES (EMPHYSEMA, ASTHMA, COPD AND ARDS)

This secnon is dedicated 10 the memory ofDr. Maria Trusso.

Ozonetherapy has not yet been tested in pulmonary disease, probably because
everybody knows that breathing air polluted with ozone is toxic to the respiratory
system (Kelly et al., 1995). Two or three papers on this topic are published every
week in international journals and this problem has been discussed in Chapters 3 and
5. However, an almost irrelevant episode that occurred about four years aga suggests
that this fact has misled us. Among our numerous ARMD patients treated with 0 3-
AHT, one, with emphysema , told us that after about fourteen sessions his dyspnea was
alleviated and he could walk up to the third floor of his apartment with little effort. I
sensed that he had given us a good tip and I took him to the Pneumology Unit where
the specialist, Dr. Maria Trusso, was bewildered by the result. Actually, at first she
imagined that the treatment for ARMD was based on breathing ozone and the proposal
to continue this sort of treatment appeared crazy to her. After 1 explained that we
simply ozonized and reinfused the patient's blood, she became interested and correctly
asked how ozonized blood eould improve lung funetion. That was a good question and
I had to use the best of my imagination to provide a few answers:
the simple one was improved blood oxygenation. Yet this is not quite valid
because , although we reinfuse hyperoxygenated blood (p02 is easily at 500 mmHg),
the infusion rate is so small (about 15 ml per minute, compared with a cardiae output
of about 5 L) that the p02 of venous blood reaehing the lungs is hardly modified.
However, if ozonetherapy enhanees the delivery of oxygen at the tissue level,
metabolie conditions may improve, even though ideas such as decreased blood
viscosity and increased 2,3-DPG levels in erythrocytes have not been definitively
demonstrated. I advaneed the hypothesis that perhaps ozonized blood acting on
endothelium could aetivate the release of prostacyclin. Only last year, we deteeted
an enhanced release of NO in HUVECs in vitro, but we have not yet evaluated
prostaeyclin. It appears that release of NO and NOthiols may represent a mechanism
for vasodilatation, although NO could also be a double-edged sword (Barnes and
Liew, 1995; Warren and Higenbottam, 1996; Jindal and Dellinger, 2000) .
A eommon denominator of asthma, emphysema, chronie obstructive pulmonary
disease (COPD) and aeute respiratory distress syndrome (ARDS) is oxidative stress. It
is demonstrated by an increase ofROS (H202) and 8-isoprostane, activation ofNF-KB
with inereased synthesis ofTNF-a, IL-6, IL-8, and inaetivation (by oxidative damage)
of o l-antitrypsin and leukoproteinase inhibitor, unable to counteraet elastase,
cathepsins and matrix metalloproteinases (Smith et al., 1997; Barnes, 2000). In 1996,
we postulated that the paradoxieal action of ozone in inducing an adaptation to COS
could be critical in readjusting the oxidant-antioxidant balance.
Finally, we searehed for other eases of emphysema and COPD . We found two
patients, a man and a woman, who after two eycles of therapy had noticed an
improvement in their performance of daily activities . This response was subjective
and could have been due to a placebo effect, but it encouraged us to make a
protocol. The Director of the Pneumology Unit agreed although he was somewhat
skeptical, but not so much as the University's Professor of Pneumology, who was
 298 CHAPTER24

absolutely antagonistic. The protocol was then prepared, submitted to the Ethical
Committee and, after revision, approved after about seven months . Unfortunately,
the health of Dr. Trusso deteriorated (she had a metastatie breast tumour) and she
died shortly afterward, leaving four young children practically alone . We lost a very
nice, energetic woman, who after accepting the idea became very enthusiastic to try
this unusual therapy .
I hoped that one of her colleagues would accept the challenge, but he thought
that the project was too laborious to develop and uncertain, so I found myself back
where I started . Nothing has been done, but luckily there has been some progress in
conventional Medicine , since COPD morbidity and mortality is increasing due to the
use oftobacco. Indeed it may soon become the third most common cause of death .
In addition to rehabilitation with exercise training, anti-smoking measures and
domiciliary oxygen therapy, new bronchodilators and appropriate antibiotics can
control acute exacerbations. After a long incubation (1957), surgical removal of the
most emphysematous parts of the lung has come of age; when the operation is
successful, short-term results are good, with marked improvement of the quality of
life (Hillerdal 1997; Bames, 2000) .
However, some of the patients do not benefit from surgery (National
Emphysema Treatment Trial Research Group , 2001) and the value and cost-
effectiveness of the volume reduction surgery remain uncertain in the long run ,
Moreover, medieal expenditures to treat COPD, associated with invalidity , represent
a significant economic and social burden for Health Authorities and society in
general. I believe that these are sufficiently good reasons to justify serious and wide-
ranging experimentation with ozonetherapy. We can take advantage of OJ-AHT and
even more of BaEX and RI (non-invasive procedures), always starting with a low
dose (20 ug/rnl) with gradual dose escalation up to 35-40 ug /rnl. Needless to say,
supporting measures, particularly oxygen therapy, remain available to the patient.

7. RENAL DISEASES
Prof. N. Di Paolo is the head physician of the Nephrology and Dialysis Unit at the
Siena Polyclinic. He is a good friend of mine and his role in developing EBOO has
been crucial . However, when I proposed to evaluate ozonetherapy in not too
advanced dialysis patients, he answered that the kidney does not have the
regenerative ability of liver and there is no hope to improve the function of a
sclerotic kidney . He may weil be right in regard to terminal patients but my idea was
to limit progression, possibly achieving regression of initial chronic diseases . A few
recent reports have further convinced me that during infective glomerulo-nephritis
or at the initial stage ofrenal failure, ozonetherapy could have a beneficial influence .
It is not worth repeating the usual mechanisms, but I wish to re-emphasize the
possibility of adaptation to COS, typically present in chronic renal failure (Ceballos-
Picot et al., 1996b; Witko-Sarsat et al., 1998). It is weil established that
haemodialysis in itself generates RaS and consequently enhances oxidative stress. I
feel that it is wrong to passively accept the concept of unavoidable irreversibility
because, in addition to potentiating the antioxidant system, ozonetherapy may switch
on angiogenesis or favour the release of unknown nephropoietins. Unscientifically,
 OZONETHERAPY IN V ARIOUS PATHOLOGIES 299

I believe that ozone is a stimulator of untapped natural resources and we know

that if we can just give a little help to Nature, she may respond in ways that are
wonderful and unsuspected by our little minds. Einstein used to say that
"imagination is more important than knowledge".
Ruggenenti et al. (200 I) have given a moral boost to my belief because they
expertly say that today "nephropathies lack a specific treatment and progress
relentlessly to end-stage renal disease". In order to delay or avoid dialysis (which
would be a great success), nephrologists should develop a combined approach to
renal diseases by pharmacologically controlling blood pressure and loss of proteins.
I now like the idea of adding ozonetherapy performed at low-rnedium ozone
concentrations for long periods. Selected patients could undergo 1-2 EBOO
treatments every week during the final hour of dialysis. The ozonizer could be
simply placed after the dialysis filter with blood exposed to very low ozone
concentration (1-2 ug/ml).
The lack of side effects, the modest adjunctive cost, the control of
hyperoxidative state and the feeling of wellness are the most obvious and eloquent
advantages. I agree that the study of gene and stem-cell biology will favour the
advancement of science and produce important therapeutic innovations, but common
sense also suggests that the possibility of stimulating natural resources should not be
neglected. Moreover , we should keep in mind that a combination of therapeutic
approaches, and not the reductionist simplification of substituting a gene, is most
likely the best way to effectively treat the multiform manifestations of a chronie
disease.

8. HAEMATOLOGICAL DISEASES
ß thalassaemia major, fairly frequent in Italy, and sickle cell anaemia (SCA),
affecting black populations, are genetic diseases leading to oxygen blood deficiency
and other serious manifestations. Ozonetherapy cannot correct the gene alteration
but in SCA it can apparently reduce the frequency of vessel occ1usive crises with
related infarctions. SCA involves a modified Hb (Hbs), which tends to crystallize
during deoxygenation ; this leads to a change in the shape of the erythrocytes , which
aggregate and cause vessel occ1usion. At least conceptually , ozonetherapy may
increase oxygenation and, as often c1aimed, may improve cell pliability, although I
am not convinced about this change. A RCT, which must be reported because they
are so rare in this field, was performed in 55 SCA patients (25 control and 30
experimental) at the National Center for Scientific Research at Havana. Ozone was
administered daily (5 days per week) for 3 weeks in 30 patients via the rectal route.
The control group received only analgesics, vasodilatators and IV saline infusion.
The results showed that the ozone-treated group displayed a rise in arterial blood
pOz, the frequency and severity of painful crises was significantly reduced (by about
50%) and there were no adverse reactions (Gomez et al., 1995). This work was
supported by a firm producing ozone generators and it is surprising that no further
experimentation has been reported.
300 CHAPTER24

SCA is a serious disease and it suffices to say that only 2% of about 120,000
affected babies born in Africa survive to the age of five . What can official Medicine
do to help patients in poor countries? Practically nothing, because transplantation
can only be perfonned in about 1% of patients: in 1999, the number of bone marrow
transplants was only 100 and 800 for SCA and ß-thalassaemia, respectively .
Moreover, 10-15% of patients do not survive.
For SCA, it would be important to have an effective and atoxic oral drug that could
be widely and easily used . So far, hydroxyurea appears useful as it reduces Hbs and
increases the percentage of HbF, but the drug is mutagenic and somewhat toxic
(Steinberg, 1999) . Clotrimazole, a specific Ca 2+-activated K-I- channel (Gardos
channel) inhibitor able to reduce the deleterious dehydration of sickle erythrocytes, is
being tested and seems promising (Brugnara et al., 1996) . Morris et al. (2000) have
reported that oral arginine administration may benefit SCA patients by increasing NO
production during a vaso-occlusive crisis. Clearly, these approaches are experimental
and only partly satisfactory and the promise of gene therapy is far from being
materialized. Thus, I do not see anything wrong in using ozone; with small generators,
patients (after careful instruction) could do horne autotreatment using RI. However,
ozonetherapy has the serious drawback that ozone must be generated and used
extempore. The unavailability of generators, medical oxygen, electricity and the need
of an almost daily use for life makes ozonetherapy a solution that cannot be practically
proposed in poor countries for SCA, malaria and HIV infection.
As always, the possibility of ozonetherapy is never mentioned by official
Medicine and 1 must presume that it is unknown. This is one of our weak points,
which can only be overcome by presenting good controlled results to peer-reviewed
international journals. However, someone has written that hydroxyurea advocates
are covering up its long-term carcinogenic potential, but I want to think that this
cannot be true . I hope that this book will serve to make this topic known and
promote unbiased clinical trials to evaluate the validity of ozonetherapy.
Finally, I must mention that I often receive calls frorn desperate people because a
relative is affected by a haematological malignancy. I have great respect for what
haematologists are able to achieve in these diseases and unless we can produce good
experimental evidence, I am not certain that the addition of ozonetherapy would be
useful. This aspect will be discussed further in the section "Cancer", although the
pathophysiology of solid tumours is immensely different from haematological ones.
For the inexpert ozonetherapist, I would like to remark that ozone cannot displaya
direct cytotoxic effect on malignant cells.

9. NEURODEGENERATIVE DISEASES

The progressive prolongation of the human life-span is accompanied by an

increase of degenerative diseases, and those of the CNS are very crippling. There is
substantial evidenee that the combination of genetie predisposition, life-Iong
oxidative damage, an excessive or poorly balanced diet , exposure to transition metal
ions, alcohol and tobaceo smoke intoxieation, lack of physical exercise and diabetes
may be responsible for neurodegenerative disorders such as Parkinson ', Menkes '
 OZONETHERAPY IN VARIOUS PATHOLOGIES 301

and Wilson's disease, senile dementia, amyotrophic lateral sclerosis, optic nerve
dysfunction, primary open angle glaueoma, neurosensorial bilateral hypoacusia and
maeulopathies (Halliwell et al., 1992; Halliwell, 2001; Yu, 1994; Ames et al., 1993;
Cohen et al., 1994; Jenner, 1994; Bondy, 1995; Carlsson et al., 1995; Jaeschke,
1995; Pardo et al., 1995; Yoritaka et al., 1996; Simonian and Coyle, 1996; Rotilio et
al., 2000; Poli and Schaur, 2000; Rotilio, 2001). Authoritative scientists, such as
Ames, Halliwell, Gutteridge, Pryor, Cross, Packer, Rotilio, ete., have suggested that
neurodegenerative diseases triggered by an uneertain primary eause are perpetuated as
the cellular redox system goes awry. The pathophysiology is quite variable: in some
cases, there is ehronic inflammation with the release of ROS and pro-inflammatory
cytokines; in other cases, we ean observe a biochemical defect such as low GSH
content or a deerease of antioxidant enzymes (GSH-Pxs, SOD, catalase) associated
with improper metal binding; in other cases, there is an excessive release of NO'
(hence ONOO) or of noradrenaline from presynaptic terminals or of glutamate with
Ca2+ influx and activation of protein kinases, phospholipases, ete. (Pardo et al., 1995;
Nakao et al., 1995; Ceballos-Picot et al., 1996a; Markesbery 1997; Aejmelaeus et al.,
1997; Sagara et al., 1998; Floyd 1999; Li et al., 1999; Perry et a\., 2000; Rotilio,
2001).
Ozonetherapists must know that there is intense research activity trying to find
drugs able to delay or block the neuronal degeneration and death : the usual
hydrophilie and lipophilie antioxidants taken in appropriate amounts via os are not
harmful but are minimally effeetive (McCall and Frei, 1999), also because only a
small percentage reach the CNS. Metal chelators may help by reducing free
transition metals and OH' formation, but one must pay attention not to exceed with
the dose. Moreover, several inhibitors of the reuptake of doparnine, of NO' synthesis
and of ionotropic reeeptors to block glutamate neurotoxicity are being tested. The
more biologically oriented approaches are attempting to use neurotrophic factors or
to transplant dopaminergic foetal cells (or, probably very soon, stern cells) into
seleeted areas (Weber and Buteher, 2001). Among neurodegenerative diseases,
Parkinson's disease is the ideal one, because the degeneration is fairly restricted to
partieular areas of dopaminergic neurons (Lang and Lozano, 1998a,b).
In spite of all this tremendous effort and biIlions of dollars spent in basic and
clinieal research, we can eonc1ude that pharmaeologicaI therapy is certainly useful
(levodopa is still the most effective therapy after three decades !) but only for a
limited time and it does not arrest progression of the disease. The combination of
several experimental therapies promises to improve on the present limitations, but
still we are fighting a virtually lost war beeause neurodegenerative diseases are
projected to surpass even cancer as the second eause of death by the year 2040
(Lilienfeld and Perl, 1993).
Can ozonetherapy be of any use?
At first glance, it seems irrational to propose a treatment of neurodegenerative
diseases based on aseries of brief and ealculated oxidative stress (therapeutie
"shock"). However, this approach (which is not a panacea) may paradoxically break
and stabilize an otherwise irreversible situation. The idea (already discussed in
Chapter 22) is that a gradual escalation ofthe ozone dose (from 15 to 40 ug/ml) may
302 CHAPTER24

be able to induce adaptation to COS, which in practical tenns means that, by

gradually receiving trace amounts of LOPs, neuronal cells under oxidative stress
may reactivate the depressed synthesis of antioxidant enzymes. Today there is no
phannacological approach able to achieve this objective, which instead can be
realized, without any biotechnological trick, simply by ozonizing blood for a few
minutes. More than ever, I persevere in my idea that if neurodegeneration is not due
to an irreversible genetic defect (like amyotrophic lateral sclerosis (ALS) or Lou
Gehrig's disease, for example), judicious administration of ozone can be helpful. In
Chapter 2, I related the awful story I had with the Editor of FRBM, who refused to
send me the offensive referees' comments on my minireview proposing the concept
of adaptation to COS . More than ever, I am convinced that excessive dogmatism in
biology and medicine can be very dull. While I am aware, and I repeat to everyone,
that ozone is intrinsically toxic and must be used with care, I do not see any risk in
evaluating this problem with 0 3-AHT, or RI, or BOEX. At worst, we will not obtain
any positive result but patients will not be harmed.
Actually, if it can be believed and in support of my stubbomness, as early as
1993 in Cuba , Rodriguez et al. performed a double blind RCT on 60 patients
affected by senile dementias: group A (30 patients) was treated with O 2-0 3 by daily
RI (50 ug /ml) for 21 days and group B with O2 only . This was an important
pioneering study . Using several psychometric tests (mental condition, capacity for
self-administered medication and evaluation of daily activities), it demonstrated that
73-90% of ozone-treated patients showed marked improvement without any
adverse effect. In 1995, at the XII IOA Congress in Lilie, Gomez Moraleda
summarized several studies carried out in Cuba since 1990. Use of OrAHT in optic
nerve dysfunction, primary open angle glaucoma, cochleo-vestibular syndrome and
ischaemic cerebro-vascular disease yielded an improvement ranging frorn 50 to 100%.
Therefore, in Europe and the USA , we are already a decade too late and if
ozonetherapy is really useful , to how many patients have we denied this possibility?
Our inadequacy and short-sightedness is unforgivable!
If we are ever able to perform a study , it will be important not only to evaluate
the clinical effects but also to c1arify the mechanism of action. At the moment, I can
only advance a few speculations: ozonetherapy can simultaneously improve blood
flow and oxygen supply to hypoxie tissue, thus stimulating aerobic glycolysis in
hypofunctional cells, which by resuming normal metabolism might restore the
normal ATP content and GSH /GSSG ratio . LOPs generated during lipoperoxidation
of plasma or absorbed from the rectal mucosa (R!) or the skin (BOEX) will be
diluted in the plasma pool and in part can pass through the blood-brain barrier to
reach the sites of neurodegeneration and upregulate the cellular synthesis of
antioxidant enzymes, which is the crucial step to readjust the impaired cell redox
system. The release of neuronal growth honnones and the activation of resident stern
cells remain speculative, but they are not too far-fetched ideas . The possibility that
Alzheimer's disease, associated with adeposition of insoluble ß-amyloid
aggregates, reflects an NO· /superoxide imbalance has been entertained by Thomas
et al. (1996) . The therapeutic implication is that a prevalence of NO· over
superoxide is advantageous and may inhibit aggregation. This may be achieved by
 OZONETHERAPY IN VARIOUS PATHOLOGIES 303

the administration of exogenous SOD mimetics andJor antioxidants but,

interestingly, 0 3-AHT could correct the imbalance by inducing SOD and the
production of NO' at the same time . Two cautionary annotations appear to be in
order : the first is that functional recovery may be achieved only in initial or not too
advanced patients, and secondly an optimal 03-AHT schedule has not yet been
worked out, although it appears reasonable to start with a low 0 3 concentration (15-
20 ug/rnl) and slowly raise it (in 3-4 weeks) to 35-40 ug /ml per ml ofblood. For RI,
I would suggest beginning with 5 ug /ml and proceed to a maximum of 30 ug/ml and
a volume of 600 ml gas . In this case, I think that the concentration (50 ug/ml) used
constantly by Rodriguez et al. (1993) is excessive and frequently causes intestinal
cramps . If an improvement really occurs, it may be necessary to continue the
treatment (once weekly or biweekly?) for life. It must be explained and understood
that one cycle of ozonetherapy cannot be the "eure": all cells have a more or less
long biochemical memory and must be stimulated by LOPs at intervals of 1-2
weeks . Our study on ARMD has been very instructive in this sense. Retinal
maculopathies deserve a short discussion on their own. Open studies carried out
using OrAHT in both ARMD and retinitis pigmentosa showed an improvement of
visual activity in about 2/3 of patients (Gomez Moraleda, 1995; Diadori et al,
unpublished). Our study is described in a previous section. However, arecent
evaluation of 10 patients with retinitis pigmentosa performed in Cuba with a
regimen of electrical stimulation, 0 3-AHT and ocular surgery has not validated this
multi-technique approach. Actually it suggested that, in comparison to simple
vitamin A supplementation, this complex intervention may worsen the course of the
disease (Berson et al., 1996; Weleber, 1996). Thus , this problem remains open , since
the study examined a genetic disease and used a too complex protocol, in which it
was not possible to clarify the exclusive role of03-AHT.
Patients with neurodegenerative diseases undergoing ozonetherapy must receive
oral antioxidant supplementation (as has been specified in Chapter 22) because they
are frequently undernourished and may have a low TAS.

10. CANCER
Although a number of haematological cancers are now being treated successfully,
the common solid cancers, which are the great majority, continue to be a problem
for mankind (Bailar III and Gornik, 1997). Owing to earlier diagnoses and some
therapeutic advances, for the first time in western European countries, the total
cancer mortality was moderate1y reduced for both sexes in the period 1990-1994
(Levi et al., 1999) . However, due to prolongation of the life-span, the figures for
overall mortality from cancer (in Italy about 160,000 and in the USA about 520,000
in 1993) are still dramatic. Moreover, in the same period, cancer mortality was still
increasing in eastern European countries. This is not likely to change ovemight
because a highly desirable improvement of chemotherapeutic compounds, so far
rather unspecific and toxic , may come too slowly . An appropriate cancer prevention
campaign, aimed at early detection and the use of an appropriate diet rich in fibre
and antioxidants (Dreher and Junod , 1996; Bailar III and Gamick, 1997; Kramer and
304 CHAPTER24

Klausner, 1997), may help up to a point. Yet, on the whole, smoking is not
decreasing and has partly shifted from men to women and to Third World countries.
At least theoretically, immunotherapy (the fifth modality of cancer treatment)
aims specifically at destroying only neoplastic cells, but unfortunately these cells are
poorly immunogenic and diabolically equipped to evade or suppress the immune
system. In addition to showing the multiform conventional anti-neoplastic therapy,
Figure 101 indicates that since 1980 a considerable effort has been made to develop
new and efficient immunotherapeutic approaches, which however have failed to
achieve substantial advances (Rosenberg et al., 1987; Rosenberg, 200 I ; Bocci, 1985,
1987b, 1990b; Kim et al., 1996; Fenton et al., 1996; Wemer and Jolles , 1996; Reddy
et al., 1997; Ernst, 1997; Motzer et al., 2001) .

SURGERY 11 RADIOTHERAPY 1 I CHEMOTHERAPY I I HORMONE THERAPY

Hlgh-Intenslty chemotherapy, regtonal

1)

I\~- •.
RECENT DEVELOPMENTS
I
adminIstration wlth Inhibitors of
chemoreslstance and wlth granulopoletlns rescue

~ 1 2) Hyperthermla Therapy w ith anti sen se

oligodeoxynucleolides

I I
' - - - - - - - -- -'

3) Photodynaml c therapy
Therapy with ribozymes

I 4) Bone marrow transplantation I

S) Immunotherapy:
a) wlthlmmunomodulatory compounds (thymJc ( Anliangiogenetic therapy )
hormones, melatonln, etc .)
b) wlth Inducers or cytoklnes
c) wlth exegenuus cytcklnes (lFN ,IL-2, -12, TNFa)
wlth or wlthout adoptlve Immunotherapy (LAI(, T1L)
d) wlth gene therapy
e) wlth twnour vacctnes and dendrltlc cetls (APe) FUTURE DEVELOPMENTS
o wlth dllTerenUaUng agents (all trans RA. tamoxlfen)
g) wlth radloactlve and toxle 8I1tlbodles (the old maglc bulletI)

Figure J0 J. The various aspects ofconventional antineoplastic therapy

Immunological gene therapy works weil in experimental murine tuu.ours, but so

far has been disappointing in patients (Anderson, 1992; Bubenick, 1996; Roth and
Cristiano, 1997; Parmiani et al., 2000) . The greatest hurdle for successful cancer
therapy is a thorough understanding ofthe several mechanisms used by tumour cells
to evade the immune attack . The latest disappointment has been anti-angiogenic
therapy; in spite of a perfect rationale (Carmeliet and Jain, 2000), it works very weil
in mice (O'Reilly et aI., 1997; Boehm et al., 1997; Perletti et al., 2000) but not, as
we hoped, in human tumours, even though angiogenic inhibitors (Oehier and
Bicknell, 2000) combined with other drugs may still play an important role . Thus,
after all the untimely and deleterious propaganda of the mass media, it is not
surprising that desperate patients are always looking for other possibilities,
 OZONETHERAPY IN VARIOUS PATHOLOGIES 305

particularly in the vast field 'of complementary medical practices (Cassileth and
Chapman, 1996; Burstein et al., 1999) such as diet, nutrition and lifestyle changes,
therapeutic touch (Rosa et al., 1998), mind-body control (Flach and Seachrist, 1994)
and anthroposophie medicine based on the use of mistletoe lectins (Bocci, 1993b;
Ernst, 2001; Steuer-Vogt et al., 2001).
In June 1995, the National Institutes of Health (NIH, Bethesda, MD, USA)
included the use of oxidizing agents (ozone, hydrogen peroxide) in class 5, among
chelation and metabolie therapies, cell treatment and anti-oxidizing agents. It is
noteworthy that H20 2 has been evaluated as an anti-neoplastic agent by Zanvil Cohn
at the Rockefeller University (Nathan et al., 1979; Nathan and Cohn, 1981). Other
studies have been performed by Sasaki et al. (1967) and Samoszuk et al. (1989).
At an earlier stage, ozone was tested in cancer by Varro (1966, 1974, 1983) and
Zabel (1960). Thus, although ozonetherapy is more than 40 years old, it has been
carried out in a few private clinics in central Europe but it has never been accepted
by official Medicine and is currently despised in France, England, the USA and
barely tolerated in Italy. Several reasons, mostly right but partly wrong, have been
discussed at length previously (Chapters 2 and 11).
Is ozonetherapy useful in cancer? Varro (1983) claimed that, after undergoing
surgery, chemotherapy and radiotherapy, most of his private cancer patients
benefited from ozonetherapy , as their quality of life improved and they survived for
a long period. However, these statements were not validated by statistical data and
have no scientific value. There are other anecdotal reports of major or minor
autohaemotherapy having beneficial effects: for example, Beyerle (1996) treated
prostate cancer with "phenornenal" (?) results. For other types of cancer (throat,
ovarian, colon and breast), he comments:
"We are seeing patients who were bedridden two years ago and sent home to die. They
are becoming ambulatory. Their energy level is coming up. They are gaining weight.
And we see these spontaneous fractures in the spine are gradually disappearing.
Strength is retuming to the musculature. There is no spinal pain ".

It is unclear why Dr. Beyerle has not reported the data in a peer-reviewed
medical journal, because as presented they are worthless . His comments were
actually recorded by a journalist (Null, 1996) during an interview published, fancy
that!, in Penthouse, where certainly you can admire beautiful women! Another
confusing example was the abstract entitled "Ozonetherapy in oncology",
surprisingly selected for presentation at the 12th Ozone World Congress in Lille in
May 1995 (Baltin). If one reads the abstract, it becomes clear why ozonetherapy has
such a low reputation in the medical field. Kief (1993a), at his clinic at
Ludwigshafen (Germany), has used Auto-homologous Immunetherapy (AHIT) to
treat a variety of malignancies . AHIT was administered daily for aperiod of four
months and he claimed that it is:
"cost-effective, individually-oriented, has no-side effects, decreases pain in 70% of alI cases
and increases the life-qualityand vitality in approximately 90% ofthe cancer patients",

What AHIT really was remains a mystery (apparently a mixture of the patient's
blood and urine treated with ozone!) and, to the best of my knowledge, the German
Health Authorities have now prohibited its use. My personal experience is very
306 CHAPTER24

limited, mostly because I have found that oncologists are very reluctant to evaluate
ozonetherapy. The only three cases that I had a chance to follow (two terminal lung
carcinoma and one metastatic and ulcerated breast carcinoma) did not show, as
expected, any objective improvement after repeated o.,-AHT. My feeling is that once
the disease has reached the point ofno return, any therapy becomes practically useless .
In conclusion, today there is no evidence that ozonetherapy can be beneficial to
cancer patients because:

• Randomized, double-blind clinical trials have not been performed as they should
have been done (Ernst and Resch, 1996).
• It is unclear whether biological and/or clinical effects, if any, are due to either
oxygen or ozone or to both, or simply to blood retransfusion.
• The relevance ofthe placebo effect is unknown.
• Too often ozonetherapy is carried out together with other conventional or natural
therapies, so that any result remains questionable.

In spite of these negative conclusions, let me enumerate (Table 25) and discuss
some biological mechanisms of action that potentialIy could be activated by
ozonetherapy.

Tahle 25. Possible mechanisms ofaction ofozonetherapy in cancer.

I) Direct effect of oxygen-ozone on cancer cells in vitro and in vivo.

2) Improved oxygenation and metabolism.
3) Potential upregulation ofthe antioxidant enzymatic system with
improvement of the celIular redox potential.
4) Effects on the immune system.
5) Effects on the CNS and endocrine system. Therapy of cancer-related
fatigue .

I) Direct effect of oxygen-ozone on cancer cells in vitro and in vivo .

Solid tumours generally tend to create an ideal microenvironment for their
growth, characterized by hypoxia, increased glycolysis and a high intracellular
level of ascorbic acid . Hypoxia varies widely for different neoplasias but, on
average, oxygen partial pressure can be between 1 and 10 mmHg (normal cells
have a p02 between 40 and 50 mmHg) depending on the distance from a nutrient
vessel and the rate ofblood flow . CelIs beyond the O2 diffusion limit are anoxic
and die, but hypoxic celIs remain viable, clonogenic and resistant to therapy .
Indeed the rate ofmalignancy is positively corre1ated with the over-expression of
hypoxia-inducible factor l« (HIF-I) which, by upregulating the expression of
transferrin, VEGF, endothelin-I and inducible NO synthase, enhances
vasodilatation, neoangiogenesis and tumour metastasis (Brown and Giaccia,
1998; Zhong et al., 1999). Interestingly, glioblastoma multi forme, "the
terminator", expresses HIF-Ia very strongly. Warburg's work in the 1920s
demonstrated that, even in hypoxia, cancer celIs intensely convert glucose to
 OZONETHERAPY IN V ARIOUS PATHOLOGIES 307

lactic acid , but unless they are in anoxia their intracellular pH remains neutral
(pH 7.0-7 .2) while the pH is slightly acidic (6 .8) in the interstitial fluid . It is also
noteworthy that epithelial and haematopoietic tumour cell lines actively take up
DHA and , by reductase activity, reduce it to AR (Agus et al., 1999) . Vitamin C
has many functions (Chapter 12) but the administration of megadoses, as often
done by charlatans, may even give a metabolic advantage to malignant cells .
Throughout the years, I have found reviews on tumour hypoxia very stimulating
(Coleman 1988; Brown, 1999; Vaupel and Hockei , 2000; Hockel and Vaupel,
2001). In conclusion, improving tumour oxygenation by significantly and
constantly increasing O 2 availability and the microcirculation may slow down
tumour growth and inhibit metastatization.
Can ozone be of any help? It has been shown (Sweet et al., 1980; Van der Zee
et al., 1987; Washuttl et al., 1990) that the growth of human cancer is inhibited
by ozone in culture, suggesting that cancer cells have an impaired defence
system against ozone damage. The fact that cancer cells live bett er in a hypoxie
environment may imply that they have a rudimentary antioxidant system to get
rid of ROS . However, it remains uncertain whether this is true for all human
tumour cells in vivo , as they have high levels of AR and produce large amounts
of H 20 2 (Szatrowski and Nathan, 1991) . It also remains controversial whether
lipid peroxidation is low or high in tumours and when 4-HNE induces cell
proliferation or cell death (Dianzani 1993; Kondo et al., 1999). This is an
important point because ozonization ofblood produces LOPs which, upon blood
reinfusion, could exert important cytotoxie effects on neoplastic cells if they
have a poor defensive system. Zanker and Kroczek (1990) found that incubating
neoplastic cells in the continuous presence of a low dose of ozone «0.5 ppm) for
24 h was distinctly cytotoxic. Moreover, ozone was able to potentiate the
cytotoxicity of 5-fluorouracil (5-FU) and to increase the sensitivity in a 5-FU-
resistant colon carcinoma variant in vitro . However, I do not feel that the direct
cytotoxic action of ozone in tissue culture is important and may be misleading
because cancer cells never come in direct contact with ozone, not even when a
mad charlatan injects the gas intravenously.
In practice, a cytotoxic effect could be obtained only by intratumoural injection
of 0 2-0 J. Cutaneöus malignancies could be infiltrated with gas or ozonized H 20
or oil. Hepatic metastasis could be embolized with OrOJ via the hepatic artery
(Chapter 16).
2) Improved oxygenation and metabolism
This topic received attention in Chapter 14 (Erythrocytes) and again in this
chapter when we examined the problem in ischaemic tissues after reinfusion of
ozonized blood. Oxygenation ofblood during ozonization has a negligible value,
but the biochemical modifications induced by ozone on erythrocytes seem
important because ischaemic muscles and perhaps hypoxie tumours will receive
more oxygen. In the case of muscles, after ozonetherapy, the venous p02
decreased to 10-15 mmHg instead of 35-40, suggesting that oxygen was
delivered to the ischaemic tissue (Rokitansky et al., 1981) .
If indeed ozone is able to induce new generations of "supergifted erythrocytes",
with an increased content of ATP , 2,3-DPG, antioxidant enzymes as well as an
308 CHAPTER24

elevated G6PD (Hemandez et al., 1995; Viebahn-Hansler, 1996; Bocci, 1996c),

by continuing ozonetherapy for several months (actually for life), it may be
possible to profoundly modify their funct ional activities so that delivery of
oxygen increases and tumour tissues pass from a hypoxie to a normoxic state. If
this happens, it will dramatically change the tumour microenvironment,
probably leading neoplastic cells to a dormant or a very vulnerable state,
Vaupel and Hockel (2000) examined several possibilities to improve the oxygen
availability to neoplastic tissue: normobaric hyperoxia (100% O 2 plus carbogen),
hyperbaric hyperoxia, modification of 02-Hb affinity (shift to the right of the
Hb0 2 sigmoid curve), anaemia correction with transfusions and/or erythropoietin
administration. All of these ways are correct but more or less impractical and
transitory. Obviously, ozonetherapy was not inc1uded among these strategies,
because it is a poorly known approach. It must be c1ear that a few OJ-AHT
treatments are practically useless and that we cannot permanently change the
biochemistry of mature circulating erythrocytes. I feel that this a crucial point
to keep in mind. It is only during the phase of erythroblast differentiation in the
bone marrow that we can modify the cellular biochemical machinery by means
of the LOP messengers produced during ozonization of blood just before
reinfusion. Considering the erythrocyte half-life, a schedule of two sessions per
week of OJ-AHT (225 ml blood and maximum OJ concentration of 40 ug /ml per
ml of blood) and the dilution and catabolism of LOPs, it may be possible in 6
months (48 sessions) to substitute the normal erythrocyte population with a
majority of "supergifted" erythrocytes able to normalize oxygen levels in
neoplastic tissues . After each 03-AHT, a new cohort of young erythrocytes will
appear in the blood pool , replacing old and less effective erythrocytes.
Moreover, by acting on endothelial cells, LOPs enhance NO and NOthiol
formation, which may further increase the O 2 supply by improving the
tumour microcirculation. The consequence of a desirable downregulation of
HIF-I will be an inhibition of angiogenesis and tumour metastasis.
An additional (and not trivial) effect may be a generalized metabolic improvement
involving the immune and neuro-endocrine system, with a tolerable quality of life
for the patient. This is not a wild hypothesis: it already has some experimental
support and the possibiJity of being fully tested by measuring tissue p02 and
biochemical parameters of erythrocytes. The ideal methodological approach is OUf
EBOO and secondarily BOEX and 03-AHT (Chapter 16).
3) Potential upregulation of the antioxidant enzymatic system with improvement of
the cellular redox potential.
Ozone is a strong oxidizer and decomposes in a few seconds when it com es in
contact with blood. Oxygen per se is practically inactive: it does saturate Hb but
although there is an increase of solubilized O 2 in the blood water, no formation
of lipoperoxides has been detected. In contrast, when ozone dissolves in the
plasmatic water, it gives rise immediately to a cascade of ROS and LOPs
(Chapter 13). All of these compounds are unselective and in theory can damage
blood cel1 components. This is probably the reason why conventional Medicine
considers ozonetherapy a dangerous approach, more Iikely to be toxie than
therapeutical1y advantageous. However, it must be considered that if ozonization
 OZONETHERAPY IN VARIOUS PATHOLOGIES 309

of blood is carried out when the ozone dose per gram of blood is precisely
known, the reservoir of non-enzymatic and enzymatic antioxidants is capable of
minirnizing any possible damage to blood cell components. It has already been
explained (Chapters 13 and 14) that during the ozonization procedure a
"calculated" oxidative stress must occur in order to generate a certain amount of
ROS and LOPs, which act as crucial signalling molecules to elicit biochemical
and immunological responses i.e. the therapeutic "shock". This means that the
oxidative stress ought to be strong enough to trigger signals above the threshold
level (as otherwise they would be ineffective) but also be abated in a very short
time by the antioxidant system. Thus ozonetherapy is a procedure that involves a
"calculated", very transient oxidative stress capable of inducing cellular
responses without adverse effects. The exciting novelty is that ozonetherapy,
cautiously carried out with a scaling-up of ozone doses, stimulates the increase of
cellular antioxidant enzymes (SOD, GSH-Pxs) capable of inhibiting chronic
oxidative stress. What now seems a paradoxical effect of ozone has been described
since 1984 as an adaptive response. Olivieri et al. (1984) were probably the first to
observe that human lymphocytes undergoing very low doses of ionizing radiation
become resistant to a high dose of x-rays , Other studies cited in Chapter 22 clearly
showed that animals kept in a hyperoxygenated environment could survive by
upregulating the expression and activity of antioxidant enzymes.
This interesting new phenomenon of oxidative stress adaptation may explain, at
least in part, why ozonetherapy has a therapeutic activity in ischaemic,
degenerative and autoimmune diseases, and possibly in cancer in which a
persistent oxidative stress has been noted as a factor favouring the progression of
invasion and metastasis (Toyokuni et al., 1995).
4) Effects on the immune system .
It is obvious that, due to its strong disinfectant effect, ozone kills bacteria,
viruses, fungi, etc., thus facilitating their phagocytosis by leucocytes. The next
step was to understand how ozone activates both the humoral and cell-mediated
immune system. We discovered that ozone acts as a mild inducer of cytokines
because the generated H2 0 2 crosses the cell membrane freely and activates the
cytoplasmic NFKB, ultimately causing the transcription of mRNAs of several
cytokines (Los et al., 1995; Sen and Packer, 1996). Since the production of
several ILs, TNFa and IFNs is very small and transient , it appears unlikely that
induction occurs via stimulation of a membrane lectin. In fact, in the same
experimental conditions, the use of amitogen (PHA) that persistently activates a
cascade of protein kinases allows the synthesis of cytokines in amounts 1000
times higher than when using ozone. Similarly, the proliferation index of BMC
barely increases after blood exposure to ozone. This interpretation is in line with
the results of Schreck et al. (1991), who found that human lymphocytes can
express specific mRNAs after a transient exposure to 30-100 u M H20 2 •
It has taken several years of research to understand how ozone works and why
even a small activation of BMC can be useful in an immune-depressed patient. If
ozone acted as amitogen upon blood reinfusion, causing the massive release of
cytokines, we would have noted a frightening clinical response similar to that
observed after intravenous injection of LPS, characterized by shivering,
310 CHAPTER24

hyperthennia, hypotension and malaise (Mackensen et al., 1991). Moreover, the

disadvantage of a toxic syndrome that few patients are able to tolerate is associated
with the disruption of the cytokine network , which is usually deleterious .
In contrast, ozonetherapy never causes any side effects, only very rarely a
transient tiredness usually followed by a sense of well-being. Nonetheless, after
homing in their microenvironments, activated BMC may prime or stimulate
neighbouring cells, thus slowly upregulating the immune system . Obviously a
single session has no effect, and I would like to propose the same protocol
suggested previously to induce "supergifted" erythrocytes. Even a simplistic
calculation justifies this way of thinking. The immune system comprises about
1012 cells dispersed in various organs . Yet for each treatment, we can at best
transiently activate only about 6 x IOR cells , for which we do not know the life-
span or the pattern of redistribution. It is useful to recall that in the course of
adoptive immunotherapy, a fairly critical number of about 10 10- 11 LAK have to
be reinfused after exposure to IL-2 ex vivo (Rosenberg et al., 1987). An aspect
deserving some attention is the possibility that, during exposure of the patient's
blood ex vivo to ozone, circulating neoplastic cells (from breast , gastric, prostate
and colon cancers) could undergo oxidation and become a potential autovaccine.
This hypothesis is not too far-fetched because a) tumour cells have been detected
in blood (Pantel et al., 1999; Riethmuller et al., 1999); b) they are more sensitive
to ROS and LOPs than normal blood cells , and c) they could be taken up by
APe. Moreover, HSP-peptide complexes may elicit a CTL response and tumour
immunity (Tamura et al., 1997; Wells and Malko vsky, 2000) .
5) Effect on the CNS and endocrine system. Therapy of cancer-related fatigue .
On the basis of casual observations, a functional interaction between the nervous
system , the endocrine glands and the immune cells has been suspected for
decades. Immunological and neuroendocrinological studies (Blalock, 1984;
Payne and Krueger, 1992; Reichlein, 1993; Jones and Kennedy, 1993) have
c1early shown that these apparently distinct systems are indeed highly integrated.
This topic is beyond the scope of this review, but I cannot fail to comment about
the pleasant feeling of euphoria and well-being reported by the majority of
patients with chronic hepatitis and AMRD during OrAHT. This effect must be
kept in mi nd because cancer patients are orten plagued by fatigue . At long
last this severe complication is receiving attention and "Cancer" has published a
supplement on the topic (92, n.6, September 15, 200 I).
Would this effect also occur in cancer patients? Does ozonetherapy trigger a
psychoneuroimmunological effect via the release of a cascade of endocrine
secretions, namely of CRH, ACTH, cortisol, DHEA, growth honnone,
endorphins, melatonin, etc .? Could this effect be due to the withdrawal of a large
blood volume or to the reinfusion of ozonized and oxygenated blood with the
stimulatory effect ofLOPs on the endothelial bed?
It is not difficult to envisage that a change in the homeostatic balance is bound to
evoke a multi-organ response that could positively influence the psychological
status ofthe patient, hence the immune response (Fig . 102) (Flach and Seachrist,
1994). How important is the placebo effect (Chapter 25) remains undetennined
because even a slight increase of cortisol may improve the mood of the patient
 OZONETHERAPY IN VARIOUS PATHOLOGIES 311

(Coleman, 1992). Aseries of circumstances, e.g. being attentively cared for and
observing the AHT steps with "energized" blood being reinfused, compose the
so-called "Hawthorne's effect", which may have a boosting effect and evoke a
psycho-hormonal-immune response, the significance of which cannot be under-
estimated (Cassileth et a1., 1991; Cassileth and Chapman, 1996; Trussel, 1999).
It is weil documented that a few cancer patients have been miraculously healed
after a pilgrimage to Lourdes!

COGNmvE STIMUlI
(phys lcll , ehomlell, omDthle>

D.

Figure 102. Modern psychoneuroimmunology is based on the interactions among the

CNS, endocrine system and immune system. Release ofcytokines by the immune system
influences both the CNS and endocrine system. Thus it has become possible to understand
the genesis and relevance ofthe placebo effect
312 CHAPTER24

Another important question is whether ozonetherapy should be carried out in the

morning or in the aftemoon. Intuitively, I would favour the aftemoon (4-8 PM)
because the normal circadian rhythm ought to be least disturbed (Bocci , 1985b).
However, only experimental data can define the optimal time of the day, although
there is the practical problem that most ofthe work must be done in the moming .

As exemplified in Figure 103, the war on cancer is won when all cells have been
killed. There is no doubt that tumour debulking with surgery or other therapies (Fig .
10 I) is essential, because a large tumour load or extensive metastasis enhances the
anergie state (Elgert et al., 1998) and reduces the chance of a eure . Figure 103
schematically indicates that the primary tumour could be either eradicated or more
or less extensively removed. The former case is rare because haematogenous
dissemination of individual tumour cells occurs at early stages of the malignancy, as
has been clearly shown by immunocytochemical detection of epithelial tumour cells
in bone marrow (Riethmuller et al., 1999).

death

_·....
.:-
..
detection
. ....
..
--------_. ...· .. level

relapse
.'
...
·
..
minimal residual disease

cornplete ablation and eure

o
Time
Figure J03. Tumoural mass reduction by cytoreductive therapy

Thus, if only about 104 neoplastic cells have been disseminated, there is hope of
either destroying them or preventing metastatic growth if the surveillance of the immune
system remains active. Today conventional medicine offers several approaches
 OZONETHERAPY IN VARIOUS PATHOLOGIES 313

attempting to achieve this goal (Fig. 10I). The most promising appear to be
immunotherapy and the various forms of gene therapy. However, all these approaches
are still experimental and it may take several years before they are validated.
If metastases are present, the problem is far more complex and chemotherapy is
widely used with mixed results; indeed the side effects frequently impoverish the
quality oflife.
Can ozonetherapy be more useful than chemotherapy in metastatic cancer? It is
very difficult to answer this quest ion because the few anecdotal reports are not valid .
Only unbiased, randomized, double-blind clinical studies for several cancer types,
possibly carried out in several oncological institutions, can ultimately prove whether
ozonetherapy can really be useful . Due to the lack of serious biological and clinical
research in the past , this approach remains in limbo today and is totally disregarded
by conventional oncology, particularly by chemotherapists. This is very
unsatisfactory, mostly because, in spite of some progress, the death rate remains
high and real breakthroughs are not yet in sight . Because I feel that this is one of the
most important issues, I have tried to objectively review several, albeit hypothetical,
reasons to pursue the evaluation of ozonetherapy, not as a procedure able to eure
the neoplasia but rather as a means to stabilize its progression, particularly in
elderly patients susceptible to the serious side effects of high-dose chemotherapy.
In the last few years, I have made a useless effort to explain that this approach has
a rational basis and can be carried out in a scientific and reproducible fashion. The
ozone dose can be precisely adjusted to the blood volume or patient's weight, and
optimal ozone concentrations for the proposed methods are based on experimental data
and not on homoeopathic or imaginary beliefs. Almost needless to say, a lot of basic
work remains to be done, particularly in order to define the molecular and
immunological modifications of erythrocytes and leukocytes . Analysis of the
adaptation to COS may weil be able to show that ozone can profoundly modify the
biochemistry and functionality of these ceUs in order to create an environment
hostile to cancer cells. In my opinion, this a new line of thought stating that the cell
malignancy can be tamed simply through the use of a potent biological modifier .
However , only reliable c1inical data can ultimately inform us about the validity of the
approach; indeed we have often observed that an improvement of immunological
parameters is not necessarily paralleled by a complete response and prolonged survival
(Bocci, 1987b; 1990b; Reddy et a1., 1997).
What will be the future of this approach? As usual, we are facing the same story .
It looks unpromising unless we carry out controlled c1inical trials. At the moment,
on the basis of my frustrating experience, I doubt very much that we will be able to
perform them, due to the disinterest and skepticism of oncologists. Obviously the
pressure of pharmaceutical companies does not allow them to experiment with
anything other than cytotoxic drugs. Although I have a great admiration for the
scientific strides in biology and medicine, I feet that the biased attitude of most
oncologists towards ozonetherapy is wrong and unjustified. On the other hand, most
physicians performing ozonetherapy in private practice are unable to perform a
c1inical t)-(al and the habit of mixing other therapies makes any conclusion
impossible.Donsequently, the only hope is that serious and concerted efforts will be
made in the next few years . For the moment, however, it seems that, because of the
 314 CHAPTER 24

laek of dialogue and eooperation, a potential therapeutie advantage will eont inue to
remain in Iimbo , perhaps to the patient's disadvantage.

11. ORTHOPAEDIC DISEASES. THE PROBLEM OF BACK-ACHE

In the last deeade, a number of orthopaedie surgeons (Riva Sanseverino, 1989;

Siemsen, 1995) have begun to treat aeute and ehronie polyarthritis (osteoarthritis of
the hip, knee, interphalangeal joints, saeroiliae joint, ete.), tendinitis, myofaseial pain,
epicondilitis and earpal tunnel syndrome with intra-artieular or peri-articular
insufflation of small volumes of Or03 (5-10 ml in one or three sites with 0 3
eoneentrations from 5 to 15 ug /ml) with very eneouraging results . In Morton's disease
(neurorna), up to six infiltrations of O 2-03 (4 ml eaeh at 20 ug /rnl have yielded great
pain relief. In a review article , Siemsen (1995) reported that applieation of medieal
ozone in aeute and ehronie painful diseases ofthe joints is a eomplementary method of
treatment to obtain rapid pain relief, deeongestion, disappearanee of oedema, reduetion
of loeal temperature and inereased mobility. If performed by an expert orthopaedie
surgeon, the treatment is not risky and eauses only transitory loeal pain that disappears
in 5-10 min without any other adverse effeet.
The pathophysiology of these diseases is eomplex and eharacterized by the
softening and even destruetion of the artieular eartilage, with inereased matrix
degradation due to eollagenase and proteoglyeanases. The enzymes may be seereted
by aetivated ehondroeytes and monoeytes, whieh release IL-I and TNF o ; Synthesis
of POs inereases several fold and there is a natural attempt to maintain a
biomeehanieally adequate matrix. In eontrast to RA, pannus does not develop . Joint
pa in may be aggravated by eoneomitant synovitis.
Drug therapy is symptomatie, aiming to reduee pain and disability. Inhibitors of
eyelooxygenase I are in wide use, with aceompanying gastritis, ete . and are being
substituted with inhibitors of eyclo H. Loeal injeetion of glueoeortieoids into a given
joint ean be earried out no more than twiee per year.
Beeause eonventional medieine does not provide a "eure", patients seareh for
eomplementary therapies. On the basis ofthe pathology, ozonetherapy should be the last
treatment to perform, beeause ozone (a potent oxidant) injeeted into the synovial spaee
should elicit further inflammation or degeneration. Therefore, it is incredible that, after
initial but tolerable pain, ozone produees great relief for a long time . By now,
innumerable patients have been treated and we eannot doubt the results . Obviously
ozone is not a "miraculous" medicine and we must try to understand how it aets.
On several oeeasions, I have asked orthopaedie surgeons to eollaborate with us
beeause I think it would be interesting to examine the synovial eontent before and
after ozonetherapy. So far, this has not been possible, either beeause most patients
are treated privately or beeause it is difficult to eolleet sampIes. Thus I can only
advanee a few speeulations.
Onee ozone dissolves in the synovial fluid , it reaets with free proteins, enzymes,
proteoglyeans and ehondroeytes and may elicit:
 OZONETHERAPY IN V ARIOUS PATHOLOGIES 315

a) Inactivation and inhibition of the release of proteolytic enzymes and of

endogenous ROS.
b) Stimulation of the proliferation of chondrocytes (probably via H202 ) and
fibroblasts, with increased synthesis of matrix and possibly of articular
cartilage. Induction of the synthesis of antioxidant enzymes (SOD, GSH-Pxs
and catalase) may be a crucial event as an adaptive response to COS and to
ozone. That is the reason why I would start infiltrating ozone at low doses.
c) Release of bradykinin and synthesis of inflamrnatory PGs is probably inhibited,
with reabsorption of oedema and pain relief.
d) An increased release of IL-I soluble receptor or of other soluble receptors and
antagonists able to neutralize IL-I, IL-8, IL-12, IL-15 and TNFa.,just to name a
few possible culprits.
e) Conversely the release of imrnunosuppressive cytokines, such as TGF-ßI and IL-
10, may inhibit inflammation. Among several growth factors, TGFß I is interesting
because it modulates the expression of integrins and stimulates the synthesis of
matrix proteins such as collagen and glycosaminoglycans (TrippeI, 1995; Qi and
Scully, 1997). 1fthis is the case, the long period ofremission can be explained.

These are just hypothetical ideas, which shouldbe verified by examining the
synovial fluid and bioptic fragments to c1arify these really paradoxical ozone
effects. Ozone never ceases to surprise us!
In RA, the use of0 3-AHT at high doses has been suggested (Chapter 14). Yet it
remains untested whether the association of one treatment (per week) of 03-AHT at
a low-medium ozone dose would improve the outcome in osteoarthritis.
Low back pain is a very disturbing symptom that can affect, at least for a while,
up to about 80% of the worId's population. Luckily, in most cases, physicaI
therap ies (exercise, manipulation therapy, etc.) can solve the problem (Cherkin et
al., 1998; Samanta and Beardsley, 1999). However , if a herniated disc (protrusion of
the nucleus puIposus through the annulus fibrosus) is present and causes
considerable pain , it must be removed with the least invasive procedure.
Up to the I970s, the typical orthopaedic operation removed the compression but
often destabilized the mechanical and functional stability of the vertebral colurnn .
Thus it has been substituted by a mini-invasive intervention. This trend was
accentuated by chemonucleolysis, introduced by Smith in 1969. However, the
intradiscal injection of chymopapain and collagenase, potent enzymes able to digest
the components of the nucleus pulposus, has been abandoned because of occasional
risk of allergie reactions and the exorbitant cost of the pure enzymes. Subsequently,
Onik et al. (1987) introduced the alternative concept of aspirating the degenerated
disc including part of the herniated material, thus reducing the abnormal pressure
and relieving the nerve root compression. This technique is still in use with a
success rate of about 75% (Bocchi et al., 1998). There are other variants of this type
of approach, the latest being nucleoplasty.
In 1988, Verga , a private ozonetherapist, noted pain relief after infiltrating
trigger points in myalgias with O 2-0 3 and proposed to use an indireet technique by
injecting the gas into the points localizable in the paravertebral muscle (locus
316 CHAPTER24

dolendi) corresponding to the metamer of the hemiated disc , This approach is now
widely used by many ozonetherapists in Italy and it can be defined as the indirect
approach, or as I call it: "chemical acupuncture" (Bocci, 1998a).
The "chymopapain model" probably inspired a neurosurgeon, Jucopilla et al.
(1995), to test whether intradiscal injection of ozone would be nucleolytic and
beneficial. This can be defined as the direct intradiscal injection of 0 2-03' More
recently, another indirect variant has been introduced by the epidural injection of O 2-
0 3 in correspondence to the lesion. This is being performed by anaesthesiologists and
seems promising. The use of O2-0 3 to treat back pain syndrome is now widely used in
Italy, while it is unknown abroad . As it is a minimally invasive treatment with a
negligible cost and rare side effects, it is worth trying before surgical intervention . At
our University, on the basis of our protocol , over 100 patients have been treated and
about 80% have shown marked improvement (Bocchi et al., 2000) . Thus there are as
many as three technical approaches, which are exemplified in Figure 104.

EP CA ID
Figure J04. Schematic view ofa transverse section of the lumbar region: NP: nucleus
pulposus. MC: medullarycana!. The arrows indicate the three possible routes O!Or03
administration. ID: intradiscal; CA: "chemical acupuncture " in the paravertebral muscle,
PM and EP: epidural injection
 OZONETHERAPY IN V ARIOUS PATHOLOGIES 317

The Direct Approach

A clear view of the L4-L5 intersomatic space with the needle just before direct
insufflation of O 2-0 3 is shown in Figure 105 (Andreula, 2001; Simonetti et al., 2001).
The direct approach is carried out under radioscopic control and an expert can do it in
about 10min. After a rest of 10-15 min, the patient can get up and often he/she is
amazed by the disappearance ofthe pain, as occurs after nucleoplasty. Ifnecessary, the
application can be repeated a.second time before changing the approach.

Figure 105. The intradiscal approachfor direct injection o/OrOJ into the nucleus pulposus.
The radiograph (above) shows the correct positioning 0/ the needle in a frontal scan
(Andreula , 2001). Discographie view (below) 0/ a transverse lesion in the fibres ofthe
annulus in disc L4-L5. in continuity with ascending disc herniation (Simonetti et al.. 2001)

Good results have been obtained after either intradiscalor intraforaminal

injection of a variable volume (3-15 ml) of gas at an 0 3 concentration of 27-30
318 CHAPTER24

ug/rnl. Several thousand patients have been treated, with a success rate of 54-86'%
(Alexandre and Fumo, 1998; Jucopilla et a1., 2000; Bonetti et a1., 200 I; Fabris et al.,
200 I; Leonardi et al., 2001 a; Petralia et al., 200 I) . It remains unc1ear how ozone
acts . One real possibility, previously discussed at length (Bocci 1998a, 1999), is that
ozone dissolves in the interstitial water and reacts immediately, generating a cascade
of ROS, among which HzOz and possibly the hydroxyl radical, OHo, whieh is most
reactive. The hydroxyl radieal appears to react with carbohydrates and amino acids
composing proteoglycans and collagen type land 11, major components of the
degenerate nuc1eus pulposus, leading to its breakdown (MeCord, 1974; Curran et a1. ,
1984; Hawkins and Davies, 1996; Bocci et al., 2001b; Leonardi et a1. , 2001b). These
studies, as weil as those performed on human blood, have been carried out using the
Electron Paramagnetie Resonance (EPR) spin trapping technique (Ueno et al.,
1998). Consequently, reabsorption of hydrolytic products and water may lead to
progressive shrinkage and disappearance of the hemiated material. Reduced
mechanical irritation decreases the sensitivity of nerve axons, but nociceptors are
also excited by endogenous algesie substances released during perineural ischaemia
or neural inflammation present in the spinal ganglion and neural roots (WiIIis,
1995). Thus, more than the mechanical compression as primum movens, it is the
inflammatory reaction that sustains chronic pain by releasing PLA z, several
proteinases and cytokines. The continued release of ROS, PGE z, serotonin,
bradykinin, cathepsins, IL-I , IL-6, substance P, etc ., causes oedema, possibly
demyelination and increased excitability of nociceptors (Fields, 1986). Indeed , it has
been observed that even a large hemia can be painless. Moreover, the hemia may
remain after an operation (as seen radiographically), but the pain disappears once the
inflammatory disorder dies down . Interestingly, epidural injections of the anti-
inflammatory methylprednisolone transitorily improve leg pain and sensory deficits
in patients with sciatica due to a hemiated disc (Carette et al., 1997).
So, how does ozone act? We are again facing the ozone paradox : although OHo can
degrade the degenerated material and reduce pressure, it often exerts a rapid "anti-
inflammatory action", partieularly because only a few ml of gas can be introduced
inside the nucleus pulposus and most of the gas invades the intraforaminal space . This
may mean that ozone rapidly blocks inflammatory reactants and stimulates the
restitutio ad integrum. What is even more surprising is that this change remains stable
(unlike corticosteroids) and it does not necessarily coincide with the disappearance of
the herniated material. In fact, CAT or NMR controls in 612 patients, 5 months after
treatment, showed that the hemia disappeared in 226 (37%), was reduced in 251 (41%)
and was unmodified in 135 (22%). After another 5 months, CAT/NMR controls were
performed again in 200 (of251) patients in whom the hernia was reduced : a further
reduction and improvement was noted in 44 patients (22%) . In 120 patients (of 135) in
whom the hernia was unmodified, there was an improvement in 11.6'% (14 of 120)
(Alexandre et al., 2000) .
Thus the ozone effect is deployed in successive phases: there is an initial rapid
change, probably with disappearance of oedema and improvement of circulatory and
metabolic conditions, followed by stasis and then a further improvement possibly
due to release of TGFß 1 and bFGF (Silver and Glasgold, 1995; TrippeI, 1995),
 OZONETHERAPY IN V ARIOUS PATHOLOGIES 319

favouring the reorganization of the residual nucleus pulposus with incipient fibrosis.
So far, attempts to examine the histopathological changes have been inconclusive .
A few problems have been reported. In young patients, it is often very difficult to
introduce more than 1-2 rnl of gas inside the nucleus pulposus, so that the gas is
released into the intraforaminal space. I have been wondering if, in these cases, a
preliminary aspiration of the nucleus followed by the gas introduction might
improve the result. Apparently, the intraforaminal administration of gas yields good
results even in the case of sclerotic hernias (Fabris et a1., 2001). Side effects are very
rare: one patient had a transient lipothymia and one reported by Alexandre et a1.
(1999) presented amaurosis fugax (bilateral blindness which reversed after about 24
hours) after cervical discolysis in a young athlete (Chapter 21).

The Indirect Approach, or " Chemical Acupuncture "

Use of the paravertebral muscles as a route for infiltration of Or03 is shown in
Figure 106 (taken from Tabaracci, 2001).

Figure 106.The iliac crests are palpated and the transiliac line is determined to identify the
L4 spinous process, the interspinous spaces are identified by selecting the space
corresponding 10 the herniated disc. Roughly 2 cm are calculated bilaterally to the spinous
process (above). Once the needle is inserted througb thefasciae, material is aspirated while
holding the needle still and a 20 ug/ml concentration 0/ an oxygen-ozone mixture is injected
velY slowly up to a maximum 0/10 ml per infiltration . Aspiration is repeated during
infiltration (below) (Tabaracci, 2001)
320 CHAPTER24

This approach, which seems technically simple, has become very popular in Italy .
Indeed some physicians think they can become ozonetherapists ovemight and start
to inject a patient with an excessive dose of ozone, which might kill hirn owing to a
complex neurovegetative over-reaction. This has happened on ce and that is why it is
important to have precise guidelines and mies for the practice of ozonetherapy.
In reality, it is an easy approach consisting in one or several (up to four)
injections of 5-1 0 rn1 of O 2-0 3 per site. The ozone concentration must not exceed
15-20 IJglml because it is painful . At first, it is wise to test the patient's reactivity
with an injection of sterile saline and then start with 10 ug/ml ozone. The injection
must be done very slowly into the trigger points corresponding to the metamers of
the hemiated disk. The length of the needle varies (from G22 to G25) depending on
the patient's obesity. Usually two symmetrical injections (total dose 10-20 ml gas
with at most 200-400 ug ozone) repeated twice per week for about 5-6 weeks (I0-12
sessions) are sufficient; ifnot, the patient is unresponsive to this approach .
I repeat that injection of O 2-03 e1icits a sharp pain lasting a few minutes and the
injection must be done very slowly to avoid any risk of embolization. If we act
carefully, we can avoid serious adverse effects, such as sud den hypotension,
bradycardia, mydriasis, intense perspiration and cardiac arrest (vasovagal reflex) .
Any serious ozonetherapist must be prepared for this emergency, which is very rare
but can happen (see Chapter 36) .
The results of a number of studies vary somewhat (Cinnella and Brayda-Bruno,
2001), but they can be summarized as : about 40% optimal, 35-40'% marked
improvement 15-25% minimal or no result. Gionovich et al. (2001) compared three
approaches:

A) Paravertebral injection of0 2-03 75% Satisfactory response

B) Peridural injections with desamethasone 55% Satisfactory response
C) Paravertebral injection of'buvipicaine 0.25% 70% Satisfactory response

The term "chemical acupuncture" was coined (Bocci, 1998a) because we must
c1arify the role of the needle, oxygen and ozone. It was proposed to compare this
procedure against a waiting-list control, two placebo controls (one with oxygen
alone and another without any gas) and a standard-treatment control. Gionovich et
al. have now shown that, as expected, even an anaesthetic has some effect. Owing to
an unexpected, unintentional incorrect use of the medical generator (delivering
medical oxygen only), we can now give a reasonable answer to the above-mentioned
uncertainty. Torri et al. (1999) treated a group of 66 patients with O 2-03 and a group
of 30 patients with oxygen alone. Interestingly, excellent or good responses were
observed in 86% of patients of both groups but the O 2-03 group showed a
statistically significant improvement of some c1inical parameters. This suggests that
the needle and oxygen together already have a therapeutic role, wh ich is potentiated
by the addition of ozone.
Then the question is: how does ozone injected intramuscularly work? The gas
infiltrates the musele and after 24 hours some gas bubbles move towards the
vertebral canal (as seen radiologically). It was postulated that ozone will reach the
site of the hemiated material and will Iyse it. This is an untenable idea : ozone
 OZONETHERAPY IN V ARIOUS PATHOLOGIES 321

dissolves rapidly into the interstitial water of the muscle and will generate H202
within a few minutes; by inhibiting amyelinic fibres (C-nociceptors), the H202 will
activate the anti -nociceptive system via the descending antinociceptive system (Fig .
107). As occurs during acupuncture (Ceccherelli et al., 1995), the introduction ofthe
needle, reinforced by the pressure of O 2-03 , induces strong inhibition of nociceptors,
perhaps a prolonged stunning due to H 202 • It is known that an algic stimulation of
the skin and muscles can reduce pain through the mechanism of diffuse noxious
inhibitory control (DNIC) . That is why the needle + H 202 + oxygen pressure can be
translated into chemical acupuncture.

Figure J07. The scheme indicates the mechanismsfor the control ofalgesie signals. By
releasing endorphins (End.), the enkephalinergic interneuron may inhibit the presynaptic
connection ofa neurocyte (C) of a spinal ganglion which, under compression ofa herniated
disc, stimulates the release ofsubstance P (SP). Endorphins ean inhibit the transmission of
the algesie signal to neuron D. hence to the ascending spinal-thalamicfibres. The
monoam inergic 01' serotoninergic neuron A. as a component ofantinociceptive descending
fibres , ean reinforce the analgesie effect of neuron B
322 CHAPTER24

This mechanism is likely correct because too low 0 3 concentrations (3-10 ug/rnl)
or gas volumes (1-2 ml) are ineffective, whereas too high 0 3 concentrations (above 20
ug/rnl) or gas volumes can cause lipothymia . It is unclear whether pre-infiltration with
an anaesthetic reduces the effect of ozone . We do not know whether the generated
H20 2 causes irreversible damage and death of the nociceptors, with a consequent
increase of the activation threshold . Furthermore, it is not known if this means a
blocked release of algesie compounds, with a simultaneous release of endorphins .
In conclusion, the probable mechanisms playing a role are the following :

a) release of endorphins blocks transmission of the noxious signal to the thalamus

and cortex.
b) Hypostimulation (elevation of the activation threshold) linked to the oxidative
degeneration of C-nociceptors. H 2 0 2 and LOPs may act like capsaicin.
c) Activation ofthe descending antinociceptive system.
d) Simultaneous psychogenic stimulation of the central analgesie system induced
by the gas injection (elicitation of a placebo effect).
e) The localized oxygenation and analgesia permit muscle relaxation and
vasodilatation, and thus a reactivation of muscle metabolism, by favouring
oxidation of lactate, neutralization of acidosis, increased synthesis of ATP, Ca 2;
reuptake and reabsorption of oedema.

Once again, by reactivating natural defence mechanisms, the use of oxygen-

ozone surprisingly seems to solve a painful problem.
We have prepared a protocol proposing to evaluate the loeal effect (paravertebral
muscles) ofa solution ofH 2 0 2 diluted in a 5% glucose solution. We may be able to
ascertain if H20 2 is the compound that acts on nociceptors and evokes the analgesie
response. Samanta and Beardsley (1999) wondered what was the best way forward
to treat low back pain, but they did not know and eould not mention the O 2-0 ,
approach. If American and English orthopaedic surgeons read this book and try this
approach, they may produce new and interesting results, useful for science and
above all for patients.

12. CHRONIC FATIGUE SYNDROME (CFS) AND FIBROMYALGIA

I am grateful to Prof. R. Mareolongo, Director of the Institute of Rheumatology,
Siena University, for his broad-rnindedness and interest in evaluating ozonetherapy
in RA, CFS and fibromyalgia. Some three years ago, we submitted a eomplex
protocol to the Ethical Committee, which approved it after a few revisions.
However, the study on RA never took off because I was personally very doubtful
about the ozone dose and because Prof. Marcolongo did not agree to suspend
conventional medication in these patients. CFS and fibromyalgia are diseases
charaeterized by poorly understood signs and symptoms, with severe fatigue and a
eontinuous flu-like syndrome that profoundly disables patients. Since 1990, I have
fotlowed the abundant medicalliterature on CFS and I have realized that I could not
even attempt to give a short synthesis of an ambiguous aetiology and
 OZONETHERAPY IN V ARIOUS PATHOLOGIES 323

pathophysiology. Recent papers have added some new ideas and the interested
reader may consult them (Komaroff, 2000; Manu , 2000; Reid et al., 2000; Spence et
al., 2000; Natelson, 2001 ; Powell et al., 2001 ; Prins et al., 2001; Wessely, 2001).
Conventional treatment is based on antidepressants, low-dose glucocorticoids,
exercise (in contrast, prolonged rest seems harmful), immunotherapy and oral
nicotinamide adenine dinueleotide to increase the generation of ATP . Benefits are
limited and there are adverse effects . Cognitive behavioural therapy performed by
skilled therapists appears to be an effective intervention without harmful effects . In
this book, I have reported that 03-AHT often yields a feeling of well-being and
euphoria . This is quite true, even though we can only speculate about the reasons for
these positive effects . In search of a good therapy , CFS patients go from one
physician to another. Last year, at the hospital of Conegliano Veneto, they treated
six patients diagnosed with CFS. 03-AHT was carried out twice per week for 8
weeks and the physician in charge assured me that four patients showed a
"remarkable improvement". He could not give information about the follow-up.
In our hospital, Dr. Cosentino treated one patient with a modest outcome. I
apologize for such a crude report but, given the severity of the disease, I would not
hesitate one second to do 03-AHT if the patient wanted to try it. I would suspend
any other drug and c1early explain that ozonetherapy may readjust the metabolie,
immunological and hormonal derangements, causing only the discomfort of the
venous puncture.
Fibromyalgia is another mysterious disease . The American College of
Rheumatology (ACR) has established a procedure for examining 18 tender points on
the patient. A tentative diagnosis can be made if at least 11 ofthese points elicit pain
when pressed. In Italy, fibromyalgia is considered a disease causing considerable
socio-economic problems, since it affects about 6 million people between 30 and 60
years of age. The aetiology is unknown but initially seems caused by a
psychosomatic factor later complicated by biochemical modifications in the musele,
possibly similar to those found in CFS (Fulle et al., 2000), and neuro-psycho-
immune endocrine disorder.'
Two studies have been carried out in Italy and they were reported at the IMOS
congress last year at Siena University (November 2-4, 2000). Unfortunately, they
have not yet been published and I will give a short summary. From 1988 to 2000,
Dr. Salvatore Loconte (Andria, Bari) has treated 150 patients by infiltrating 5 mI gas
directly on the trigger points (0 3 concentration: 5-10 ug/ml) and perforrning a cycle
of 03-AHT with about 150 ml blood and a total ozone dose of 4500 Ilg (30 ug/ml),
He is a private ozonetherapist and cannot do a control but he has elaimed to achieve
total remission in about 60% of patients and partial improvement in 15%.
A RCT has been performed in the Institute of Rheurnatology of our University
on 40 women (age 30-50) diagnosed as having fibromyalgia on the basis ofthe ACR
criteria . The scope of the study was to evaluate the effect of A) AHT with O2-03 (20
patients, with 0 3 concentrations scaling up from 20 to 40 ug/ml , twice per week for
a total of 16 treatments), B) AHT with O 2 alone (10 patients), and C) simple AHT
without gas (10 patients). Several standard end-points were tested before treatment,
after 8 weeks and I month thereafter.
324 CHAPTER24

Patients of group C did not show any improvement and are now under Or03
treatment. Three patients of group B (30%) showed good improvement. Seven
patients of group A (35%) showed excellent improvement, while one (5%) had good
improvement. Cosentino et al. (2000) conc1uded that 03-AHT has therapeutic
validity and no side effects. However, in comparison to conventional
automedication, it is time-consuming for hospital personnel.

13. TRAUMNBURN INJURIES AND EMERGENCY SURGERY.

OZONETHERAPY BEFORE TRANSPLANTAnON
OR BEFORE ELECTIVE SURGERY.

Regarding the first topic, I never managed to convince the chief doctor of intensive
therapy of the potential usefulness of AHT performed with O 2 and 0 3 at low
concentrations (15-25 ug/ml of blood) in patients with permanently cannulated
central or peripheral veins . They are mostly concemed about the legal aspect of
using a non -validated and somewhat controversial therapy in high-risk patients.
When I visited Russian hospitals, I was told that they do not worry about it and use
ozonetherapy to disinfect traumatic and war wounds, bums (due most frequently to
flames) , radiation injuries and abdominal surgery after stomach or intestinal
perforations. Disinfection with ozonized bidistilled water and application of
ozonized oil has been found to be most useful in bums. It is unfortunate that they
abundantly use ozonized saline instead of ozonized blood for systemic treatment. On
this point, our opinions are greatly divergent. I cannot agree with their assertion that
ozonized saline is as effective as blood, because on several occasions we have noted
the multidirectional potentialities of ozonized blood. Serious trauma, bums and
peritonitis lead more or less rapidly to systemic alterations of several organs,
particularly the cardiopulmonary (AROS), coagulative (DIC) and renal systems.
Because of an adverse series of metabolic impairments, these alterations cause the
patient's death . Thus, using all the most appropriate conventional supporting
therapies combined with OrAHT (every 3-4 hours throughout the day), I "feel" that
we could save some lives .
The second topic is less tragic, but no less serious. I have often wondered if a
cardiac patient waiting for a heart transplant might gain increased resistance to
infections and to immunesuppression (unavoidably linked to deep anaesthesia and
surgery) ifhe could undergo two OJ-AHT per week (at low 0J concentrations: 20 to
40 ug/ml) for 3-8 weeks before transplantation. This strategy is all too obvious and
may induce a sort of ischaemic preconditioning or, to use language comprehensible
to most people, the adaptation to chronic oxidative stress (Chapter 22). During heart
transplantation, all organs (particularly the CNS, retina and kidneys) undergo a
bland ischaemia-reperfusion syndrome, which in unlucky cases may have dire
consequences even if the operation is technically perfect. Thus prophylactic
ozonetherapy, with little effort and expense, might be useful.
The final point worth pursuing involves the scheduled operation for application
of a prosthesis, particularly joint implants. In particular, as a precaution, coxo-
femoral surgery requires the collection of I or 2 standard units of blood from the
patient. Discussing this problem with several orthopaedic surgeons, J found that at
 OZONETHERAPY IN V ARIOUS PATHOLOGIES 325

least three were interested in evaluating whether performing at least four 03-AHT
(ozone at low concentrations) during the 2 weeks before the operation and then
every day immediately after it for 4-5 days (using the predeposits as weil) would
reduce the complications by enhancing healing and the patient's mood. I presented a
protocol to our Ethical Committee, which was approved. However, no trial has
started as yet because the orthopaedic surgeons do not have supporting personnel to
do AHT.

In summary, I regret that this long and dreary chapter is rather

inconclusive. On the whole, I will feel great if two out of ten proposed clinical trials
begin and if we can finish one study . The pace is too slow and I wish that we had
already made incisive advances so that I could indicate optimal doses and schedules.
Nevertheless, we have some ideas and a working hypothesis on how to proceed.
It is almost unnecessary to repeat that our good will is not sufficient and unless
there is a concerted effort by official Medicine and govemment authorities, we will
remain at the stage of "if", "perhaps" and "speculations". This is sad, not so much
for me because I have always tried to do my best, but for the patients who will not
have the advantage of recovering more rapidly and improving their health. National
health authorities, which are always complaining about the increasing costs of
medical assistance, could have an economical advantage if ozonetherapy was
widespread and organized in a systematic way in all public hospitals. The savings
could be dedicated to improving other areas of medical assistance. Even a child
could understand this simple reasoning. Although I have no hard data to support my
contention, I am convinced that the benefit of ozonetherapy does outweigh its cost.
Obviously pharmaceutical giants or other private organizations would make less
profit, but they would continue to thrive just the same . An obvious comment is that
although the Communis' creed has failed in many ways, it has succeeded in Cuba in
truly improving health assistance for everyone. A good part of the merit, however,
has to be attributed to the versatility of ozone .
 CHAPTER25

HOW IMPORTANT IS THE PLACEBO EFFECT

IN OZONETHERAPY?

" The pla cebo ejJect is the heal ing force ofnature"
Zajiceck (1995)

Orthodox medicinehas considered a placebo to be a treatment that has no specific

effect on the illness to which it is being applied (Benson and Friedman, 1996). A
real placebo has no phannacological properties or activities and it must be inert (e.g.
starch). It may, however, exert the placebo effect when the patient feels better
simply by virtue of being given a supposed medicine, owing to his/her personal
mindlbody interaction. In the 1950s, it was clearly shown that a starch tablet could
alleviate a symptom, such as headache, as long as patients believed they were
receiving areal drug ,
The placebo effect varies among different diseases and patients. In psychiatry,
particularly concerning antidepressants, the placebo effect appears outstanding
(Enserink, 1999). Almost half of gastric uleer patients can be cured by placebo
tablets rather than cimetidine. I was really impressed by the results of two
randomized placebo-controlled studies of acute relapses of MS (Durelli et al., 1985;
Milligan et al., 1986). They used high IV doses of methylprednisolone for five
consecutive days and, after one week, found an equal improvement in:

85-92% ofpatients who received active treatments, and

33-40% ofthose who received placebo (saline).

How these "sham" group patients managed to recover (to the same degree as
most "verum" group patients) remains undefined. Most likely, the continuous "drip"
infusion of saline and the physician's attention must have positively influenced their
endogenous release of CRH-ACTH and cortisol. Zajiceck (1995) noted that the
placebo simply triggers the body's natural responses and is the manifestation of the
"Wisdom of the body", which must have evolved in a Darwinian fashion over
millions ofyears. .
Complementary (or even magical) approaches are much more susceptible to a
placebo effect than science-based ones. In ozonetherapy, the methodological
scenario of O]-AHT, and even more so EBOO and BOEX, impresses the patient,
and the invisible, yet important, fact of using agas mixture containing either OrO],
or O2 alone, or only air becomes less relevant. The kind manner and attentive
attitude, especially of the private ozonetherapist, may complete the idyllic picture in

327
328 CHAPTER25

which the patient is certainly going to regain health , which is considered the
supreme blessing (Fig. 102).
Almost needless to say, most ozonetherapists consider randornization and
evaluation of the placebo effect as unethical. As we shall see, any patient at risk must
be exempted. It is also understandable that private ozonetherapists cannot participate
in a RCT, as has been done, surreptitiously, in Italy by an entrepreneur. Actually,
ozonetherapists value the placebo effect like manna from heaven (Ernst, 1996b), and
while I am glad that patients benefit from it, I remain very concerned about the
scientific validity of the approach. Ozonetherapists frequently resent the medical
establishment, since they feel that it considers them quacks and occasionally
persecutes them because of the overwhelming power of the phannaceutical industry
and medical lobbies arid the fact that ozone is non-patentable and rather inexpensive
compared with modem therapies. A few ('f' these comments may be correct, but most
are inappropriate ; it is useless to accuse oll. ~rs in order to excuse our own inadequacy .
In writing Chapter 24, I realised (and here I agree with the skeptics) that most of
the c1inical work published on ozonetherapy in obscure journals is outdated and of
poor quality, except for a few RCTs perfonned in Cuba, Gennany and ltaly. The data
are often very promising but not scientifically documented . I believe that if we leave
ozonetherapy indefinitely in limbo, we will do a disservice to Medicine, patients and
ourselves. Therefore, in order to dissipate prejudices and critically discuss new ideas,
we must get on with serious basic and clinical investigations (Kaptchuk, 1998).
The fifth revision of the Dec1aration of Helsinki is very welcome and it gives us
the possibility of objectively deciding what is the best strategy regarding the
controversial problem of the placebo effect in our field (Christie, 2000 ; Simon,
2000) . Article 11.3 of the Declaration cannot be taken literally because it is not
consistent with the ethics of medical experimentation. As often happens , the
pendulum has swung too far in the opposite direction. Two crucial considerations
have been identified and must be obligatory: respect for patient autonomy and
beneficence on the part of the physician. I cannot agree more with the World
Medical Association (WMA) when it wams phannaceutical companies and research
organizations throughout the world (hence, also Societies of ozonetherapy) against
exploiting patients, particularly poor ones, by using them to test new treatments
from which they will never benefit. The new declaration emphasizes our duties
towards patients who have decided to undergo ozonetherapy. Against the wrong
idea of many ozonetherapists that the study of placebo is immoral, I emphasize
that ozonetherapy is an experimental approach not yet approved as an effective
medical treatment. We must offer and discuss with the patient, possibly
accompanied by relatives and an independent physician, all the best
therapeutic options and we must clarify all the pros and cons of ozonetherapy.
Once the patient feels well infonned, he may decide without coercion to sign an
infonned consent to enter a randomized c1inical trial. The ozonetherapist leading the
research must not have any financial interest and he must dec1are any possible
financial or other conflict of interest. Furthennore, the revised declaration calls for
the testing of any new treatment to be compared against the best current method
where it exists or is available, and NOT against a placebo. Obviously, this does not
strictly apply to ozonetherapy because official medicine has not acknowledged its
 PLACEBO EFFECT IN OZONETHERAPY 329

validity . Therefore, cornmon sense suggests the schedulihg of three arms for an
ozonetherapy RCT : one testing O2-03 , another testing O 2 alone and another testing
the best current medical treatment (or using a suitabie historical control). I believe
that if we reaDy want to identify the role of ozone, we must check what oxygen
alone (representing 95-98% of the gas mixture) plus the stress of treating blood
ex vivo are able to ac hieve. Obviously this can only be done for diseases in which the
patient's life is not at risk: for example, we should not include IV stage POAD . In any
case, the protocol must clearly specify that, once the study is completed, control
patients have the right to receive the best treatment identified by the study, free of cost.
 CHAPTER26

OZONETHERAPHY IN DENTISTRY

The oral cavity nonnally hosts some 20 g of comrnensal bacteria, which are weil
kept in check by the MALT . However, they can became pathogenic and are mostly
responsible for dental decay. As reported in Chapter I, Dr. E. Fisch (1899-1966) is
considered the first dentist to use ozone in his practice and to have shown to Dr. E.
Payr ( 187 1- 1946) the potent disinfectant activity of ozone . After a couple of
discussions with dentists, it has become c1ear that they have a vast annamentarium
to fight oral and dental infections. Nonetheless, since 1995 in Gennany, Filippi and
Kirschner have used ozonized water under pressure as a spray during dental
treatment and surgical operations. Obviously, one needs an ozone generator and a
reservoir of bidistilled water to freshly prepare ozonized water throughout the day.
Dr. Filippi is enthusiastic about this old-new possibility and has often asked me why
ozonized water works so weil. Obviously, the jet of water removes all purulent
material and disinfects the area. The ozone probably activates the local circulation
and may stimulate the production of the usual cytokines, promoting the healing
process . Indeed Filippi , at the 151h World Congress (IOA, 200 I), reported that the
application of ozonized water in the oral cavity significantly accelerated wound
healing in comparison to placebo treatment.
However, direct use of the gas is prohibited because one must never breathe
ozone , although now a new invention has circum vented the problem .
In aseries ofpapers, Prof. E. Lynch's group (Baysan et al., 7000; Baysan A. and
Lynch E., "Management ofroot caries using a novel ozone delivery system in vivo",
subrnitted for publication) has shown that primary root carious lesions (PRCLs) can
be successfully treated with a novel ozone delivery system able to avoid any toxie
risk. The system includes a source of ozone and a dental handpiece with a
removable silicon cup for exposing the tooth's lesion to the gas. Escape of ozone is
prevented by the tightly fitting cup including a resilient edge for sealing the edge of
the cup against the selected area on the tooth. The tooth's lesion is exposed to ozone
for aperiod of 10 sec sufficient to kill all micro-organisms in the PRCL.
Interestingly 60% of PRCLs become hard after ozone application supporting the
observation that the use of an oxidant on root dentine carious lesions improves
rernineralisation.
In conclusion this new treatment regime using ozone appears extremely
effective , without any side effects and can be considered a valid alternative to
conventional "drilling and filling" for the management of PRCLs . It will be
interesting to follow this revolutionary and promising lead in the near future.

331
 CHAPTER27

OZONETHERAPHY IN COSMETOLOGY

It seems ironical that, although ozonetherapy may eventually be accepted and used
in important pathologies, in Italy it is mostly known for its application in
cosmetology. This is due to the myopic and selfish vision of a few ozonetherapists,
who have caused this approach to be discredited. This trend has been favoured
during the last decade by the continuous opening of new beauty centres , making
large profits . It is sad to think that, while every day in the world 600 million people
are starving, in the so-called developed countries a huge amount of money is being
spent to delay skin ageing or mask small imperfections.
There are two problems that mainly afflict women and that require the attention
of most ozonetherapists: one is the constantly increasing obesity and, particularly for
aesthetic reasons , localised lipomatosis; the second is chronic panniculitis. The first
problem can easily be prevented, in most cases, with an appropriate diet and healthy
lifestyle . However, multiple symmetric lipomatosis is areal disease, found mainly in
men. It is characterized by the formation of multiple lipomas, primarily present in
the nape of the neck (Madelung collar) and in the supraclavicular, deltoid and
abdominal regions . However, most women worry about localized layers of fat
around the pelvis and on the thighs (steatopygic Venus). This excess of fat can now
be removed in aesthetic medical centres by several techniques: surgery , but more
frequently liposuction, carboxytherapy and ozonetherapy.
There is no doubt that ozone acts efficientiy as a lipolytic agent .
In Chapter 13, we mentioned that once ozone is dissolved in the interstitial water,
lipids are the preferential substrate for attack; they are broken down to a number of
derivatives, such as lipoperoxides, hydroperoxides and small molecular weight LOPs.
The methodology is simple : injections of 5-10 ml O 2-0 3 (0 3 concentrations must
range from 2-3 to a maximum of 7-8 ug/ml) per site (abdomen, thighs, hips and
gluteal areas) are carried out subcutaneously in the various areas as a mosaic , once a
week. Five-eight sessions are generally sufficient to markedly and homogeneously
dissolve the excessive fat. Using a disposable ozone-resistant (polypropylene,
siliconated) 50 ml syringe, the gas can be applied in 5-10 sites at a time . Practical
needles are the 26-27 G x 12 mm. During each session, no more than 100 or 200 rnl
(20 or 40 sites respectively) may be injected very slowly and with extreme care to
avoid the risk of embolization. Side effects may include a transitory slight burning
sensation at the site of injection and occasional ecchymosis. After the treatment, the
patient must rest for about 20 rnin and a gentle massage may relieve possible pain .
The death of a 30 year-old women occurred three years aga in Italy, apparently after
receiving too many SC injections, for a volume of 600 rnI! As usual, it seems that
the ozonetherapist was ill-prepared for the job.

333
334 CHAPTER27

The total dose of ozone ranges from 200-2000 ug and does not elicit any toxicity;
indeed it may give a sense ofwellness. However, this aspect has not been evaluated . We
have very successfully treated two male Madelung disease patients using the EBOO
approach (Di Paolo et al., 2000). In Chapter 24, I mentioned that a complication during
HAART (due to protease inhibitors) is the appearance oflipodystrophy, and this may be
a rational reason to use ozonetherapy in addition to HAART.
There are several types of pathological panniculitis. I would say that the least
pathologieal is the chronie type, which today worries so many women who wish to
remain sexually desirable. The etiopathogenesis remains unclear but hereditary
factors, an excessively fat-rich diet, a sedentary life and smoking combine to
pro duce an ugly cutaneous appearance (like an orange peel) on the thighs, hips and
gluteal areas. It may start as a rnicrovascular disturbance that slowly induces an
uneven fibrosclerotic process, with intercellular oedema, frequent venous ectasis,
occasional microhaemorrhages and abnormal lipocytes. It can be defined as an
oedematous-fibro-sclerotic panniculitis (OFSP), according to Agostini and Agostini
(1994) . The skin is no Ionger smooth and the patient may report slight pa in during
palpation. It is really nothing very serious, but the ugly appearance of the skin
pro duces patients, i.e. bread and butter for many ozonetherapists.
Ozonetherapy is performed with 20-40 SC injections of 10-5 ml gas each,
respectively, for a total gas volume of 200 ml once a week for 5-8 weeks.
Depending on the stage of the panniculitis, the ozone concentration has been
differentiated as : tough-type - 2 ug /ml; soft-type - 1.5-2 .6 ug/ml ; oedematous-type -
3-4 ug /ml . However, I realty must laugh at the finesse of these details, because I
seriously doubt that these cosmetologists have such precise ozone gene rators to
select these concentrations. Most of them use portable generators of a firm that
produces very poor quality apparatuses; they lack a photometrie control and, even
w;..m new, produce very imprecise ozone concentrations. Every year at our course
on ozonetherapy, several ozonetherapists come with their portable generators to
check the real concentration on the basis of the iodometrie method. Luckily, we
always find rar lower ozone concentrations than expected : 1-2 instead of 20 and
17-19 instead of 70 ug/ml! I always tell them a true story: several years ago, after a
lecture in which I had pointed out the serious problern of unreliability of ozone
generators, one famous ozonetherapist working in Milan looked very worried. In a
very reserved way, he asked me what might be the reason why , during the last year,
he injected the gas as usual in many women but with no success at alt . So r asked
hirn : when did you last check your instrument? He said: I have never checked it!
This means simply, I replied, that your generator does not produce ozone any longer
and you inject only oxygen or air. He thanked me very profusely saying that r had
saved his workjust in time.
r have often said that ozonetherapy is vexed by several problems: the serious
control and maintenance of generators is a crucial one and, only recently after
several warnings, some ozonetherapists have become aware of this (Chapter 6 and
7) . Health authorities do not understand and care about this problem either.
Moreover, poor quality generators easily undergo corrosion and , if air mixes with
oxygen, they may produce a very toxie mixture containing NOx .
 OZONETHERAPY IN COSMETOLOGY 335

Coming back to the treatment, I insist that gas injections must be done very
slowly with Iittle pressure, taking care not to be inside a vein to avoid embolization.
Always for cosmetic reasons, small superficial telangiectasis can be sclerotized
by first blocking the blood flow and then slowly injecting 1-3 m1 of gas (at high 0 3
concentration : 80 ug/ml), remaining still for 30-60 sec. A compressive bandage
must be left for one day. Almost needless to add, for the topical treatment of these
unaesthetic features, there are many products prepared as gel or cream containing
either ozonized oil or other substances, which are fairly effective and quite
expensive.
 CHAPTER28

OZONETHERAPHY IN VETERINARY MEDICINE

There is a constantly increasing interest in the use of ozone by veterinarians,

especially for:

a) general disinfection of industrial facilities for animaI slaughtering , collection of milk

and production of cheese. As a disinfectant, chlorine is praetically as effective as
ozone, but it leaves derivatives and an unpleasant smell (Kawamura et al., 1986);
b) disinfeetion of waterworks in cattle-horse-sheep-pig-chicken-rabbit breeding
facilities, for drinking water, water for washing equipment and animals (when
necessary) and, most importantly, for the appropriate treatment of waste water. This
is a real problem affecting many countries (even the USA) because contaminated
water and anima! excrement will sooner or later pollute the groundwater.
c) therapy for pet dogs, eats , monkeys and commercially valuable animals, such as
race horses and pedigree cows . This ehapter will deal with this aspeet.

After speaking with several veterinarians, I have eome to the eonclusion that ,
pro vided the ozone is used judiciously, ozonetherapy is very useful ; it effeetively
resolves chronie and often very difficult problems, with no toxieity and in a shorter
time than eon ventional therapy.
Regarding the routes of administration, IV and IA are very rarely used or, at
most, only a few ml ofgas are injeeted very slowly. IM and SC routes are frequently
used during various infeetions, the ozone coneentration never exeeeding 15 ug/ml.
10 ml of gas can be injected per site .
Reetal insufflation, via a 20-30 em long polyethylene eatheter, is often earried
out during intestinal and systemic infeetions in pet animals beeause it is easy to
perform and not painful. The gas volume may range from 25 to 600 ml depending
on the size of the animal, while the 0 3eoneentration ranges from 20 to 35 ug/ml ,
Minor 0 3-AHT is also frequently performed in pet animals: 3-5 ml blood are
exposed to 3-5 m1 gas (0 3 concentration 40-80 ug/ml) and promptly reinjected IM.
For major 0 rAHT, as in humans a volume ofblood corresponding to 1120-1125 of
the whole mass is collected from a superficial vein in Na Citrate 3.8% (1.5 ml every
9 ml). It is then mixed, without bubbling, with an equal gas volume (ozone
concentrations : eats and dogs, 10-40 ug/rnl; sheep and pigs, 20-50 ug/ml; horses and
cows , about 1.5 L of blood + 1.5 L of gas, 0 3 concentration 20-50 ug/ml), It is
suggested to always use the up-dosing system.
Intra- or peri-artieular injections: 1-3 ml of gas (0 3 concentration 20 ug/rnl)
possibl y preceded byflushing with ozonized water. After the gas, 1 ml of sterile

337
338 CHAPTER28

ozonized oil can be injected. These administrations are perforrned for infectious
arthritis and coxo-femoral arthrosis in dogs.
Intra-Iesional (abscesses, superficial tumours, osteomyelitis, cysts) gas in;ection :
initially at a high ozone concentration (60-80 ug/ml) followed by progressively
lower concentrations (40-10 ug/ml) . If necessary, the lesion can be washed with
ozonized water.
Bagging, for cutaneous infections (pyoderrnitis, decubitus, chronic wounds and
ulcers, mycosis, etc.): the animal is placed inside a robust polyethylene bag
containing some water, with the head weil insulated outside. Depending on the
seriousness of the illness , the ozone concentration may vary from 60 to 20 ug/ml for
15-20 min. Bagging must be repeated for several days until the infection recedes .
Intravaginal and endometrial (vaginitis, endometritis) administration with ozonized
water followed by gas (0) concentration 20 ug/ml) and sterile ozonized oil.
Intra-urethral and bladder infections in cats and dogs are treated with gentle
washing with ozonized water, insufflation of gas at an ozone concentration of 15
ug/ml (volume 20-50 ml).
Intramammary injection: gas can be slowly injected at a low concentration (10-
20 ug/rnl) followed by sterile ozonized oil (Silva et al., 1999) .
In conclusion, ozone doses and schedules are generally similar to those used in
human patients.
What is the daily activity of a veterinarian using ozonetherapy? It may start with
a few operations to sterilize cats and dogs . Obviously under aseptic conditions, the
use of freshly ozonized water (20 ug/rnl) and the moistening of surgical wounds
with sterile ozonized oil prevents infections and allows rapid healing. Rectal
insufflation of 20-30 ml gas (0) : 20 ug/rnl) helps to wake the animal very quickly
after anaesthesia. The same strategy is applied for any operation to be perforrned in
dogs after dirty traumatic accidents. Fistulae are insufflated with ozonized water and
gas and then with ozonized oil, and they heal very rapidly. Minor 03-AHT seems to
accelerate the recovery . Ticks can be removed completely if ozonized oil is applied
some 15 min before removal.
Dogs frequently arrive with an ear infection due to foreign bodies, which may
have already perforated the tympanie membrane. After removal and insufflation of
ozone (20 ug/ml), it is useful to instil some ozonized oil. A similar tactic is used for
eye infections, and a few drops of oil also help to eure the infection. Ozonized oil for
animal use must have a low peroxide concentration to avoid irritation and pain .
Operations in valuable large animals, and perhaps also in a zoo, are rare but do take
place . Scrollavezza et al. (1997a) have found that major OrAHT (1-1.5 L blood + 1-
1.5 L of gas with an ozone concentration of 20-50 ug/ml) perforrned the day before
the operation, just before anaesthesia, and then for 4-5 consecutive days is a
tremendous help for the quick recovery of horses. Indeed this animal is notoriously
at high risk, particularly during anaesthesia. The same strategy is used in cows for
post-partum paralysis (Scrollavezza et al., 1997b; Silva et al., 1999). Post-partum
endometritis can be advantageously treated with gentle insufflation of ozonized
water and application of a couple of pessaries containing ozon ized oil.
 OZONETHERAPY IN VETERINARY MEDlCINE 339

Finally, I must mention that as soon as it was leamed that O]-AHT, or even RI
with ozone, stimulates the competitive performance of horses and dogs, someone
began to dope these animals . I hope that this is not true but, as I will briefly discuss
in the next chapter , it may well be.
 CHAPTER29

OZONE AS DOPING IN ATHLETES

The problem of doping in athletes has become an almost daily issue . It is often
carried out with dangerous compounds or, in any case, with biological compounds,
such as hormones and EPO , which may improve the physical performance but
unavoidably have side effects.
I have heard rumours that some bicycle racers have used ozone. If they do, I am
convinced that they use rectal insufflation of O2-0 3 just before the competition.
However, one would need at least a portable generator and, as far as I know, in Italy
the athletes are now operating under strict contro!. RI of Or03 would be
undetectable and it could represent a fairly good support. Yet, in my opinion, it is a
form of doping and must be prohibited. The International Sport Association says
that
"blood doping, w'hich is the administration of blood, red blood cells, artificial oxygen
carriers and related blood products to an athlete including the use ofEPO, is prohibited."

There have been a few studies in athletes to check if ozonetherapy really improves
the physical performance: one by Jakl in Vienna (reported by Viebahn, 1999) and the
other by Gionovich et al. (1995) in Italy. Both have used 03-AHT with ozone
concentrations of 30-40 ug/rnl and have concluded that ozonetherapy induces a
significant improvement ofphysical activity. I regret I could read only the two abstracts,
since the full papers have not yet been published, as often occurs in this field.
My lab is on the same floor as the "Sport Medicine Unit" and I asked the
Director if we could organise a serious study of this topic. However, he adamantly
refused to collaborate, adding that he cannot participate in any form of study
possibly related to doping. Obviously, I was not interested in the possibility of
doping anyone, only in ascertaining, by means of reliable parameters, how and if
oxygen or oxygen-ozone are able to influence oxygen transport and delivery.
As far as I can judge, some sport competitions (bicycling, marathon, tennis) are
now stretched to the extreme of physical capabilities and it has been found that an
excessive effort induces significant oxidative stress, immunosuppression and even
cardiovascular damage. I wonder if all these athletes should not be helped, in the light
of day, by professional sport physicians with suitable, weil controlled measures so that
they will not undergo an awful stress and will no longer have any reason to be doped .

341
 CHAPTER30

OZONE AS A REJUVENATING AGENT

"Ifthe degeneration ofthe senile alterations are diseases, a day will come when iI will
be possible to C-11re them. The question is certainly not whether the injections (of
aqueous testicular e:ctracts) rejuvenate, the question is 10 know ifone can approximate
Ihe strength ofa younger person , and 10 me that appears certain. "
C.E. Brown-Sequard, Physiology and Normal Pathology 1:719 (1889)

In a eonsumer society, when well-off people believe that the power of money is
almost infinite, it is unavoidable that we hope to buy extra time for our terrestrial
life. Everyone knows that the life expeetaney in Europe has inereased throughout the
last eentury from an average of 47 to about 78. The advent of vaeeines, antibioties,
vitamins, a low-fat and low-ealorie diet rieh in antioxidants (Youngman et al., 1992;
Ames et al., 1993), a regimen of moderate physieal exereise and the avoidanee of
smoking and drinking have been the main faetors in lengthening the life span and in
improving the quality of life.
There has been an inereasing knowledge of the ageing proeess and we have
beeome aware that chronie oxidative stress, the formation of advaneed glyeosylation
end substanees (AG ES), shortening of telomeres, ehronie exposure to pollutants, a
stressful lifestyle and the physiologie decline of circulating hormones are alt factors
that, to different extents, playa role in ending life.
Ouring the last three deeades , the theory that hormonal decline may be an
important cause of ageing has gathered momenturn, with the postulation that
hormonal replaeement may result in a rejuvenating process (Seeman and Robbins,
1994). Thus numerous hormones have been proposed and variably tested : estrogen,
which produces numerous benefits in post-menopausal women (Grady et al., 1992;
Peterson, 1998); growth hormone (Rudman et a1., 1990); dehydroepiandrosterone
(OHEA) and OHEA-sulphate, a sort of mother steroid (Bilger, 1995; Baulieu and
Robel, 1998); melatonin (Reiter, 1991) and, last but not least, testosterone for
androgen defieieney (Morley and Perry III, 2000) in ageing men.
Many experiments have been eondueted in rodents, frequently using very high
doses of hormones. However, it remains unclear whether the results obtained in
these non-primate models ean be extrapolated to human beings, also beeause rodents
often have a different hormonal pattern frorn man . Several studies in humans have
shown benefieial aetions of some hormones : prevention of osteoporosis,
improvement of memory and of the HOLILOL ratio due to estrogen; inereased
energy and sex drive during testosterone replaeement therapy; an apparent
improvement of mental aetivities after OHEA, promoted to the role of a
neurosteroid. Nevertheless, improvement of the quality of life is not a eonsistent
finding and many quest ions remain to be explored, mostly beeause long-term

343
344 CHAPTER30

therapy may be associated with serious adverse effects. Another problem is that, in
order to achieve striking results, enthusiastic c1inicians tend to administer
pharmacological doses of a single hormone, thus possibly disrupting the
physiological equilibrium with unforeseeable consequences. Indeed it remains
unclear what is the optimal method of hormone replacement, although slow-release
patches and creams are probably better than oral administration or injection. Without
minimizing the importance of this approach, I must conclude that we have not yet
reached the stage of an equilibrated and optimised exogenous therapy , which is
conceptually difficult to individualize.
The justification of this prologue can be found in the following question : is there
any possibility of inducing a harmonious release of hormones and how might this be
achieved? Throughout the book , I have reported (Chapter 16 and 25) that most
patients report a feeling of euphoria and a sense of wellness after ozonetherapy. Is
this simply due to faith in this medical treatment (the power of the mindl), or are the
generated messengers actually able to modify the secretion and release of several
hormones? We ought to have answered this question a long time ago; indeed it would
not be too difficult to evaluate, before and after 0rAHT, the complete hormonal
pattern and cycling in the plasma throughout the day . This study would be very
enlightening and might help to understand why the patient feels better after OJ-AHT
and to identify the best time of the day to perform it. On the other hand I never heard
saying that ozonetherapy consistently improves sexual desire and performance and this
is surprising because one of the most powerful hook for catching media attention is
sex . I can only report that a few vasculopathic (III and IV stage) patients informed us
that, after a few EBOO treatments, they noticed areturn of early morn ing penile
erection. This may be due to improved oxygenation or/and enhanced DHEA
secrection.
Another thing that has always puzzled me is why and how ozonetherapy relieves
pain . Is it able to enhance the effects of some endogenous neurotransmitters such as
serotonin and dopamine, similar to the effects of endorphins observed after intense
physical exercise (Viru and Tendzegolskis, 1995)?
1t has been postulated (Chapter 22) that ozonetherapy can paradoxicall y
strengthen the antioxidant defences against a transitory and controlled oxidati ve
stress. The exciting possibility is that, by performing two brief cycles (6-8
treatments per cycle) of ozonetherapy each year (around March and October), we
may be able to delay ageing. Low doses of ozone should be used for either OJ-AHT
(15-30 ug/ml) or RI (5-20 ug/rnl) or BOEX (0 .2-1 ug/rnl) .
We have noted that ageing is a multifactorial process and consequently
administration of a single hormone, while temporarily beneficial , is unlikely to be
useful in the long run . Longevity, and even better "longevityfreefrom disability and
fun ctional dependence" as Hayflick (2000) has written, may be more rationally
achieved by the yearly repetition of agentie, yet paradoxical, treatment Iike
ozonetherapy, which is probably able to simultaneously reactivate several functions ,
such as antioxidant defences, T-cell mediated functions, the network of enzyme
repair, a sustained and balanced hormonal release, with the inherent benefits of more
energy, improved mood and memory, prevention of cancer and atherosclerosis, and
retention of sexual activity . However, I refuse to dream that ozone will represent the
 OZONETHERAPY AS A REJUVENATING AGENT 345

etemal fountain of youth (as hoped for melatonin) nor that it will prolong the life-
span by some 15-20 years so as to have an extra decade of a good and productive
life . After all , the earth already hosts 6 billion people and it is far better to give space
and opportunity to young people rather than to maintain too many almost
mummified centenarians.
 CHAPTER31

OZONE AS A DRINKING WATER DISINFECTANT

The antiputrefactive activity of chlorine has been known since 1774 and this
halogenous gas is still in use, although its toxicity and other drawbacks now limit its
application as a water disinfectant. It has been used as agas, or as a calcium
hypochlorite, or as chloramines, but in any case the "active" chlorine is represented by
a mixture of HOCI and OCr. HOCl is a powerful oxidizing agent that can react with
thiol groups and thioethers (cysteine and methionine), haeme proteins, nucleotides,
DNA, PUFAs and cholesterol. It is weil known that HOCI produced by the
myeloperoxidase (an enzyme present in phagocytesj-Hjöj-Cl' system is responsible
for killing a wide range of pathogens in vivo. The in vitro bactericidal activity of
HOCl is conditioned by various factors, such as pH, the excessive presence of organic
materials, metals, etc.; although it is effective, several chlorinated compounds remain,
which have unsatisfactory organoleptic characteristics .
Ozone is now substituting chlorine as a potent drinking water disinfectant able to
inactivate several human pathogens, e.g. as many as 63 different bacteria
(Salmonella , Shigella, Vibrio, Campylobacter jejuni, Yersinia enterocolitica,
Legionella, etc.), some 15 viruses (polio- , echo-, Coxsackie viruses, etc.), some 25
fungi and mould spores (Aspergillus, Penicillium , Trichoderma , etc.), several yeast
varieties, and up to 13 fungal pathogens (Altemaria, Monilinia, Rhizopus, etc.) .
More recently, due to contamination of groundwater with faecal material, the
problem of disinfection has become more complex, since encysted protozoa, such as
Giardia lamblia, Cryptosporidium parvum oocysts and helminth eggs (Ascaris suum
and Ascaris lumbricoides), require a much Ionger time of contact with ozone than
bacteria and viruses. Every year Cryptosporidium causes outbreaks of sickness,
which can be fatal for elderly and very ill patients (AIDS).
Water is rapidly becoming a precious commodity and wastewater from cities,
animal breeding (particularly cattle, sheep, swine) and industrial plants must be
reused for irrigation in order to increase agricultural production. This happens most
frequently in underdeveloped countries, but also in the USA and Italy, and poses a
health risk by causing serious gastro-intestinal diseases (Stein and Schwartzbrod,
1990; Ayres et al., 1992; Johnson et al., 1998; Orta de Velasquez et al., 2001) . Toze
(1999) has reported that, in countries with poor sanitation systems, about 250
million people are infected each year by waterbome pathogens, with about 10
million deaths. The oxidation of organic and inorganic materials during ozonization
(gas to water phaser'occurs via a combination of molecular ozone and OH·. Water
companies throughout the world are evaluating several methods to optimize the
various steps of the water-treatment process, which varies in different countries
depending on the quality of the water (concentration of organic matter, turbidity, salt

347
348 CHAPTER 31

content) (Kadokawa et al., 2001 ; Evans et al., 2001; Courbat et al., 2001 ; Hijnen et al.,
2001). Ozone appears very effective in inactivating most bacteria and viruses, while
protozoan cysts and helminth eggs are far more resistant; only by using realistic
ozonization conditions can one achieve a moderate degree ofinactivation (Graham and
Paraskeva, 2001; Lewin et al., 2001). This is an important problem that requires more
intensive sanitation ofwastewater, particularly from animal breeding.
Another aspect for prevention of outbreaks of intestinal infections is the
possibility of using ozone as an antimicrobial agent in direct contact with food and
fruits . On June 26, 2001, the V.S. Food and Drug Administration (FDA)
formally approved the use of ozone, in the gaseous and aqueous phase, as an
antimicrobial agent for the treatment, storage and processing of foods . This is
good news and raises the hope that, as soon as we can provide reliable data on the
c1inical benefit of ozonetherapy, this approach will be assimilated into orthodox
Medicine. It must be mentioned that, in addition to the dis infection of drinking
water, the use of ozone can also improve its organoleptic properties. In fact , it
enhances the eoaguIation and flocculation process, oxidizes bad taste and odour
compounds (as weil as iron and manganese), and improves particle removal in filters
or through bioactive granular activated carbon. The effieacy of ozone has now been
validated by more than 3,000 municipal water treatment plants around the world .
In his keynote speech at the 15th World Congress of the IOA (September 11-15,
2001, London) entitled "Century 21 - pregnant with ozone", Dr. R.G. Rice pointed out
that, besides the classical applications for ozone, there are a great many more uses, those
in agrieulture, food proeessing and medieal therapy being very active and promising.
 CHAPTER 32

OZONE DISINFECTION
TO PREVENT NOSOCOMIAL INFECTIONS

During the last decade, the resistance of pathogens to antibiotics has increased to a
point where we no longer have an effective drug for some strains . This is a complex
story, partly due to the extensive use of antibiotics in animal food and the improper
use in patients. The result is dramatic because almost every month, we hear of a
series of deaths due to incontrollable infections breaking out in hospitals after more
or less complex operations and in intensive therapy units. With some approximation,
it seems that several thousand deaths could be avoided each year if we could
eliminate the resistant bacteria. The problem is so important that some 1000 papers
per year report relevant data (Aitken and Jeffries, 2001; Guerrero et al., 2001; Kollef
and Fraser, 2001; Olsen et al., 2001; Shiomori et al., 2001; Slonim and Singh, 2001;
Stephan et al., 2001; Stover et al., 200 I; Wenzel and Edmond, 2001) .
At the end of the previous chapter, I mentioned that Rice (2001) had reported
new applications for ozone, to which most bacteria are unable to become resistant.
Applications for ozone can be divided into two phases :

• the gas to gas phase,

• the gas to water phase (liquid phase-ozone).

The Gas to Gas Phase

The first phase is widely used to remove as many as 272 organic odours and
pollutants: these range from acrolein to bathroom smells, body odours, cigarette
smoke , decaying substances, ether, exhaust fumes, faecal and female odours,
hospital odours, medicinal odours , mould, putrefying substances, sewer odours,
toilet odours, waste products, etc. Ozone is proficiently used in hospital wards and
nursing hornes to get rid of the smells caused by incontinent patients. In air
conditioning systems (cooling towers, etc.), a small amount of ozone rids the
recirculating air of odours, bacteria (Legionella pneumophila, etc.) and viruses.
Moreover, ozone is providential for fumigation ofbedding, bedclothes and treatment
of air in operating rooms . Ozone is effective but it is necessary to take precautions:

a) to allow enough time, even days if necessary, for the ozone gas (which is less
active and slower than aqueous solubilized ozone) to be in contact with the
contaminants to be oxidized and destroyed;

349
350 CHAPTER32

b) when confined spaces are treated with gaseous ozone, people must not be present.
The ozone generator must be regulated by a timer, which can be operated by every
user . Ozone release must stop weil before people re-enter the facility;
c) prior to returning the air mixed with ozone into the atrnosphere, the gas mixture
must pass through an ozone destructor. Personnel can usually re-enter an area
treated with ozone, after appropriate de-aeration, after a short while ;
d) to prevent lung toxicity, an ozone monitor must be instalIed to check for any
residual ozone concentration.

Ozone fumigation ofbedding, bedclothes and any other object can be carried out
according to the instructions given by Inui and Ichiyanaghi (200 I). Ozone is used in
conjunction with a negative ion generator and, if necessary, a heater to control mites
and ticks.
Several pharmaceutical firms in the USA have recently started to package
pharmaceutical products in an ozone-containing atmosphere to maintain a sterile
packaged product line .

The Gas to Water Phase (Liquid Phase-Ozone)

Uses involving liquid phase-ozone have been adopted in the USA by a number of
laundries to effectively launder and sterilize various linens used in health care
facilities . It seems that, although this process is not energy efficient, it does extend
linen life by 25-50%1. Moreover, ozone washing provides a good alternative to
conventional linen processing, since it is more effective in preserving the
environment from contaminated water . All these innovative technologies increase
health care costs, but the quality of service is improved and, more importantly,
nosocomial infections can be minimized.
A full report informing about how to improve safety in hospitals can be found online
at http:// www.ahrq.gov/making health care safer: a critical analysis ofpatient safety.
 CHAPTER 33

CAN OZONE BE USEFUL FOR BANKED BLOOD

OR FRESH FROZEN PLASMA?

"Blood transfusion is like marriage; it should not be entered upon lightly , unadvisedly
01' wantonly, 01' more often than ts absolutely necessary"
Seal ,l976

There are a few problems involved with this question. The first is to examine whether
ozonization of blood, blood fractions or plasma is able to further reduce the risk of
infection. Although it is unlikely that zero-risk transfusion will ever be achieved, the
safety of blood has been greatly improved by carefully checking eligible blood donors
at various levels and then applying sensitive and precise screening tests. The
achievement has been quite remarkable in the USA (Schreiber et al., 1996; Glynn et
al., 2000) but there remains the risk of infectious blood collected during the window
period (Ling et al., 2000). Moreover, two-thirds ofthe countries throughout the world,
in which the number of infected donors is significant, do not yet have appropriate
systems to ensure a safe blood supply (WHO, 2000).
Ozone is a very good disinfectant but, being a potent oxidant, may damage plasma
protein components and blood cells. It remains doubtful that we can ozonize whole
blood to the extent of destroying pathogenic agents without affecting the function of
cells, particularly after a storage period. There is one report (by Mattassi, 1985) that, to
prevent the transmission of HBV, Wehrli (in 1957) treated 10,000 blood donations
with ozone (unknown concentration and dose) and did not record any infection. For
many reasons, this result is superficial and hard to believe unless, by a stroke of luck,
the ozone inactivated virus particles in the plasma and made then immunogenic,
creating a sort of vaccine. There are also a number of patents in this regard. The one
by Y.c. Zee and D.C. Bolton (U.S. patent no. 4,632,980, December 30, 1986) does
not, in my opinion, give the assurance of absolute sterilization of blood according to
today's standards. A second one by R. Schmitthaeusler (Eur. pat. appl. 0261032,
March 23, 1988) regards either a resuspended cryoprecipitate containing fibrinogen,
fibronectin, factor XIII, some albumin and IgG or a sampie of fresh plasma. This
patent, after appropriate checking, may be taken into consideration.
As far as blood is concemed, it may be worthwhile re-examining the problem
after LD. This important step, enforced in the UK since November 1999
(Williamson, 2000), offers many benefits : it removes leukocyte-associated viruses
and bacteria, and it avoids TRAU, possible HLA alloimmunization and
immunomodulation, and the release of cytokines, which (with traces of LPS) may
cause febrile reactions . It has taken several decades to understand and remove
the prejudice that blood, as the "gift of Iife", should not be touched. Yet it is

351
352 CHAPTER33

now c1ear that carefully and freshly leukocyte-depleted blood has a far higher
therapeutic index and safety than the original sampIe. Thus the remaining
erythrocytes and plasma could undergo careful ozonization in a glass (ozone-
resistant) container and perhaps then we may able to achieve total viral inactivation .
The addition of antioxidants (GSH and AR) may restore anormal TAS and improve
storage (Dumaswala et a1. , 1999, 2000). Obviously, it will be necessary to examine
several virological markers, the activity of protein components, and biochemical
markers of erythrocytes after ozonization and subsequent storage.
A second intriguing problem concems the possibility that ozonization may
rapidly restore a suitable 2,3-DPG concentration in stored erythrocytes before
transfusion. 2,3-DPG levels decrease more rapidly in erythrocyte concentrates than
in plasma, but they are almost nil after either 12 or 27 days storage at 4°C,
respectively. After 20 days storage, transfused erythrocytes recover their ATP , K
and 2,3-DPG content within 3-24 hours in vivo (Beutler et al., 1969; Valeri and
Hirsch, 1969; Beutler and Wood, 1969) . However, during the first few hours, their
2,3 -DPG concentration remains low and it does not allow a satisfactory oxygen
release. Thus, the issue has been raised whether ozonization prior to transfusion
accelerates the resynthesis of 2,3 -DPG to the point of representing areal c1inical
advantage. Hoffmann and Viebahn (2001) reported that ozonization (50 ug/rnl 0 3
per ml for 35 sec) of cold erythrocyte concentrate stored for 27 days , using the
microbubble flask, accelerates the 2,3-DPG synthesis by 10-30% in comparison to
contro!. It seems that an ozone concentration of 50 ug/ml is critical, since a lower
concentration is ineffective and higher ones cause extensive haemolysis. That ozone
may enhance the synthesis of 2,3-DPG remains a controversial topic, but it is
nonetheless interesting. However, in practical terms, the cost-benefit of this
additional step remains to be assessed.
A third problem of future practical importance is the viral inactivation of fresh
frozen plasma (FFP). Pamphilon (2000) precisely reviewed the current options for
viral inactivation of FFP, i.e, treatment with solvent-detergent, or methylene blue , or
psolaren S-59 and ultraviolet A light exposure. These treatments, as well as others
under development, seem to be effective and probably atoxic: the first one (S-D) is
widely used in the USA, the second one (MB) is used in Europe and the third one
(S-59-UVA) is not yet licensed. Since all of them are expensive treatments, I
wonder why ozonization of FFP has not been mentioned, not even to say that it is a
bad idea . Recently, I proposed to our Blood Centre to evaluate the pros and cons of
ozonization of LD plasma in glass containers, since ozone should not cause the
release of xenocompounds after the reaction in plastic bags . Evaluation of this
inexpensive approach will take some time but, if coagulation factors and protein
functions are not compromised, viral inactivation is likely to be optimal. In Chapter
34, it will be made c1ear that ozonized FFP derived from LD blood may become a
good option to substitute ozonized blood.
 CHAPTER34

IS THERE ANY WAY TO SUBSTITUTE

OZONIZATION OF BLOOD?

Direct ozonization of blood appears to be the most effective procedure to obtain the
biological and ciinical effects of ozone. However, this requires the handling ofblood
and its reinfusion. Some physicians and nurses in infectious diseases units are often
reluctant to perform 03-AHT because they are afraid of accidentally pricking
themselves with an infected needle. In poor countries, National Health Services
cannot afford to buy the autotransfusion sets, which cost about ten dollars each . In
affluent countries, it is deplorable that some ozonetherapists do not like "to waste"
their time and thus look for a rapid procedure. For these reasons, I have often been
asked ifthere is any way to substitute OrAHT.
In 1994, I spent several months searching for a suitable solution and, after a long
screening, I found a few possibilities. The first was to ozonize physiological saline
(Oj-saline)! In fact, we tested several isotonic and isoionic solutions, with or without
glucose, but eventually realized that simple saline (NaCI:O.9%) could trap more
ozone and derivatives than any other solution. We also tested plasma expanders such
as the weil known Emagel and hydroxy ethyl starch (HES) solution. Emagel
solution could be weil ozonized but I was very concemed that the oxidation of
polypeptides could produce immunogens, which during repeated infusion could
eventually cause an anaphylactic reaction. The HES solution also gave us a bad
surprise. Initially, the idea that HES might trap 0 3 molecules seemed interesting.
Yet when we tested ozonized HES solutions with BMC cultured in vitro, we
observed a striking ozone concentration-dependent cell death . The result was
interpreted as due to cell uptake of 03-HES particies, followed by excessive
intracellular oxidation during HES degradation and ozone release. Thus, it is far
preferable to solubilize ozone in plasma (exogenous oxidation), as occurs when
blood is exposed to O 2-03.
In spite of the simplicity and rapidity of preparation, I disliked the concept of 0 3-
saline; although it could contain some H202 , a variable amount of HOCI could also
be formed. Moreover traces of Fe2+ can allow the formation of OH·. It is possible I
made the mistake of ozonizing saline with an excessive amount of ozone (80 ug/ml),
but I deemed it useful to prepare a fairly strong solution to be injected very slowly,
possibly with a very thin needle (027), in patients lacking a venous access. I tried it
on myself twice in a large vein and, in spite of considerable blood dilution, the next
day I feit a painful irritation along the venous path up to the axilla . The vein tended
to harden and I concluded that Oi-saline was somewhat caustic and could cause a
chemical phlebitis.

353
354 CHAPTER34

The following year, I visited the regional hospital in Niznhy Novgorod and I was
surprised that Russian doctors were using Oj -saline extensively and c1aiming good
results . However, they ozonized the saline with only 1.5-2 ug/rnl, i.e. with a
concentration 40-50 times lower than mine . We had a heated discussion whether 0 3-
saline could be as efficacious 03-AHT and, against my doubts, two doctors asserted
that it was . Yet the lack of comparative data and of control solutions did not allow to
reach any conclusion.
In Russia, they continue to perform Oj-saline in many hospitals, as can be noted from
their papers presented at the 2001 10A meeting in London. They remained convinced
that Oj-saline is efficacious and they agreed that the ozonization has to be very weak. I
suspect that they have weak 0 3 generators and no easy access to blood autotransfusion
sets. In any case, my firm opinion is that we should not use this procedure .
By 1997, we became sure that H202 is one of the most important ROS , as an
early ozone messenger (Bocci et al., 1998a) . This helped to remind me that a
solution of H 202 had been used by Dr . LN. Love , working in St. Louis (USA). He
published a note entitled "Hydrogen peroxide as aremedial agent" (Love, 1888). At
that time , he could only instil a diluted solution of H20 2 into the nostrils of patients
affected by diphtheria, whooping cough and tonsillitis, obtaining beneficial effects. I
believe that Dr. Love had a wonderful insight into a problem that has taken several
decades to c1arify, i.e. that phagocytes can win their battle against pathogenic
bacteria only ifthey can deliver O 2•• , H 202 , NO· and HOCI. Today, everyone knows
that the topical use of a 3.6% solution of H202 is very useful for the disinfection of
wounds. Subsequently, Dr. C.H . Farr (1993) promoted the use of IV administration
of a dilute solution of H 202 in several iIInesses, very sirnilar to those treated with
ozonetherapy. Needless to say, H 202 must be considerably diluted before contact
with blood in order to avoid dangerous oxygen embolism and damage to
endothelium. Dr . Farr is acknowledged as one of the founders of bio-oxidative
therapy, included among the complementary medical approaches by the NIH .
The precise formulation of the H 202 solution for IV administration, first
elaborated by Dr. Farr, consists of a few steps that I have simplified and improved:

1) A 15% stock solution is prepared by diluting 30% reagent grade H 202 with an
equal volume of apyrogenic, sterile bidistilled water. The sterile container is
stored in the dark at +2°C.
2) In order to prepare the final solution when needed, it is necessary to dilute 0.5 ml
of the sterile 15% H 202 solution with 250 ml of 5% sterile glucose solution. I
would like to recommend: a) to withdraw the 0.5 rn1 without the use of a metal
needle because iron (from the needle) will contaminate the solution and enhance
formation of OH·; b) to filter the 15% stock solution through a 0.22 um filter
and to directly inject 0.5 ml, via a plastic spike, into the 5% glucose solution
flask; c) to never dilute H202 into saline, to avoid the risk of HOCI formation .
My procedure eliminates one step and avoids iron contamination. Moreover, the
solution must be infused via an angiocath (plastic catheter) . The final "202
concentration is equivalent to 0.03%, is isotonic and suitable for direct slow
(2-3 hr) IV infusion. It may be worthwhile reminding physicians, who like to
 SOLUTIONS SUBSTITUTING OZONIZED BLOOD 355

make strange solutions, to avoid mixing the 0.03 % H202 solution with
antioxidants (vitamin C, GSH), amino acids, minerals, etc., to avoid negative
interference. Depending on the stage and type of disease, treatments can be
carried out daily , every other day or twice weekly.

I have been told that, for serious illnesses, Dr. Farr has slowly infused a five-fold
greater concentration (0.15%, i.e. 2.5 ml of the 15% H202 solution diluted into 250
ml of 5% glucose solution), with "excellent results". In order to avoid toxicity and to
allow adaptation to COS, I would suggest a gradual increase of the total volume
(from 125 to 250 ml) and an increase of the concentration to 0.09%, at most. I have
tried on myself two 250 rnl infusions at 0.03 % without any adverse effects, in
contrast to Oj-saline. Dr. Farr has performed IV infusions of a 0.03% solution in
very many patients affected by several diseases. Yet so far my protocol has not been
accepted by any c1inician in Italy.
The IV administration of H202 solutions in arterial and heart ischaemia and in
cancer has been reported by Ursche! Jr. (1967) . Interesting studies on the
antitumoural effects ofH 202 have been reported by Sasaki et al. (1967), Nathan and
Cohn (1981) and Symons et al. (200 I). While this approach has been widely used in
the USA, Canada and Mexico, it has not been used in Russia , Germany or Italy.
However, I believe it may be more effective and less toxic than Oj-saline.
To start with, it would be interesting to compare laboratory and c1inical results
by testing the classical OJ-AHT and the H202 solution in chronic limb ischaemia and
chronic C hepatitis. Such a study appears very difficult because to achieve c1ear
statistical significance, it may be necessary to evaluate thousands of patients . The
crucial question is: can the H20 2 solution satisfactorily substitute OJ-AHT or other
approaches using ozone?
The proposal of H202 is not senseless, particularly since we know that H202 is
one of the early ozone messengers. However, it may be less effective because late
products, like LOPs, may not be generated in vivo owing to rapid reduction of H202 .
Moreover, although certainly not presenting the same risk as direct gas infusion
(Chapter 16), direct IV infusion of H202 solution involves similar uncertainties. In
fact, infusion can never be precisely related to the venous blood flow, with the
inherent consequence that it may be either toxic or useless if antioxidants quench the
H 202 totally before it diffuses intracellularly. Nonetheless, I believe that this
approach deserves to be tested because if it works :

• Ozone generators, with all their problems and cost , would become superfluous.
Electric energy is unnecessary.
• The cost of the H202 solution is almost negligible. Preparation of the solution is
simple, weil standardized and reliable , and the solution is more stable than
ozone . Moreover, it can be transported everywhere and can be injected with a
small angiocath into any patient at horne.
• One needs reagent grade H202 (30%), sterile bidistilled water, a 5% glucose
solution , an antibacterial filter and a few plastic disposable tools . The advantage
is that the therapy can be performed in poor countries in the most remote corners
356 CHAPTER 34

of the Earth, particularly to alleviate diseases. r will do the best I can to promote
its application by the WHO, which probably has not been sufficiently informed
about it.

In 1993, Dr . Farr reported that injection of a 0.03% HzOz solution into joints and
muscles relieved pain quickly. This paradoxical result is similar to the one r
discussed after ozone injection (Chapter 24, Orthopaedic diseases). Last year, the
Ethical Committee of Siena University approved my protocol for the IM
administration of a 0.15-0 .30% solution (49-98 ,..M HzOz). Preliminary results have
shown that these concentrations are suitable (depending on the patient's reactivity)
for IM injection (5 rnI .per site) into trigger points present in paravertebral rnuscles,
as a substitute for gas injection (0 3 at 20 ug/ml) , in patients with backache. In
Chapter 24, the effect of so-ca lied "chernical acupuncture" with OZ-03 was
attributed to the local release of HzOz acting on nociceptors and eliciting the
analgesic response. I am hopeful that this study will c1arify the role of HzOz as an
"antinociceptive" drug.
If the HzOz-glucose solution is not acceptable, two possibilities remain :

a) fresh frozen plasma (FFP);

b) a lipid emulsion made ofmedium- and Iong-ehain fatty acids and phospholipids,
eurrently used for total parenteral nutrition.

After blood, FFP seems a reasonable solution because it contains all the basic
reactants preferred by the solubilized ozone. However, as blood cells are absent, the
formed HzOz will not diffuse into them and will not activate metabolie pathways ex
vivo . As noted in Chapters 13 and 14, HzOz will be reduced in a couple of minutes
after ozonization and the infused plasma will contain late ROS and LOPs and will
have a reduced T AS . It is unlikely that it will be as effective as ozonized blood. Yet
perhaps if altemated with HzOz solution, it may represent a good compromise.
However, while HzOz solution is sterile, FFP can still transmit infections, in spite of
a highly redueed risk . To enhance its validity, FFP should be obtained after strict
screening and controls and only from LD blood. Moreover, it should be subjected to
one ofthe currently used and expensive methods to ensure viral inactivation, such as
solvent-detergent or methylene blue treatment, unless the ozonization process has an
equivalent potency (Chapter 33) . 1fthis can be proved, it would be useful and reduce
the cost. Even so, there remains the problem of the availability of FFP , as it is
widely employed to obtain precious plasma components.
The final option is a lipid emulsion. There are several already employed for
parenteral nutrition. Indeed we have spent some time evaluating one, which I will
simply indicate as LE, rich in phospholipids, partly unsaturated medium and long-
chain triglycerides, glycerol and water. It is isotonic, practically ion -free and
obviously sterile. When exposed to OZ-03, ozone dissolves as usual, reacts
immediately with PUF As and forms ROS and LOPs, which by mixing with blood
during reinfusion may at least partly activate blood cells . Thus, it shows advantages
and is a promising solution. After obtaining permission from the Ethical Committee
 SOLUTIONS SUBSTITUTlNG OZONIZED BLOOD 357

and the Ministry of Health in April 1998, we conducted a preclinical study to assess the
toxicity in rabbits (manuscript in preparation). Initially, we investigated which ozone
dose (20, 40, 60, 80 ug/ml) would be most suitable for the ozonization of LE. More
recently, we examined the effect of 5, 11 and 21 treatments (within 56 days) (slow
infusion via the ear marginal vein) of LE exposed to O 2-03 or only O 2 • Results showed
that a medium ozonization (40 ug/ml of LE) markedly enhanced (in comparison to
control) the animal 's body weight (mean increase of 550 g). Haematological parameters,
TBARS, PTG and TAS plasma levels did not show abnormal variations. Histological
examinations performed at the end ofthe experimental period on many organs from each
rabbit group failed to show any pathological variations.
We are now characterizing the chemical change in composition of LE after
ozonization. This line of research is interesting and we will take a step forward if we
can use ozonized LE in patients, thus avoiding the problem of blood handling.
Moreover, we envisage the possibility of dissolving a precise volume of filtered
15% H 20 2 solution directly in the LE, thus excluding the use of ozone and extending
its therapeutic use to poor countries. This study is in progress in our laboratory
because we feel important to develop a useful possibility for patients who are not
treated today . I would like to remind that hardly 10% of the world population
receives proper medical attention and we ought to make an effort to help the
remaining majority.
 CHAPTER 35

HYPERBARIC OXYGEN THERAPY (HOT)

VERSUSOZONETHERAPY

I have realized that oxygen-ozone therapy is unknown to many physicians and they
often ask me if it is a sort of HOT. HOT is a medical procedure by which 100%
medical oxygen (Kindwall, 1993; Tibbles and Edelsberg, 1996; Leach et al., 1998)
is delivered at 2-3 times (usually 2.6) the atmospheric pressure (l atmosphere = 760
mmHg ) at sea level. In physiological conditions, at this level with normal air, the
p02 in the alveolar space (0 2:14%) is equivalent to 100 mmHg and the p02 of
arterial blood is about 98 mmHg ; Hb is fully saturated to Hb 40S and there is about
0.3 ml per decilitre of O 2 solubilized in the plasma. Tissues at rest extract from
blood an average of about 25% O 2 (i.e. 5-6 ml of 02/dL), so that venous blood has a
p02 of about 40 mmHg and Hb 40S , having released at least one molecule of O2,
becomes Hb 406 . Thus the amount of O2 physically dissolved in the plasma is grossly
insufficient for the requirements of the tissues and the necessary 5.5 ml of O2 derive
from deoxygenation of Hb40S ' In the hyperbaric chamber, administering 100% O 2 at
3 atmospheres, the O2 solubilized in plasma is as much as 6 ml/dL and the Hb is
fully saturated with O 2 • In this situation, the dissolved O2 content is suffieient to
satisfy the cellular requirements and Hb40S hardly release any O2 .
Rapid deeompression (say from 4-5 to 1-2 atmospheres) causes deeompression
sickness due to nitrogen dissolved in plasmatic water, which suddenly forms inert gas
bubbles that cause disseminated embolization. The diver can be saved if rapidly placed
in the hyperbarie ehamber, because during slow deeompression the nitrogen is
replaced by oxygen and slowly expired while the oxygen is metabolized by the tissues.
Carbon monoxide (CO) poisoning is a cause of death all over the world (Ernst
and Zibrak, 1998) due to the fact that CO binds to Hb with an affinity 240 times that
of O2. In the presence of CO, the Hb0 2 dissociation curve shifts to the left and
changes to a more hyperbolie shape , with the result of impaired release of O2 at the
tissue level, where CO also binds to myoglobin.
The hyperbaric chamber ean save the intoxieated subject by delivering O 2
dissolved in the plasma to anoxie tissues and by aecelerating the dissociation of
COHb : its half-life decreases from about 300 min while air is breathed to about 20
min with hyperbaric 100% O2. Moreover, HOT allows the dissociation of CO from
cytoehrome C oxidase, thus improving the eel1ular energy state. The immediate
administration of normobaric oxygen to a CO-intoxicated patient is certainly useful,
because the half life of CO-Hb is only about 60 min and tissue oxygenation is
improved , but it is not as effeetiv e as HOT .

359
360 CHAPTER35

On rare occasions, haemorrhagic shock may cause intensive anaemia, unable to

satisfy the metabolic demands of tissues : if suitable blood is not available or blood
transfusion is not allowed for religious reasons, HOT may temporarily compensate
for the lack of erythrocytes. These three examples suffice to iIIustrate the unique
importance of HOT.
Adverse effects are rare and partly due to typical oxygen toxicity (optic
symptoms in about 20% of patients) , which can be prevented by administration of
antioxidants and by shortening the period of hypoxia (OuBois, 1962). In addition to
the high cost of installing a HOT facility, the oxygen presents a fire hazard. Indeed,
in the last decade, there have been two tragic explosions in Italy: one in Naples in a
single-place chamber and another in Milan in a multi-place chamber with several
deaths . These accidents should never occur , as the chamber should be regularly
filled with inert air. In comparison, oxygen-ozone therapy does not present risks,
unless a mad ozonetherapist directly injects the gas IV, a procedure that is
prohibited. Moreover, the cost of the material for ozonetherapy is negligible.
There are fundamental differences between HOT and ozonetherapy. Although
the bulk ofthe gas mixture is represented by 95-99% oxygen, ozonetherapy does not
aim to oxygenate blood directly. Indeed, with all the procedures (03-AHT, EBOO,
BOEX and RI), the arterial p02 hardly increases in vivo. Yet if ozone is used
properly, it has many virtues: disinfectant and immunomodulatory (cytokine release)
activities, increased delivery of oxygen to hypoxic tissue through vasodilatation
(NO·, CO) and possibly a shift of the Hb0 2 dissociation curve to the right (the
venous p02 may fall to 20 mmHg) , release of growth factors (POOF, TOF-ß I, etc.)
thus enhancing tissue healing , possibly hormonal release due to a sudden
homeostatic change and/or a placebo effect and, most importantly, a generalized
metabolic improvement with enhancement ofthe antioxidant defence.
Another significant difference is that ozonetherapy induces fairly Iong-lasring
and interconnected metabolie changes , while the effects of HOT, being due mainly
to a transitory oxygen hyperconcentration, are of shorter duration . Interestingly,
increased ONA damage was detected immediately at the end ofthe first HOT, while
no effect was found one day later. Oennog et al. (1996) suggested that further HOT,
under the same conditions, mayaIso increase antioxidant defences . This suggestion
is now supported by interesting experimental data (Kim et al., 200 I). The finding of
significant oxidative base damage after the first HOT treatment reinforces my
conviction that ozonetherapy should always start with a very low dose followed by a
gradual increase to rninimize any possible damage.
An objective comparison of the therapeutic efficacy of HOT versus
ozonetherapy is not possible, mostly because valid RCTs of ozonetherapy are hard
to find, while there are many publieations dealing with HOT . However, even though
as many as 64 different disorders seemed to be improved with HOT, in most ofthem
the evidence to warrant its c1inical use was insufficient (Kindwall, 1993). There is
only one paper comparing rheological parameters (but not clinical efficacy) between
HOT and ozonetherapy: Verrazzo et al. (1995) c1aimed that only the latter approach
caused a significant increase of erythrocyte filterability and a decrease of blood
viscos ity. On the basis of our data, these results need to be confirmed.
 HYPERBARIC OXYGEN VERSUS OZONETHERAPY 361

In Table 26, I attempt to summarize the diseases for which either HOT or
ozonetherapy are used and to express an opinion, based on personal experience and
not on hard data, about which of the two approaches seems more beneficiaI.

Table 26. Diseases for wb ich HOT and ozonetherapy are used.

HOT OZONETHERAPY
I) Arterial gas embolism +++
2) Decompression sickness +++
3) Severe CO poisoning and smoke inhalation +++
4) Severe blood-loss anaemia +++
5) Clostridial myonecrosis (gas gangrene) +++ ?
6) Compromised skin grafts and flaps + +++
7) Prevention of osteo-radionecrosis + +++
8) Radiation damage + +++
9) Refractory osteomyelitis + +++
10) Necrotizing fascitis + +++
11) Traumatic ischaemic injury + +++
12) Thermal bums + +++
13) Chronic ulcers and failure ofwound healing + +++
14) Multiple sclerosis +?
15) Chronic fatigue syndrome + ++
16) HIV-AIDS +? +
17) Senility ++
Legend : + Iittle ++ modest +++ good activity --- no activity

It may seem that I favour ozonetherapy and the reason is that, in these affections,
ozonetherapy is really more effective. In most cases, we can apply both parenteral
administration, in the form of OJ-AHT, EBOO, BOEX and RI, and topical
application, either as OrOJ gas mixture (bagging and dynamic insufflation) or
ozonized water and oil. The combination favours an incredible synergistic efTect,
which acts on several targets. Indeed this explains the efficacy of ozonetherapy
where there are several components at work simultaneously (infection,
inflammation, cell necrosis, ischaernia, dysmetabolism, impaired healing, etc.).
Several ofthese affections have been discussed in Chapters 24 and 30 .
Bevers et al. (1995) proposed HOT (20 sessions at 100% O2 at 3 bar for 90 rnin)
for patients with severe radiation-induced haematuria. Yet Dr . R. Dall' Aglio
recently solved this problem with only three applications of ozone gas (once
weekly!). It is regrettable that in 1996 Dr. Bevers failed to accept my proposal to
conduct a comparative, controlled study.
HOT was proposed for patients with AIDS (Bocci, 1987a) and a subsequent
study showed a transitory improvement of the quality of life ("Hyperbaric Oxygen
Therapy for the Treatment of Debilitating Fatigue Associated With HIV/AIDS",
Janac, vol.4, issue 3, July-Septernber, 1993) . There is no doubt that HOT has a
precise and unique rationale in affections no. 1 to 5. In all other diseases, the use of
362 CHAPTER 35

HOT is not weil supported and the risks of transferring the patient, who often Iives
far away from the site ofthe chamber, discourage its use.
The purpose of this chapter was to clarify that ozonetherapy is very versatile,
practical, inexpensive, without side effects and quite heneficial in several affections
Iisted in Tahle 26. I would like to believe that orthodox physicians, rather than being
hiased against ozonetherapy, simply do not know about it nor how to perform the
therapy. I live with the hope that , in the future, we will ahle to help many patients
much hetter than today .
 CHAPTER36

ACUTE CARE DURING OR AFTER OZONETHERAPY

B. BIAGIOLI AND V. BOCCI

In spite of intrinsic toxicity (Chapter 5), ozone, if properly used, is not toxic and ·can be
useful. Consequently, adverse effects of ozonetherapy are rare and modest. However,
owing to a lack of control, charlatans (and not real ozonetherapists, who should be
physicians with definitive knowledge of ozonetherapy) have in the past directly injected
the gas via the IV route and killed six patients . This should never happen again because
since 1983, the European Society of Ozonetherapy has prohibited this malpraetice. It is
unfortunate that Regulatory Agencies in many countries are barely interested in
ozonetherapy and do not enforce precise regulation. Death is due to oxygen embolism
because the amount of ozone is small and dissolves and reacts rapidly with plasmatic
water. It has been amply shown that ozone can be administered via numerous routes
described in Chapter 16. Exposure of blood to O 2-03 ex vivo, as occurs in classical
autohaemotherapy or during extracorporeal circulation or with the interface of skin
(body exposure) or the rectal mucosa (rectal insuftlation), cannot lead to embolism or
other problems and one needs only to control the ozone concentration and total dose.
Modest and rare adverse effects were described in Chapter 2 I.
On the other hand, IM or SC administration of Or03 can be fatal, although rarely.
During the last three years in Italy, we recorded two deaths: one during lipodystrophy
treatment owing to an excessive volume of gas injected subcutaneously and another due
to gas injection into the paravertebral muscles in the attempt to elicit an antinociceptive
response in a patient with backache. Regrettably , we do not have the final autopsy report,
but we have been told that the latter case was likely due to a vago-vasal reflex with
cardiac arrest. We know that in Medicine any invasive procedure, even ofmodest entity,
can induce an abnormal and risky response . However, IM administration of Or03 has
the peculiarity of causing a transitory, burning pain and the ozonetherapist must be able
and ready to control it promptly. Ozonetherapy Societies, rather than increasing in
number (there are now three in ltaly!), should cooperate and establish guidelines and
instruct ozonetherapists for any emergency. One must also consider that the
ozonetherapist treating a chronic Iimb ischaernia patient must be aware of the risk that,
during treatment, the patient can develop an ictus or cardiac infarction not directly linked
to the ozone treatment but to the generalized cardiovascular pathology .
Thus, first of all, it is essential that the ozonetherapist performs a complete evaluation
of the patient. He must know the medical history and make an objective and extensive
examination of the respiratory, cardiovascular and reflexogenic activities of the patient.
By talking with hirn, he must also appreciate if he has a calm or anxious temperament
and he must be informed of any medication taken by the patient , particularly regarding
coagulation disorders, circulatory and pulmonary problems. It is weil known that sudden

363
364 CHAPTER36

death due to acute central or peripheral circulatory failure may happen at any time,
irrespective ofthe ozonetherapeutic treatment and due to a precarlous situation unknown
to the patient (Myerburg and Castellanos, 1997; Engelstein and Zipes, 1998).
Ozonetherapy has been found to be useful in neurodegenerative diseases and perhaps
also in pulmonary diseases such as emphysema and COPD . Therefore, before starting
any treatment, the ozonetherapist must consider the risk of a sudden respiratory arrest or
a worsening respiratory activity.
If oxygen transport and delivery become insufficient or absent for aperiod longer
than 4-5 min, the CNS can undergo irreversible damage and cerebral death. However, if
the ozonetherapist is prepared for this dramatic circumstance, he can promptly apply
basic life support (BLS) and save the patient. Indeed, it seems that at least some of the
recent deaths cited above could have been avoided if the ozonetherapist had the ability to
irnmediately apply BLS instead of wasting precious time calling for help that arrived
after 20-30 min.
Detailed descriptions of resuscitation guidelines (2000) are readily available and can
be consulted at either the Web site: http://www :resus.org.ukIpageslbls.htm or in
Curnmins's book (1994) and the llcor advisory statements (1997). For these, the site is
http ://www.americanheart.org/ScientifidstatementsiI 997/049705 .html.
For the sake of space, only the essential elements can be reported here: first, check
the responsiveness of the patient by gently shaking bis shoulder and asking loudly two or
three times "are you all right?". If he/she is unconscious, it is immediately necessary to
open the airway by appropriately tilting the head and lifting the chin. Then one must
look, listen or feel if the patient is breathing. Next, one must check if there is a sign of
cardiac arrest, i.e. an absent carotid pulse. Unfortunately, an inexpert physician may be
unable in 50% of cases to detect a carotid pulse and, in any event, the assessment of the
circulation must be no longer than 10 sec. If there is no sign of circulation, the reseuer
must start appropriate ehest compressions at a rate of about 100 times a minute (just a
little less than 2 compressions per second) by counting aloud. Rescue breathing and ehest
compression must be combined according to precise rules until qualified help arrives or
the patient shows signs oflife. At any time during all these phases, one must ask for help
because BLS is very exhausting.
Obviously, reading these notes does not really help and it is strongly advised to
follow a BLS course with appropriate training. The ozonetherapist in bis private clinic is
advised to repeat all the basic steps from time to time. Moreover, he must have a face
mask ready, several sizes oftracheal tubes, the Ambu, medical oxygen and possibly an
automated external defibrillator to be used if the rhythm is ventricular fibrillation or
pulseless ventricular tachycardia.
The ozonetherapist must also be ready to do 2-3 IV bolus injections of epinephrine (1
mg) diluted with saline every 3 min. The avaiJability of solution buffers, antiarrhythmics,
atropine and corticosteroids is highly recommended. In some cases of strong pain, an
injection of morphine (l0 mg) or valium is very useful.
Since our first course on ozonetherapy, we have included a full four hours dedicated
to these resuscitation guidelines, which must be supplemented by suitable training.
As we mentioned, if performed correctly, ozonetherapy per se tends not to cause
problems. However, ozonetherapists must be able to overcome any emergency.
 CHAPTER37

ORTHODOX MEDICINE VERSUS COMPLEMENTARY

MEDICINE: A CONFLICT THAT MAY BE RESOLVED
WITH APPROPRIATE RESEARCH

" Ofall the ills that suffe ring man endu res
The largest fra ction liberal nature eures ,
Of the remainlng, 'tts the smaJlest part
Yields 10the effort ofj udicious art"
Oliver Wendel! Holmes, 1892

It is already late moming but a gentle breeze mitigates the heat. Under a large
baobab, almost covering the sparse huts of a Kenyan village, lies a woman with a
desperate look on her face. Another woman, wearing a dress with splendid colours,
sits next to her talking and making large gestures so as to dispel fears and worries .
She is a highly respected healer, weIl known from Nanyuki to Maralal. Her
ancestors called her to become an "isangomas", a diviner, and she has helped and
saved many women who wanted to die. Her power to understand and care is
immense and, with her ritual, she will deliver new strength to the sad woman, who
will regain the desire to live. After a little while, a noisy jeep stops nearby in a cloud
of dust. A tall, robust black man with a white coat gets out of the car and silently
waits until the healer is ready to talk to him. He is also a weIl known doctor , who
sees his patients every two-three days and gives them different coloured pills. There
is not a shadow of contrast between the physician and the healer. She knows she has
no power to treat malaria, pneumonia or trachoma and he has no cure for mental
patients . Yet both of them care very much about suffering souls .
In Italy or in England, a GP works hard to write prescriptions and fill out forms
and he hardly has the time to say Hello! and gaze at the patient's face . Hopefully, he
knows his patient 's problem, but he can spare only eight minutes to talk and tell him
that he has to undergo a sophisticated test. After one or two weeks, the patient is
caIled for the exam at the regional hospital , but the specialist has no time to see him
because he is terribly busy pushing buttons on a very expensive apparatus, which
immodestly visualizes and describes in detail what is wrong. After another week, the
patient will go back to the hospital and will get a sealed envelope to take back to his
GP, who has little time to explain the problem. Often the patient will have to go
back to the hospital for additional exams and after further time a diagnosis will be
made. Then , perhaps, the patient needs an operation and, in such a case, a good
move would be to have a private examination by the surgeon . This may cost 200
pounds, but it is money weIl spent because now the patient has really been examined
by the surgeon, who has had time to see the colour ofhis eyes .

365
366 CHAPTER37

If the patient does not need an operation and is unhappy about taking untrusted
pills, he decides to see a famous physician, who privately practices homeopathy and
aromatherapy. At long last , he has the pleasure of seeing another human being
sitting near hirn, who kindly asks many questions, discusses the problem at length
and evaluates the pros and cons of a wonderful therapy that he will follow with eare
and faith . Ifthe disease is not too serious, the patient will soon be better and happily
will tell friends he has found a very good doctor with admirable bed-side manners.
I beg both the reader's and the physician's pardon for this Iittle story. Yet, in reality,
it summarizes the long ordeal which many patients report these days of super-
technological medicine. Unfortunately, I don't think I have exaggerated what happens
every day. With the permission of a friend of ours, Dr. Michael Alms, M.B. Ch.B
(Bristol), M.Ch.Orth (Liverpool) and FRCS (UK and Canada), now living in Vancouver,
BC, Canada, I would like to report the letter he sent to the editor of the British Columbia
Medical Journal and to us after bis wife's death some IO months ago.
Sir,
One often hears the claim that in Canada we enjoy the best healthcare system in the
world , and that we should protect it. On the other hand the British National Health
Service is sometimes criticised as an efficient but impersonal and uncaring sausage
machine. I regret to say that in its hospital services that is sometimes true. It was with
satisfaction, therefore, that nearly forty years ago I experienced the establishment in
Canada of universal insurance of private practice by the srate . As a consultant surge on
working in teaching hospitals in Saskatchewan, where our present health care system
was introduced, I feit that we had combined the best of two worlds . I have now retired
in Vancouver and am no longer a doctor but a patient , and a patient 's relative and I am
beginning to wonder what has happened to that ideal.
It is alarming to disco ver that one may wait over three months to consult a specialist, or
four months to have a CAT-scan to determine ifone is suffering a malignant proce ss. lt
is demeaning on visiting a specialist for the first time to be taken by the receptionist to a
small empty room and invited to take off one's clothes and wait for the arrival of an
unknown doctor. I have not worked in the National Health Ser vice for a long time but
not even in that impersonal scene did patients in the service that I worked in wait that
long or be treated so inconsiderately.
My wife was recently admitted to a Vancouver teaching hospital in the terminal stages
of an illness. She was presumably under the care of a consulting internist but I do not
believe that he ever saw her . She was certainly never seen by hirn in the four days after
she was admitted, for members of her family were there ever y minute of the day and
night and never met hirn. I am satisfied that she was weil cared for by the resident
physicians in training who may have been guided off-stage in the managernent of the
technical problems encountered. They might have benefited from learn ing how a trusted
physician deals with a dying patient and her worried relatives. Instead they learned how
to work the sausage machine.
One hundred years ago the doctor would have had nothing effective to offer my wife.
He could have done little more than sit by her bed, hold her hand, and comfort her . He
would have reassured and consoled us, her relatives. He might have prescribed some
useless potions. Scientific medicine has long discredited those potions but do we have
to throw out the baby with the bath-water? There is still a need for the personal touch ,
the reassurance that the doctor in charge is a friend that is on one 's side in difficult
times . There are still times when that is all that is left.

I feel that Dr. Alms' letter needs no comment, only that we must really think
seriously why an increasing number of people are interested in Complementary
Medicine. In saying this, I am in good company beeause several authoritative
 ORTHODOX VERSUS COMPLEMENTARY MEDICINE 367

experts on this topic (Astin, 1998; Eisenberg et a1., 1993, 1998; Sugarman and Burk,
1998; Ernst, 1996a, 2000; Ernst and Resch, 1996; lwu and Gbodossou, 2000) have
stated the same thing. In England, the House of Lords Select Committee on Science
and Technology (2000) has recently examined the heterogeneous world of
Complementary Medieine and has made (6th report) several recommendations to the
Govemment. The Govemment will likely consider them in tenns of voters ' opinions
but not with regard to funding badly needed RCTs.
I believe that objective basic and clinical research is fundamental in order to
demonstrate that a complementary approach is important and useful, so that
eventually it can be included in orthodox Medicine (Chapter 24).
Unfortunately, most complementary approaches are based on more or less arcane
theories that defy any scientific demonstration (at least at the present time). Only a
few approaches, such as acupuncture (Hsu and Diehl, 1998; Galloway, 2001),
oxygen-ozone therapy and phytotherapy (or herbalism), are conceptually easy to
understand and can be experimentally proved.
Throughout this book, I have tried to show that, by reacting with body fluids,
ozone induces weil known chemical reactions and that reactants, such as ROS and
LOPs, can activate a number of metabolic and immunological pathways. I am
convinced that this is a crucial advantage, which, after appropriate clinieal
investigations, will show the advantage of ozonetherapy over the frequently
reductionist approach of conventional medicine for the treatment of several diseases .
With this, I do not deny the practical significance of conventional medicine; actually
I am in favour, whenever possible, of a combined treatment.
The paradoxieal concept that ozone, one of the most potent oxidants, becomes
(with appropriate doses and schedules) a generator of antioxidant defences, and may
be able to improve and block the progress of degenerative diseases, is quite
fascinating. After three decades of medical use in over ten million people, the
often extolled toxicity of ozone in patients appears to be a colossal blunder, due
to ignorance of biological mechanisms and the body's defence capabilities and,
worst of all, to prejudice. It is time that all those who have expressed the opinion
that "ozone is toxic any way you deal with it" start to think and humbly revise their
position. I regret that Prof. Ernst, in his brief review (2000), did not include
ozonetherapy in the list. Yet it is true that at present in England, ozonetherapy is less
known than iridology and chelation therapy, which according to Ernst have yet to
produce convincing benefits.
We cannot lower our guard concerning the toxicity of any of the complementary
approaches : there is no doubt that ozone is toxic for the respiratory system and, if
used improperly, presents some risks. Acupuncture, although rarely hannful, is not
completely safe either (Ernst and White, 1997). Moreover, herbs must be carefully
controlled for their activity and possible toxie contamination (Nortier et a1., 2000;
Escher et a1. , 2001).
There remains plenty of work to do and all of us who are seriously interested in
the progress of these approaches must accelerate the pace of basic and clinical
studies, so that they may soon become a solid part of integrated Medicine (Rees and
Weil, 2001). Besides the importance of research, it will be necessary to establish
guidelines, which will have to be revised from time to time, and we will have to
368 CHAPTER37

conduct serious teaching programmes, as we actually already do. Moreover, in order

to implement and extend these approaches, it will be necessary both to publish
scientific reports in good peer-reviewed journals and to inform orthodox physicians
of our progress at pertinent meetings. In this way, we will increase the number of
patients who will take advantage of our work.
I will be very gratified if, during my lifetime, I see the introduction of
ozonetherapy into public hospitals around the world .
 CHAPTER38

DOES OZONETHERAPY HAVE A FUTURE

IN MEDICINE?

"Nihtl est verttatis luce dulcius"

Cicero (106 - 43 B.C.), Acad ., 2, 31
(Nothing is better than the truth)

In answering this question, I have mixed feelings and I would be interested to know
the reactions of the readers in the near future . Table 27 sumrnarizes the most
important problems plaguing ozonetherapy.

Table 27. Why ozonetherapy has not yet been accepted by official medicine in the west?

• Conceptual and methodological mistakes in using ozone .

• Incomplete knowledge of the biological mechanisms of action.
• Inadequacy of scientific research in the biological and clinical
fields . (Publications either as abstracts or in unknownjournals).
• Lack of standardized procedures, therapeutic dosages and schedules .
• Insufficient clinical data .
• Relevance ofthe placebo effect ?
• Emphasis of ozone toxicity .
• Lack of financial support.
• Technical and practical difficulties.

There have been unfavourable circumstances, like the tendency to monopolize

the market with poor and imprecise ozone generators, that have delayed progress
and led to unreproducible, if not negative, results. Unfortunately, ozone is a labile
gas and must be produced extempore with precise and reliable generators (Chapter
6). Our dependency on an instrument is a drawback but, at least in Italy, we have
now won the battle and today ozonetherapists can choose among three or four good
instruments delivering precise ozone concentrations, measured photometrically.
Further studies on alternative solutions delivering bioxidants may even give us more
freedom and extend the application of bioxidative therapies (Chapter 34) into
underdeveloped countries, where too many patients remain untreated. Still other
countries in Europe and the rest of the world must be alerted about the dangerous
invasion of poor instrumentation and poor techniques, which jeopardize
standardisation of the classical 03-AHT. In addition to the prohibition of toxic
plastic bags for autotransfusion (on the basis of experimental data), it is necessary to
convince all ozonetherapists to use Y2 L glass botdes and that any process leading to

369
370 CHAPTER38

foaming will damage blood . Unfortunately, deplorable commercial interests prevent

awareness ofthese problems.
However, a good ozone generator and an unprepared ozonetherapist also produce
bad ozonetherapy. Indeed, physicians who already practise or would Iike to practise
ozonetherapy must be properly trained in fairly extensive teaching courses (at least
60 hours of theory and practice during three weekends of 20 hours each) , organized
by either a university or by a public health organization. Since 1999, we have done
this at Siena University and most of the physicians attending our courses have
repeatedly thanked me for having leamt how ozone acts and not simply to push a
few buttons or to carry out a technique. So far, the Italian Ministry of Health's
disinterest and avoidance of its responsibilities regarding ozonetherapy has allowed
the prevalence of short (4-5 hours in one day) courses organized by firms, with the
obvious interest of selling their instruments and products. A couple of times, I have
been appalled to observe what is done during these so-ca lied "refresher courses" :
most of the time is spent in showing technical details of how to inject the gas in
patients with Iipodystrophy or how to carry out O}-AHT in a few minutes (!!) and in
emphasizing the performance of a particular ozone generator, which of course is
praised as the best on the market. Most of the beginner physicians are inexpert and
only eager to start making money , so they easily fall prey to the cunning salesman .
The newly promoted ozonetherapist is only interested in knowing the ozone doses
(recipes!) for the various treatments and not why and how ozone acts. Around Italy,
there are some 500 old portable generators that were unreliable even when they were
new . Thus, one cannot expect any progress, but at best only a placebo therapy .
This situation triggers an involutional process that enhances the skepticism of
orthodox physicians and perpetuates an even poorer, and only technically orientcd,
teaching of ozonetherapy. I must say that my complaints regarding the excessi ve
pervasion of a business-oriented mentality is not restricted to ozonetherapy but
extends as weil to herbalism and pharmaceutical industries, where the eagemess to
quickly make a profit often leads to intoxicated or dead patients. The justification
that money is needed to further develop research is hardly valid and, in the case of
ozonetherapy, is simply not true, since practically nothing is invested in research .
This creates another vicious circle where the results of the few, poor quality studies
cannot be published in valid scientific journals. In the recent past, reports of great
ozonetherapeutic successes have appeared in Penthouse and other non-scientific
magazines. Today, Internet allows one to advertise the existence of wonderful
centres of alternative medicine, where miraculous recoveries frorn all of mankind's
diseases can be achieved by super-holistic treatments. Once again, I must note that
the theatricality is not restricted to ozonetherapists, but extends to famous
oncologists photographed while injecting IL-2 or TNFa into a frightened cancer
patient! Even worse is the fact that unscrupulous, outlaw technicians (engineers,
electricians, etc.) not only go around the world selling their ozone equipment but
also publish booklets and teach how to do intravenous infusions of Oz-O}. It is
unbelievable that the FDA prohibits ozonetherapy carried out by real physicians but
allows far worse activities. All of this shows the degree of depravation reached by
some professional and non-professional people to "make money" and "win fame" at
the expense ofhelpless patients.
 DOES OZONETHERAPY HAVE A FUTURE IN MEDICINE? 371

Luckily there are some positive aspects. I will not bore the reader further by
describing them in detail, but I would like simply to say that :

a) we now have some ideas about how ozone works after dissolving in body fluids .
b) An increased release of oxygen in ischemic tissues with enhanced cell
metabolism, the release of autacoids, the possible activation of resident stern
cells are just a few possibilities that can be experimentally tested to explain the
c1inical results in vasculopathies.
c) Most importantly, we now know for sure that ozone used within the therapeutic
window IS NOT TOXIe.
d) We are also convinced that ozone is areal drug and it must be used with all the
relative precautions.
e) Ozone, one of the supreme oxidants, can induce upregulation of the antioxidant
defences and likely correct a chronie oxidative stress. This possibility is most
interesting and unexpected because during the last thirty years thousands of
excellent papers have provided the concept that ageing and many other human
afflictions are due to the continuous and progressive oxidative stress. While this
is perfectly true , it has somehow clouded our critical judgement leading to the
conclusion that any other stress should be avoided. Ozonetherapists, with all
their crude empiricism, have not helped to reach a realistic perception of this
therapy. I would plead with scientists and clinicians to abandon the prejudice and
consider the profound difference between the endogenous oxidative stress and
the ozonetherapeutic "shock".
f) We have scientifically developed two new techniques (EBOO and BOEX) that
are far more powernd than the old 0 3-AHT and RI. While BOEX is still under
study, EBOO has already provided important clinical results. It is even more
striking that even the ozonization of 5 L of blood in I hr does not show any
toxicity. Throughout the years, I have tried all the ozonetherapeutic procedures
on myself many times and thus I represent living proof of the lack of any
toxicity. The skeptics and those who disparage the use of medical ozone are
challenged to disprove scientifically these results.
g) We must perform RCTs in selected diseases, for which we have good evidence
of ozone's activity. In order to convince skeptics, the results must be more than
adequate and be published in peer-reviewedjournals.

However, I do not expect that the existing skepticism will fade overnight.
Authoritative scientists know all too weil that free radicals are dangerous, but ozone
induces only abrief and calculated oxidative stress that should not be confused
with all the pathologies maintained by a chronic oxidative stress.
History shows that we have to revise our ideas from time to time and that not all
dogmas have a long life in Biology and Medicine. Until 1987, we thought that
gaseous moleeules such as NO· and CO (the silent killer) were nasty molecules, but
now we know that in physiological concentrations they have crucial functions.
Indeed Perutz (1996) considered the discovery of NO to be one of the most relevant
in Physiology. Although it is unlikely that cells synthesize 0 3 (unlike what occurs in
372 CHAPTER 38

the stratosphere), why should we not think that judicious amounts of 0 3 can be as
useful as CO and NO?
I agree with everyone that ozone can be a toxic molecule. At the Verona
Congress (1999), when I moderated a round table entitled "The ozonetherapy
dilemma", I tried to play down the contrast between OPPONENTS and
PROPONENTS by comparing ozone to the ambivalent character described by
Robert Louis Stevenson in his novel of 1866 "The Strange Case of Dr. Jekylt and
MI'. Hyde". Figure 108 shows both characters, masterly played by Spencer Tracy in
the c1assic 1941 Victor Fleming movie . By night, MI'. Hyde behaved as a cold-
blooded murderer, Iike ozone when breathed 01' incubated with cells with low
antioxidant content, while during the day Dr. Jekyll was an amiable and valid
physician, like ozone when used carefully as a drug .

Figure 108. Ozone has become a controversial gas : it is providential in the stratosphere
by blocking UV radiation but is toxicfor the respiratory system and plants in the troposph ere.
What is less known, but no less important, is that a judicious use ot ozone can he ve,y useful
as a therapeutic agent. 71IUs ozone can be symbolically personified by a respectable DI'.
Jekyll (left) and an odious MI'. Hyde (right)

If I confess to an audience of physicians that I believe ozone can be a useful

drug , I often feel I am exposing myself to derision: in these days of great
biotechnological achievements, what good can come from a small toxic moleeule?
And yet, in spite of alt these important biological successes, several cytokines, novel
genetic approaches and angiogenetic proteins continue to disappoint us, either
because we do not use them properly 01' because a disease like cancer cannot be
combated by reductionist approaches. I have asked myself if ozonetherapy, defined
as a "barbaric procedure", is an obsolete enterprise that would be best forgotten in
the third rnillennium. However, the fact that, after millions of years , Nature
maintains and proficiently uses simple molecules such as O 2. ' , H20 2, NO· and HOCI
cheers me up. Thus ozone remains to accomplish its controversial and not yet fully
understood activities.
The idea that, with a little help, the organism can repair itself is almost as old as
man . After a long dark period, Paracelsus (1493 -1541) revitalized it during the
Renaissance with the concept of "archeus" 01' the "spirit of life" . The use of ozone as
a drug allows us to transform the nebulous expression into reality : after its reaction
by biomolecules, a variety of compounds (H 202 being one of the earliest) are
generated and are able to turn on impaired 01' blocked biochemical pathways. A
serious disease involves the dysfunction of more than one organ and tends to set up
 DOES OZONETHERAPY HAVE A. FUTURE IN MEDICINE? 373

a hypo-anergie state . Orthodox Medieine has priority; however, if it does not

sueceed, ozonetherapy may intervene and it may or may not be helpful (Fig. 109).
fnfeetious agents. toxie cornpounds,
radiations. drugs, xenobioties
v

DEATH

Figure 109. Today, ozonetherapy is not meant to substitute con ventional Medi cine unless
we ean demonstrate a specific excellence. In practice. it ean comp lement orthodox Medicine
if the latter does nOI provide satisfactory results

Ozonetherapy seems to induee a simultaneous resuseitation of funetions that had

gone wrang: from metabolie aetivation to immunomodulation and from hormonal
release to restoration of antioxidant defenees. 1 believe that the strength of
ozonetherapy is to reaetivate and re-equilibrate physiological activities.
Metaphorically, I Iiken ozone to either a sort of metabolie (and atoxic) "shock"
or to an orchestra conductor capable of bringing dissonant players to order, It
remains our duty to provide hirn with the best possible musical score, i.e. with
the optimised dose and treatment schedule.
We have plenty of work ahead to prove unequivoeally the validity of
ozonetherapy. Yet if we ean do that, the future will be bright. An eneouraging
thought is that if we ean prove the usefulness of bioxidative therapy with H2 0 2 alone
or eombined with other solutions (Chapter 34), we ean expand our field of action
and provide billions of people in poor eountries with valid medieal treatment.
In September 2001, after the New York tragedy, we again fell into one of our
darkest periods. However, as usual , this is the end result of our past negligence in
failing to reeognize the right of any individual given the gift of life to express
himself. Of all anirnals, human beings have developed the best mental capabilities
but, as happens in a diseased body , our intelligence has gone astray .
We have amassed useless mountains of gold, we have invented terrific anns for
mass destruction, we have polluted the Earth, we have invented religions beeause we
needed the hope of a wonderfullife after death . It has turned out that religions divide
374 CHAPTER38

humanity into opposite parties! Are human beings really intelligent? There is only
one religion that commands us to honour life every day , while we are really alive. I
know that is a vain hope but let us try in the future to be less selfish , to talk, to help
each other, so that when death comes, we will dissolve happily, knowing that we did
our best.
The question : His ozonetherapy therapeutic?" (Bocci , 1998b) remains open and I
am rather pessimistic that will be answered. However I know that some Western
countries, the United States and Japan have powerful medieal resources and in a
couple of years could examine the three main possibilities of oxygen-ozone therapy :
infections, vascular diseases and cancer.
IF commercial interests and prejudice will not prevent this research and IF
results will prove useful, oxygen-ozone therapy could commence in all hospitals and
be quickly extended in less developed countries. Will this dream come true?
January 1i" 2002
 CHAPTER 39

APPENDIX:
THE OPTIMIZED PROCEDURE OF 03-AHT

I . INTRODUCTION
AHT by exposing human blood to UV irradiation in the presenee of oxygen was
firstly earried out in 1954 by Wehrly and Steinbart. However it is Wolff's (1974)
merit to have defined the proeedure by exposing blood direetly to agas mixture
eomposed of medical oxygen-ozone (Or03) and to have perforrned preliminary
clinical studies. It is necessary to remember that up to 1990 the ozonization was
carried out in neutral glass bottles that are ozone resistant.
Unfortunately, later on 0 3-AHT has never undergone the necessary
standardization so that several variants of the original procedure have been used
generating an enorrnous confusion.
A critical examination of the various methodologies used in the last decade for
carrying out 03-AHT in Italy and Germany has pointed out serious pitfalls that are
potentially risky for the patient. It has been ascertained that, very often, blood is
collected and reinfused via the same tubing with, or even without, the appropriate
filter indispensable for blood transfusion. In order to avoid coagulation and hence
the possibility of either needle clogging or infusion of a coagulum, the exposure of
blood to O 2-03 is too brief (30-40 sec) and we have demonstrated that this period of
time is insufficient for the complete solubilization of0 2-03 in blood.
In Italy another worrisome problem is the widespread use of plastic (PVC)
autotransfusion bags that , while suitable for storing blood, release various plastic
microparticles and phthalates into the blood even during a short exposure to O 2-0 3
(Valeri et al., 1973; Thomas et al., 1978; Callahan et al., 1982; Estep et al., 1984;
Labow et al., 1986; Quinn et al., 1986; Whysner et al., 1996). As it has been noted in
patients undergoing dialysis, the mutagenic and toxic activity of these compounds is
a matter ofgrave concern (Lawrence, 1978; Divincenzo et al., 1985). For all ofthese
reasons, the use of a new device is now strongly recommended and this paper
reports a new system that is practical, flexible and atoxie.

2. MATERIALS AND METHODS

We have tested several samples ofplastic bags largely used in Italy for storing blood
and inflow-outflow tubing in polivinyl chloride-di(2ethylesil)phthalate (PVC-
DEHP) . All of these bags are authorized by the Ministry of Health to store blood but
not to be insufflated with OrO): Bags are made of PVC for a maximum content of

375
376 CHAPTER39

55% while for achieving a good elasticity additional materials amount to about 45%•.
With small differences the composition is the following :

a) about 40% of DEHP

b) about 1% of Zn 2 ethyl esanoate
c) about I% Ca or Zu stearate
d) about 1% N,N l diacyl ethylen diamine
e) 5 - 10% of epoxidated soya bean oil or simi lar

While all bags are sterile and suitable for storing blood, they are NOT
chemically inert when a strong oxidant such as 0 3 is insuffIated into the bag .
Particularly DEHP and butyl-glycobutyl phthalate (BGBP) are immediately released
and bound extensively to plasma lipids . The plasma is likely to yield a higher
content of DEHP than physiological saline. In line with the criteria expressed by the
European Pharmacopea (1997), we carried out the investigation by using sterile
physiological saline that is considered the optimal "medical device" for evaluating
release and size ofplastic particles (2,5, 10,20 and 25 ~ size), phthalates and other
compounds. Sampies were numbered and a11 the following tests have been carried
out in a blind fashion . The code was open after the final results were available.
Particles were measured by an automatic counter (Royco) by Dr. G. Gavioli and
collaborators at Braun Carex, Mirandola (Modena, Italy) while several chemical
compounds among which phthalates were detected by HPLC by a specialized Institute
(Istituto di Ricerche Agroindustria, Director: Dr. G.C. Angeli, Modena , Jtaly).
The proliferation index (PI) of blood mononuclear cells (BMC) has been
assessed after isolation of BMC from human blood of normal donors. PBMC were
isolated by Ficoll-Hypaque (Sigma Chemical Co ., St. Louis, MO) gradient
centrifugation, washed twice in RPMI-1640 medium supplemented with 20 mM
HEPES buffer, spun down at low speed to remove platelets, and resuspended in
RPMI-1640 medium supplemented with 2 mM HEPES, 10% heat-inactivated fetal
calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin and 100 ug /ml
streptomycin (all from Life Technologies, Gaithersburg, MD) at the final
concentration of I x 106 viable celis/mI. Cell viability was assayed by the trypan
blue exclusion technique and light microscope observation.
Aliquots (0.1 ml) of BMC suspension were added per well in triplicate wells to
96-well flat bottomed tissue culture plates (Costar, Cambridge, MA). BMC were
cultured without stimulation or stimulated with PHA at a final concentration of 5
ug /rnl (Sigma Chemical Co.). After 12 hours incubation, either control saline, or
ozonized saline in a glass syringe, or in blood bags was added to the culture medium
in a I : 4 proportion. Thereafter incubation continued for 40 and 64 hours . Cell
proliferation was evaluated by a colorimetric immunoassay (Boehringer Mannheim ,
Mannheim, Germany) based on BrdU incorporation. Briefly, after either 40 and 64
hours of incubation at 37° with 5% CO 2 in air and 100 % humidity, the cells were
labelIed with BrdU for 6 hr (10 IU/well) . The cells were then fixed, anti-BrdU-POD
antibody added and the immune complexes detected by the subsequent substrate
reaction. The proliferative index (PI) was obtained, calculating the ratio between
 ApPENDlX 377

PHA-stimulated cells and unstimulated ones, after subtraction of the corresponding

blanks .
It is emphasized that all tests were carried out with the same procedure and
timing used during a conventional autohaemotherapy.
All tests were performed in double blind fashion by two external firms
specialized in the pertinent assays . PI and all other analyses were assessed in the
Institute of General Physiology, University of Siena . Results were expressed as
mean± SO.

3. RESULTS ANO DISCUSSION

These can be summarized as folIows :
Table 28 reports the number of plastic particles ranging in size among 2, 5, 10, 20
and 25 J.l in either the control saline (test no .10), or in saline withdrawn from blood
bags with no exposure to O 2-0 3 (test no.l) or in saline as before but exposed to O 2-
0 3 (70 ug/ml per ml saline, ratio I :I) for 10 min (test no.2), or in saline from other
PVC bags, control (test no. 15) or in saline exposed to O2-0 3 for 10 min (test no.
16). It appears very clear that the number ofplastic particles released from different
PVC blood bags far exceed the number of control sampIes . According to the
European Pharmacopea values of particles released after ozonization exceed the
maximal tolerated value of 3.3-10.7 fold. All the saline sampIes collected from the
plastic bags after ozonization showed by HPLC exarnination, several compounds as
phthalates, caprolactamate and linear chain hydrocarbons not readily identifiable.
Interestingly, the same examination of tubing in PVC-OEHP normally used for
collecting blood and insufflating O2-03 do not show an abnormal release of plastic
particles (Table 29) even though the ozone exposure was prolonged for 30 min. This
is not surprising because tubings have far less additives than bags . Thus as the time
of contact with O2-0 3 is very transient, these tubings could still be used although we
have preferred to substitute them with a new brand made up of more resistant
material (PVC additioned with tri (2-ethylesil-trismellitate, TEHT, C33Hs406) known
as Staflex TOTM. Material released from this type of tubing is less than 100 fold
than from tubing PVC-OEHP so that this new type is absolutely safe .
Besides the potential risk propounded by plastic particles and chemical
compounds during the reinfusion of ozonized blood , we thought important to
investigate whether BMC withdrawn from the bags show any modification ofthe PI.
Aseries of analyses carried out after two different periods of incubation (40 and 64
hours) clearly show a consistent depression of the PI that can be as high as 27.2%
(Table 30) . Taking into account the small volume of ozonized saline added to the
culture medium this value is possibly underestimated and therefore is worrisome.
This negative effect is not directly due to 0 3 but rather to unknown compounds
released into the saline during ozonization of the blood bags. It is obvious that we do
not want the same phenomenon occurring in vivo and moreover, owing to the
variety of compounds released from the plastic material, we don't know which is
(are) the compound(s) responsible for the inhibition.
378 CHAPTER39

Tahle 28 . Numb ers ofplastic particles (size of 2. 5, 10, 20 and 25 u) countedfor each ml of
physiological saline after the indicated tests.

Test n. Size (u)

2 5 J() 20 25
10) Physiological saline (contro!) 195 ' 2Y 6' I" 0,7 '
I) Saline in blood bags not exposed 376+ 61' 13' r O,S'
to O 2-03
2) Saline in blood bags exposed to 10707+" 839 H ' 51 ++ 26" 21 "
O 2-03 (70 ug /rnl) for 10 min
15) Saline in PVC bags not exposed to 230+ 28' 7
4
I' I'
O 2-03
16) Saline in PVC bags exposed to 3343'+ 381 '';- 71'+' 36 " 26
H

O 2-03 (70 ug/ml) for 10 min

Maximal values of particles 1000 100 25 3 2,5

allowed today
Values found in excess 3,3-10,7 3,8-8,4 2-J 9-12 8,1-10

, Average values after 3 tests

H· Average values after 6 tests

Tahle 29. Numbers ofplastic particles (size of2. 5. 10. 20 and 25 u) countedfor each 1111
ofphysiological saline after tests in PVC tubings in current use.

Test n. Size (u)

2 5 ]0 20 25
11) Physiological saline (control) 481* 86 23 3,5 2
15) Saline exposed to 70 ug /ml 0 3 509 87 25 3,2 2
for 30 min at 37°C
in the PVC tubings

Maximal values of particles 1000 100 25 3 2,5

allowed today

* All values are the mean ofB tests.

 ApPENDIX 379

Table 30. Evaluation ofthe p roliferation index (PI) of human isolated blood mononu clear
cells after 40 and 64 hours of incubation in culture medium after addition (see Methods) 0/
physiological saline (PS) collectedfrom control orfrom ozonized saline (80 ug/ml) in glass
syringes (OS) orfrom saline previously ozonized
in a blood bagfor either 10 min (A) orfor 12 hours (8) .

GSO j BagA BagB GSO-, * Bag A * BagB *

ControI ControI Control Control Control Control

Hours 40 Ratios : 0,1062 0,0882 0,09 1 0, 1217 0,1058 0,0975

0, 1155 0, 1155 0, 1155 0,1293 0, 1293 0,1293

PI + 0,919 0,763 0,7 88 0,941 0,818 0,754

-17% -14,3% -13,1% -19.9%

hours 64 Ratios: 0,1057 0,1054 n.d. 0,2414 0,2394 0,1758

0,1164 0,1164 0,2560 0,2560 0,2560

PI++ 0,9080 0,9055 0,9430 0,9351 0,6867

-0,3% -0,8% -2 7,2%

* Witli PHA (5 jJg/ml) addition

+ Av erage 0/4 determ inations
+ + Averag e Q(8 det ermination s
n.d.: not determined

For all of these reasons, the use of a new deviee is now strong ly reeornrnended
(Fig. 110). This is eomposed of a) a neutral 500 ml glass bottle (sterile and under
vaeuum), b) a new atoxie tubing for eolleeting blood and insufflating sterile-filtered
O 2-03 via an antibaeterial (0,2 /-I), hydrophobie ozone-resistant filter and e) an
appropriate tubing with filter that is used , firstIy for infusing saline, and seeondly for
retuming the ozonized blood to the donor.
It is importan t that the exposure of blood to Or03 lasts at least 5 min beeause
mixing of blood must be gentle to avoid foaming. Beeause blood is very viseous, it
takes about 5 min to aehieve a eomplete and homogenous equilibriurn . It ean be noted
that the p02 slowly reaehes supraphysiologieal values (up to 400 mmHg) and then it
remains eonstant. On the other hand, 0 3 dissolves in the water of plasma but then
reaets instantaneously so that all ofthe 0 3 dose is praetieally exhausted within 5 min.
The ozonetherapists must follow this proeedure for avoiding either negative
effeets on the patients, or being found guilty of medieal malpraetiee.
380 CHAPTER39

Ildterlly G Ig
WIlh Lu~-Ick ecm ectlon _
Segme nt A _ __
~
L

Segment B

Figu re 110. A sehematte drawing ofthe several comp onents necessary 10pe/f orm 0 3-AHT
with a glass bottle.

4. SUMMARY
A critical examination of the various methodologies used in the last decade for
carrying out AHT in Italy and Germany has pointed out serious pitfalls that are
potentially risky for the patient. It has been ascertained that , very often , blood is
collected and reinfused via the same tubing with, or even without, the appropriate
filter, indispensable for blood transfusion. In order to avoid coagulation, hence the
possibility of either needle clogging or infusion of a coagulum, the exposure of
blood to O 2-0 3 is too brief (30-40 sec) and we have demonstrated that this period of
time is insufficient for the complete solubilization of O 2-0 3 in btood . The p02
reaches at best values of about 90 mmHg that is far below the supraphysiological
values (about 400) determined after 5 min of gentle mixing. Another worrisome
problem is the widespread use ofptastic autotransfusion bags that, while suitable for
storing blood, release various plastic cornpounds into the blood even during a short
exposure to O 2-0 3 , As it has been noted in patients undergoing dialysis, the
mutagenic and toxic activity of these compounds is a matter of grave concern. For
all of these reasons, the use of a new device is now strongly recommended. This is
composed of a neutral 500 ml glass bottle (sterile and under vacuum of about 0,9
bar), a new atoxic tubing for collecting blood and insufflating sterile-fiItered OrOj
and an appropriate tubing with another filter that is used , first1y for infusing saline ,
and secondly for returning the ozonized blood to the donor.
 REFERENCES

Abe , H., Ikebuchi K ., Shimbo M ., and Sekiguchi S., 1998, Hypotensive reactions with a white cell-
reduction filter : activation of kallikrein-kinin cascade in a patient, Transfusion 38:411 -412.
Abraham, N . G., Drummond G. S., Lutton 1. D., and Kappas A , 1996 , The biological significance and
physiological role ofheme oxygenase, CelloPhys iol. Biochem . 6 :129-168 .
AcseIl , J. R., and Riley J. B., 1993 , lmmunology of the extracorporeal circuit components, Adv.
Nel/l'oimmunol.3:227-241.
Adler, R., 1996, Mechanisms ofphotoreceptor death in retinal degenerations. From the cell biology ofthe
1990s to the ophthalmology of the 21st century?, Arch , Ophthalmol. 114:79-83.
Aejmelaeus, R. T., Holm P., Kaukinen U., Metsä -Ketel ä T. 1. A , Laippala P., Hervonen A L. 1., and
Alho H. E. R., 1997, Age-related changes in the peroxyl radical scavenging capacity of human
plasma, Free rad. Biol. Med. 23 :69-75 .
Agostini, G., and Agostini S., 1994, Contributo alla conoscenza e al trattamento della parmiculopatia
edemato-fibro-sclerotica, in Proceedings: VII National Meeting ofOzonetherapy, Roma , 1994 .
Agus , D. 8., Vera J. c., and Golde D . W ., 1999 , Stromal cell oxidation: a mechanism by which tumors
obtain vitamin C, Cancer Res. 59:4555-4558 .
Aiello, L. P., Pierce E. A , Foley E. D., Takagi H., Chen H., Riddle L., Ferrara N ., King G. L., and Smith
L. E. H., 1995 , Suppression ofretinal neovascularization in vivo by inhibition of vascular endothelial
growth factor (VEGF) using soluble VEGF-receptor chimeric proteins, Proc . Not. Acad. Sei. USA
92 : I0457-1 0461. .
Aitken, c., and Jeffries D. J., 2001 , Nosocomial spread ofviral disease, Clin. Mtcrob iol. Rev . 14:528-546 .
Akaike , T., Suga M ., and Maeda H ., 1998, Free radicals in viral pathogenesis: molecular mechanisms
involving superoxide and NO , Proc . Soc . Bxp. Biol. Med. 217 :64-73.
Akdis, C. A ., and Blaser K., 2001 , Mechanisms of interleuk in-IO-mediated immune suppression,
Immunology 103 :136.
Akdis, C. A., Blesken T., Akdis M., Wüthrich B., and Blaser K., 1998, Role ofinterleukin 10 in specific
immunotherapy,.I. Clin. Invest . 102:98-106 .
Akey, D ., and Walton T. E., 1985 , Liquid-phase study of ozone inactivation of Venezuelan Equine
Encephalomyelitis virus , Appl. Environ. Microbiol. 50:882-886.
Al Sa'd oni , H., and Ferro A, 2000 , S-Nitrosothiols: a class ofnitric oxide-donor drugs, Clin. Sei. (Coleh.)
98:507-520 .
Alexandre, A, Buric 1., Cor ö L., Rigobello L., and Scopetta S., 2000, Dis colisi percutanea mediante 0 20 3
intradiscale, in Proceedings: I Congres so lMOS. ltalia . Siena , 2-4 novembre 2000, pp.7-8 .
Alexandre, A, and Fumo G., 1998 , Discolisi percutanea mediante 0 203 nell'ernia discale lombare, in
Lombalgie e lombo sciatalgie. Criteri di diagno si e cura (F . Ceccherelli, and A. Ricciardi, Eds .),
Edizioni Libreria Cortina, Torino, pp .367 -377 .
Alexandre, A., Pentimalli L., Rigobello L., and Cor ö N ., 1999 , Amaurosi fugax in un caso di discolisi
cervicale mediante 0 20 3, in L'Ozonoterapia nel 2000 (F. Ceccherelli, and F. Giron, Eds .), Edizioni
Libreria Cortina, Torino, pp .141-144 .
Alkhatib, G ., Combadiere c., Broder C. c., Feng Y., Kennedy P. E., Murphy P. M., and Berger E . A,
1996, CC CKR5 : a RANTES , MIP-Ialpha, MIP -Ibeta receptor as a fusion cofactor for rnacrophage-
tropic HIV -I, Scien ce 272 :1955-1958.
Allen , R. G., and Tresini M., 2000, Oxidative stress and gene regulation, Free rad. Biol. Med. 28:463-
499 .
Allikmets, R., Shroyer N . F., Singh N ., Seddon J. M., Lewis R. A., Bernstein P. S., Peiffer A., Zabriskie
N. A, Li Y ., Hutchinson A ., Dean M., Lupski J. R" and Leppert M., 1997 , Mutation ofthe Stargardt
disease gene (ABCR) in age-related ma cular degeneration, Science 277 :1805-1807.
Altman, N ., 1998, Oxygen healing therapies, Healing Arts Press, Rochester, Vermont, USA, pp .I-254.
Amato, G ., 2000 , Uso dell'ozonoterapia mediante grande autoemotrasfusione nella terapia dell'angina
abdominis, in Proceedings: [ Congre sso [MOS, Italia, Siena. 2-4 novembre 2000, p.1O.

381
382 REFERENCES

Amato, G., Sacchetta A ., Borrelli E., and Bocci V ., 2000, Ruolo dell'ozonoterapia medi ante grande
autoernotrasfusione nel trattamento delle epatiti croniche post-epatite virale (11 parte), in
Proceedings: I Congresso IMOS, Italia, Siena. 2-4 novembre 2000, p.ll.
Amersi , F., Buelow R ., Kato H., Ke B., Coito A J., Shen X. D., Zhao D., Zaky 1., Melinek J ., Lassman C.
R., Kolls J. K., Alam 1., Ritter T ., Volk H. D., Farmer D. G.. Ghobrial R. M., Busuttil R W. , and
Kupiec-Weglinski 1. W., 1999, Upregulation of heme oxygenase-I protects genetically fat Zucker rat
livers from ischemia/reperfusion injury,.J. Clin. Invest 104 : 1631-1639.
Arnes, B . N ., Shigenaga M. K., and Hagen T. M ., 1993 , Oxidants, antioxidants, and the degenerative
diseases of aging, Pro c. Nat. Acad. Sei. USA 90 :7915 -7922.
Anderson, C; 1992, Gene therapy researcher under fire over controversial cancer trials, Nature 360:399-
400 .
Anderson, K. c., and Weinstein H. J., 1990, Transfusion-associated graft-versus-host disease, N. Eng!. .J.
Med.323:315-321.
Anderson, M. M., Hazen S. L., Hsu F. F., and Heinecke 1. W., 1997, Human neutrophils employ the
rnyeloperoxidase-hydrogen peroxide-chloride system 10 convert hydroxy-amino acids into
glycolaldehyde, 2-hydroxypropanal, and acrolein,.I. Clin. Invest. 99:424-432 .
Anderson, M. T. , Staal F. 1. T., Gitler c., Herzenberg L. A ., and Herzenberg Le. A., 1994 , Separation of
oxidant-initiated and redox -regulated steps in the NF-kB signal transduction pathway, Proc. Nat.
Acad. Sei . USA 91: 11527 -11531 .
Andreone, P., Gramenzi A., Cursaro C; Sbolli G., Fiorino S., Di Giamrnarino L., Miniero R ., D'Errico
A ., Gasbanini G., and Bemardi M., 1999, Interferon-a plus ribavirin in chronic hepatitis C resista.nt
to previous interferon-a course: results ofa randomized multicenter trial ,.J. Hepatol. 30 :788-793 .
Andreoni, K. A , Kazui M., Cameron D. E., Nyhan D., Sehnert S. S ., Rohde C. A ., Bulkley G. B., and
Risby T. H., 1999, Ethane: a marker of lipid peroxidation during cardiopulmonary bypass in humans,
Free rad. Biol. Med. 26:439-445 .
Andreula, C. F., 200 I , Emie discali lombosacrali e patologia degenerativa correlata. Trattamento
interventisrico spinale con chemiodiscolisi con nucleoptesi con 0) e infilrra zione periradi colare e
periganglionare, Riv. Neuroradiol. 14:81-88 .
Ankoma-Sey, V., 1999 , Hepati c Regeneration-Revisiting the Myth of Prometheus. News Physiol Sei.
14 :149-155.
Anonymous, 1996, The power ofplacebo, Brtt. Med. J. 313 :1569-1570 .
Antonelli, G., Bagnato F., Pozzilli c., Simeoni E., Bastianelli S. , Currenti M ., De Pisa F., Fieschi c.,
Gasperini C., Salvetti M., and Dianzani F., 1998 , Development of neutralizing antibodies in patients
with relapsing- remitting multiple scleros is treated with [FN -beta1a. .I. Interferon Cytokine Res.
18 :345-350.
Ant onelli , G., Blalock 1. E., and Dianzani F., 1985, Generation of a soluble [FN -g indu cer by oxidation 01'
galactose residues on macrophages, CelloImmunol. 94 :440-446.
Aris, R. M ., Christian D., Heame P. Q ., Kerr K., Finkbeiner W. E., and Balmes 1. R., 1993, Ozone-
induced airway inflammation in human subjects as deterrnined by airway lavage and biopsy, Anter.
Re v. Respir. Dis . 148 :1363 -1372 .
Arnason, B. G. W., 1993, Interferon beta in multiple sclerosis, Neurologv 43:641-643 .
Arrigo, A P., 1999, Gene expression and the thiol redox state, Free rad. Biol. Med. 27:936-944.
Arsalane, K., Gosset P., Vanhee D., Voisin C; Hamid Q., Tonnel A. B., and Wallaert B., 1995 , Ozone
stimulates synthesis of inflammatory cytokines by alveolar macrophages in vitro, Am er. .I. Respir.
Cell Molec . Biol. 13:60-68 .
Arvin, AM ., and Prob er C. G., 1997, Herpes simplex virus type 2 - a persistent problern. N. Eng]. .J.
Med. 337 :1158-1159.
Assoian, R. K., Komoriya A ., Meyers C. A, Miller D. M ., and Sporn M. S, 1983 , .J. Biol . Chem .
258 :7155-7160.
Astin, 1. A , 1998 , Why patients use alternative medicine: results of anational study, .lAMA 279 : 1548-
1553 .
Aubourg, P., 1936 , Colibacillose aigue, colibacillose cronique: ameliorations c1iniques notables par un
traitement d'ozone, Bull. Med. Paris [40 :644-654.
Aubourg, P., 1940 , Ozon in der Chirurgie, Mem, Acad. Chir 65 :1183-1192.
Auphan, N., DiDonato J. A, Rosette c., Helmberg A., and Karin M ., 1995, Immunosuppressi on by
glucocorticoids: inhibition of NF-k B activity through induction of IkB synthesis, Sci ence 270 :286-
290 .
 REFERENCES 383

Ayres , R. M., Stott R., Mara D. D., and Lee D. L., 1992, Wastewater reuse in agriculture and the risk of
intestinal nematode infection, Parasttot. Today 8 :32-35.
Babior, B. M., 2000, Phagocytes and oxidative stress, Am. .I. Med. 109 :33-44.
Back , T., 1998, Pathophysiology of the ischemic penumbra--revision of a concept, Cell Mol . Neurob iol.
18:621 -638 .
Badwey, 1. A, and Karnovsky M. L., 1980 , Active oxygen species and the functions of phagocytic
leukocytes, Annu. Rev. Biochem. 49 :695-726 .
Baeuerle, P. A., and Henkel T., 1994 , Function and activation ofNF-kB in the immune system, Annl/.
Rev.lmml/nol.12:141 -179 .
Bailar, J. c., 111, and Gomik H. L., 1997, Cancer undefeated, N. Engl. .1. Med . 336:1569-1574.
Balkanyi, A, 2001 , Herpes Zoster - a treatment concept with ozone, in Proceedings 0/ the 15th Ozone
World Congress, London, UK. lIth-15th September 2001. Medical Therapy Conference (IOA 2001,
Ed .), Speedprint Mac Media Ltd, Ealing, London, UK, pp .89-93.
Baltin, H., 1995, Ozontherapie in onkology., Proceedings ofthe 12th Ozone World Congress, May 1995.
Lilie :41.
Barber, E., Menendez S., Leön O. S., Barber M. 0 ., Merino N ., Calunga J. L., Cruz E., and Bocci V .,
1999, Prevention of renal injury after induction of ozone tolerance in rats submitted to wann
ischaemia., Mediat. Inflamm. 8 :37-41.
Barnes, P. J., 2000, Chronic obstructive pulmonary disease, N. Engl. .I. Med. 343 :269-280 .
Barnes, P. J., and Karin M., 1997, Nuclear factor-kappaB: a pivotal transcription factor in chronic
inflammatory diseases, N. Engl. .I. Med. 336 :1066-1071 .
Barnes, P. J., and Liew F. Y., 1995, Nitric oxide and asthmatic inflammation, Immunol. Today 16:128-
130.
Bates, D. V., 1996, Air pollution: time for more clean air legislation?, Brit. Med. J. 312 :649-650.
Baulieu, E.-E ., and Robel P., 1998, Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate
(DHEAS) as neuroactive neurosteroids, Pro c. Nat. Acad. Sei. USA 95:4089-4091.
Bayram, H., Sapsford R. J., Abdelaziz M. M., and Khair O. A, 2001, Effect of ozone and nitrogen
dioxide on the release of proinflammatory mediators from bronchial epithelial cells of nonatopic
nonasthmatic subjects and atopic asthmatic patients in vitro ,.I. Allergy Clin. Immunol. 107:287-294.
Baysan, A., Whiley R. A , and Lynch E., 2000 , Antimicrobial effect of a novel ozone- generating device
on rnicro - organisms associated with primary root carious lesions in vitro, Caries Res. 34:498-501.
z.r.
Beal , R. W. , 1976 , The rational use ofblood, Aust. N. SI/I'g. 46 :309-313 .
Beaven, D. W. , 1989 , Alternative medicine a cruel hoax-your money and your life", N. Z. Med. J.
102:416-417.
Beck, L. S., DeGuzman L., Lee W . P., Xu Y., Siegel M. W ., and Arnento E. P., 1993, One systemic
administration of transfonning growth factor- bl reverses age- or glucocorticoid-irnpaired wound
healing,.I. Clin. Invest. 92 :2841-2849 .
Beck, N . B., Koenig J. Q., Luchtel D. L., Altman L. c ., Orsborn M . T ., and Kenney 1. S., 1994, Ozone
can increase the expression of intercellular adhesion molecule-I and the synthesis of cytokines by
human nasal epithelial cells, Inhal . Toxicol. 6:345-357 .
Becker, S., Jordan R. L., Orlando G. S., and Koren H. S., 1989, In vitro ozone exposure inhibits mitogen-
induced Iymphocyte proliferation and IL-2 production., J. Toxicol . Environ. Health 26 :469-483 .
Beckman, K. B., and Ames B. N., 1997, Oxidative decay ofDNA,.1. 8iol. Chem . 272:19633-19636.
Beckman, K. B., and Arnes B. N ., 1998, The free radical theory of aging matures, Physiol Rev. 78 :547-
581.
Bell , S., and Kamm M. A, 2000, Antibodies to tumour necrosis factor alpha as treatment for Crohn's
disease, Lancet 355 :858-860 .
Belluzzi, A, Brignola c., Campiert M., Pera A, Boschi S., and Miglioli M., 1996, Effect ofan enteric-
coated fish-eil preparation on relapses in Crohn's disease, N. Engl. .I. Med. 334: 1557-1560.
Beltrani, V . S., 1999 , The c1inical spectrum of atopic dennatitis,.1. Allergy Clin. lmmunol. 104 :S87-S98 .
Bendinelli, M., Vatteroni M. L., Maggi F., and Pistello M., 1999, Hepatitis C virus . Biology,
pathogenesis, epidemiology, c1inical description, and diagnosis, in Viral hepatitis: diagnosis,
therapy, and prevention (S . Specter, Ed .), Humana Press Inc ., Totowa,NJ, pp.65-127 .
Benes, S. E., Murphy T. M., Anderson P. D., and Houpis J. L. J., 1995 , Relationship of antioxidant
enzymes to ozone tolerance in branches of mature ponderosa pine (Ptnus ponderosai trees exposed to
long-term, low concentration, ozone fumigation and acid precipitation , Phys iol. Plant. 94 :124-134.
384 REFERENCES

Benesch, R., and Benesch R. E., 1967 , The effect of organ ic phosphates from the human erythroc yte on
the allosteric properties ofhemoglobin, Biochem . Biophvs. Res. Commun. 26 :162-1 6 7.
Benson, H., and Friedrnan R., 1996 , Hamessing the power of the placeb o efTect and renaming it
"remembered wellness ", Annu. Rev. Med. 47 :193-1 99 .
Benzie, 1. F., and Stra in 1. J., 1999 , Ferric reducing/anti oxidant power as say : direct measure of total
antioxidant activity of biol ogieal fluid s and modified version for simultaneous measurement of total
antioxidant po wer and ascorbic acid concentrati on, Methods Enzymol. 299 : 15-27 .
Bergamini , A , Capozzi M., Ghibelli L., Dini L., Salanitro A, Milanese G., Wagner T., Beninan S .,
Delfin a Pesce c., Amici c., and Rocchi G ., 1994, Cystamine potentl y suppress es in vitro H1V
replication in acutely and chronicall y infected human cells,.I. Clin. Invest. 93 :2251-2257.
Berger, T. M., Polidori M . c., Dabbagh A ., Evans P. 1., Halliwell B ., Morr ow J. D., Roberts L. J., and
Frei B., 1997 , Antioxidant activity of vitarnin C in iron-overloaded human plasma ,.1. Biol. Chem.
272 :15656-15660 .
Bergers, G., Javaherian K., Lo K.-M ., Folkman J., and Hanahan 0 ., 1999, Effect s of angi ogenesis
inhibitors on multistagecarcinogenesis in mice, Scien ce 284 :808-812.
Bergqvist, 0. , 1999, Salvage of critically ischaemic limbs , Lancet 354 :1920-1921.
Berlell, B. A, Omar O. H. M ., Sahakian 1. A , Levine R. L., and Stadt man E. R., 1991, Oxidation of
proteins by ozone, FASER.I. 5:A 1524.
Berliner, 1. A , and Heinecke 1. W ., 1996 , The role of oxidized lipoproteins in atherogenesis, Free ra d.
Binl. Med . 20 :707-727.
Berson, E. L., Rernulla J. F. C., Rosner B., Sandberg M. A., and Weigel-DiFranco c., 1996, Evaluation of
patients with retinitis pigmentosa receiving electric stimulation, ozonared blood, and ocular surgery
in Cuba , Arch . Ophthalmol. 114 :560-563 .
Bess on , G., and Bogoussla vsky J., 1995, C urrent and future options for the pre venti on and treatment of
srroke, CNS Drugs 3 :351-362 .
Beutler, E., Meul A , and Wood L. A , 1969, Depletion and regeneration of 2,3-diphosphoglyceric acid in
stored red blood cells, Transfusion 9: 109-115 .
Beutler, E., and Wood L., 1969, The in vivo regeneration of red cell 2,3 diph osph oglyceric acid (DPG)
after transfusion of stored blood,.1. Lab Clin. M ed. 74 :300-304.
Bevers, R. F. M ., Bakker D. 1., and Kurth K. H., 1995 , Hyperbane oxygen treatment for haem orrhagi c
radiation cystitis, l.ancet 346 :803-805 .
Beyerle, 199 6, eited by Null , 1996 (Oz one: a wide -spectrum realer, Penthouse Magazine January).
Bi1ger, B., 1995, Fore ver young, The Seiences September/October:26-30.
Bilodeau, c., and Chevrier N ., 1998, Endogenous ascorbate level modulates ozone tolerance in Euglena
gra cilis cells, Plant Physiol. Bioch em. 36 :695-702 .
Bisett i, P., Mattassi R., D'Angelo F., Col ombo R., Miccol is M., and Sanfilippo F., 1988, Effetti dell'ozono
su diversi pararnetri emat ochimici, Minerva Mesoterapeutica 3 :11-13 .
Bish op, G. A ., Ramirez L. M., Baccarn M., Busch L. K., Pederson L. K., and Tomai M. A ., 200 I , The
immune response modifier resiquimod mirnies CD40-induced B cell activation, Cell Immun ol. 208 :9-
17.
Blalock, J. E., 1984, The immune system as a sensory organ,.1. Immunol. 132 :I 067-1 070 .
Block, J. A, and Sequeira W., 2001, Raynaud's phenomenon, Lancet 357 :2042-2048 .
Bocchi , L., Cervelli C ., and Ferrata P., 1998, La nucleoaspirazione, in Lombalgie e lombosciaralgie.
Criteri di diagnosi e cura (F. Ceccherelli, and A . Ricciardi , Eds.) , Edizioni Libreria Cortina, Torino,
pp .285-293 .
Bocchi, L., Cervelli c., and Ferrata P., 2000 , L'ossigeno-ozono terapia nel tratt amento delle patologie
vertebral i lombari, in Pro ceedings : I Congresso IMOS. ltalia, Siena . 2-4 novembre 2000. p.15 .
Boc ci, v., 1981a , Deterrninants of erythrocyte ageing: a reappraisal. , Brit . .I. Haemat ol. 48 :515-522.
Boeci, V., 1981b, Pharmacokinetic studies of interferons, Pharmacol. Ther. 13 (3) :421 -440 .
Boeci, V., 1981c, Produetion and role of interferon in physiological conditions , Biol. Rev. 56 :49-85 .
Bocci, V., 1985a, Immunemodulators as local hormones: new insights regarding their clinical utilizati on .,
.I. Biol. Resp. Modit. 4 :340-352 .
Bocci, V., 1985b, Administration of interferon at night ma y increase its therapeutic index., Cancer Drug
Dei. 2:313-318.
Bocci , V., 1987a , May hyperbaric oxigenation be useful to patients with AIDS ?, .I. Bi ol . Regulat .
Homeost. Agent. 1:201.
 REFERENCES 385

Bocci, V., 1987b , Metabolism of pr.otein anti cancer agents. (Updated and reprinted in 1994 in Int.
Encyclopedia of Pharmacology and Therapeutics, Sec .140 Anticancer Drugs , Oxford: Pergamon
Press, pp . 387-436), Pharmacol . Ther. 34 :1-49.
Bocci , V., 1988a, Central nervous system toxicity of interferons and other cytokines, .J. Blol. Regulat.
Homeost. Agent. 2:107-118 .
Bocci , V., 1988b , Roles of interferon produced in physiological conditions. A speculative review,
Immunology 64: 1-9.
Bocci , V., 1990a, Catabolism of therapeutic proteins and peptides with implications for drug delivery.,
Advan. Drug Del ivery Rev. 4: 149-169 .
Bocci, V., 1990b, Tumor therapy with biological response modifiers. Why is progress slow?, EOS-J.
Immunol. Immunopharmacol. 10 :79-82 .
Bocci, V., 1991a, Absorption of cytokines via oropharyngeal-associated lymphoid tissues. Does an
unorthodox route improve the therapeutic index of interferon?, Clin. Pharmacokinet. 21 :4 11-4 17.
Bocci , V., 1991b, Interleukins. C1inical pharrnacokinetics and practical implications, CUn.
Pharmacokinet. 21 :274-284.
Bocci , V., I992a , Ozonization of blood for the therapy of viral diseases and immunodeficiencies . A
hypothesis, Med. Hypotheses 39 :30-34 .
Bocci, V., I992b , Physicochemical and biologic properties of interferons and their potential uses in drug
delivery systems., Grit. Rev. Ther. Drug Carr. Syst . 9:91-133 .
Bocci , V., 1992c , The neglected organ : bacterial flora has a crucial immunostimulatory role, Perspect.
Biol. Med.35:251 -260 .
Bocci, V., 1993a, Interferon . Una storia recente ed antichtssima. Fisiopatologia e clintca del slstema
Interfe ro n, Antea Edizioni, pp .I-205 .
Bocci, V., 1993b , Mistletoe (viscum album) lectins as cytokine inducers and immunoadjuvant in tumor
therapy. A review,.I. Biol. Regulat. Homeost. Agent. 7: 1-6.
Bocci , V., 1994a, A reasonable approach for the treatment of HIV infection in the early phase with
ozonetherapy (autohemotherapy). How inflammatory cytokines may have a therapeutic role, Mediat.
Inflamm. 3 :315-321.
Bocci , V., 1994b, Autohaemotherapy after treatment ofblood with ozone. A reappraisal.,.I. Int . Med. Res .
22:131-144.
Bocci , V., 1995a, Immunologische Aspekte - eine kritische Betrachtung aus italianischer Sicht -, in Ozon-
Handbuch. Grundlagen. Prävention. Therapie (E. G. Beck, and R. Viebahn-H änsler, Eds.), Ecomed ,
Landsberg, pp.I-16 .
Bocci, V ., 1995b, Ozonetherapy today , in Proceedings Ozone In Medicine. 12th World Congress of the
International Ozone Associatton. 15th to 18th May 1995. Lille, France (International Ozone
Association, Ed.), Instaprint S.A., Tours, pp. 13-28 .
Bocci, V., 1996a, Does ozone therapy normalize the cellular redox balance?, Med. Hypotheses 46 :150-
154.
Bocci, V., 1996b, Has Ozonetherapy any future in the West? (Some critical comments)., Practice oI
Ozonetherapy 1:4-9.
Bocci, V., 1996c, Ozone as a bioregulator. Pharmacology and toxicology of ozonetherapy today,.J. Blol .
Regulat .Homeost. Agent. 10 :31-53.
Bocci, V., 1996d, Ozone: a mixed bless ing . New mechanisms of the action of ozone on blood cells make
ozonated major autohaemotherapy (MAH) a rational approach, Fors ch. Komplement ärmed. 3 :25-33 .
Bocci , V., 1998a , Ipotetici meccanismi di az ione dell'ozono nel trattamento dei conflitto discoradicolare,
in Lombalgie e lombosciatalgie. Criteri di diagnos i e cura (F . Ceccherelli, and A. Ricciardi, Eds .),
Edizioni Libreri a Cortina, Torino, pp.331-340.
Bocci , V., 1998b , Is ozonetherapy therapeutic ?, Perspect. Biol. Med. 42 :131-143 .
Bocci, V., 1998c, Ozonetherapy as a possible biological response modifier in cancer, Fors ch.
Komplementürmed. 5:54-60 .
Bocci , V., 1999a, Biological and clinical effects of ozone. Has ozonetherapy a future in medicine ?, Brit .
.I. Biomed. Sc/. 56:270-279 .
Bocci , V., 1999b , Ozonetherapy as a complementary medical approach. Where are we and where do we
need to go?, in Proceedings of the Int. Ozone Symposium, 21 and 22 October 1999. Basel.
Switzerland (IOA - EA 3G Ed .), Bauer Druck AG, Basel, pp.353-374.
Bocci, V., 2000, Oss igeno-ozono terapia , Casa Editrice Arnbrosiana, Milano, pp.I-324.
386 REFERENCES

Bocci, V ., Aldinucci C., Borrelli E., Corradeschi F., Diadori A. , Fanetti G., and Valacchi G., 200la,
Ozone in medicine, Ozone-Sci. Eng . 23:207-217 .
Bocci, V., Borrelli E., Corradeschi F., and Valacchi G., 2000, Systemic effects after colorectal
insuffiation of oxygen-ozone in rabbits, Int. .J. Med. Biol. Environ . 28 :109-113 .
Bocci, V., Carraro F., Naldini A., Paulesu L., and Pessina G. P., 1990 , Roles of interferons in
physiological conditions and for the control of viral diseases, in Mtcrobtological, chemotherapeutical
and immunological problems in high risk patients (E. Garaci , G. Renzini , F. Filadoro, A. L.
Goldstein, and J. Verhoef, Eds .), Serono Symposia Publication from Raven Press, New York,
pp .243-250.
Bocci , V., Di Paolo N., Borrelli E., Larini A. , and Cappelletti F., 200lc, Ozonation of blood during
extracorporeal circulation 11 . Comparative analysis of several oxygenators-ozonators and selection of
one type ., Int . J. Artif. Organs 24 :890-897 .
Bocci, V., Di Paolo N ., Garosi G., Aldinucci c., Borrelli E., Valacchi G., Cappelli F., Guerri L., Gavioli
G ., Corradeschi F., Rossi R., Giannerini F., and Di Simplicio P., 1999b, Ozonauen ofblood during
extracorporeal circulation. I. Rationale, methodology and prehminary studies, lI/I. .J. Artif. Organs
22 :645-651.
Bocci, V. , Luzzi E., Corradeschi F., and Paulesu L., I 994a, Studies on the biological effects of ozone: 5.
Evaluation of immunological parameters and to1erability in normal volunteers receiving ambulntory
autohaemotherapy., Biotherapy 7 :83-90 .
Bocci, V., Luzzi E., Corradeschi F., Paulesu L., and Di Stefano A., 1993a , Studies on the biological
effects of ozone: 3. An attempt to define conditions for optimal induction of cytokines, Lymphokine
Cytokin e Res . 12: 121-126.
Bocci, V., Luzzi E., Corradeschi F., Paulesu L., Rossi R., Cardaioli E ., and Di Simplicio P., 1993b ,
Studies on the biological effects of ozone: 4. Cytokine production and glutathione levels in human
erythrocytes.,J. Biol . Regulat. Homeost. Agent. 7 :133-138.
Bocci, V., Luzzi E., Corradeschi F., and Silvestri S., 1994b, Studies on the biological effects of ozone: 6.
Production of transforrning growth factor bl by human blood after ozone treatment,.J. Btol . Regulat.
Homeost. Agent. 8:108-112 .
Bocci, V., and Paulesu L., 1990, Studies on the bioJogical effects of ozone I. Induction of interferon
gamma on human leucocytes, Haematologica 75 :510-515.
Bocci, V., Pessina G. P., Paulesu L., Muscettola M ., and Valeri A ., 1988, The lymphatic route. V.
Distribution of human natural interferon-ß in rabbit plasrna and lymph., J. Interferon Res. 8 :633-640.
Bocci , V ., Pogn i R., Corradeschi F., Busi E., Cervelli c., Bocchi L., and Basosi R., 2001 b, Oxygen-ozone
in orthopaedics: EPR detection of hydroxyl free radic als in ozone-treated "nucleus pulposus"
material, Riv. Neuroradiol. 14:55-59 .
Bocci , V., Russi M ., and Rita G, 1967 , Recovery and identification of interferon in the rabbit urine,
Expertentta 23: 1-5.
Bocci, V ., Valacchi G., Corradeschi F., Aldinucci C., Silvestri S., Paccagnini E., and Gerli R, 1998a,
Studies on the biological effects of ozone: 7. Generation of reactive oxygen species (ROS) after
exposure ofhuman blood to ozone,.I. Biol. Regulat. Homeost. Agent. 12:67-75.
Bocci, V., Valacchi G., Corradeschi F., and Fanetti G., 1998b, Studies on the biological effects of ozone:
8. Effects on the total antioxidant sratus and on interleukin-8 production, Med iat. Inflamm. 7 :313-
317 .
Bocci, V., Valacchi G., Rossi R., Giustarini D., Paccagnini E., Pucci A. M ., and Di Simplicio P., 1999a ,
Studies on the biological effects of ozone: 9. Effects of ozone on human platelets, Platelets 10:110-
116.
Bocci, V ., Venturi G., Catucci M ., Valensin P. E., and Zazzi M., 1998c, Lack of efficacy of ozone
therapy in HIV infection , Clin. Microbiol. Infec. 4 :667-669 .
Boehm, T., Folkman J., Browder T ., and O'Reilly M . S ., 1997, Antiangiogenic therapy of experimental
cancer does not induce acquired drug resistance, Nature 390:404-407 .
Boker, T., Fang T., and Steinmetz R., 1993, Refractive error and choroidal perfusion characteristics in
patients with choroidal neovascularization and age-relared macular degeneration, GerJ. Ophthalmol.
2:10-13 .
Bondy, S . C; 1995, The relation of oxidative stress and hyperexcitation to neurological disease, Pro!'.
Soc, Exp. Biol. Meel. 208 :337-345.
Bonetti, M., Cotticelli B., Valdenassi L., and Richelmi P., 2001, Analisi dei risultati dopo trattamento con
O 2-0 3 nelle emie intra ed extra foraminali lombari, Riv. Neuroradiol. 14 :89-92 .
 REFERENCES 387

Boni, c., Bertoletti A., Penna A, Cavalli A., Pilli M., Urbani S., Scognamiglio P., Boehme R.,
Panebianco R., Fiaccadori F., and Ferrari C., 1998, Lamivudine treatment can restore T cell
responsiveness in chronic hepatitis B,.I. Clin. Invest 102 :968-975 .
Bosch-Morell, F., Flohe L., Marin N ., and Romero F. J., 1999, 4-hydroxynonena1 inhibits glutathione
peroxidase: protection by glutathione, Free rad. Bio/. Med. 26:1383-1387.
Bosia, A., Pescarmona G. P., Loesche W ., and Spangerberg P., 1989, Role of glutathione in blood p1ate1et
function, in Glutathione - Chemical. biochemical and medical aspects (Part B) (D. Dolphin, R.
Poulson, and O. Avramovic , Eds.) , John Wi1ey & Sons, New York, pp.235-289.
Bowry, V. W., and Stocker R., 1993, Tocopherol-mediated peroxidation: The pro-oxidant effect of
vitamin E on the radical-initiated oxidation of human low-density lipoprotein, J. Amer. Chem. Soc.
115:6029-6040.
Boxer, L. A., and Smolen J. E., 1988, Neutrophil granule constituents and their release in health and
disease, Hematol . Oneol. Clin. N. Amer. 2:101-134.
Brandes, M. E., Allen J. B., Ogawa Y., and Wahl S. M., 1991, Transforming growth factor b1 suppresses
acute and chronic arthritis in experimental animals.,.I. CUn. Invest. 87:1108-1113.
Brayda-Bruno, M., and Cinnella P., 1998, 11 trattamento dell'ernia discale con infiltrazioni di ossigeno-
ozono in paravertebrale, in Lombalgie e lombosctatalgie. Criteri di diagnosi e cura (F . Ceccherelli,
and A Ricciardi, Eds .), Edizioni Libreria Cortina, Torino, pp .361-365 .
Bressler, N . M., Bressler S. B., and Fine S. L., 1988, Age-related macular degeneration, SlIIV.
Ophthalmol.32:375-413.
Bridgeman, M. M., Marsden M., MacNee W., Flenley D. C., and Ryle A. P., 1991, Cysteine and
glutathione concentrations in plasma and bronchoalveolar lavage fluid after treatment with N-
acetylcysteine, Thorax 46 :39-42.
Broeckaert, F., Arsalane K., Hermans C., Bergamaschi E., Brustolin A, Mutti A, and Bernard A, 1999,
Lung epithelial darnage at low concentrations of ambient ozone, Lancet 353 :900-90 I .
Brouard, S., Otter bein L. E., Anrather J., Tobiasch E., Bach F. H., Choi AM., and Soares M. P., 2000 ,
Carbon monoxide generated by heme oxygenase I suppresses endothelial cell apoptosis, .I. Exp. Med.
192:1015-1026.
Browder, T., Folkman J., and Pirie-Shepherd S., 2000, The hemostatic system as a regulator of
angiogenesis,.I. Biol. Chem. 275:1521-1524.
Brown , J. M., 1999, The hypoxic cell : a target for selective cancer therapy--eighteenth Bruce F. Cain
Memorial Award lecture, Cancer Res. 59:5863-5870.
Brown, J. M., and Giaccia A J., 1998, The unique physiology of solid tumors: opportunities (and
problems) for cancer therapy, Cancer Res. 58 :1408-1416.
Bruckdorfer, K. R., 1998, Lipid oxidation products and vascular function, Free rad. Res. 28:573-581.
Brugnara, c., Gee 8. , Armsby C. c., Kurth S., Sakamoto M., Rifai N., Alper S. L. , and Platt O. S., 1996,
Therapy with oral c1otrimazole induces inhibition of the Gardos channel and reduction of erythrocyte
dehydration in patients with sickle cell disease,.I. Clin. Invest 97 :1227-1234 .
Bubenlk, J., 1996, Cytokine gene-modified vaccines in the therapy of cancer, Pharmacol. Ther. 69 :1-14.
Buckley, R. D., Hackney J. D., Clark K., and Posin c., 1975, Ozone and human blood, Arch . Environ .
Health 30:40-43 .
Buege, J. A., and Aust S. D., 1994, Microsomallipid peroxidation, Meth. Enzymol. 233:302-310.
Bulinin, V.!., SoIod N. V., and Moshurov I. P., 1995, The first experience of chronic abscesses and
pleura emphyemas treatment by the method of ozonization, in The ozone in biology and medicine.
2nd all Russian scientific-practicol conference, September 6-8, 1995. Russian assoctation of
ozonetherapy, Reshetnikovskaya street 2, Nizhni Novgorod, 603006 Russia, p.20 .
Burdon, R. H., 1986, Heat shock and the heat shock proteins, Biochem. .I. 240:313-324.
Burstein, H. J., Gelber S., Guadagnoli E., and Weeks 1. c., 1999, Use of alternative medicine by women
with early-stage breast cancer, N. Engl . .I. Med. 340 :1733-1739.
Bustamante, J., Lodge J. K., Marcocci L., Tritschler H., Packer L., and Rihn B. H., 1998, a-lipoic acid in
liver metabolism and disease, Free rad. Bio/. Med. 24:1023-1039.
Butler, A. R., Flitney F. W., and Williams D. L., 1995, NO, nitrosonium ions, nitroxide ions, nitrosothiols
and iron-nitrosyls in biology: a chemist's perspective, Trends Pharmacol. Sei. 16:18-22 .
Byvoet, P., Balis J. U., Shelley S. A, Montgomery M. R., and Barber M. 1.,1995, Detection ofhydroxyl
radicals upon interaction of ozone with aqueous media or extracellular surfactant : the role of trace
iron, Arch. Blochern. Biophys. 319:464-469 .
388 REFERENCES

Cadenas, E., and Davies K. J., 2000 , Mitochondrial free radical generation, oxidative stress , and aging,
Free rad. Biol. Med . 29:222 -230 .
Caglayan, S., and Bayer R, 1995, Does ozone change the erythrocyte function ? Effects of oxidative
stress on erythrocyte deformability and fragility , in Proceedings 12/h Ozon e World Congress, Ml(F
15/h /0 18th, 1995. Lilie , France (International Ozone Association, Ed.), Lille, p.29.
Calabrese, E. J., and Baldwin L. A , 2001, Hormesis: U-shaped dose responses and their centrality in
toxicology, Trends Pharmacol. Sei. 22 :285-291.
Calder , P. c., 1998, Fat chance of immunomodulation, Immunol. Today 19:244-247 .
Callahan , J. T, Collecutt M. F., Lightbody J. R., and Faragher B. S., 1982, Alteration of human red blood
cells stored in plastic packs, Transfusion 22: 154-157 .
Campbell, D. E ., Fryga A. S., Bol S., and Kemp A. S., 1999, Intracellular interferon-gamma (IFN-g)
production in normal children and children with atopic dermatitis , Clin. Exp. 111I11I111I01. 115 :377-3 82.
Cangiano, c., Laviano A , Muscaritoli M., Meguid M. M., Cascino A., and Rossi Fanelli F., 1996, Cancer
anorexia: new parhegenie and therapeutic insights , Nutrititton 12:S48 -S51.
Cannistra, S. A , and Niloff J. M., 1996, Cancer ofthe uterine cervix , N Engl . J. Med. 334 :I030-1 038.
Canoso, R T , Rodvien R., Scoon K., and Levine P. H., 1974, Hydrogen peroxide and platelet function ,
Blood 43 :645-656 .
Cantin, AM., Fells G. A , Hubbard R. c., and Crystal R G., 1990, Antioxidant macromolecules in the
epitheliallining fluid ofthe normal human lower respiratory tract,.I. Clin . Invest . 86 :962-971 .
Cardile, V ., Jiang X., Russo A., Casella F., Renis M., and Bindoni M., 1995, Effects of ozone on some
biological activities of cells in vitro, Cell Biol. Toxicol. 11:11-21.
Carette, S., Leclaire R., Marcoux S., Morin F., Blaise G. A, St.-Pierre A , Truchon R , Parent F.,
Levesque J., Bergeron V., Montm iny P., and Blanchette c., 1997, Epidural corticosteroid injections
for sciatica due to hem iated nucleus pulposus, N Engl . J. Med. 336 :1634-1640 .
Carlsson, L. M., Jonsson J., Edlund T, and Marklund S. L., 1995, Mice lacking extracellular superoxide
dismutase are more sensitive to hyperoxia, Proc . Nat. Acad. Sei. USA 92 :6264-6268 .
Carmeliet, P., and Jain R K., 2000, Angiogenesis in cancer and other diseases, Na/ure 407 :249-257 .
Carpendale, M. T , Freeberg J., and McLeod Griffiss J., 1993, Does Ozone alleviate AIDS diarrhea? ,.I.
Clin. Gastroenterol . 17:142-145 .
Carpendale, M. T F., and Freeberg J. K., 1991, Ozone inactivates HIV at noncytotoxic concentrations,
Anttvir . Res. 16 :281-292 .
Carr , A., and Frei B., 1999, Does vitamin C act as a pro-oxidant under physiological conditions", FAS ER
J .13:1007-1024.
Carta, A., Braccio L., Belpoliti M., Soliani L., Sartore F., Gandolfi S. A., and Maraini G., 1998, Self-
assessment of the quality of vision : association of questionnaire score with objecti ve c1inical tests ,
CW7'. Eye Res. 17:506-511.
Cassatella, M. A, 1995, The production of cytokines by polymorphonuclear neutrophils , Immunol. Todav
16:21-26.
Cassileth, B. R., and Chapman C. c., 1996, Alternative and complementary cancer therapies, Cancer
77 :1026-1034 .
Cassileth, B. R., Lusk E. J., Guerry D ., Blake A. D., Walsh W. P., Kascius L., and Schultz D. J., 1991,
Survival and quality oflife among patients receiving unproven as compared with conventional cancer
therapy , N. Engl. J. Med.324 :1180-1185.
Ceballos-Picot, 1., Merad-Boudia M., Nicole A., Thevenin M., Hellier G., Legrain S., and Berr C; I 996a,
Peripheral antioxidant enzyme activities and selenium in elderl y subjects and in dementia of
Alzheimer's type-place ofthe extracellular glutathione peroxidase, Free rad. Biol. Med. 20:579-587 .
Ceballos-Picot, 1., Witko-Sarsat V., Merad-Boudia M., Nguyen A. T , Thevenin M., Jaudon M. c.,
Zingraff J., Verger c., Jungers P., and Descamps-Latscha B., 1996b, Glurathione anti oxidant system
as a marker of oxidative stress in chronic renal failure , Free rad. Binl. Med. 21:845-853.
Ceccherelli, F., Gagliardi G., Matterazzo G., Rossato M., and Giron G., 1995, La riflessoterapia per
agopuntura, in La riflessoterapia per agopuntura (P . Procacci, A. Di Massa, F. Ceccherelli, and R.
Casale , Eds.), Edizioni AI.RAS. , Padova , pp.49-77 .
Cerny, A, and Chisari F. V., 1999, Pathogenesis of chronic hepatitis C: immunological features of
hepatic injury and viral persistence, Hepatology 30 :595-601.
Chae, H. Z., Kim K., and Kim I.-H., 1999, The novel antioxidant enzyme, thioredoxin peroxidase, and
mammalian peroxiredoxins, in Redox regulotion of cell signaling and its clin ical appliration (L.
Packer, and J. Yodoi , Eds.), Marcel Dekker, Inc., New York, pp.85-9 2.
 REFERENCES 389

Chanock, S . J., EI Benna J., Smith R..M ., and Babior B . M., 1994 , The respiratory burst oxidase,.!. Bio/.
Chem. 269 :24519-24522 .
Chen , K. Y., Ko S. C ., Hsueh P. R., Luh K. T., and Yang P. C ., 2001 , Pulmonary fungal infection:
emphasis on microbiological spectra, patient outcome, and prognostic factors, Chest120 :177 -184.
Chen, L. c., and Qu Q ., 1997, Formation of intracellular free radicals in guinea pig airway epithelium
during in vitro exposure to ozone, Toxicol. Appl. Pharmacol. 143:96-101.
Chen, Q . M ., Tu V . c., Wu Y., and Bahl J. J., 2000a, Hydrogen peroxide dose dependent induction of cell
death or hypertrophy in cardiomyocytes, Arch . Biochem . Biophys . 373 :242-248.
Chen, Z., Oberley T . D., Ho Y., Chua C. C., Siu S ., Hamdy R. c., Epstein C . 1., and Chua B. H., 2000b,
Overexpression of CuZnSOD in coronary vascular cells attenuates myocardial ischemia/reperfusion
injury, r-e« rad. Biol. Med . 29:589-596 .
Cheng, K. N ., Pacy P. 1., Dworzak F., Ford G . C; and Halliday D., 1987, Influence of fasting on leueine
and muscle protein rnetabolism across the human forearm determined using L-[l -13C,15N]leucine as
the tracer, Clin. Sei. 73 :241-246 .
Cherkin, D. C ., Deyo R. A., Batt ie M ., Street J., and Barlow W ., 1998, A comparison ofphysicaltherapy,
chiropractic manipulation, and provision of an educational booklet for the treatment of patients with
low back pain, N. Engl . J. Med. 339 :1021-1029 .
Chevion, S .. and Chevion M., 2000, Antioxidant status and human health. Use of cyclic voltammetry for
the ev;luation of the antioxidant capacity of plasma and of edibleo plants, Ann. N. Y. Acad. Sei.
899 :308-325 .
Chew, E. Y., 1995, Nutritional supplement use and age-related macular degeneration, Curr. Opin.
Ophthalmol. 6: 19-24 .
Chiechanover, A., and Schwartz A . L., 1998, The ubiquitin-proteasome pathway: the complexity and
myriad functions ofproteins death, Proc. Nat. Acad. Sei. USA 95 :2727-2730 .
Cho , H. Y., Zhang L. Y., and KJeeberger S . R., 2001 , Ozone-induced Jung inflammation and
hyperreactivity are mediated via tumor necrosis factor-alpha receptors, Am. .1. Physiol Lung Cell Mol.
PhysioI280 :L537-L546.
Chong, B . H., 1995 , Heparin-induced thrombocytopenia, BI'. J. Haematol. 89:431-439 .
Chow, C. K., and Kaneko J. J., 1979, Influence of dietary vitamin E on the red cells of ozone-exposed
rats, Environ. Res. 19:49-55.
Chow, C. K., and Tappel A. L., 1973 , Activities of pentose shunt and glycolytic enzymes in lungs of
ozone-exposed rats., Arch . Environ . Health 26:205-208.
Choy, E. H., and Panayi G . S., 2001 , Cytokine pathways andjoint inflammation in rheumatoid arthritis,
N. Eng/. J. Med. 344 :907-916 .
Christian, D. L. , Chen L. L. , Scannell C. H ., Ferrando R. E., Welch B . S ., and Balmes 1. R., 1998, Ozone-
induced inflammation is attenuated with multiday exposure, Amer. J. Respir. Crit. Care Med.
158:532-537 .
Christie, B., 2000, Doctors revise declaration of Helsinki, Brit. Med. J. 321 :9 13.
Chua, C. C., Hamdy R. C., and Chua B. H. L., 1998 , Upregulation of vascular endothelial growth factor
by H,02 in rat heart endothelial cells, Free rad. Biol. Med. 25:891-897 .
Chun, T . W ., Davey R. T ., Jr., Engel D., Lane H. C ., and Fauci A. S., 1999, Re-emergence ofHIV after
stopping therapy, Nature 401 :874-875 .
Chun, T . W ., and Fauci A. S ., 1999 , Latent reservoirs of HIV : obstacles to the eradication of virus, Proc.
Not!. Acad. Sei. U. S. A 96:10958-10961.
Cines, D. B., Pollak E. S ., Buck C. A. , Loscalzo J., Zimmerman G. A., McEver R. P., Pober 1. S., Wick
T . M ., Konkle B. A ., Schwartz B. S ., Bamathan E. S., McCrae K. R., Hug B . A. , Schmidt A. M. , and
Stern D . M., 1998, Endothelial cells in physiology and in the pathophysiology of vascular disorders,
Blood 91 :3527-356 1.
Cinnella, P., and Brayda-Bruno M ., 2001 , La nostra esperienza nel trattamento dei conflitti disco-
radicolari e delle radicolopatie post-chirurgiche con ossigeno-ozono terapia infiltrativa
paravertebrale, Riv. Neuroradial. 14:75-79.
Ciocca, D. R., Oesterreich S., Chamness G. c., McGuire W . L., and Fuqua S . A., 1993 , Biological and
c1inical implications of heat shock pro tein 27 ,000 (Hsp27): a review,.f. Natl. Cancer Inst. 85 :1558 -
1570 .
Clemens, M . R., and Waller H. D ., 1987, Lipid peroxidation in erythrocytes, Chem. Phys. Lipids 45 :251-
268 .
390 REFERENCES

Clerici, M., Bevilacqua M., Va go T ., Villa M. L., Shearer G. M ., and Norbiato G., 1994, An
immunoendocrinological hypothesis 01' HIV infection, Lancet 343 :1552 -1553 .
Clerici, M., and Shearer G. M ., 1993, A THI TH2 switch is a critical step in the etiology 01' HIV infection,
Immunol. Today 14 :107-111.
Clerici, M., and Shearer G. M., 1994 , The Th 1-Th2 hypothesis 01' HIV infection : new insights, Immunol,
Today 15 :575-581.
Clinton, S. K., 1998, Lycopene: chemistry, biology, and implications for human heallh and disease, Null'.
Rev. 56:35-51.
Cocchi, F., DeVico A. L., Garzino-Demo A., Arya S . K., Gallo R. C ., and Lusso P., 1995 , Identification
01' IV\NTES, MIP-I alpha, and MIP-I beta as the major HIV -supperessi ve factors produced by CD8 +
Tcells, Science 270 :1811-1815 .
Cohen, M. V ., and Downey J. M., 1996, Myocardial preconditioning promises to be a novel approach to
the treatment 01' ischemic heart disease, Annu. Rev. Med. 47 :21-29.
Cohen, S. M., Olin K. L., Feuer W. J., Hjelmeland L., Keen C L., and Morse L. S , 1994, Low
glutathione reductase and peroxidase activity in age-related macular degeneration, Brit. .I.
Ophthalmol. 78 :79 1-794.
Coleman, C N ., 1988 , Hypoxia in tumors : a paradigm for the approach to biochemical and physiologic
heterogeneity, J. Natl . Cancer Inst, 80:310-317 .
Coleman, R. E., 1992 , Glucocorticoids in cancer therapy, Biotherapy 4:37-44.
Comporti, M ., 1998a, Lipid peroxidation and biogenic aldehydes: from the identification 01' 4-
hydroxynonenal to further achievements in biopathology, Free rad. Res. 28 :623-635 .
Comporti, M., 1998b , Lipid peroxidation as a mediater 01' chemical-induced hepatocyte death, in
Toxicology ofthe liver (G . L. Plaa, and W . R. Hewitt, Eds .), Taylor & Francis, pp.221-257.
Cooke, E. D., Pockley A. G ., Tucker A. T., Kirby J. D. T., and Bollon A. E., 1997, Treatment 01' se vere
Raynaud's syndrome by injection 01' autologous blood pretreated by heating, ozonati on and exposure
to ullraviolet light (H-O-U) therapy, Int. Angiol. 16:250-254.
Copello, M., Egula F., Menendez S ., and Menendez N., 2001, Ozone therapy in patients with retinitis
pigmentosa, in Proceedings of the 151h Ozone World Congress, London, UK, l lth-lSth September
20111, Medical Therapy Conference 00,.1. 2001 , Ed.), Speedprint MacMedia LId, Ealing, London ,
UK ,pp.117-130.
Coppola, L., Giunta R., Verrazzo G., Luongo C, Samrnartino A , Vicari o c., and Giugliano D., 1995 ,
Influence 01' ozone on haemoglobin oxygen affinity in type-2 diabetic patients with peripheral
vascular disease: in vitro studies, Diabete Metab. 21 :252-255.
Coppola, L., Verrazzo G., Giunta R., and Luongo c., 1992, OxygenJozone therapy and haemoreological
parameters in peripheral chronic arterial occlusive diseases, Thromb. Atheroscler. 3 :83-89.
Corley, P. A , 1995 , HIV and the cortisol connection: a feasible concept 01' the process 01' AIDS , Med.
Hypotheses 44:483-489.
Cosentino, R., Manca S., De Stefano R., Frati E., Harnmoud M ., ManganeIli 5., and Marcolongo R.,
2000 , Efficacia dell'ozonoterapia nella sindrome fibromialgica, in Proceedings: I Cong resso IMO.\'.
Ital ia, Siena. 2-4 novembre 2000 , p.30 .
Cotgreave, I. A., and Gerdes R. G., 1998 , Recent trends in glutathione biochernistry. Glutathione-protein
interactions: a rnolecular link between oxidative stress and cell proliferation?, Biochem. Biophvs. Res .
C0/l1mun.242 :1-9 .
Courbat, R., Urfer D., Wallher J. L., and Mironova T. A., 2001 , Optimisation 01' disinfection with ozone
at full-scale in Nizhny No vgorod, Russia, in Proceedings of the 151h Ozon e World Congress,
London, UK, Illh-15lh September 2001 , Vohtme 1(10,.1. 2001 , Ed .), Speedprint Mac Media LId.
Ealing, London, UK, pp .235-249.
Cracowski, J. L., Devillier P., Durand T., Stanke-Labesque F., and Bessard G., 2001 , Vascular biology 01'
the isoprostanes,.1. Vase. Res . 38 :93-103 .
Criegee, R., 1975, Mechanisms 01' ozonolysis, Angew. Chem. 111/. Ed. 14:745-75 2.
Crile, G., 1963 , The effects 01' heat and radiation on cancers implanted on the feet 01' mice, Cancer Res.
23 :372-380 .
Cross, C. E., Motelunk P. A., Bruener B. A ., Jones D. A., Kaur H., Ames B. N, and Halliwell B, 1992a.
Oxidative damage to plasma constituents by ozone, FEHS Lell . 298 :269-272 .
Cross, C. E., Reznick A. Z., Packer L., Davis P. A ., Suzuki Y. J., and Halliwell B., 1992b, Oxidative
darnage to human plasma proteins by ozone, Free rad. Res . Commun. 15:347-352.
 REFERENCES 391

Cruickshanks, K . J., Klein R., and Klein B. E ., 1993, Sunlight and age-related macular degeneration . The
Beaver Dam Eye Study, Arch. Ophthalmol. 111 :5 14-5 18.
Cru tzen , P. J., 1971, Ozone production rates in an oxygen, hydrogen, nitrogen-oxide armosphere, J.
Geophys. Res . 76:7311 -7327 .
Crystal, R. G., 1995, Transfer of genes to humans: early lessons and obstacles to success, Science
270 :404 -410 .
Csermely, P., Schnaider T., Soti C. , Prohaszka Z., and Nardai G., 1998 , The 90-kDa molecular chaperone
family : structure, function , and c1inical applications. A comprehensive review, Pharmacol. Ther.
79 :129-168.
Csonka, c., Pataki T., Kovacs P., Muller S. L., Schroeter M. L., Tosaki A., and Blasig I. E., 2000, Effects
of oxidative stress on the expression of antioxidative defense enzymes in spontaneously hypertensive
rat hearts, Fl'ee rad. Biol. Med. 29 :612-619.
Csonka, c., Varga E ., Kovacs P., Ferdinandy P., Blasig I. E., Szilvassy Z., and Tosaki A., 1999 , Heme
oxygenase and cardiac function in ischemic/reperfused rat hearts, Free rad. Biol. Med. 27 :119-126.
Cummings, K. 1., Lee S. M ., West E. S., Cid-Ruzafa J ., Fein S. G., Aoki Y., Sulkowski M . S., and
Goodman S. N ., 2001, Interferon and ribavirin vs interferon alone in the re-treatment of chronic
hepatitis C previously nonresponsive to interferon : A meta- analysis of randomized trials, .lAMA
285 :193-199.
Cummins, R. 0 ., 1994, Textbook ofadvanced cordtue lije suppon, Scientific Publishing American Heart
Association, Dallas,Tx.
Curran, S. F., Amoruso M . A., Goldstein B. D., and Berg R. A. , 1984, Degradation of soluble collagen by
ozone or hydroxyl radicals, FEBS Lett. 176 :155-160 .
Curtin, B. J., 1985, The myoptas, Harper & Row Publishers, Philadelphia.
Curtis-Prior , P., Vere D., and Fray P., 1999 , Therapeutic value of Ginkgo biloba in reducing symptoms of
decline in mental function,J. Pharm. Pharmacol. 51 :535-541.
Cuzzocrea, S., Tan D. X ., Costantino G ., Mazzon E., Caputi A. P., and Re iter R. J., 1999, The protective
role of endogenous melatonin in carrageenan-induced pleurisy in the rat, FASEB J. 13 :1930-1938.
D'Am ico, D. 1., 1994, Diseases ofthe retina, N. Engl . .I. Med. 331 :95-106.
Dale, J. 1., Ruckley C. V. , Harper D. R., Gibson B., Nelson E. A. , and Prescott R. J., 1999 , Randomised,
double blind placebo controlled trial of pentoxifylline in the treatment of venous leg ulcers, Brit.
Med. J. 319 :875-878.
Darzins, P., Mitchell P ., and Heller R . F., 1997, Sun exposure and age-related macular degeneration . An
Australian case- control study, Ophthalmology 104 :770-776 .
Das , D ., Bandyopadhyay D., Bhattacharjee M., and Banerjee R. K., 1997 , Hydroxyl radical is the major
causative factor in stress-induced gastric ulceration, Free rod. Biol. Med. 23 :8-18 .
Daschner, F. D., 1997, Hepatitis C and human immunodeficiency virus infeetion following ozone
autohaemotherapy, EIII'. J. Clin. Mtcrobiol. Infe ct. Dis . 16:620 .
Davey, R. T., Jr ., Murphy R. L., Graziano F. M ., Boswell S. L., Pavia A. T ., Caneio M ., Nadler J . P.,
Chaitt D . G ., Dewar R. L., Sahner D. K., Duliege A. M. , Capra W . B ., Leong W . P., Giedlin M. A.,
Lane H. C., and Kahn J. 0., 2000, Immunologie and virologic effects of subcutaneous interleukin 2
in combination with antiretroviral therapy: A randomized controlled trial, .lAMA 284: 183-189.
Davies , A ., 2000, The practical management ofclaudication, Brit. Med. .I. 321 :9 11-9 12.
De La Paz, M. A., Zhang J., and Fridovich 1., 1996, Red blood cell antioxidant enzymes in age-related
macular degeneration, BI'. .I. Ophthalmol. 80 :445-450.
De Maio, A., 1999, Heat shock proteins : facts, thoughts, and dreams, Sho ck II :1-12 .
De Maria, N ., Colantoni A., Fagiuoli S ., Liu G.-1., Rogers B. K., Farinati F., van Thiel D. H., and Floyd
R. A., 1996, Association between reactive oxygen species and disease aetivity in chronic hepatitis C,
Free rad. Biol. Med. 21 :291-295.
De Monte, A ., and van der Zee H., 2001, Ozone auto-haemotherapy in lower limb ulcerations, in
Proceedings of the 15th Ozone World Congress, l.ondon, UK, l lth-LSth September 20(H. Medical
Therapy Conference (lOA 2001 , Ed.), Speedprint MacMedia LId, Ealing, London, UK, pp .148 -157 .
de Zwart, L. L., Meerman 1. H. N ., Commandeur J. N . M., and Verrneulen N. P. E., 1999, Biomarkers of
free radical damage applications in experimental animals and in humans, Free rod. Biol. Med.
26 :202 -226.
DeForge, L. E., Fantone 1. c., Kenney J. S ., and Remick D . G., 1992 , Oxygen radical scavengers
selectively inhibit interleukin 8 production in human whole blood,.I. Clin. lnvest. 90:2123-2129 .
392 REFERENCES

Dei Bello, B., Paolicchi A., Comporti M., Pompella A., and Maellaro E., 1999 , I-Iydrogen peroxide
produced during gamma-glutamyl transpeptidase activity is involved in prevention of apoptosis and
maintainance ofproliferation in U937 cells, FASEB.I. 13 :69-79 .
Dei Corso, A., Dal Monte M ., Vilardo P. G., Cecconi 1., Moschini R., Banditelli S., Cappiello M ., Tsai L.,
and Mura U., 1998 , Site-specific inactivation of aldose reducrase by 4-hydroxynonenal, Arch.
Biochem. Biophys. 350:245-248.
Dei Principe, D., Menichelli A., De Matteis W. , Di Corpo M. L., Di Giulio S., and Finazzi -Agro A. ,
1985 , Hydrogen peroxide has a role in the aggregation ofhuman platelets, F EBS Lell . 185 :142-146 .
DeLange, R. J., and Glazer A. N., 1989, Phycoerythrin fluorescence-based assay for peroxy radicals : a
screen for biologically relevant protective agents, Anal. Biochem. 177 :300-306.
Delgado, J., 1991, Tratamiento con ozono dei herpes zoster, CENIC Ciencias Biologicas 20 :) 60-162.
DeMartino, G. N ., and Slaughter C. A., 1999, The proteasome, a novel protease regulated by multiple
mechanisms,.J. Biol. Chem. 274 :22123-22126.
Deng , 1-1., Liu R., Ellmeier W., Choe S., Unurmaz D., Burkhart M ., Di Marzio P., Mannon S., Sutton R.
E., Hili C. M ., Davis C. B., Peiper S. c., Schall T. J., Littman D . R., and Landall N . R., 1996 ,
Identification of a major co-receptor for prirnary isolates of BIV-I , Nature 38 t :66 1-666.
Dennog, c., Hartmann A., Frey G., and Speit G., 1996, Detection of DNA darnage after hyperbaric
oxygen (I-IBO) therapy, Mutagenesis II :605-609.
Desikan, R., Neill S . J., and I-Iancock J. T., 2000, I-Iydrogen peroxide-induced gene expression in
Arabidopsis thaliana, Free rad. Biol. Med. 28 :773-778.
Devlin, R. B., McDonnell W . F., Mann R., Becker S., House D. E., Schreinemachers D., and Koren H. S.,
1991, Exposure of humans to arnbient levels of ozone for 6.6. hours causes cellular and biochemical
changes in the lung , Amer.. .J. Respir. Cell Molec. Biol. 4 :72-81.
Di Mascio, P., Kaiser S., and Sies H., t 989 , Lycopene as the rnost efficient biological carotenoid singlet
oxygen quencher, Arch. Biochem. Biophys. 274 :532-538 .
Di Paolo, N ., Bocci V ., Garosi G., Borrelli E., Bravi A ., Bruci A., Ald inucci C; and Capetondo L., 2000,
Extracorporeal blood oxigenation and ozonation (EBOO) in man . Preliminary report. , Im . .I. An!1.'
Organs 23 :131-141.
Diadori, A., Bocci V., Carraro F., Nuti A., Corradeschi F., Ferrari G ., Sabatini L., Sil vestri S., and
Frezzotti R., 1999, Ozonetherapy and age related macular degeneration : a pilot study. in
L'Ozonoterapia nel 2000 (F. Ceccherelli , and F. Giron, Eds .), Edizioni Libreria Cortina, Torino,
pp.33-45.
Diadori, A., Nuti A., Ferrari G., Corradeschi F., and Bocci V., 1996, Ozone therapy: a new perspective in
ophthalmology., Vision Res . 36(suppl.):418.
Dianzani, F., 1999, Chronic hepatitis B, biological basis for new therapeutic strategies, .I. Biol. Regulat.
Homeost. Agent. 13 :71-79 .
Dianzani, F., Santiano M., Ramenghi U., CapoBianchi M . R., and Antonelli G., 1985, Membrane events
leading to interferon-g induction by antigens, Proc. Soc. Exp. Biol. Med. 178 :139-142.
Dianzani, M. U., 1993, Lipid peroxidation and cancer, CI';' Rev. Oncol. Hematol . 15:125-147.
Dianzani, M . U., 1998, 4-Bydroxynonenal and cell signalling, Free rad. Res . 28 :553-560.
Diaz, S., Menendez S., Eng L., and Femandez 1., 1995 , No increase in sister chromatid exchanges and
micronuclei frequencies in human Iymphocytes exposed 10 ozone in vitro, in Proceedings Ozone in
Medicine 12th World Congress ofthe International Ozone Association. 15th /0 l Sth May 1995 , Lilie
France (International Ozone Association, Ed .), Instaprint S.A. , Tours, pp .43-52 .
Didier, C ., Pouget J. P., Cadet J., Favier A., Beani J. c., and Richard M. J., 2001, Modulation of
exogenous and endogenous levels of thioredoxin in human skin fibroblasts prevents DNA damaging
effect of ultraviolet A radiation, Free rad. Biol . Med. 30 :537-546.
Dinarello, C. A. , 1999, IL-18 : a Tln-inducing, proinflammatory cytokine and new member of the IL-I
family.v, Allergy C1in. Immunol. 103 :11-24 .
Dismukes, G. c., Klimov V. V ., Baranov S . V., Kozlov Y. N ., DasGupta J., and Tyryshkin A ., 2001 , The
origin of atmospheric oxygen on Earth : the innovation of oxygenic photosynthesis, Proc. Na/I. Acad.
Sei. U. S. A 98 :2170-2175.
Divincenzo, G. D., Hamilton M. L., Mueller K. R., Danish W. 1-1 ., and Barber E. D., 1985, Bacterial
mutagenicity testing of urine from rats dosed with 2-ethylhexanol derived plasticizers, Toxicologv
34 :247 -259 .
Dockrell, H. M ., and Playfair J. H., 1983 , Killing of blood-stage murine malaria parasites by hydrogen
peroxide,ln/ect. Immun. 39 :456-459.
 REFERENCES 393

Dong, Z., Lavrovsky Y. , Venkatachalam M. A., and Roy A. K ., 2000, Heme oxygenase-I in tissue
pathology: the Yin and Yang , Am . J. Patho/. 156 : 1485-1488.
Dormandy, J. A, and Rutherford R. S ., 2000, Management of peripheral arterial disease (PAD). TASC
Working Group. TransAtlantic Inter-Society Concensus (TASC),J. Vase. Surg. 31 :SI-S296.
Doroshow,1. H., 1995, Glutathione peroxidase and oxidative stress, Toxicol. Leu. 82/83 :395-398.
Drago, F., Marino V ., and Marino A ., 1993, Neuroprotezione e danno neuronale, Boll . Agg . Farm .
Oculare :5.
Draper, H. H., Csallany A S., and Hadley M ., 2000, Urinary aldehydes as indicators of lipid peroxidation
in vivo, Free rad. Biol. Med . 29:1071-1077.
Dreher, D., and Junod A F., 1996 , Role of oxygen free radicals in cancer development, Eur. J. Cancer
32A :30-38.
Drew, S., and Davies E., 2001, Effectiveness of Ginkgo biloba in treating tinnitus: double blind, placebo
controlled trial, Brit. Med. J. 322:73 .
Drury, J. A, Nycyk J. A, and Cooke R. W. 1., 1997, Pentane measurement in ventilated infants using a
commercially available system, Free rod. Bio/ . Med. 22 :895-900 .
Du Bois, A. 8. , 1962 , Oxygen toxicity, Anestesiology 23:473-477 .
Duda, P. W., Schmied M. c., Cook S. L., Krieger J. 1., and Hatler D. A., 2000, G1atiramer acerate
(Copaxone) induces degenerate, Th2-polarized immune responses in patients with multiple sclerosis,
J. Clin . Invesll05:967-976 .
Dumaswala, U. J., Wilson M. J., Wu Y. L., Wykle J., Zhuo L., Douglass L. M., and Daleke D. L., 2000,
G1utathione loading prevents free radical injury in red blood cells after storage, Free rod. Res .
33 :517-529 .
Dumaswala, U. 1., Zhuo L., Jacobsen D. W., Jain S. K., and Sukalski K. A, 1999, Protein and lipid
oxidation ofbanked human erythrocytes : role of glutathione, Free rad. Biol. Med . 27 :1041-1049.
Durelli , L., Cocito A , and Riccio A ., 1985, High-dose intravenous methylprednisolone in the treatment of
multiple sclerosis. Clinical-immunologic correlations, Neurology 36 :238-243 .
Dusheiko, G . M., 1995 , Treatment and prevention of chronic viral hepatitis, Pharmacol. Ther. 65:47-73 .
Dworkin, R. H., 1999 , Prevention of postherpetic neuralgia, Lancet 353 :1636-1637.
Ehhalt , D. H., 1999, Tropospheric ozone, chemistry and man -induced trends, in Proceedings of the Int.
Ozone Symposium, 21 and 22 October 1999. Basel, Switzerland (IOA - EA 3G Ed .), Bauer Druck
AG, Basel, pp .89-105 .
Eisenberg, D. M., Davis R. S ., Ettner S. L., Appel S., Wilkey S., Van Rompay M ., and Kessler R. C .,
1998, Trends in alternative medicine use in the Uni ted States, 1990-1997: results of a follow-up
national survey,JAMA 280 :1569-1575.
Eisenberg, D . M., Kessler R. c., Foster c ., Norlock F. E., Calkins D. R., and Delbanco T. L., 1993,
Unconventional medicine in the Uni ted Stares . Prevalence, costs, and patterns of use ., N. Engl . J.
Med . 328 :246-252 .
Elgert, K. D., Alleva D. G ., and Mullins D. W ., 1998, Tumor-induced immune dysfunction: the
macrophage connection, J. Leukocyte Biol . 64 :275-290.
Elliott, M . J., Maini R. N ., Feldmann M., Kalden J. R., Antoni c., Smolen J. S., Leeb S., Breedveld F. c.,
Macfarlane J. D ., Bijl H. , and Woody J. N., 1994, Randomised double-blind comparison of chimeric
monoclonal antibody to tumour necrosis factor a (cA2) versus placebo in rheumatoid arthritis, Lancet
344: 1105-1110.
Ellis , R. 1., and van der Vies S. M., 1991, Molecular chaperones, Annu. Rev . Biochem. 60 :321-347.
Engelstein, E. D., and Zipes D. P., 1998, Sudden cardiac death, in The heart., arteries and veins (R. W.
Alexander, R. C. Schlant, and V. Fuster, Eds .), McGraw-HiIl, New York,NY, pp .1081-1112.
Enserink, M., 1999, Can the placebo be the cure?, Science 284:238-240.
Ernst , A., and Zibrak 1. D., 1998, Carbon monoxide poisoning, N. Engl. J. Med. 339 :1603-1608.
Ernst , E., 1996a, Complementary medicine: from quackery to science?, J. Lab . Clin . Med. 127:244-245 .
Ernst , E., 1996b, The power ofplacebo, Brit. Med . J. 313 :1569-1570.
Ernst , E., 1997, Thymus therapy for cancer? A criteria-based, systematic review, EIII'. J. Cancer 33:531-
535 .
Ernst, E., 2000, The role ofcomplementary and alternative medicine, Br/l. Med. J. 321 :1133-1135.
Ernst, E., 2001 , Mistletoe for cancer", Bur. J. Cancer 37 :9-11.
Ernst , E., and Cohen M. H., 2001, Informed consent in complementary and alternative medicine, Arch.
Intern . Med. 161 :2288-2292.
 394 REFERENCES

Ernst , E., and Resch K. L., 1996 , Evaluating specific effectiveness of complementary therapies - a
position paper, Forsch. Komplement ärmed. 3 :35-38.
Ernst, E., and White A., 1997, Acupuncrure: safety first, Brit. Med. .I. 314 :1362.
Escher, M., Desmeules 1., Giostra E., and Mentha G ., 2001, Hepatitis associated with Ka va, a herbal
remedy for anxiety, Brit . Med. .I. 322 :139.
Estep, N. T ., Pedersen R. A., Miller T. 1., and Stupar R. K., 1984, Characterization of erythrocyte quality
during the refrigerated storage of whole blood containing di-(2-ethylhexyl) phthalate, Blood 64 :1270-
1276 .
Esterbauer. H., 1996 , Estimation ofperoxidative darnage. A critical review, Pothol. Biol. 44 :25-28 .
Esterbauer. H., Schaur R. J., and Zollner H., 1991, Chemistry and biochemisrry of 4-hydroxynonenal,
malonaldehyde and related aldehydes, Free rad. Biol. Med. 11:8 1- 128 .
European Pharmacopoeia, 1997, pp.153-167.
Evans, H., Bauer M., Luckman 1., and Page M., 2001 , An assessment of the benefits afforded by the
continuous versus intermittent operation of ozone for drinking water treatment, in Proceedings ofthe
15/h Ozone Wor/d Congress. London , UK. llth-15/h Sept ember 2001, Volume I (IOA 2001 , Ed .),
Speedprint MacMedia LId, Ealing, London, UK , pp .219-234.
Evans, 1., and Wormald R., 1996, Is the incidence of registrable age-related macular degenernnon
increasing?, Br. .I. Ophthalmol. 80 :9-14.
Evans, M. D., and Pryor W. A., 1994 , Cigarette smoking, ernphysema, and darnage to ar-proteinase
inhibitor, Amer . J. Phys iol. 266 :L593-L611.
Ezekowitz, R. A., Mulliken 1. 8. , and Folkman J., 1992 , Interferon alfa-2a therapy for life-threatening
hemangiomas ofinfancy, N Eng!. .I. Med. 326 :1456-1463 .
Fabris, G ., Tonunasini G., Petralia B., Lavaroni A, De Nardi F., De Luca G., Biasizzo E., and laiza F.,
2001, L'ossigeno-ozono terapia intra-foraminale, Riv. Neuroradiol. 14:61-66 .
Färber, 1. L., Kyle M. E., and Colernan J. B., 1990 , Biology of disease . Mechanisms of cell injury by
activated oxygen species, Lab. Invest . 62 :670-679 .
Farci, P., Shimoda A , Coiana A , Diaz G ., Peddis G., Melpolder J. c., Strazzera A., Chien D. Y., Mun oz
S. J., Balestrieri A ., Purcell R. H., and Alter H. J ., 2000, The outcorne of acute hepatitis C predicted
by the evolution ofthe viral quasispecies , Science 288 :339-344.
Farr, C. H., 1993, Protocol for the intravenous administration of hydrogen peroxide, International Bio-
Oxidative Medicine Foundation, Oklahoma City, pp .29-31 .
Fauste, N., Laird A. D., and Webber E. M., 1995, Role of growth factors arid cytokines in hepatic
regeneration, FASEB .1. 9:1527-1536.
Feldmann. M., and Maini R. N ., 2001 , Anti-TNF alpha therapy of rheurnatoid arthritis : wha t have we
learned?, Annu. Rev.Tmmunol. 19:163-196.
Fellay, J., Boubaker K., Ledergerber B., Bernasconi E., Furrer H., Bauegay M., Hirsche) B., Vernazza P..
Francioli P., Greub G., Flepp M., and Telenti A., 2001, Prevalence ofadverse events associat ed with
potent antiretroviral treatment: Swiss HIV Cohort Study, Lancet 358:1322 -1327.
Feng, Y., Broder C. c., Kennedy P. E., and Berger E. A , 1996 , HIV -I entry cofactor: functional cDNA
cloning of a seven-transmembrane, 0 protein-coupled receptor, Science 272 :872-877 .
Ferrara, N ., and Davis-Srnyth T., 1997, The biology of vascular endothelial growth factor, Endocr. Rev.
18 :4-25.
Ferrara, N ., Houck K., Jakernan L., and Leung D. W ., 1992, Molecular and biological properties of the
vascular endothelial growth factor family of proteins, Endocrine Rev, 13: 18-32 .
Ferri , A., Gabbianelli R., Casciati A., Celsi F., Rotilio G., and Carri M . T., 2001 , Oxidative inactivation
of calcineurin by CU,Zn superoxide dismutase 093A, a mutant typical of familial arnyotrophic lateral
sclerosis,.J. Neurochem. 79 :531-538 .
Fetner, R. H., 1958 , Chromosorne breakage in Vicia faba by ozone, Nature 181:504-505.
Fetner, R. H., 1962 , Ozone-induced chromosome breakage in human cell cultures, Na/ure 194:793-794.
Fields, H . L., 1986, La generazione ectopica di impulsi negli afferenti primari, in n dolore: meccanism i di
insorgenza e trattamento terapeutico McOraw-Hill , pp .126-129.
Filippi, A., 2001, The influence of ozonised water on the epithelial wound healing process in the oral
ca vity , in Pro ceedings ofthe 15/h Ozone World Congress. London, UK. l l th-lith Sept ember 20/1/.
Medical Therapy Conference (lOA 2001, Ed .), Speedprint MacMedia LId, Ealing, London, UK,
pp.l09-116.
 REFERENCES 395

Filippi, A ., and Kirschner H ., 1995, Ozoniertes Wasser zur Desinfektion und Prophylaxe in der Zahn-
Mund-Kieferheilkunde, in Ozon-Handbuch. Grundlagen . Prävention. Therapie (E . G. Beck , and R.
Viebahn-H änsler , Eds .), Ecomed, Landsberg, p.V-12.2 I-V-12.2 12.
Fiocchi, C., 1998, Inflammatory bowel disease: etiology and pathogenesis, Gastroenterology 115 :182-
205 .
Fiocchi, C., 1999, From immune activation to gut tissue injury: the pieces of the puzzle are coming
together, Gastroenterology 117: 1238-1241.
FitzGerald, G. A. , and Patrono c., 200 I, The coxibs, selective inhibitors of cyclooxygenase-2, N. Engl. .I.
Med . 345:433-442.
Flach , J., and Seachrist L., 1994, Mind-body meld may boost immunity,.I. Not . CancerInst. 86 :256-258.
Fleischer, AB ., Jr ., 1999 , Treatment of atopic dermatitis: role of tacrolimus ointment as a topical
noncorticosteroidal therapy,.I. Allergy Clin. Immunol. 104 :S 126-S 130 .
Flohe, L., Günzler W . A , and Schock RH., 1973 , Glutatione peroxidase. A selenoenzyme, FEBS Leu.
32 :132-134.
Flohe , L., Brigelius-Flohe R., Saliou c., Traber M. G., and Packer L., 1997, Redox regulation of NF -
kappa B activation, Fsee rod. Biol. Med. 22 : 1115-1126.
Floyd , R. A, 1999, Neuroinflammatory processes are important in neurodegenerative diseases: an
hypothesis to explain the increased formation of reactive oxygen and nitrogen species as major
factors involved in neurodegenerative disease development, Free rad. Biol. Med. 26 :1346-1355.
Foksinski, M., Bialkowski K ., Skiba M., Ponikowska 1., Szmurlo W., and Olinski R., 1999 , Evaluation of
8-oxodeoxyguanosine, typical oxidative DNA darnage. in Iymphocytes of ozone-treated
arteriosclerotic patients, Mutat. Res . 438 :23-27.
Forsmark-Andree, P., Lee C.-P ., Dallner G., and Ernster L., 1997, Lipid peroxidation and changes in the
ubiquinone content and the respiratory chain enzymes of submitochondrial particles, Free rad . Bio/.
Med.22 :391-400.
Forum, 1998, Genes and ozone, Environ. Health Perspect. 106 :1\130-AI31.
Foster, W. M., Brown R. H., Macri K., and Mitchell C. S., 2000, Bronchial reactivity of healthy subjects :
18-20 h postexposure to ozone,.I. Appl. Physiol 89 : 1804-1810.
Framingham Eye Study, 1980, Macular degeneration, SIIIV. Ophthalmol. 24(Suppl.):428-457.
Frarnpton, M. W ., Pryor W. A , Cueto R., Cox C., Morrow P. E., and Utell M. J., 1999 , Ozone exposure
increases aldehydes in epithelial Iining fluid in human lung, Am ..1. Respir. Crit Care Med. 159 :1134 -
1137 .
Fraser, A, McCarthy N ., and Evan G. 1., 1996 , Biochemistry of cell death, Curr. Opin. Neurobiol. 6:71-
80 .
Freeman, B. A., Miller B. E., and Mudd 1. B., 1979, Reaction of ozone with human erythrocytes, in
Assessing toxic effects of environmentul pollutants (S. D. Lee, and J. S . Mudd, Eds .), Ann Arbor
Science Publishers, Ann Arbor, Mich, pp .151-171.
Frei , S ., 1999 , On the role of vitamin C and other antioxidants in atherogenesis and vascular dysfunction,
Pro c. Soc. Exp . Biol. Med. 222 :196-204.
Frei, S ., England L., and Ames S . N ., 1989, Ascorbate is an outstanding antioxidant in human blood
plasma, Proc. Natl. Acad. Sei. U. S. A 86 :6377-6381.
Frei, S ., Forte T. M., Arnes S. N., and Cross C. E., 1991, Gas phase oxidants ofcigareUe smoke induce
lipid peroxidation and changes in lipoprotein properties in human blood plasma. Protective effects of
ascorbic acid , Biochem. .1. 277 ( Pt 1) :133-138.
Fremont, L., 2000, Biologreal effects ofresveratrol , Life Sei. 66 :663-673 .
Fridovich, 1., 1995 , Superoxide radical and superoxide dismutases, Annu. Rev. Biochem. 64 :97-112.
Friedman-Kien, A. E., Eron L. J., Conant M., Growdon W., Badiak R , Bradstreet P. W., Fedorczyk D .,
Trout J. R., and Plasse T. F., 1988 , Natural interferon alfa for ..treatrnent of condylomata acuminata.,
.IAMA-.I. Am. Med. Assn. 259 :533-538.
Friedman, E., 1997, A hemodynamic model of the pathogenesis of age-related macular degeneration, Am .
.J. Ophthalmol. 124 :677-682.
Fuchs, 1., and Kern H., 1998 , Modulation of Uv'-light-induced skin inflammation by D-alpha-tocopherol
and L-ascorbic acid: a c1inical study using solar simulated radiation, Free rad. Biol. Med. 25: 1006-
1012.
Fujimaki , H., 1989 , Impairment of humoral immune responses in mice exposed to nitrogen diox ide and
ozone rnixtures, Environ. Res. 48:211 -217.
396 REFERENCES

Fukagawa, N. K., 1999 , Aging : is oxidative stress a marker or is it causai?, Proc. Soc. Ex». Biol. Med.
222 :293-298 .
Fukunaga, K., Nakazono N ., Suzuki T., and Takama K., 1999, Mechanism of oxidative darnage to fish
red blood cells by ozone, lUBMB. Life 48:631-634 .
Fulle, S., Mecocci P., Fano G., Vecchiet 1., Vecchini A ., Racciotti D., Cherubini A., Pizzigallo E.,
Vecchiet L., Senin U ., and Seal M. F., 2000 , Specific oxidative alterations in vastus lateralis muscle
of patients with the diagnosis of chronic fatigue syndrome, Free rad. Biol. Med. 29 :1252-1259.
Fung, W . E ., 1991, Interferon alpha 2a for treatment of age-related macular degeneration , Amer. .I.
Ophthalmol. 112 :349-350.
Furchgott. R. F., and Zawadzki J. V., 1980, The obligatory role of endothelial cells in the relaxation 01'
arterial sm ooth muscle by acetylcholine, Nature 288 :373-376.
Gabriel.Xl, Blauhut B., Greul R., Schneewels S. , and Roggendorf M., 1996, Transmission 01'hepatitis C
by ozone enrichment ofautologous blood, Lancet 347:541.
Gaetani , G. F., Kirkman H. N., Mangerini R., and Ferraris A. M., 1994, Importance of catalase in the
disposal 01' hydrogen peroxide within human erythrocytes, Blood 84 :325 -330.
Gaetani, G. F., Rolfo M., Arena S., Mangerini R., Meloni G. F., and Ferraris A . M ., 1996 , Active
involvement of catalase during hemolytic crises of favism , Blood 88 :1084 -1088 .
Galbraith, R., 1999 , Heme oxygenase: who needs it", Proc. SO(:. Exp. Biol. Med. 222 :299-305 .
Galleano, M ., and Puntarulo S ., 1995, Role of antioxidants on the erythrocytes resistance to lipid
peroxidation after acute iron overload in rats, Biochim . Biophvs . Acta 1271 :32 1-326.
Galloway, J., 200 I, Gerring the point across, Nature 409:560 -561.
Garber, G. E., Cameron D. W., Hawley-Foss N ., Greenway D., and Shannon M. E., 1991 , The use 01'
ozone-trealed blood in the therapy of H[V infection and immune disease : a pilot study of safety and
efficacy., AIDS 5:98[-984.
Garland, C . J., Plane F., Kemp B. K., and Cocks T. M., 1995 , Endothelium-dependent hyperpolarization :
a role in the control of vascular tone, Trends Pharmacol. Sei. 16:23-30.
Gassmann , M., and Wenger R. H., 1997 , H[F-I , a mediator of the rnolecular response to hypoxia, News
Physiol. Sei. 12 :2[4 -218 .
Gaston, 8. , 1999 , Nitric oxide and thiol groups, Btochim. Biophys . ANa 14J I :323- 333.
Ghiselli, A., Serafini M., Natella F., and Scaccini C .; 2000, Total antioxidant capacity as a tool to assess
redox status: critical viel'.' and experimental data, Fre e rad. Biol. Med. 29: I 106-11 14.
Giannini , c., Giannelli F., Monti M., Careccia G., Marrocchi M. E., Laffi G., Gentilini P.. and Zignego A .
L., 1999, Prevalence of mixed infecton by different hepatitis C virus genotypes in patients with
hepat itis C virus-related chronic liver disease,.I. Lab . Clin. Med. 134:68-73 .
Ginn-Pease, M . E., and Whisler R. L., 1998 , Redox signals and NF -kB activation in T cells, Fre e ra d.
Biol. Med. 25 :346-361.
Giovannucci , E., Ascherio A., Rimm E. S. , Stampfer M. J., Colditz G. A ., and Willen W . c., 1995, Intake
of carotenoids and retinol in relation to risk of prosrate cancer,.J. Na/I. Cancer Inst. 87 :1767 -1776.
Girouard, S ., Levine G., Goodrich K., Jones S., Keyserling H , Rathore M., Rubens C; Williams E., and
Jarvis W ., 200 I, Pediatric Prevention Network: a multicenter collaboration to improve health care
outcomes, Am . .I. Infect. Control 29 :158-161.
Gjonovich, A ., Sattin G. F., Brusomini A., and Preciso G ., 1995 , Effetto dell'ossigeno-ozono terapia sulla
soglia anaerobica dell'atleta: considerazioni preliminari, in Ant "Incontri di terapia antalg ica ".
Monseitee. 30.9.95, pp.230-245 .
Gj ono vich, A ., Sattin G. F., Girotto L., Bordin M., Gallo L., and Preciso G., 2001 , Lombalgie ribelli :
l'ossigeno-ozono terapia a confronto con altre rnetodiche, Riv. Neuroradial. 14:35-38 .
Gleave, M . E., Elhilali M., Fradet Y., Davis 1., Venner P., Saad F., Klotz L. H., Moore M . J., Paton V.,
Saj amonde A., and Canadian Urologie Oncology Group, 1998, Interferon gamma-I b compared with
placebo in rnetastatic renal -cell carcinorna, N. EI/gI. .J. Med. 338:1265-1 271 .
Glynn, S. A., Kleinman S. H., Schreiber G. B., Busch M. P., Wright D. 1., Smith J. W., Nass C. C; and
Williams A. E., 2000, Trends in incidence and prevalence of major transfusion-transmissible viral
infections in US blood donors , 1991 to 1996. Retrovirus Epidemiology Donor Study (REDS), .lAMA
284 :229 -235 .
Goheen , S. c., O'Rourke L., and Larkin E. c., 1986, Ozone and the peroxidation of polyunsatured fatty
acids in vivo, Environ. Res . 40 :47-57 .
Goldman. M., 1996 , Cancer risk oflow-Ievel exposure, Science 271 :[821-1822.
 REFERENCES 397

Goldstein, B . D ., and Balchum O. J., .1967, Effect of ozone on lipid peroxidation in the red blood cell .,
Proc . Soc. Exp . Biol . Med. 126 :356-359.
Gomez Moraleda, M . A., 1995, Ozone therapy in the functional recovery from diseases involving damage
to central nervous system cells, in Pro ceedings Ozone in Medicine 12th World Congress 0/ th
International Ozon e Association , 15th to 18th May 1995, Lille, France. (International Ozone
Association, Ed .), 1nstaprint S .A ., Tours, pp .I II-123 .
Gomez, M., Espinosa E., and Caplan J. A , 1995, Application of medicinal ozone/oxygen in patients with
sickle cell anemia, Townsend Letterfor Doctors January:48-52.
Gonet , B., 1994, Free radical aspects of the ozone influence on blood, Phystol. Chem . Phys. Med . NMR
26 :273-286.
Gooch, P. C., Creasia D. A., and Brewen J. G., 1976 , The cytogenetic effects of ozone: inhalation and in
vitro exposures, Environ. Res. 12: 188-195.
Gorre, M. E., Mohammed M., Ellwood K., Hsu N ., Paquette R., Rao P. N ., and Sawyers C. L., 2001,
Clinical resistance to STI -571 cancer therapy caused by BCR-ABL gene mutation or amplification,
Science 293:876-880 .
Gottlieb. N., 1999 , Cancer treatment and vitamin C: the debate lingers, J. Natl. Cancer Inst. 91:2073-
2075 .
Gornicki. A., and Gutsze A ., 2000, In vitro effects of ozone on human erythrocyte membranes: an EPR
study , Acta Bio chim. Polon . 47 :963-971.
Grady, D., Rubin S. M ., Petitti D. B ., Fox C. S. , Black D ., Ettinger B ., Ernster V. L., and Cummings S.
R., 1992 , Hormone therapy to prevent disease and prolong life in postrnenopausal women, Ann .
Intern. Med . 117 :1016 -1037 .
Grafton, G., and Thwaite L., 2001, Calcium channels in Iymphocytes, Immunology 104 :119-126.
Graham, A, 1998, Cellular thiol producnon and oxidation of low -density lipoprotein, Free rad. Res .
28:611-621.
Graharn , N ., and Paraskeva P., 2001 , Recent studies of ozone disinfection of secondary municipal
effluents, in Proceedings 0/ the 15th Ozone World Congress, London, UK. llth-15th September
20OJ. Volume I(IOA 2001, Ed .), Speedprint MacMedia LId, Ealing, London, UK , pp.276-291.
Grainger, D. J ., Mosedale D. E., and Metcalfe J. c., 2000, TGF-beta in blood: a complex problem,
Cytokine Growth Factor Rev. 11 :133-145.
Greenwald, R. A. , 1990 , Superoxide disrnutase and catalase as therapeutic agents for human diseases . A
critical revi ew ., Fre e rad . Biol . Med . 8 :201-209 .
Gregory, C. Y., and Bird A. c., 1995, Cell loss in retinal dystrophies by apoptosis. -death by informed
consent!, Br. .I. Ophthalmol. 79 :186-190 .
Grisham, M . B ., Granger D. N ., and Lefer D . J., 1998 , Modulation of leukocyte-endothelial interactions
by reactive metabolites of oxygen and nitrogen: relevance to ischemic heart disease, Free rad. Biol.
Med.25:404-433.
Gubitz, G ., and Sandercock P., 2000, Prevention of ischaemic stroke, Brit. Med . .I. 321: 1455-1459 .
Guerrero, A, Torres P., Duran M. T ., Ruiz-Diez B., Rosales M., and Rodriguez-Tudela J. L., 2001 ,
Airborne outbreak of nosocomial Scedosporium prol iticans infection, Lancet 357 :1267 -1268.
Guerrero, R. R., Rounds D. E., Olson R. S., and Hackney J. D., 1979 , Mutagenic etfects of ozone on
human cells exposed in vivo and in vitro based on sister chromatid exchange analysis, Environ. Res .
18:336-346.
Gulick, R. M ., Mellors J. W ., Havlir D., Eron J. J ., Meibohm A., Condra J. H., Valentine F. T., McMahon
D., Gonzalez C ., Jonas L., Emini E. A., Chodakewitz J. A ., Isaacs R., and Richman D. D., 2000, 3-
year suppression of HIV viremia with indinavir, zidovudine, and lamivudine, Ann. Intern . Med.
133 :35-39.
Gutteridge, J. M., and Halliwell B., 1990 , Reoxygenation injury and antioxidant protection: a tale of two
paradoxes, Arch . Biochem. Bioph ys. 283 :223-226 .
Haas, A. F., Wong J. W ., Iwahashi C. K., Halliwell B., Cross C. E., and Davis P. A ., 1998, Redox
regulation of wound healing? NF-kappaB activation in cultured human keratinocytes upon wounding
and the effect oflow energy HeNe irradiation, Free rod. Biol. Med . 25:998-1005 .
Hack , V., Breitkreutz R., Kinscherf R., Rohrer H., Bartsch P., Taut F., Benner A ., and Droge W., 1998 ,
The redox stäte as a correlate of senescence and wasring and as a target for therapeutic intervention,
Blood 92:59-67 .
398 REFERENCES

Haddad , E. B., Salmon M., Koto H., Barnes P. J., Adeeck 1., and Chung K. F., 1996 , Ozone induetion of
cytokine-induced neutrophil chemoartractant (CINC) and nuclear factor-kappa b in rat lung :
inhibition by corticosteroids, FEBS t.eu. 379 :265-268 .
Hahn , M ., Fennerty M . B ., Corless C. L., Magaret N ., Lieberman D. A., and Faigel D. 0 ., 2000 ,
Nonin vasive tests as a substitute for histology in the diagnosis of Helieobacter pylori infection,
Gastraintest. Endosc. 52 :20-26.
Halliwell, B., 1994, Free radicals and antioxidants : a personal view, NlIIr. Rev. 52 :253-265.
Halliwell, B., 1996 , Antioxidants in human health and disease, Annu. Rev. NlIIr. 16:33 -50.
Halliwell, B., 1999a, Antioxidant defence mechanisms: from the beginning to the end (of the beginning),
Free rad. Res. 31:261 -272.
Halliwell, B., I 999b, Vitamin C : poison, prophylactic or panacea", Trends Biochem. Sei. 24 :255-259 .
Halliwell, B., 200 I, Role of free radicals in the neurodegenerative diseases: therapeutic impliealions for
antioxidant treatment, Drugs Aging 18:685-716.
Halliwell, B., and Gutteridge J. M . C; 1990 , The antioxidants oE human extracellular fluids, Arch.
Biochem. Biophys. 280 :1-8 .
Halliwell, B., and Gutteridge J. M. c., 1999 , Free radtcal in biology a/1(1 medicine, Oxford University
Press, Oxford.
Halliwell, B., Gutteridge 1. M. C., and Cross C. E., 1992, Free radicals, antioxidants, and human disease:
where are we now?, J. Lab . Clin. Med. 119 :598-620.
Halliwell, B., Zhao K. , and Whiteman M ., 2000, The gastrointestinal traet : a major site of antioxidant
action?, Free rad. Res. 33 :819-830.
Hamilton, M. L., Van Remmen H., Drake J . A., Yang H., Guo Z. M., Kewitt K., Waller C. A., aud
Richardson A., 2001, Does oxidative damage to DNA increase with age", Proc. Natl. Acad. SI'i. U. S.
A 98 :10469-10474.
Harnilton, R. F. Jr., Li L., Eschenbaker W . L., Szweda L., and Holian A ., 1998, Potential involvemenr of
4-hydroxynonenal in the response ofhuman lung cells to ozone, Amer . J. Physiol. 274 :L8-Ll6.
Hanauer, S . B., 1996, Inflammatory bowel disease, N. Engl . J. Med . 334 :841-848 .
Hanauer, S. B., and Dassopoulos T., 2001 , Evolving treatment strategies for inflamrnatory bowel disease,
Annu. Rev. Med. 52 :299-318 .
Handelman. G. 1., Dratz E. A., Reay C. c., and van Kuijk 1. G., 1988 , Carotenoids in the human macula
and whole retina, Invest Ophthalmol. Vis. Sei. 29 :850 -855.
Haniflin , J. M., and Tofte S. 1., 1999, Update on therapy of atopic dermatiti s, .I. Allergy Clin . lmmunol.
104 :S123-S125 .
Hannuksela, M. L., and Ellahham S ., 200 I, Benefits and risks of sauna bathing, Am . J . Med. 110 : 118-126.
Hardwick, C., 1940 , The indications for and technique oE whole-blood inkljections, Practitioner 144 :79-
82.
Harman, D., 1956 , A theory based on free radical and radiation chernistry, J. Gerontol. 1 I :298-300.
Harris, N. R. P., and Bishop L., 1999, Changes in global stratospheric ozone, in Proceedings of'the tnt.
Ozon e Symposium. 21 and 22 Ocrober 1999. Basel , Switzerland (10.-\ - E.-\,G Ed .), Bauer Druck
AG, Basel , pp .67 -76 .
Harrison, P., Savidge G . F., and Cramer E. M., 1990, The origin and physiological relevance of alpha-
granule adhesive proteins, BI'. .I. Haematol. 74 : I 25 -130.
Hatch, G . E. Jr ., 1991, Comprehensive treatise on pulmonary toxicologv (R. Patent, Ed .), CRC Press,
Boca Raten, Florida, pp.61 7-632.
Hawkins, C. L., and Davies M . J., 1996, Direct detection and identification of radioals generared during
the hydroxyl radical-induced degradation of hyaluronic acid and related rnaterials, Free rad. Biol.
Med. 21 :275-290.
Hayflick, L., 2000, The future of ageing, Nature 408 :267 -269 .
Heathcote, E. J., Shiffrnan M . L., Cooksley W . G., Dusheiko G. M ., Lee S . S ., Balart L., Reindollar R.,
Redd y R. K ., Wright T. L., Lin A, Hoffrnan J., and De Pamphilis J., 2000 , PeginterEeron alfa-Za in
patients with chronic hepatitis C and cirrhosis, N. Engl. .I. Med. 343 :1673-1680.
Heinecke, J. W ., 1999, Mechanisms of oxidative darnage by myeloperoxidase in atherosclerosis and other
inflammatory disorders, .I. Lab. Clin. Med. 133 :32 I -325 .
Heng, H., Rucker R. B., Crotty J., and Dubick M. .-\., 1987, The effects of ozone on lung , heart , and Iiver
superoxide dismutase and glutathione peroxidäse acti vities in the protein-deficient rat , Toxirol. LeI/.
38 :225 -237.
 REFERENCES 399

Hennekens, C. H., Buring J. E., and Peto R., 1994, Anrioxidant vitamins - benefits not yet proved, N.
Engl. J. Med. 330:1080-1081.
Hemandez , F., Menendez S., and Wong R., 1995, Decrease of blood cholesterol and stimulation of
antioxidative response in cardiopathy patients treated with endovenous ozone therapy, Free rad . Biol.
Med.19:115-119.
Hemandez. F., Alvarez 1., Coreho 1., and Gonz älez M., 2001, Changes in glutathione antioxidant pathway
components, HLA-DR and IgE in blood from asthma patients treated with ozone therapy, in
Proceedings ofthe 15th Ozone World Congress, London , UK. llth-15th September 2001 . Medical
Therapy Conference (I0A 2001, Ed.), Speedprint MacMedia LId, Ealing , London , UK, pp.l31-141.
Hemnann, M., Voll R. E., and Kalden J. R., 2000, Etiopathogenesis of systemic lupus erythematosus,
Immunol. Today 21:424-426 .
Herzenberg , L. A, De Rosa S. c., Dubs J. G., Roederer M., Anderson M. T., Ela S. W., Deresinski S. c.,
and Herzenberg L. A., 1997, Glutathione deficieney is associated with impaired survival in HIV
disease , Pro c. Natl. Acad. Sei . U. S. A 94 :1967-1972 .
Hiatt, W. R, 2001, Medieal treatment of peripheral arterial disease and c1audication, N. Engl. J. Med.
344 :1608-1621.
Hightower , L. E., 1980, Cultured animal cells exposed to amino acid analogues or puromycin rapidly
synthesize several polypeptides, .1. Cello Physiol. 102:407-424.
Hijnen, W. A. M., Bosklopper Th. G. J., Hofrnan J. A. M. H., Boseh A. D., and Medema G. J., 2001,
Improvement of the disinfection efficiency of the full-scale ozonation of the River -Iake waterworks
of Amsterdam water supply , in Proceedings ofthe 15th Ozone World Congress, London, UK. l l th-
15th September 20()]. Volume I (IOA 2001, Ed .), Speedprint MaeMedia LId, Ealing, London, UK,
pp.250-261.
Hillerdal, G., 1997, New principles for the treatment of diffuse pulmonary emphysema, J . Intern . Med .
242:441-448 .
Hirsch, K. R, and Wright T. L., 2000, The dilemma of disease progression in hepatitis C patients with
normal serum aminotransferase levels , Am. J. Med. 109 :66-67 .
Ho, D. D., 1997, Perspectives series : host/pathogen interactions. Dynamics of HIV-I replication in vivo,
.I. C1in. Invest 99 :2565-2567.
Hobbs, A J., Fukuto 1. M., and Ignarro L. J., 1994, Formation of free nitric oxide from L-arginine by
nitrie oxide synthase: direct enhancement of generation by superoxide dismutase, Proc. Nat . Aead.
Sei. USA 91:10992-10996 .
Hockel , M., and Vaupel P., 2001, Tum or hypoxia : definitions and current elinical , biologie , and
moleeular aspects,.I. Natl. Can cer Inst. 93 :266 -276 .
Hodgson , H. J., 1996, Keeping Crohn's disease quiet , N. Eng! . .I. Med. 334 :1599 -1600.
Hoffmann. A , and Viebahn R., 200 I , The influence of ozone on 2,3-diphosphoglyeerate synthesis in red
blood eell concentrates, in Proceedings of the 15th Ozone World Congre ss, London, UK. llth-15th
September 2001 . Medical Therapv Conference (IOA 2001 , Ed.), Speedprint MacMedia LId, Ealing,
London , UK, pp .10-25 .
Hogg, N., 2000, Biological chemistry and c1inical potential of S-nitrosothiols, Free rad. Biol. Med.
28:1478-1486 .
Hoigne, J., and Bader H., 1979, Ozonation of water: selectivity and rate of oxidation of solutes, Ozone-
Sei. Eng . 1:73-85 .
Holmes, O. W., 1892, Complete works ofOlivet Wendell Holmes, Houghton Miffl in, Boston, p.145.
Holmgren , A., 1989, Thioredoxin and glutaredoxin systems, J. Biol. Chem . 264:13963-13966.
Hoofnagle, J. H., and Bisceglie A. M., 1997, The treatment of ehronic viral hepatitis, N. Eng!. J. Med.
336 :347-356 .
Hooper, D. c., Seott G. S., Zborek A., Mikheeva T. , Kean RB ., Koprowski H., and Spitsin S. V., 2000,
Urie acid, a peroxynitrite scavenger, inhibits CNS inflanunation, blood- CNS barrier permeability
changes, and tissue damage in a mouse model of multiple sclerosis, FASEH J. 14 :691-698.
Hooper , L. V., and Gordon J. 1., 2001, Conunensal host-bacterial relationships in the gut, Seienee
292:1115-1118.
House of Lords Select Comm ittee on Science and Technology, 6u, report 1999-2000 [HLl23]., 2000,
Complementary and Altemotive Medicine, Stationery Office , London .
Hruz, P. W., Murata H., and Mueckler M., 2001, Adverse metabolie consequences of HIV protease
inhibitor therapy: the search for a central mechanism, Am . J. Physiol Endocrinol. Metab 280:E549-
E553.
400 R EF EREN CES

Hsu, D. T., and Diehl D. L., 1998, The West ge ts the point, Lancel352 SuppI4 :SIV1 .
Hu, M .-L. , 1994 , Measu rernent of protein thiol gro ups and glut athi on e in plasma, Met h. Enzymol.
233 :380-385.
Huang, P. L., Huang Z., Mas hirno H., Bloch K. D ., Mosko witz M. A ., Bevan J. A., a nd Fishman M. C,
1995 , Hypertension in rnice lacking the ge ne for end oth elial nitric oxide syn thase, Na ture 377 :23 9-
242 .
lked a, F., Shirnomura H., Miyake M., Fuj ioka S. 1., lIoh M., Tak ah ash i A., lwasaki Y., Sakaguchi K.,
Yam am oto K., Higashi T., and Tsuji T., 2000, Earl y c1earance of ci rculating he pat itis C virus
enhanced by induct ion therapy with twice -a-da y intra ven ous injection of IFN-bela, .J. Interfero n
Cy tokine Res. 20 :831 -836.
lleor Advisory Statements , 1997, The uni versal advanced Iife suppo rt a lgorithm, Circutatio n 95 :21 80-
2 182.
Inui , Y., and Ichi yanagi 1., 200 I, "Ozo ne Clea ner fo r Bedding, Bedcl oth es, etc. ". Japanese Patent 200 1
161 79 7 A2 (Assignee: Yasun aga K.K.) .
Isaa cs, A., and Lindenmann 1., 1957 , Virus interference. I. The interfero n, Prot: Roy. So«. London Sero R
147 :147-152.
Isner, J. M. , Pieczek A., Schainfeld R., Blair R., Haley L., Asahara T., Rosenfiel d K., Raz vi S., Walsh K.,
and Sym es J. F., 1996, Clin ical evid ence of angiogenesis after arterial gene tran sfer of phVEG Flf,j in
patient with ischaemic lirnb , Lan cet 348 :370-374 .
Iuliano, L., Cola vita A. R., Leo R., Praticö D., and Violi F., 1997 , Oxygen fre e radicals and platelet
activation, Free I'Od. Biol. Me d. 22:999- I 006 .
Iwu , M. M., and Gb odos sou E., 20 00, The role oft raditional medic ine, Lancet 356 Suppl :s3 .
Jac kso n, K. A., Majka S. M. , Wang H., Pocius J., Hartley C. J., Maj esk y M. W ., Entman M. L., Micha el
L. H., Hirsch i K. K., and Goodell M. A., 200 1, Regeneration of ischemic cardiac muscle and vascular
endothelium by adult stem cells, .J. Clin. In vest 107 :1395-1402.
Jacobs, M.-T ., 1982, Untersuchung uber Z wischen falle und typische Kompl ikat ionen in der Ozo n-
Sauer stoff-Therapie ., Ozotsachrt chten 1:5.
Jacob son, M. D., 1996 , Reactive oxy gen spe cies and pr ogrammed cell death , Trends Bioch em Set 21: 83-
86 .
Jaes chke , H., 1995, Mechan isms of ox idan t stres s-induced ac ute tissu e inj ury, Pro c. SOl:. Exp. Biol. Med.
209 :104 - 11 I.
Jaffe , E. A., Nac hma n R. L., Becker C. G., and Minick C. R., 197 3, Culture of human endot helial ce lls
derive d fro m umb ilic al veins . Ident ification by morpholog ic and immunologic criteria,.J. Clin.
Invest. 52 :274 5-2756 .
Jan g, M., Cai L., Udeani G. 0. , Siowin g K. V ., Tho mas C. F., Beech er C. W., Fong H. H.. Farns worth N.
R., Kingh orn A . D., Mehra R. G., Moo n R. c., a nd Pezzut o J. M., 1997, Ca ncer chemo preve ntive
activity of resv eratro l, a natural product der ived fro m grapes, Science 275 :21 8- 220 .
Jaspers, 1., Flescher E., and Chen L. C.; 1997, Ozone-induced [L-8 exp ress ion a nd transcr ipn on facto r
bind ing in resp iratory epith elial cell s, Amer. .I. Physio l. 272: L504 -L51I.
Jenki ns , V . A., Fallo wfield L. J., Souhami A., and Sawtell M., 1999, How do doctors explain rand om ised
cli nical trials to the ir patient s?, Eur. .I. Cancer 35 :1187 -1193 .
Jenner, P., 1994 , Oxidative darnage in neu rode generat ive disease, Lancet 344 :7 96-798.
Jessell , T . M ., and Kell y D. D., 1994, Dol ore ed analgesia, in l'rinctpi di neuroselenze (E. R. Kandel, J .
H. Schw artz, and T . M. Jes sell , Eds .), Casa Ed itrice Arnbrosiana, Milan e, pp.396-41 I.
Ji, L. L., 1995 , Oxidati ve stress during exercise : implications of antioxidant nutri ents, Free rad . Biol.
M ed. 18:1079-1086.
Jia , L., Bonaventura c., Bonaventura J., and Stamler 1. S. , 1996 , S-nit rosohaem ogl obin : a dynarm e
acti vity of blo od invol ved in vasc ular contro l, Nature 380:221 -226 .
Jind al, N ., and Dell inger R. P., 2000, Inhal ati on of nitric ox ide in ac ute respiratory distre ss syndrome . J,
Lab Clin. Me d. 136 :21- 28.
Johnson , P. W., Dixon R., a nd Ross A. D., 1998, An in-vitro test for assessi ng the viability of Asea ns
suum eggs ex pos ed to various se wage treatment processes,lnl. .I. Parasitol. 28 :627- 633 .
John son , R. J., Willson R., Yarnabe H., Co us er W ., Alpers C. E., Wener M. H., Davis c., and Gretch D.
R., 19 94, Renal rnan ifestati ons of hepatitis C virus infect ion, Kidn ey Int, 46 : 1255-1263.
John ston, H. S ., 1971 , Reduction of stratospheri c ozone by nitrogen oxide catalysts fro m superso nic
trans po rt exhaust , Science 173 :517- 522.
 REFERENCES 401

Jolly, C., and Morimoto R. 1., 200Q, Role of the heat shock response and molecular chaperones in
oncogenesis and cell death,.I. Natl. Cancer Inst. 92: 1564-1572.
Jones, T . H., and Kennedy R. L., 1993, Cytokines and hypothalamic-pituitary function, Cytokine 5:531-
538.
Jomot , L., Mirault M. E., and Junod A . F., 1991, Differential expression of hsp70 stress proteins in
human endothelial cells exposed to heat shock and hydrogen peroxide, Amer. .I. Respir. Cell Molec.
Biol. 5 :265-275.
Josse, C., Legrand-Poels S., Piret B., Sluse F., and Piette J., 1998 , Impairment of the mitochondrial
electron chain transport prevents NF- kappa B activation by hydrogen peroxide, Free rad. Biol. Med.
25 :104-112.
Joyner, M. J., and Dietz N . M ., 1997, Nitric oxide and vasodilation in human limbs, .I. A[J[JI. Physiol.
83 :1785-1796.
Jucopilla, N ., Ferrarese c., Tirapelle G., Battista R., Mazzo G., and Robert A., 2000, Infiltrazioni disco-
foraminali con 02-03 nelle SDR da conflitto disco-radicolari lombari, in Proceedings: I Congresso
IMOS. Italia, Siena. 2-4 novembre 2000, p . 38 .
Kadokawa, N. , Morioka T., Motoyama N., Hashino M., Mori Y., Nishijirna W ., Okada M., and Moniwa
T., 2001, Advanced water treatment using ozone resistant microfiltration membrane, in Proceedings
ofthe 15th Ozone wona Congress. London, UK, 11th-15th September 2001. Volume I (IOA 2001 ,
Ed.), Speedprint Mac Media LId, Ealing, London, UK, pp.125-133.
Kalebic , T., Kinter A., Poli G., Anderson M. E., Meister A., and Fauci A . S., 1991, Suppression ofhuman
immunodeficiency virus expression in chronically infected monocytic cells by glutathione,
glutathione ester, and N-acetylcysteine, Proc. Nat. Acad. Sei. USA 88 :986-990.
Kalinich , J. F., Ramakrishnan R., McClain D. E., and Ramakrishnan N ., 2000, 4-Hydroxynonenal, an
end-product of lipid peroxidation, induces apoptosis in human leukemic T- and B-cell lines, Free
rad. Res. 33 :349-358 .
Kangasjärvi , J., Talvinen 1., Utriainen M., and Karjalainen R., 1994, Plant defence systems induced by
ozone, Plant Cell Environ. 17:783-794.
Kappus, H., and Diplock AT., 1992, Tolerance and safety of vitamin E: a toxicological position report,
F"ee rad. Biol. Med. 13 :55-74 .
Kaptchuk, T . 1., 1998, Powerful placebo: the dark side of the randomised controlled trial, Lancet
351:1722-1725.
KarIsson, K., and Marklund S. L., 1988 , Plasma c1earance ofhuman extracellular-superoxide dismutase C
in rabb its, J. Clin. Invest. 82 :762-766.
Karp, C. L., Biron C. A, and Irani D. N., 2000, Interferon beta in multiple sclerosis : is IL-12 suppression
the key ?, Immunol. Today 21 :24-28.
Kashiba, M ., Kasahara E., Chien K. c., and Inoue M., 1999, Fates and vascular action of S-
nitrosoglutathione and related compounds in the circulation, Arch. Biochem. Biophys. 363:213-218.
Kasting,1. F., 200 I, The rise of atmospheric oxygen, Science 293 :819-820.
Kasumjan, S. A , Lelyanov A . D., Guseva E. D., and Alexeev B. P., 1995, The ozonetherapy ofthe acute
suppurative infection, in The ozone in btology and medtcine. 2nd all Russian scienttfic-practical
confe rence. September 6-8. 1995 . Russian assoclation ofozonetherapy, Reshetnikovskaya street 2.
Nizhni Novgorod, 603006 Russia , p.16 .
Katusic, Z. S., 1996 , Superoxide anion and endothelial regulation of arterial tone, Free radal Biol. Med.
20:443 -448 .
Kawamura, K., Kaneko M., Tsuyoshi H., and Tagushi K., 1986, Microbial indicators for the efficiency of
disinfection process, Wat. Sei . Tech. 18:175-184.
Keane, J., Gershon S., Wise R. P., Mirabile-Levens E., Kasznica 1., Schwieterman W. D., Siegel 1. N .,
and Braun M. M. , 2001, Tuberculosis associated with infliximab, a tumor necrosis factor alpha-
neutralizing agent, N. Engl. .I. Med. 345 :1098-1104.
Keaney, 1. F., Jr ., Simon D. 1., and Freedman J. E., 1999, Vitamin E and vascular homeostasis:
implications for atherosclerosis, F ASEB.I. 13 :965-976.
Kelly, F. J., Mudway 1., Krishna M. T., and Holgate S. T., 1995, The free radical basis of air pollution:
focus on ozone, Resp. Med. 89 :647-656.
Keshan Disease Research Group of the Chinese Academy of Medical Sciences, 1979, Epidemiologie
studies on the etiologic relationship of selenium and Keshan disease, CUn. Med. J. 92 :477-482.
Khan, A . U., and Wilson T., 1995 , Reactive oxygen species as cellular messengers, Chem . Biol. 2:437-
445 .
402 REFERENCES

Kiang, J. G., and Tsokos G. C., 1998, Heat shock protein 70 kDa : molecular biology, biochemistry, and
physiology, Pharmacol. Ther. 80:183-201.
Kief, H., 1993a, The treatment of malignant diseases with AHIT, in Proceedings: IDA Congre ss, San
Francisco. Ca, USA (International Ozone Association, Ed .), pp.26 -31 .
Kief, H., 1993b, The treatment of neurodermatitis with the auto-homologous immune therapy, in
Proceedings: 10.'1 Congress, San Francisco. Ca. USA (International Ozone Association , Ed.), pp. 36-
42.
Kim, C. H., Choi H., Chun Y. S., Kirn G. T., Park J. W ., and Kim M . S., 2001 , Hyperbaric oxygenation
pretreatrnent induces catalase and reduces infarct size in ischemic rat my ocardium, Pflugel's AI'{:h.
442 :519 -525 .
Kim , D. K., Cho E. S., Lee B. R., and Um H. D., 2001, NF -kappa B mediates the adaptation of human
U937 cells to hydrogen peroxide, Free rod. Biol. Med. 30:563-571.
Kindwall , E. P., 1993, Hyperbaric Oxygen, Brit. Med.. 1. 307 :515-516.
Kinney, P. L., Thurston G. D., and Raizenne M ., 1996, The effects of ambient ozone on Jung function in
children: areanalysis of six summer camp studies, Environ. Ilealth Perspeer. 104 : 170-174.
Kipnis, 1., Yoles E., Porat Z., Cohen A., Mor F., Sela M., Cohen I. R., and Schwartz M., 2000, T cell
immunity to copolymer I confers neuroprotection on the damaged optic nerve : possible therapy for
optic neuropathies, PI'OC. Natl . Acad. Sei. U. S. A 97:7446-7451.
Kirby, P. K., Kiviat N ., Beckman A., Wells D., Sherwin S ., and Corey L., 1988 , Tolerance and efficacy
ofrecombinant human interferon gamma in the treatment of refractory genital warts ., Amer. .I. Med.
85: 183-188 .
Kleeberger, S. R., Levitt R. c., Zhang L. Y., Longphre M., Harkema 1., Jedlicka A , Eleff S. M .,
DiSilvestre D., and Holroyd K. J., 1997, Linkage analysis of susceptibility to ozone-induced lung
inflammation in inbred mice, Nat, Genet. 17:475-478.
Klein, R., Klein B . E., and Franke T., 1993 , The relationship ofcardiovascular disease and its risk factors
to age- related maculopathy. The Beaver Dam Eye Study, Ophthalmology 100:406-414.
Klein , R., Klein B. E., Jensen S. c., and Meuer S. M., 1997, The five- year incidence and progression of
age -related maculopathy: the Beaver Dam Eye Study, Ophthalmology 104:7-21.
Klomp, H. M., Spineemaille G. H. J . J., Steyerberg E. W ., Habbema J. D. F., van Urk H., and for the
ESES study group, J 999 , Spinal-cord stimulation in critical Jimb ischaemia : a rand omised trial. ,
Lancet 353 :1040 -1044.
Knoch, H.-G ., and Klug W ., 1990 , Ozon-Sauerstoff-Therapie der Proktitis, Med. Welt 41 :37 1-374.
Knoch, H.-G ., Roschke W., and Klug W., 1987, Ozone/oxygen therapy in proctology, Ozotvachrichten
6:51-70.
Kohen, R., Vellaichamy E., Hrbac J., Gati 1., and Tirosh 0 ., 2000 , Quantification of the overall reactive
oxygen species scavenging capacity of biological fluids and tissues, Free rad. Biol. Med. 28:871-
879 .
Kollef, M . H., and Fraser V . J., 2001 , Antibiene resistance in the intensive care unit , Ann. Intern. Med.
134 :298-314.
Komaroff, A. L. , 2000, The biology of ehrenie fatigue syndrome, .'111I. .I. Med. 108: 169-171 .
Kondo, S., Toyokuni S ., Iwasa Y., Tanaka Y., Onodera H., Hiai H., and lmarnura M., 1999, Persistent
oxidative stress in human colorectal carcinoma, but not in adenoma, Free rod. Biol. Med. 27:40 I-
410 .
Kondro, W ., 1999 , Study defines safe concentrations of ozone, I.ancet 353 :994.
Konrad, H., 1995 , Ozone therapy for herpes simplex and herpes zoster, in Proceedings Ozone in
Medicin e, 12th World Congress 0/ the International Ozone Association, 151h 10 /81h Mo)' 1995.
Lille. France (International Ozone Asso ciation, Ed .), Instaprint S.A ., Tours, pp. 187-194.
Konrad, H., 2001 , Ozone therapy for post-herperle neuralgia. A retrospective study of 55 cases, in
Proceedings 0/ the 151h Ozone World Congress, London , UK. 1Ith- 151h September 2(1t//. Medical
Therapy Conference (IOA 200 I , Ed .), Speedprinl MacMedia LId, Ealing , London, UK, pp .85-88.
Koppenol , W. H., 1998, The basic chemistry of nitrogen monoxide and peroxynitrite, Free rad. Bio!.
Med.25:385-391.
Komitzer, D., and Ciechanover A , 2000, Modes of regulati on 01' ubiquitin-rnediated prore in degradation ,
J. Ce/l PhysioI182 :1-11.
Korzets, A., Chagnac A., Weinstein T., Ori Y ., Malachi T., and Gaffer U., 1999 , H202 induces DNA
repair in rnononuclear cells : evidence for association with cytosolic Ca2 + fluxes , J. Lab Clin. Med.
133 :362-369.
 REFERENCES 403

Kosaka , H., 1999 ,Nitric oxide and hemoglobin interactions in the vasculature, Biochim. Biophys. Acta
1411 :370-377.
Kosheleva, 1. V ., Ivanov O. L., and Kulikov A. G ., 2001 , Various modes of ozone-oxygen gas mixture
application in patients with eczerna, in Proceedings ofthe 15th Ozone World Congress, London, UK.
l lth-l Sth September 2001. Medical Therapy Conference (lOA 2001 , Ed .), Speedprint MacMedia
LId, Ealing, London, UK , pp .105-108 .
Kouoh, F., Gressier B., Luyckx M., Brunet c., Dine T., Cazin M., and Cazin 1. c., 1999, Antioxidant
properties of albumin : effect on oxidative metabolism of human neutrophil granulocytes, Farma co
54 :695-699.
Kourie , 1. 1., 1998 , Interaction of reactive oxygen species with ion transport rnecharusms, Amer. J.
Phvs iol. 275 :CI-C24.
Kovacicova, G., Hanzen J., Pisarcikova M ., Sejnova D., Horn J., Babela R.; Svetlansky 1., Lovaszova M.,
Gogova M., and Krcmery V ., 2001 , Nosocomial fungemia due to amphotericin B-resistant Candida
spp . in three pediatric patients after previous neurosurgery for brain tumors, .I. Infect . Chemother.
7:45-48.
Kozumbo, W . J., Hanley N . M., Agarwal S., Thomas M. J., and Madden M. c., 1996 , Products of
ozonized arachidonic acid potentiate the formation of DNA single strand breaks in cultured human
lung cells, Environ . Mol. Mutagen. 27 :185-195.
Kraft , K., Stenkamp E., Sachinidis A., Seewald S., and Vetter H., 1998 , Effect of autohemotherapy with
ozone on cardiovascular risk factors in patients with mild hypertension, Perfusion 11:216-219 .
Kramer, B. S., and Klausner R. D., 1997 , Grappling with cancer, Defeatism versus the reality ofprogress,
N. Eng/..I. Med. 337 :931-934.
Kreuzer, T., Grube R., Wutte A. , Zarkovic N., and Schaur R. J., 1998 , 4-hydroxynonenal modifies the
effects of serum growth factors on the expression of the c-fos proto-oncogene and the proliferation of
HeLa carcinoma cells, Free rad. Biol. Med . 25 :42-49 .
Krieg , A. M ., and Wagner H., 2000, Causing a commotion in the blood: immunotherapy progresses from
bacteria to bacterial DNA, lmmunol. Today 21:521-526 .
Kristiansen, M., Graversen J. H., Jacobsen C.; Sonne 0 ., Hoffman H. J., Law S. K., and Moestrup S. K.,
200 I, ldentification of the haemoglobin scavenger receptor, Nature 409 :198-201.
Kudravcev, B. P., Miroshin S. J., and Semyonov S. V ., 1995, The use of ozonized solutions in complex
treatment of peritonitis, in The ozone in biology and medicine. 2nd all Russian Scienttfic-Practtcal
Conferen ce, September 6-8. 1995. Russian Association of Ozonetherapy, Reshetnikovskaya stret, 2 -
N izhni Novgorod, 603006 Russia, Nizhni Novgorod, p.20 .
Kume , M., Yamamoto Y., Saad S., Gomi T., Kimoto S., Shimabukuro T., Yagi T., Nakagarni M., Takada
Y., Morimoto T., and Yamaoka Y., 1996 , Ischemic preconditioning ofthe Iiver in rats: implications
of heat shock protein induction to increase tolerance of ischemia-reperfusion injury, .I. Lab Clin.
Med .128:251-258 .
Kuruvilla, A . P., Shah R., Hochwald G. M., Liggitt H. D., Palladino M. A ., and Thorbecke G. J., 1991,
Protective effect of rransforrning growth factor b, on experimental autoimmune diseases in mice.,
Proc. Nat . Acad. Sei. USA 88 :2918-2921.
Labow, R. S., Tocchi M., and Rock G ., 1986 , Centamination ofplatelet storage bags by phthalate esters,
Toxicol. Environ. Health 19:591-598.
Laham , R. J., Simons M., and Sellke F., 200 I, Gene transfer for angiogenesis in coronary artery disease,
Anl/II. Rev. Med. 52 :485-502.
Laitinen, M., Mäkinen K., Manninen H., Mats i P., Kossila M., Agrawal R. S., Pakkanen T., Luoma 1. S.,
Viita H., Hartikainen J., Alhava E., Laak so M., and Yl ä-Herttuala S., 1998, Adenovirus-mediated
gene transfer to lower limb artery of patients with chronic critical leg ischemia, HilI/I. Gene Ther.
9:1481-1486.
Lambert, H. M., Capone A . Jr ., Aaberg T. M ., Sternberg P. Jr ., MandelI B. A., and Lopez P. F., 1992,
Surgical excision of subfoveal neovascular membranes in age-related macular degeneration, Amer.. .I.
Ophthalmol. 113 :257-262 .
Landino, L. M., Crews B. c., Timmons M. D., Morrow J. D., and Mamett L. 1., 1996, Peroxynitrite, the
coupling product of nitric oxide and superoxide, activates prostagiandin biosynthesis, Proc. Nat.
Acad. Sei. USA 93 :15069-15074.
Lane, N. J., 1998, Blood ties, The Seiences 24:25-29 .
Lang , A. E., and Lozano A. M., 1998, Parkinson's disease. First of two parts, N. EI/gI. .I. Med . 339 :I044-
1053 .
 404 REFERENCES

Lang, A. E., and Lozano A. M., 1998, Parkinson's disease. Second of two parts, N. Engl . .f. Med.
339: 1130 -1143.
Larini , A., Aldinucci C., and Bocci V., 2001, Ozone as a modularer of the immune sysrem, in
Proceedings ofthe 15th Ozone World Congress, London, UK. 11th-15th September 200l. Mediral
Therapy Conference (10A 2001, Ed.), Speedprint MacMedia LId, Ealing, London, UK, pp. I-I O.
Larkin, M., 200 I , Vitamins reduce risk ofvision loss from macular degeneration, Lancet 358 :1347.
Larrea, E., Beloqui 0 ., Muiioz-Navas M. A., Civeira M. P., and Prieto 1., 1998 , Superoxide dismutase in
patients with chronic hepatitis C virus infection, Free rad. Biol. Med. 24 :1235-1241 .
Last, J. A. , Warren D. L., Pecquet-Goad E., and Witschi H., 1987, Modification by ozone of Jung tumor
development in mice, J. Nat . CancerInst, 78 :149-154.
Laughton, M . J., Evans P. J., Moroney M. A ., Hoult J. R., and Halliwell B., 1991 , Inhibition of
mammalian 5-lipoxygenase and cyclo-oxygenase by flavonoids and phenolic d ietary additives.
Relationship to antioxidant activity and to iron ion-reducing ability, Btochem. Pharmacol. 42 :1673-
1681.
LaVail , M. M ., Unoki K., Yasumura D., Matthes M. T., Yancopoulos G. D., and Steinberg R. H., 1992,
Multiple growth factors, cytokines, and neurotrophins rescue photoreceptors from the damaging
effects of constant light , Proc. Natl. Acad. Sei. U. S. A 89 :11249-11253.
Lawrence, D. A. , 1996 , Transforming growth factor-beta : a general review, Eur . Cvtokine Netw. 7 :363-
374.
Lawrence, W . H ., 1978, Phthalate esters: the question of safety, Clin. Toxicol. 13:89 .
Le Bars, P. L., Kieser M., and Itil K. Z ., 2000 , A 26-week analysis of a double-blind, placebo-controlled
tria l ofthe ginkgo biloba extract EGb 761 in dementia, Dement . Geriatr. Cog n Disord. 11:230 -237.
Leach, R. M ., Rees P. J., and Wilmshurst P., 1998 , Hyperbaric oxygen therapy, Brit. Med..I. 317:1140-
1143 .
Lechner, F., Wong D. K , Dunbar P. R., Chapman R., Chung R. T., Dohrenwend P., Robbins G ., Phillips
R., Klenerman P., and Walker B. D., 2000, Analysis of successful immune responses in persons
infected with hepatitis C virus, J. Exp. Med. 191:1499-1512 .
Lee , J. H., and Klein H. G., 1994 , Mononuclear cell adoptive immunorherapy, in Transfusion medicine I
Hematology/Oncology Clinics of North America, pp .1203-1221 .
Lee, Y . J., and Shacter E., 2000, Hydrogen peroxide inhibits activation, not activity, of cellular caspase-3
in vivo, Free rad. Biol. Med. 29:684-692 .
Leist, M., Raab 8., Maurer S., and Brtgelius-Flohe R., 1996 , Conventional cell culture media do nor
adequately supply cells with antioxidants and thus facilitate peroxide-induced gen otoxicity, Free rad.
Biul. Med . 21 :297-306.
Lenfant, C ., Torrance J., English E., Finch C. A., Reynafarje c., Ramos J., and Faura J., 1968 , Effect of
altitude on oxygen bin ding by hemoglobin and on organic phosphate levels, J. Clin. Inve st. 47 :2652 -
2656 .
Leonardi, M., Barbara C., Agati R., Simonetti L., and Giatti S., 2001a, Trattamento percutaneo dell'ernia
discale lombare con iniezione intradiscale di miscela di ozono, Riv. Neuroradial. 14 :51-53 .
Leonardi, M., Simonetti L., and Barbara c., 2001b, Effetti dell'ozono sul nucleo polposo : reperti
anatomo-patologici su un caso operato, Riv. Neuroradtot. 14:57-59 .
Le ön, O. S., Menendez S ., Merino N., Castillo R., Sam S., Perez L., Cruz E., and Bocci V., 1998, Ozone
oxidative preconditioning: a protection against cellular damage by free radicals, Mediat. Inflamm,
7:289 -294 .
Lerner, I. J., 1996, Against nonscientific treatments,.I. Lab. C1in. Med. 127:242-243 .
Letterio. J. J., and Roberts A. B., 1998 , Regulation of immune responses by TGF-b, Annll. Re.... Immunol.
16:137-161.
Leung, D. Y., 1999 , Pathogenesis ofatopic dermatitis,.I. Allergy Clin. /111111111101. 104 :S99-108.
Levi, F., Lucchini F., Negri E., Boyle P., and La Vecchia c., 1999, Cancer mortality in Europe, 1990 -
1994, and an overview oftrends from 1955 to 1994, EIII·. .I. Cancer 35 :1477-1516.
Levin, E. R., 1995, Endothelins, N. Engl. .I. Med. 333 :356-363 .
Levine, M., Conry-Cantilena C ., Wang Y., Welch R. W ., Washko P. W ., Dhariwal K. R., Park 1. B ,
Lazarev A. , Graumlich J. F., King 1., and Cantilena L. R., 1996, Vitamin C pharmacokinetics in
health volunteers: evidence for a recommended dierary allowance, Proc. Nat. Acad. Sei . USA
93 :3704-3709.
Levine, M., Daruwala R. C; Park 1. B., Rumsey S . c., and Wang Y , 1998, Does vitanun C have a pro-
oxidant effect", Nature 395:231.
 REFERENCES 405

Levy, Y., Capitant C., Houhou S., Carriere 1., Viard J. P., Goujard c., Gastaut J. A., Oksenhendler E.,
Boumsell L., Gomard E., Rabian C., Weiss L., Guillet J. G., Delfraissy J. F., Aboulker J. P.,
Seligmann M., and ANRS 048 Study Group, 1999, Comparison of subcutaneous and intravenous
interleukin-2 in asymptomatic HIV -I infection: a randomised controlled trial, Lancet 353: 1923-1929.
Lewin, N ., Craik S., Li H., Smith D. W., and Belosevic M., 2001 , Sequential inactivation of
Cryptosporidtum using ozone followed by free chlorine in natural water, Ozone-Sci. Eng. 23:411-
420 .
Lewis, L. M., Flechtner T. W., Kerkay J., Pearson K. H., Chen W. T., Popowniak K. L., and Nakamoto
S., 1977, Determination of plasticizer levels in serum of hemodialysis patients, Trans . Am. So c. Anif
Intern. Organs 23:566-572 .
Li, P. A., Liu G. 1., He Q. P., Floyd R. A ., and Siesjo B. K., 1999, Production ofhydroxyl free radical by
brain tissues in hyperglycemic rats subjected to transient forebrain ischemia, Free rad. Biol . Med.
27 :1033-1040 .
Li, X., Cobb C. E., Hili K. E., Burk R. F., and May J. M., 2001, Mitochondrial uptake and recycling of
ascorbic acid , Arch. Biochem . B iophys . 387 :143-153.
Liaw , K.-L., Glass A. G ., Manos M. M., Greer C. E., Scott D. R., Sherrnan M., Burk R. D ., Kurrnan R. J.,
Wacholder S., Rush B. B., Cadell D. M., Lawler P., Tabor D., and Schiffman M., 1999, Detection of
human papillomavirus DNA in cytologically normal women and subsequent cervical squamous
intraepitheliallesions, J. Nat . Cancer Inst. 91 :954-960.
Lilienfeld, D. E., and Perl D. P., 1993, Projected neurodegenerative disease mortality in the United States,
1990-2040, Neuroepidem iology 12:219-228 .
Linde, K ., Clausius N. , Ramirez G., Melchart D., Eitel F., Hedges L. V., and Jonas W . B., 1997, Are the
c1inical effects of homoeopathy placebo effects? A rneta-analysis of placebo-controlled trials, Lancet
350 :834-843 .
Linde , K., Ramirez G ., Mulrow C. D., Pauls A., Weidenhammer W ., and Melchart D., 1996, St John's
wort for depression--an overview and rneta -anal ysis of randomised clinical trials , Brit. Med . J.
313 :253-258.
Ling, A. E., Robbins K. E., Brown T. M., Dunmire V ., Thoe S. Y ., Wong S. Y., Leo Y. S., Teo D.,
Gallarda J., Phelps B., Chamberland M. E., Busch M . P., Folks T. M., and Kalish M. L., 2000 ,
Failure of routine H1V-1 tests in a case involving transmission with preseroconversion blood
components during the infectious window period, .lAMA 284 :210-214 .
Linnane, A. W ., Degli Esposti M., Generowicz M., Luff A. R., and Nagley P., 1995, The universality of
bioenergetic disease and amelioration with redox therapy, Bio chim, Biophys. Acta 1271 :19 1-194 .
Lippman, M., 1989 , Health effects of ozone, a critical review, .I. Am . Air Poltut. Control Assoc. 39 :672-
695 .
Liu, W., Kato M., Akhand A . A ., Hayakawa A ., Suzuki H., Miyata T. , Kurokawa K ., Hotta Y., Ishikawa
N ., and Nakashima 1., 2000, 4-hydroxynonenal induces a cellular redox status-related activation of
the caspase cascade for apoptotic cell death,.1. Cell Sei. 113 ( Pt 4) :635-641.
L1oyd, A. R., and Oppenheim J. J., 1992 , Poly's lament : the neglected role of the polymorphonuclear
neutrophil in the afferent limb of the immune response, lmmunol. Today 13:169-172.
1.0, A. c., Houenou L. J., and Oppenheim R. W ., 1995 , Apoptosis in the nervous system: morphological
features, methods, pathology, and prevention, Arch . Histol. Cytol . 58 :139-149 .
Loconte, S., 2000, La sindrome fibromialgica primaria, in Proceedings: I Congresso IMOS. Italia , Siena,
2-4 novembre 2000 , p .40.
Leng, N. c., Suh J., Morrow J. D., Schiestl R. H., Murthy G. G., Brain J. D., and Frei B., 2001, Ozone
causes lipid peroxidation but little antioxidant depletion in exercising and nonexercising hamsters, ,I.
Appl. Physiol91 :1694-1700.
Loprete, F., 1999 , Utilizzo dell'ossigeno-ozonoterapia nel trattamento della malattia varicosa e sue
complicanze, in L 'Ozonoterapia nel 2()OO (F. Ceccherelli, and F. Giron , Eds .), Edizioni Libreria
Cortina, Torino, pp .129 -135.
Los, M., Dröge W" Stricker K., Baeuerle P. A .• and Schulze-Osthoff K., 1995 , Hydrogen peroxide as a
potent activator ofT lyrnph ocyte functions, ElII'. .I. lmmunol. 25 :159-165 .
Love, I. N., 1888 , Peroxide ofhydrogen as aremedial agent, .lAMA :262-265.
LovelI, D. J., Giannini E. H., Reiff A., Cawkwell G. D., Silverrnan E. D., Nocton J. J., Stein L. D.,
Gedalia A., Ilowite N . T., Wallace C. A.. Wlutrnore J., and Finck B. K., 2000, Etanercept in children
with polyarticular juvenile rheumatoid arthritis. Pediatric Rheumatology Collaborative Study Group,
N. Engl . .I. Med. 342:763-769.
406 REFERENCES

Ludewig, 8., Ochsenbein A. F., Odermatt B., Paulin D., Hengartner H., and Zinkernagel R. M., 200 0,
Immunotherapy with dendritic cells directed against tum or antigens shared with normal host cell s
results in severe auto immune disease,.I. Exp. Med. 191:795-804.
Lum, H., and Roebuck K. A., 2001 , Oxidant stress and endothelial cell dysfuncti on, Am. .I. Ph vsiol Cell
Physiol 280 :C7 I9-C74 I.
Lundstrom, T., and Sobel J., 2001, Nosocomial cand iduria: a review, Clin.Jn fect. Dis. 32 :1602-1607.
Luong o, C., Sammartino A., Lauritano S., Coppola L., Lauritano L., Luongo M., and Terullo A ., 2000.
Trattamento e monitoraggio dei potenziali redox nelle membrane cellulari nello studio delle infezi oni
da HCV . in Pro ceedings: I Congress o IMOS. Italia , Siena . 2-4 novembre 2000. pp. 42-43.
Lusi s, A. J., 2000 , Atherosclerosis, Natu re 407 :233-241.
Luthman, M., and Holmgren A., 1982, Rat Iiver thioredoxin and thioredoxin reductase : purification and
characterization, Biochemistry 2 I :6628-6633.
Maccarrone, M., Veldink G. A., Vliegenthart J. F. G., and Finazzi-Agro A. , 1997, Ozone stress modulares
amine oxidase and lipoxygenase expression in lentil tLens cultnarisi seedlings, FEBS Lett, 408:241 -
244 .
Machlin, L. J., and Bendich A., 1987, Free radical tissue damage: protective role of antioxidant nutrients,
FASEB.l.l :441-445.
Mackensen, A ., Galanos c., and Engelhardt R., 1991, Treatment of cancer patients with endotoxin
induces release of endogenous cytokines., Pathobiology 59:264-267 .
Macular photocoagulation group , 1986, Argon laser photocoagulation for neovascular maculopathy: three
years results for randomized clinical trials , Arch. Ophthalmol. 224 :493 -501 ,
Macular photocoagulation group, 1991, Argon laser photocoagulation for neovascular maculopathy aller
five years: results for randomized c1inical trials, Arch. Ophthalmol. 109:1109-1114 .
Madden, M. C; Friedmen M., Hanley N., Siegier E., Quay J., Becker S., Devlin R., and Koren H., 1993,
Chemical nature and immunotoxicol ogical properties of arachidonic and degradation prodnct s
formed by exposure to ozone, Environ. Health Perspect. 101:154-164.
Maddox , K., and Back R. F., 1935, An enquiry into the value of autohaem otherapy in juvenile asthrna ,
Arch. Dis. Chi/d. 10:381-388.
Maeda , K., Naganuma M., and Fukuda M., 1991, Effects of chr onic exposure ultra violet -A including 2%
ulrra violet-B on free radical reduction systems in hairless mice , Photochem . Photobiol. 54 :737-740.
Maggi , E., Mazzetti M., Ravina A., Annunziato F., de Car1i M., Piccinni M. P., Manetti R., Carbonari M.,
Pesce A. M., and Dei Prete G., 1994, Ability of HIV to promote a TH I to THO shift and to repl icate
preferentially in TH2 and THO cells , Science 265 :244-248 .
Magnani, M., Rossi L., Bianchi M., Serafini G., and Sto cchi V. , 1988 , Role of hex okinase in the
regulati on of erythrocyte hexose monophosphate pathway under oxidati ve stres s, Bioch em. Biophvs .
Res. Commun. 155 :423-428 .
Maier , D., and Kurzmann G. P., 1977, Die bestimrnung hoher ozon-konzentrati onen , W1b 21: 125-1 28.
Maini , R., St Clair E. W ., Breed veld F., Furst D., Kalden 1., Weisman M., Srnolen J., Emery P., Harriman
G., Feldmann M., and Lipsky P., 1999, Infliximab (chimeric anti-turnour necro sis factor alpha
monoclonal antibody) versus placebo in rheumatoid arthritis patients recei ving concomitant
methotrexate: a randomised phase lIItrial. ATTRACT Study Group, Lane el354 :1932-1939 .
Makino, Y., Okarnoto K., Yoshikawa N., Aoshima M., Hirota K., Yodoi J., Umes ono K., Makino 1., and
Tanaka H., 1996, Thioredoxin : a redox-regulating cellular cofactor for glnc ocorticoid hormone
action . Cross talk between endocrine control of stress response and cellular antioxidant defense
system ,.I. Clin. Invest 98:2469-2477 .
Malaguarnera, M., Laurino A., Di F., I, Pistone G., Castorina M., Guccione N., and Rampello L., 2001,
Neuropsychiatrie effects and type of IFN-alpha in chronic hepatitis C, J. Interferon Cytokine Res .
21:273-278.
Manns, M. P., McHutchison 1. G., Gordon S. c., Rustgi V . K., Shiffman M., Reind ollar R., Goodman Z.
D., Koury K., Ling M., and Albrecht J. K., 2001 , Peginterferon alfa -2b plus ribavirin compared with
interferon alfa-zb plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial, Lancet
358 :958-965.
Mann , P., 2000, Chronic fatigue syndrome: the fundamentals still appl y, Am . .I. Med. 108:172-173 .
Maresca, M., Colao C., and Leoncini G., 1992, Generat ion of hydrogen peroxide in resring and acti vated
platelets, Cell Bio ehem . Fund. 10:79-85 .
Marin , J., and Rodrlguez-Martlnez M. A. , 1997 , Role of vascular nitric oxide in physi ological and
pathological conditions, Pharmacol . Ther. 75 :111-134.
 R EFER EN CES 407

Markes bery , W . R., 1997 , O xidat ive stress hypothe sis in Alzheime r's dise ase, Free rad. Bio/. Med.
23:1 34 -14 7.
Marme r, R. H., and Parks S., 1998, Ozone treannent in ret init is pigmentosa : effect on color per cept ion
and blood gasses, Ann. Op htha lmol. 30 :16 1-1 63.
Marshall. K. A , Re ite r R. J., Poeggel er B., Aru om a O. 1., and Halli well B., 1996, Evaluation of the
anti oxi da nt activity of melato nin in vitro, Free rad. Biol. Med. 21 :307-3 15.
Ma rtin, P., 1997 , W ound heal ing-aiming for perfect skin regeneration , Science 276 :75-81.
Ma rtindale, W ., and Capper K. T., 1952, The extra pharmacopoeia, The Phannaceutical Press, London,
pp .I -81 6.
Massc helein, W . J., 1996 , lod ometric method for the detenninati on of ozone in a process gas, in Ozon -
Handb uch . Gru ndlage n. Präv ent ion . Therap ie (E. G. Beck, and R. Viebahn-H änsler, Eds .), Ecomed,
Lan dsberg, pp .I-3.
Mat oba, T., Shimokawa H., Nakash ima M., Hirakawa Y ., Muk ai Y ., Hirano K., Kanaide H., and
Takeshit a A ., 20 00, Hydrogen per oxide is an endothel ium -derived hyperpolar izing factor in mice , J.
Clin. Invest 106 :1521-1530.
Matos , H. R., Di Mascio P., and Medeiros M . H., 2000, Protective effect of Iycopene on lipid
peroxidati on and oxidative DNA dam age in cell culture, Arch . Bio chem. Biophy s. 383 :56-59 .
Mat sum oto, A., Sakurai S ., Shinriki N. , Suzuki S ., and Miura T., 2001 , Therapeutic effects of ozonized
olive oi l in the treatment of intractable fistula a nd wound after surgical operation, in Proceedings of
the 15th Ozone World Co ngress, London , UK, 11th-15th September 2001 . Medical Therapy
Conference (lOA 200 I , Ed .), Speedpri nt MacMedia Ltd, Ealing, Lond on, UK , pp .77-84.
Mat sun o, K., Miura T., and Shinriki N ., 1997 , The effect of ozone on platelet activatio n, The 13th Ozone
World Congress, Oct. /99 7. Kyot o. .Iap an :1-4.
Mattassi, R., 1985, Ozonoterapia , Organ izzazione Editoria le Medico Fannaceut ica, Milano , pp .I-179.
Mattassi, R., Bassi P., D'An gelo F., Frauchin a A, and Sbrascini S., 1985 , Ozo ne as therapy in herpes
simplex and herpes zoster dise ase s, in Medical appltcations ot ozo ne (1. LaRaus , Ed .), International
Ozo ne As soc iatio n, N orwalk, pp .134 -1 37 .
Ma ttassi, R., D'Angelo F., Bisetti P., Colombo R., an d Va ghi M. , 1987 , Terapi a con ozono per via
parenterale nelle arteri opat ie oblit eran ti perifer iche : rneccanism o bioch imico e ris ultati clin ici, Il
Giorn ale Di Chirurgia VIII :I 09 - 111.
Maxwell, S ., Cru ick shank A , and Tho rpe G., 1994 , Red wine and antioxidant acti vity in serum, Lancet
344 :193-194 .
May, J. M ., Qu Z.-c., Wh itesell R. R ., and Cob b C. E., 1996 , Ascorbate rec ycl ing in human erythrocytes:
ro le of GSH in redu cing deh yd roascorbate, Free rad. Biol. Me d. 20:543-55 1.
May, M. J., an d Ghos h S., 1998 , Sign al transd ucti on through NF-kB,Imnntno/. Today 19:80-88 .
Mayer, D. 1., 20 00 , Acupuncture : an e vidence-based re view of the cl inical literature, Annu. Rev . Me d.
51 :49-63.
Ma yne, S. T., 1996 , Beta-car otene, car otenoids, an d disease pre vention in hum ans, FASEB .I. 10 :690-70 I .
McCall, M. R., and Fre i B., 1999, Ca n a ntiox idant vitamins materiall y redu ce oxidat ive damage in
hum ans", Free rad. Biol . Med. 26: 1034 -1053.
McCord , J. M ., 1974 , Free radi cals and inflamm ation: protection of synovial fluid by superoxide
disrnutase, Science 185 :529 -531.
McC ord , J. M., 1995, Superox ide rad ica l: co ntro versies, contradictions, and paradoxes, Prot: Soc. Exp .
Biol. Med. 209 :112-117.
Mc Cord , J. M ., and Fridovich 1., 1968, Th e reduction of cytochrome c by milk xan thine oxidase,.1. Biol.
Chem. 243:5753-5760 .
Mc Cord, J. M., and Fr idovich 1., 1969 , Supero xide dismutase . An enzymic function for erythrocuprein
(hemoc uprein), .I. B iol. Chern. 244 :6049-6 055.
McDonnell, W . F., 1991 , Intersubject varia bility in human acute ozone responsiveness,
Phurmacogenetics I : l1 0-113 .
Mc Hutchison, J. G., Gordon S. c., Sch iff E. R., Sh iffman M . L., Lee W . M ., Rustgi V . K., Goodman Z.
D., Ling M.-H ., Cort S., Albrecht 1. K., and for the Hepatitis Interventional Therapy Group, 1998 ,
Interferon alfa -2b alone or in co mbination with riba virin as initial trea tment for chronic hepatitis C,
N. Engl. .I. Me d. 339:148 5-14 92 .
M cl nnes , I. B., an d Liew F. Y ., 1998, Interleukin 15: a proinfl ammatory role in rheumatoid arthritis
synovitis, Immun ol. Today 19 :75-79.
408 REFERENCES

McKenzie, W. H., Knelson 1. H., Rumaro N . J., and House D. E., 1977, Cytogenetic effects of inhalerl
ozone in man, MII/at. Res. 48 :95-102 .
McMillan, G ., 2001, Minimizing the threat ofnosocomial infection, .1AAPA. 14 :55-8 ,61,69.
Meadows, J., and Smith R. C; 1987, Uric acid protects erythrocytes from ozone-induced changes,
Environ . Res. 43 :410 -416.
Meadows, 1., Smith R. c., and Reeves J., 1986, Uric acid protects rnembranes and Iinolenic acid from
ozone-induced oxidation, Biochem. Biophys. Res. Commun . 137:536-541 .
Mecocci, P., Polidori M. C ., Troiano L., Cherubini A, Cecchetti R., Pini G., SIraalman M., Monti D.,
Stahl W ., Sies H., Franceschi c., and Senin U., 2000, Plasma antioxidants and longevity: a study on
healthy centenarians, Free rad. Biol. Med. 28 :1243-1248.
Meewes, C ., Brenneisen P., Wenk 1., Kuhr L., Ma W ., Alikoski 1., Poswig A. , Krieg T., and Scharffetter-
Kochanek K., 2001, Adapti ve antioxidant response protects dermal fibroblasts from UVA- induced
phototoxicity, Free rad. Biol, Med . 30 :238-247 .
Mehlman. M . A , and Borek c., 1987, Toxicity and biochemical rnechanisms of ozone, Environ. Res.
42 :36-53 .
Meister, A , 1994, Glutathione-ascorbic acid antioxidant system in anirnals, .1. Biol. Chem. 269 :9397-
9400 .
Mendiratta, S., Qu Z.-C., and May J. M., 1998a, Erythrocyte ascorbate recycling : antioxidant effects in
blood, Free rod. Biol . Med. 24:789-797 .
Mendiratta, S., Qu Z.-C ., and May J. M ., 1998b, Enzyme-dependent ascorbate rec ycling in human
erythrocytes : role ofthioredoxin reductase, Free rad. Biol. Med . 25 :221-228 .
Menendez, F., et al, 1991, Ozonoterapia en la artritis reumatoidea, CENIC Ciencias Biologicas 20:144-
151.
Menendez, S., 1995, Onicomycosis treated with ozonized oil, in Proceedings Ozone in Medtcin e. 12th
World Congress of the International Ozone Association. 15th to 18th May 1995. Lilie, France
(International Ozone Association, Ed .), Instaprint S .A., Tours, pp.279-282.
Menendez, S., er al , 1995, Application of ozonized oil in the treatment of infantile giardiasis , in
Proceedings Ozone in Medictne. 12th World Congress of the International Ozone Assoctation, 15th
to 18th May 1995, Lilie. France (International Ozone Association, Ed .), Instaprint S.A ., Tours,
pp .297-300.
Menzel, D. B ., 1984, Ozone: an overview of its toxicity in man and animals ,.1. Toxicol. En vtron . Health
13 :183-204.
Merz, T., Bender M. A., Kerr H. D., and Kulle T. J., 1975, Observations of aberrations in chromosomes
of lymphocytes from human subjects exposed at a concentration of 0.5 ppm for 6 and 10 hours,
Mt/tat. Res. 3 :299-302 .
Meydani, S. N., Wu D., Santos M. S., and Hayek M. G. , 1995 , Antioxidanrs and immune response in
aged persons: o verview ofpresent evidence, Amer. .I. Clin. Nutr. 62 : 1462S-1476S .
Mian, E. U., and Agostini G., 1988, Principi di balneoterapia ozon izzata , Edizioni Dedalus, pp .I-146.
Miceli , S ., 1999, Ossigeno-ozonoterapia in patologie c1iniche diverse: nostra esperienza presso l'Ospedale
dei Bianchi Corleone, in L'Ozonoterapia nel 2000 (F. Ceccherelli, and F. Giron, Eds .), Edizioni
Libreria Cortina, Torino, pp .59-63 .
Michel, T., and Feron 0 ., 1997, Perspective series: nitric oxide and nitric oxide synthases. Nitric oxide
synthases : which, where, how, and why?,.I. Clin . Invest. 100 :2146-2152.
Micheli, V. , Valacchi G., Jacomelli G., Diadori A ., Carraro F., and Bocci V ., 2000, Prolonged
ozonetherapy modi fies some bioehernreal properties of the erythrocyte, in Proceedings: I Cong resso
IMO.'). Italla. Siena. 2-4 novembre 2000, pp . 45 -46 .
Michie, H. R., Manogue K. R., Spriggs D. R., Revhaug A ., O'Dwyer S ., Dinarelle C. A , Cerami A.,
Wolff S. M ., and Wilmore D. W., 1988, Detection of c irc ulating tumor necrosi s factor after
endotoxin administration, N. Eng/. .!. Med . 318:1481 -1486.
Miller, M. J. S., and Grisham M. B., 1995, Nitric oxide as a mediater of inflammation? - You had bell er
believe it, Mediat. Inflamm . 4 :387-396 .
Miller, N . 1., Rice -Evans C., Davies M. J., Gopinathan V ., and Milner A , 1993 , A novel method for
rneasuring antioxidant capacity and its application to mon itoring the antioxidant status in premature
neonates, Clin . Sei. 84 :407-412 .
Milligau. N. G., Newcombe R., and Compston D. A S., 1986, A double-blind controlled trial of high -
dose methy1prednisolone in patients with multiple sclerosis. 1: clinical effects,.J. Neurot. Neurosurg.
Psychiatry 50 :511-516 .
 REFERENCES 409

Minetti, M., Mallozzi C., Di Stasi A. M. M., and Pietraforte D., 1998, Bilirubin is an effective antioxidant
of peroxynitrite-mediated protein oxidation in human blood plasma, Arch. Biochem. Biophys.
352 :165-174 .
Miroshin , S. J., and Kontorshikova C. N. , 1995, The use of ozonetherapy technology in the treatment of
modem war surgical trauma, in The ozone in biology and medieine. 2nd all Russ ian scienttfic-
practical conferen ce, Septmber 6-8, 1995. Russian association of ozonetherapy, Reshetnikovskaya
street 2, Nizhni Novgorod, 603006 Russia, p.16 .
Miura, T., Suzuki S., Sakurai S., Matsumoto A., and Shinriki N., 2001, Structure elucidation of ozonated
olive oil, in Proceedings of the 15th Ozone World Congress, London, UK. 11th-15th September
20tH. Medical Therapy Conference (IOA 2001 , Ed.), Speedprint MacMedia LId, Ealing, London,
UK, pp .72-76.
Miyazaki, K., Noda Y., Terada M., and Wakasugi H., 1999, Elevated serum levels of thioredoxin in
patients with chronic and /or malignant Iiver diseases, in Redox regulation of cell signaling and its
clinical applicatlon (L. Packer, and 1. Yodoi , Eds.), Marcel Dekker, Inc ., New York, pp .235-250.
Moertel , C. G., Fleming T. R., Creagan E. T., Rubin J., O'Connell M. J., and Ames M. M., 1985, High-
dose vitamin C versus placebo in the treatment of patients with advanced cancer who have had no
prior chemotherapy. A randomized double- blind comparison, N. Engl. J. Med.312 :137-141.
Mohle, R., Green D., Moore M. A., Nachman R. L., and Rafii S., 1997, Constitutive production and
thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and
platelets, Pro c. Natl. Acad. Sei . U. S. A 94 :663-668 .
Molina , M. J., 1999, The impact of human activities on atmospheric ozone , in Pro ceedings of the Int.
Ozone Symposium , 21 and 22 October 1999. Basel. Switzerland (lOA - EA)G Ed .), Bauer Druck
AG , Basel, pp .33-38 .
Molina , M. J., and Rowland F. S., 1974, Stratospheric sink for chlorofluoromethanes: chlorine atom
catalyzed destruction of ozone, Nature 249 :810-814 .
Moncada, S., 1992, Nitric oxide gas : mediator, modulator, and pathophysiologic entity ., .I. Lab . Clin .
Med.120 :187-191.
Montaner, J. S., and Mellors J. W ., 2001, Antiretroviral therapy for previously treated patients, N. Engl. .I.
Med. 345 :452-455 .
Moore , K., and Roberts L. J., II, 1998, Measurement of lipid peroxidation, Free Rad. Res . 28 :659-671 .
Moraleda, G., 1995, Ozone therapy in the functional recovery from diseases involving damage to central
nervous system cells (Optic nerve dysfunctions, glaucoma , retinitis pigmentosa and maculopathies),
in Proceedings 12th World Congress ofthe International Ozone Associa üon , Lille, Fran ce, 15-18
may 1995, pp.III -123 .
Morgan, D. L., Furlow T . L., and Menzel D. B., 1988, Ozone-initiated changes in erythrocyte membrane
and loss of deformability, Environ. Res. 45 : I08-117 .
Morley, 1. E., and Perry H. M., I1I, 2000, Androgen deficiency in aging men : role of testosterone
replacement therapy,.I. Lab Clin. Med. 135 :370-378.
Morris , C. R., Kuypers F. A., Larkin S., Sweeters N., Simon J., Vichinsky E. P., and Styles L. A., 2000 ,
Arginine therapy: a novel strategy to induce nitric oxide production in sickle cell disease, Br. .I.
Haematol. 111 :498-500.
Morrow, J. D ., Awad J. A., Boss H. J., Blair I. A., and Roberts L. J., 1992, Non-cyclooxygenase-derived
prostanoids (F2- isoprostanes) are formed in situ on phospholipids, Proc . Natl. Acad. Sei . U. S. A
89 :10721 -10725 .
Morrow, J. D., Frei B., Longmire A. W ., Gaziano 1. M., Lynch S. M., Shyr Y., Strauss W . E., Oates 1. A.,
and Roberts L. J., 1995, Increase in circulating products of lipid peroxidation (F2- isoprostanes) in
smokers. Smoking as a cause of oxidative damage, N. Engl. .I. Med. 332 :1198-1203 .
Morrow, J. D ., and Jackson Roberts L., 1997, The isoprostanes: unique bioactive products of lipid
peroxidation, Prog. Lip id Res. 36 :1-21.
Mosmann, T. R., and Sad S., 1996, The expanding universe of T-cell subsets: Thl , Th2 and more,
Immunol. Today 17:138-146.
Motzer, R. J., Rakhit A ., Thompson J. A., Nemunaitis J., Murphy B. A., Ellerhorst J., Schwartz L. H.,
Berg W . J., and Bukowski R. M., 200 I, Randomized multicenter phase II trial of subcutaneous
recombinant human interleukin-12 versus interferon-alpha 2a for patients with advanced renal cell
carcinoma,.I. Interferon Cytok ine Res. 21 :257-263.
Moulin , V., 1995, Growth factors in skin wound healing, EIII'. J. Cell Biol. 68 :1-7.
410 REFERENCES

Mudd, 1. 8., Dawson P. J., and Santrock J ., 1997, Ozone does not react with human erythrocyte
rnembrane lipids, Arch. Biochem. Btophys. 341 :25 1-258.
Murphy-Ullrich, J. E., and Poczatek M., 2000, Activation of latent TGF-beta by thrombospondin-L :
rnechanisms and physiology, Cytokine Growth Factor Rev. 11:59-69.
Murphy, W . 1., and Longo D. L., 2000, Growth hormone as an immunomodulating therapeutic agent,
Immunol. Today 21 :2 11-2 13.
Murry, C. E., Jennings R. B., and Reimer K. A., 1986 , Preconditioning with ischemia: a del ay of lethai
cell injury in ischemic myocardium, Ctrculation 74:1124-1136.
Musselman, D. L., Lawson D. H., Gumnick J. F., Manatunga A. K., Penna S., Goodkin R. S., Greiner K.,
Nemeroff C. B., and Miller A H., 200 I, Paroxetine for the prevention of depression induced by high -
dose Interferon alfa, N. Eng/. .J. Med. 344 :961-966.
Mustacich, D., and Powis G., 2000 , Thioredoxin reductase, Biochem . .J. 346 Pt I :1-8.
Mustafa, M. G., 1990 , Biochemical basis of ozone toxicity, Free Rad. Biol. Med. 9:245-265.
Mustoe, T . A., Pierce G. F., Thornason A., Gramales P., Sporn M. B., and Deuel T. F., 1987 , Accelerated
healing of incisional wounds in rats induced by transforming growth factor-beta, Setent-e 237 : 1333-
1336.
Myerburg, R. J., and Castellanos A., 1997 , Cardiac arrest and sudden death, in Heart diseas e: 0 textbook
ofcardiovascular medictne (E . Braunwald, Ed .), W .B.Saunders, Philadelphia,PA, pp .742-779.
Nakano, A., Cohen M. V., and Downey J. M., 2000, Ischemic preconditioning : from basic rnechanisrns to
clinical applications, Pharmacol. Ther. 86:263-275.
Nakao, N ., Frodl E. M., Widner H., Carlson E., Eggerding F. A., Epstein C. J ., and Brundin P., 1995,
Overexpressing Cu /Zn superoxide dismutase enhances survival of transplanted neurons in a rat
model of Parkinson's disease, Nature Medicine 1:226-231.
Namiki, A., Brogi E., Keamey M ., Kim E. A., Wu T., Couffinhal T., Varticovski L. , and Isner J. M.,
1995, Hypoxia induces vascular endothelial growth factor in cultured human endothelial cells , .J.
8iol. Chem. 270:31189-31195.
Natelson, B. H., 200 I , Chronic fatigue syndrome, .lAMA 285:2557-2559.
Nath, K. A., Haggard 1. 1., Croatt A . J., Grande 1. P., Poss K. D., and Alarn J., 2000, The indispensability
of heme oxygenase-I in protecting against acute heme protein -indu ced toxicity in vivo, Am . J,
Pathol.156:1527-1535.
Nathan, c., 1997, Inducible nitric oxide synthase: what difference does it make", .J. Clin. Invest,
100 :2417-2423 .
Nathan, C. F., Brukner L. H., Silverstein S . C ., and Cohn Z. A. , 1979a, Extracellular cytolysis by
acti vated macrophages and granulocytes. I. Pharmacologic triggering of effector cells and the release
ofhydrogen peroxide,.J. Exp. Med. 149:84-99.
Nathan, C. F., and Cohn Z. A., 1981, Antitumor effects of hydrogen peroxide in vivo, J. Exp. Med.
154 :1539-1553 .
Nathan, C. F., Silverstein S. C; Brukner L. H ., and Cohn Z. ,.1. ., 1979b , Extracellular cytolysis by
activated mac rophages and granulocytes. 11. Hydrogen peroxide as a mediater of cytotoxicity,.f. Exp.
Med.149 :100-113 .
National Emphysema Treatment Trial Research Group, 2001, Patients at high risk of death afte r 11Ing-
volume-reduction surgery, N. Engl. .I. Med. 345 :1075-1083.
Nayak, M. S., Kita M., and Marmor M. F., 1993, Protection of rabbit retina frorn ischernic injury by
superoxide dismutase and catalase, Invest Ophthalmol. Vis. Sei. 34 :2018-2022 .
Neubaus, 0 ., Farina C ., Yassouridis A., Wiendl H., Then B. F., Dose T., Wekerle H., and Hohlfeld R.,
2000, Multiple sclerosis : comparison of copolymer-I- reactive T cell lines from treated and untreated
subjects reveals cytokine shift from T helper 1 to T helper 2 cells, Proc. Natl. Acad. Sei. U. S. A
97 :7452-7457.
Neumann, A . U., Lam N . P., Dahari H., Gretch D. R., Wiley T. E., Layden T . J., and Perelsen ,-\. S .,
1998, Hepatitis C viral dynamics in vivo and the anti viral efficacy of interferon-a therapy, Science
282 :103 -107 .
Newsome, D. A. , Swartz M., Leone N . c., Elston R. c., and Miller E., 1988 , Oral zinc in macular
degeneration, Arch. Ophthalmol. 106 :192-198 .
Nicolaides, N ., 1974, Skin lipids:their biochemical uniqueness, Science 186 :19-26 .
Nolte, P., 1999, Christian Friedrich Schoenbein empiricism and speculation, in Proceedings ofthe 111/.
Ozone Symposium. 2/ and 22 October /999. Basel . Switzerland (10,.1. - EA..G Ed .), Bauer Druck
AG, Basel , pp.15-30.
 REFERENCES 411

Nortier , J. L., Martinez M. C., Schmeiser H. H., Arlt V. M., Bieler C. A., Petein M., Depierreux M. F., De
Pauw L., Abramowicz D., Vereerstraeten P., and Vanherweghem J. L., 2000 , Urothelial carcinoma
associated with the use of a Chinese herb (Aristolochia fangchi), N. Engl . .I. Med. 342 :1686-1692.
Nortvedt, M. W ., Riise T ., Myhr K. M., Nyland H. 1., and Hanestad B. R., 1999, Type I interferons and
the quality of life of multiple sclerosis patients. Results from a clinical trial on interferon alfa-2a,
Mllit. Sc/er . 5:317-322 .
Novogrodsky, A., Stenzel K. H., and Rubin A. L., 1977, Stimulation of human peripheral blood
Iymphocytes by periodate, galactose oxidase, soybean agglutinin, and peanut agglutinin : differential
effects ofadherent cells,J.lmmllnol. 118 :852-857 .
Nowak, D., Kalucka S., Bialasiewicz P., and Krol M., 2001, Exhalation of H202 and thiobarbituric acid
reactive substances (TBARs) by healthy subjects, Free Rad. Biol. Med. 30:178-186.
Null, 1996, Ozone: a wide-spectrum realer, Penthouse Magazine January.
Nylandsted, J., Rohde M., Brand K., Bastholm L., Elling F., and Jaattela M., 2000, Selective depletion of
heat shock protein 70 (Hsp70) activates a tumor- specific death program that is independent of
caspases and bypasses Bel- 2, Proc . Nat!. Acad. Sei. U. S. A 97 :7871-7876 .
O'Dell . J. R., 1999, Anticytokine therapy--a new era in the treatment of rheumatoid arthritis?, N. Engl . J.
Med.340:310-312.
O'Farrelly, C., and Crispe I. N., 1999, Prometheus through the looking glass: reflections on the hepatic
immune system ,lmmllnol. Today 20 :394-398 .
O'Neill, C. A., van der Vliet A., Hu M.-L., Kaur H., Cross C. E., Louie S., and Halliwell B., 1993,
Oxidation ofbiologic molecules by ozone: the effect ofpH, J. Lab. Clin. Med. 122:497-505 .
O'Reilly, M. S., Boehm T., Shing Y., Fukai N ., Vasios G., Lane W . S., Flynn E., Birkhead J. R., Olsen B.
R., and Folkman J., 1997, Endostatin: an endogenous inhibitor of angiogenesis and tumor growth,
Cel/ 88 :277-285.
O'Reilly, M. S., Holmgren L., Shing Y., Chen c., Rosenthai R. A., Moses M., Lane W. S., Cao Y., Sage
E. H., and Folkman J., 1994, Angiostatin : a novel angiogenesis inhibitor that mediates the
suppression of metastases by a Lewis lung carcinoma, Ce1l79 :315-328 .
Oehler , M. K., and Bicknell R., 2000, The promise of anti-angiogenic cancer therapy, BI'. J. Cancer
82:749-752.
Okabe, N., 2001, The pathogenesis ofCrohn's disease, Digestion 63 Suppl1 :52-59 .
Olivieri, G., Bodycote J., and Wolff S., 1984, Adaptive response of human Iymphocytes to low
concentrations ofradioactive thymidine, Science 223:594-597 .
Olsen, S. J., DeBess E. E., McGivern T . E., Marano N ., Eby T., Mauvais S., Balan V . K., Zirnstein G.,
Cieslak P. R., and Angulo F. J., 2001, A nosocomial outbreak of fluoroquinolone-resistant
salmonella infection, N. Engl . .I. Med. 344 :1572-1579.
Olwin , J. H., Ratajczak H. V., and House R. V., 1997, Successful treatment of herpetic infections by
autohemotherapy,.1. Altern. Complement Med. 3:155-158.
Onik, G., Maroon J., Helms c., SchweigeI J., Mooney V., Kahanovitz N ., Day A., Morris J., McCulloch
J. A., and Reicher M., 1987, Automated percutaneous discectomy: initial patient experience. Work in
progress, Radiology 162 :129-132 .
Organisciak, D. T., Darrow R. A ., Barsalou L., Darrow R. M., and Lininger L. A., 1999, Light-induced
damage in the retina : differential effects of dimethylthiourea on photoreceptor survival, apoptosis and
DNA oxidation, Photochem. Photobiol. 70 :261-268 .
Organisciak, D. T., Darrow R. A., Barsalou L., and Lininger L. A., 1997, Light induced oxidative damage
and apoptosis in the retina, Invest Ophthalmol. Vis. Sei. 38 .
Orta de Velasquez, Ma. T., Rojas Ma. N ., Martlnez J. L., and Monje 1., 2001, Destruction of helminth
eggs (Ascaris sllllm) by ozone, in Proceedings ofthe 15th Ozone World Congress, London, UI(, I lth-
15th September 2001, Medi cal Therapy Conference (lOA 2001, Ed.), Speedprint Mac Media Ltd,
Ealing, London , UK, pp .63-71.
Otterbein. L. E., Kolls J. K., Mantell L. L., Cook J. L., Alam J., and Choi A. M. K., 1999, Exogenous
administration of heme oxygenase-I by gene transfer pro vides protection against hyperoxia -induced
lung injury,.I. Clin.Tnvest. 103 :1047-1054 .
Owry, T. D., Day B. J., and Crapo 1. D., 1996, Extracellular superoxide dismutase: a regulator of nitric
oxide bioavailability, Lab Invest 75 :617-636 .
Packer, L., Roy S., and Sen C. K., 1997, Alpha-lipoic acid : a metabolic antioxidant and potential redox
modulator of transcription, Adv. Pharmacol. 38:79-101 .
412 REFERENCES

Palace, V. P., Khaper N., Qin Q ., and Singal P. K ., 1999, Antioxidant potentials of vitanun A and
carotenoids and their relevance to heart disease, Free Rad. Biol. Med. 26 :746-761 .
Palmieri, B., Manno M., and Palmieri G., 1999, Trallamento deI dolore osseo metastaneo con infiltrazioni
di ozono. Osservazioni c1iniche preliminari ., in Abstracts III Giornate Trivenete di Ossigeno -Ozono
Terapia, Padova 8-90ttobre 1999 (F . Ceccherelli , Ed.), Padova.
Pamphilon, 0 ., 2000, Viral inactivation offresh frozen plasma, BI'. J. Haematol. 109 :680-693 .
Pannen, B. H. 1., Köhler N ., Hole B., Bauer M., Clemens M . G., and Geiger K. K., 1998, Prote ctive role
of endogenous carbon monoxide in hepatic rnicrocirculatory dysfunction after hemorrhagic shock in
rats, J. Clin . Invest. 102: 1220-1228.
Pantel , K., Cote R. 1., and Fodstad 0 ., 1999, Detection and c1inical importance of rnicrometastatic
disease,.1. Nat . Cancer Inst. 91 : 1113-1124.
Pardo, C. A., Xu Z., Borchelt D. R., Price D. L., Sisodia S. S., and Cleveland D. W., 1995, Superoxide
dismutäse is an abundant component in cell bodies, dendrites, and axons of rnotor neur ons and in a
subset of other neurons, PI'Oc' Not. Acad. Sei. USA 92 :954-958.
Parmiani, G., Rodolfo M., and Melani c., 2000 , Immunological gene therapy with ex vivo gene -modified
tumor cells : a critique and a reappraisal , Hum. Gene Ther. 11: 1269-1275.
Parola, M., Bellomo G., Robino G., Barrera G., and Dianzani M. U., 1999, 4-Hydroxynonenal as a
biological signal : molecular basis and pathophysiological implications, Antiox. Redox Signal. 1:255 -
284 .
Parola, M., Robino G., Marra F., Pinzani M., Bellomo G ., Leonarduzzi G., Chiarugi P , Camandola S.,
Poli G., Waeg G., Gentilini P., and Dianzani M. U., 1998, HNE interacts directly with lNK isoforms
in human hepatic stellate cells ,.!. Clin. Invest, 102 :1942-1950.
Patel , R. P., 2000 , Biochemical aspects of the reaction of hemoglobin and NO : implications for Hb-based
blood substitutes, Free Rad. Biol . Med. 28 :1518-1525.
Patterson, C ., and Runge M. S., 2000, Therapeutic myocardial angiogenesis via vascular endothelial
growth factor gene therapy: moving on down the road , Circulation 102 :940-942.
Pauleikhoff, 0 ., Barondes M. 1., Minassian 0 ., Chisholm I. H., and Bird A. c., 1990, Drusen as risk
factors in age-related macular disease, Am . .!. Ophthalmol. ]09 :38-43 .
Pauleikhoff, 0 ., and Koch 1. M., 1995, Prevalence of age-related macular degeneration, Curr. Opin.
Ophthalmol.6:51-56 .
Paulesu, L., Luzzi E., and Bocci V ., 1991, Studies on the biological effects of ozone: 2. Induction of
tumor necrosis factor (TNF-a) on human leucocytes, Lymphokine Cytokine Res. 10:409-412.
Payne, L. C; and Krueger 1. M., 1992, Interactions of cytokines with the hypothalamus-pituitary axis , J.
Immunother. 12:171-173 .
Payr, E., 1935, Über Ozonbehandlung in der Chirurgie, Miinch . Med. Woch ensehr. 82 :220-29 1.
Pell, E. 1., Schlagnhaufer C. 0 ., and Arteca R. N ., 1997, Ozone-induced oxidative stress: mechanims of
action and reaction , Phys iol. Plant. 100:264-273 .
Pellinen, R., Palva T., and Kangasjarvi 1., 1999, Subcellular localization of ozone-induced hydrogen
peroxide production in birch (Betula pendula) leaf cells, Plant J. 20 :349-356.
Pendino, K. 1., Laskin J. 0., Shuler R. L., Punjabi C. 1., and Laskin D. L., 1993, Enhanced production of
nitric oxide by rat alveolar macrophages after inhalation of a pulmonary irritant is associated with
increased expression of nitric oxide synthase,.I. Immunol, ISI :7 196-7205.
Peng , 1., Iones G. L., and Watson K., 2000 , Stress proteins as biomarkers of oxidative stress: effects of
antioxidant supplements, Free Rad. Biol. Med. 28 :1598-1606.
Peralta, C; Leon O. S., Xaus c., Prats N., Jalil E. c., PIaneIl E. S., Puig-Parellada P., Gelpi E., and
Rosello-Catafau 1., 1999, Protective effect of ozone treatment on the injury associated with hepatic
ischemia-reperfusion : antioxidant-prooxidant balance, Free Rad. Res. 31:191-196 .
Peralta, C., Xaus C ., Bartrons R., Leon O. S., Gelpi E., and Rosello-Catafau J., 2000, Effect of ozone
treatment on reactive oxygen species and adenosine production during hepatic ischemia-reperfusion,
Free Rad. Res . 33 :595-605 .
Perdue, M. H., 1999, Mucosal inununity and inflammation 111. The mucosal antigen barrier: cross talk
with mucosal cytokines, Amer. J. Phystol. 277 :G I-G5.
Perletti, G., Concari P., Giardini R., Marras E., Piccinini F., Folkman 1., and Chen L., 2000, Antitumor
activity of endostatin against carcinogen-induced rat primary mammary tumors, Cancer Res.
60 :1793-1796.
Perutz, M. F., 1996, Tak ing the pressure off, Nature 380 :205-206.
 REFERENCES 413

Peterson, L. R., 1998 , Estrogen replacement therapy and coronary artery disease, Curr. Opin. Cardiol.
13:223-231 .
Perralia, 8., Tommasini G., Lavaroni A, and Fabris G ., 2001 , A tutto gas! 11 "mal di schiena" curato con
l'ozonoterapia, Riv. Neuroradiol. 14:71-73.
Pianko, S., and McHutchison J., 1999 , Chronic hepatitis B: new therapies on the horizon", Lancet
354 :1662 -1663 .
PickrelI , J. A, Hahn F. F., Rebar A. H., Horoda R. A , and Henderson R. F., 1987 , Changes in collagen
metabolism and proteinolysis after repeated inhalation exposure to ozone, Exp. Mol. Pathol. 46 :159-
167.
Pierce, G. F., Tarpley J. E., Tseng J., Bready J., Chang D., Kenney W. C., Rudolph R., Robson M. c.,
Vande Berg J., Reid P., Kaufman S., and Farrell C. L., 1995 , Detection of platelet-derived growth
factor (PDGF)-AA in actively healing human wounds treated with recombinant PDGF-BB and
absence of PDGF in chronic nonhealing wounds,.I. Clin. Invest. 96: 1336 -1350 .
Piguet , B., Palmvang I. B., Chisholm I. H., Minassian D., and Bird A. c., 1992 , Evolution of age-related
macular degeneration with choroidal perfusion abnormality, Am. .1. Ophthalmol. 113:657-663.
Pisetsky, D. S., 2000, Tumor necrosis factor blockers in rheumatoid arthritis, N. Engl. .I. Med. 342 :810-
811.
Pizarro, T. T., Michie M . H., Bentz M., Woraratanadharm J., Smith M. F., Jr ., Foley E., Moskaluk C. A .,
Bickston S. J., and Cominelli F., 1999, IL-18 , a novel immunoregulatory cytokine, is up-regulated in
Crohn's disease: expression and localization in intestinal mucosal cells,.I. Immunol . 162:6829-6835.
Podda, M., Traber M. G ., Weber C., Yan L.-J., and Packer L., 1998, U'V-irradiation depletes antioxidants
and causes oxidative darnage in a model ofhuman skin, Free Rad. Biol. Med. 24:55-65.
Poli , G., and Parola M., 1997, Oxidative darnage and fibrogenesis, Free Rad. Biol. Med. 22:287-305.
Poli, G., and Schaur R. J., 2000, 4-Hydroxynonenal in the pathomechanisms of oxidative stress, IUBMB.
Life 50 :315-321.
Polidori, M. c., Stahl W ., Eichier 0 ., Niestroj 1., and Sies H., 2001, Profiles of antioxidants in human
plasma, Free Rad. Biul. Med.30:456-462.
Polignano, R., Vannucchi P. L., and Degl'Innocenti R., 2000, Efficacia antisettica dell'acqua ozonizzata
su germi isolati da lesioni croniche della cute, in Proceedings: I Congresso IMOS. Italia , Siena . 2-4
novembre 2000, pp .49-50.
Polman. C. H., and Uitdehaag B. M ., 2000, Drug treatment of multiple sc1erosis, Brit. Med. .I. 321 :490-
494 .
Pomerantz, R. J., 1999 , Res idual HIV -I disease in the era of highly active antiretroviral therapy, N. Engl.
J. Med. 340 :1672-1674 .
Popovsky, M. A ., Chaplin H . c., Jr ., and Moore S. B., 1992 , Transfusion-related acute lung injury: a
neglected , serious complication of haemotherapy., Transfusion 32 :589-592.
Poumaras, C. J., Miller 1. W ., Gragoudas E. S., Husain D., Munoz J. L., Tolentino M. J., Kuroki M., and
Adarnis A. P., 1997, Systemic hyperoxia decreases vascular endothelial growth factor gene
expression in ischemic primate retina, Arch. Ophthalmol. 115 :1553 -1558.
Poutsiaka, D . D ., 2001 , Antirnicrobial resistance in the chronically critically ilI patient, Clin. Chest Med.
22 :87-103, viii .
Powell , P., Bentall R. P., Nye F. 1., and Edwards R. H., 2001 , Randomised controlled trial of patient
education to encourage graded exercise in chronic fatigue syndrome, Brit. Med.. .1. 322 :387-390.
Poynard, T., Bedossa P., Opolon P., and for the OBSVIRC M. C. a. D. g., 1997, Natural history of liver
fibros is progression in patients with chronic hepatitis C, Lancet 349 :825-832.
Poynard, T ., Marcellin P., Lee S. S., Niederau c., Minuk G . S., Ideo G., Bain V., Heathcote J., Zeuzem
S., Trepo c., Albrecht J ., and for the International Hepatitis Interventional Therapy Group (IHIT),
1998 , Randornised trial of interferon a2b plus ribavirin for 48 weeks or for 24 weeks versus
interferon a2b plus placebo for 48 weeks for treatrnent of chronic infection with hepatitis C virus,
Lancet 352 :1426-1432.
Prashar, S., Pandav S . S., Gupta A ., and Nath R., 1993, Antioxidant enzymes in RBCs as a biological
index of age related rnacular degeneration, Acta Ophthalmol. (Copenh) 71 :214 -218 .
Prather, M., Midgley P., Rowland F. S., and Stolarski R., 1996, The ozone layer: the road not taken,
Nature 381 :55 1-553.
Present , D. H., Rutgeerts P., Targan S., Hanauer S. B., Mayer L., van Hogezand R. A ., Podolsky D. K.,
Sands B. E., Braakman T., DeWoody K. L., Schaible T. F., and van Deventer S. J. H., 1999 ,
414 REFERENCES

Infliximab for the treatment of fistulas in patients with Crohn's disease, N. Engl. J. Med. 340 :1398-
1405 .
Prins , J. B., Bleijenberg G., Bazelmans E., Elving L. D., de Boo T. M ., Severens J . L., van der Wilt G. J.,
Spinhoven P., and van der Meer J. W ., 2001, Cognitive behaviour therapy for chronic fatigue
syndrome: a multicentre randomised controlled trial, Lancet 357 :84 1-847.
Prows, D. R ., Shertzer H. G., Daly M. J ., Sidman C. L., and Leikauf G . D., 1997, Genetic analysis of
ozone-induced acute Jung injury in sensitive and resistant strains of mice, Not. Gellet. 17:471-474 .
Pryor, W. A ., 1992, How far does ozone penetrate into the pulmonary air/tissue boundary before it
reacts?, Free Rad. Biol . Med . 12:83-88.
Pryor , W. A , 1993 , Ozone in all its reactive splendor, J. Lab. Clln . Med. 122 :483-486.
Pryor, W. A ., 1994, Mechanisms of radical formation from reactions of ozone with target molecules in
the lung, Free Rad. Blol. Med . 17:451-465 .
Pryor, W . A , 2000, Vitamin E and heart disease: basic science to clinical inter vention trials, Free Rad.
Biol .. Med. 28 :141-164.
Pryor, W. A, Squadrito G. L., and Friedman M., 1995, The cascade mechanism to explain ozone toxicity:
the role of lipid ozonation products, Free Rad. Blol .. Med. 19:935-941 .
Pullar, J. M ., Vissers M. C., and Winterboum C. c., 2000, Living with a killer: the effects of
hypochlorous acid on mammalian cells, IUBMB. Life 50 :259-266 .
Purasiri, P., Mckechnie A, Heys S . D., and Eremin 0 ., 1997, Modulation in vitro of human natural
cytotoxicity, lymphocyte proliferative response to mitogens and cytokine production by essent ial
fatty acids, Immunology 92:166-172.
Puskas, F., Gergely P., Jr., Banki K., and Perl A , 2000, Stimulation of the pentose phosphate pathway
and glutathione levels by dehydroascorbate, the oxidized form of vitamin C, FASEB.I. 14 :1352 -
1361.
Qi, W.-N ., and Scully S. P., 1997 , Extracellular collagen modulates the regulation of chondrocytes by
transforming growth factor-bl ,.J. Orthopaed. Res . 15:483-490.
Quinn, M. A , Clyne J. H., Wolf M. M ., and Crvickshank D., 1986 , Storage of platelet concentrates - an
in vitro study offour types ofplastic packs, Pathology 18 :331-335 .
Rahman, 1., Clerch L. B., and Massaro D., 1991 , Rat lung antioxidant enzyme induction by ozone, Amer.
J. Physiol. 260 :L412-L418.
Rahrnan, 1., Massaro G. D., and Massaro D., 1992 , Exposure of rats to ozone: evidence of damage to
heart and brain, Free Rad. Biol. Med. 12 :323-326 .
Ranieri, A , D'Urso G., Nali C; Lorenzini G., and Soldatini G. F., 1996, Ozone stimulates apoplastic
antioxidant systems in pumpkin leaves, Phvsiol. Plant . 97 :381-387 .
Ranieri, A., Petacco F., Castagna A ., and Soldatini G. F., 2001, redox state and peroxidase system in
sunflower plants exposed to ozone, Plant Sei. 159: 159-167 .
Ranjbar, S ., and Holmes H., 1996 , Influence of hydrogen peroxide on the in vitro infectivity of human
immunodeficiency virus , Free Rad. Blol . Med. 20 :573-577 .
Rapp, L. M., Singh S. H., and Choi J. H., 1997 , Lipid peroxidation pre cedes apoptosis in retinas damaged
by spectral light exposure, Inve st Ophthalmol. V/So Sei. 38 .
Rasterayeva, M ., Struchkov S., Belova A., Peretaygin S., and Khroulev S., 200 I , Ozonetherapy in
complex treatment ofneuropathy in bum patients, in Proceedings ofthe 15th Ozone World Cong ress ,
London. UK. 11th-15th September 2001 . Medical Therapy Conference (10.-\ 200 I, Ed .), Speedprint
MacMedia LId, Ealing, Lond on, UK, pp .159 -162 .
Re , R., Pellegrini N ., Proteggente A, Pannala A., Yang M., and Rice-Evans c., 1999, Antioxidant
activity applying an improved ABTS radical cati on decolorization assay, Free Rad. Biol.. Med.
26:1231-1237 .
Reddy, S. P., Harwood R. M., Moore D. F., Grimm E. A., Murray J. L., and Vadhan-Raj S ., 1997,
Recombinant Interleukin-2 in combination with recombinant interferon-g in patients with advanced
malignancy: a phase I study,.I. Immunother. 20 :79-87 .
Rees , L., and Weil A, 2001 , Integrated medicine, Brit . Med. J. 322:119-120.
Reeve, V. E., and Tyrrell R. M., 1999, Heme oxygenase induction mediates the photoimmunoprotective
activity of UVA radiation in the mouse, Proc. Natl . Acad. Sei . U. S. A 96 :9317 -9321 .
Reichard, 0 ., Norkrans G., Fryden A., Braconier l -H ., Sönnenborg A., Weiland 0 ., and for the Swedish
Study Group, 1998 , Randomised, double-blind, placebo-controlled trial of interferon a-2b with and
without ribavirin for chronic hepatitis C, Lancet 351 :83-8 7.
Reichlin, S ., 1993 , Neuroendocrine-immune interactions, N. ElIgi. .J. Med. 329 :1246-1253 .
 REFERENCES 415

Reid, S., Chalder T., Cleare A., Hotopf M., and Wessely S., 2000, Chronic fatigue syndrome, Brit. Med..
.I. 320:292 -296 .
Reiter, R. J., 1991, Pineal melatonin : cell biology of its synthesis and of its physiological interactions,
Endocr. Rev. 12:151-180 .
Remick, D. G., and Villarete L., 1996 , Regulation of cytokine gene expression by reactive oxygen and
reactive nitrogen intermediates, .1. Leukoc. Biol. 59 :471-475.
Renaud, B., and Brun -Buisson C., 200 I, Outcomes of primary and catheter-related bacteremia. A cohort
and case- control study in critically ill patients, Am . .I. Resp lr. Crit Care Med. 163: 1584-1590 .
Rice-Evans, C., and Miller N. 1., 1994, Total antioxidant status in plasma and body fluids, Meth. Enz imol.
234:279-293 .
Rice , R. G., 1999, Evolution of drinking water treatment with ozone, in Proceedings 0/ the Int. Ozone
Symposium , 21 and 22 October 1999, Basel. Switzerland (I0A - EA 3G Ed .), Bauer Druck AG ,
Basel , pp .241-252.
Rice, R. G., 2001 , Century 21 - Pregnant with ozone, in Proceedings ofthe 15th Ozone World Congress,
London, UK. 11th-15th September 2001 . Volume I (I0A 2001, Ed .), Speedprint MacMedia LId,
Ealing, London, UK, pp .I-19 .
Richter, c., Gogvadze V., Laffranch i R., Schlapbach R., Schweizer M., Suter M., Walter P., and Yaffee
M., 1995, Oxidants in mitochondria: from physiology to diseases, Biochim. Biophys. Acta 1271:67-
74.
Richter, C; Park J. W., and Ames B. N., 1988, Normal oxidative damage to mitochondrial and nuclear
DNA is extensive, Proc. Nat . Acad. Sei. USA 85 :6465-6467 .
Riethmüller, G., Klein C. A., and Pantel K., 1999, Hunting down the seminal cells of c1inical metastases,
Immunol . Today 20:294-296 .
Ritossa, F., 1962, A new puffing pattern induced by temperature shock and DNP in Drosophila,
Experientia 18:571-573 .
Riva Sanseverino, E., 1989, Knee-joint disorders treated by oxygen-ozone therapy, Eur. Medicophysica
25 :163- 170.
Riva Sanseverino, E., Meduri R. A., Pizzino A., Prantera M., and Martini E., 1990, Effects of oxygen -
ozone therapy on age -related degenerative retinal maculopathy., Panminerva Med. 32 :77- 84.
Roberts , A. B., Sporn M. B., Assoian R. K., Smith J. M., Roche N. S., Wakefield L. M., Heine U. 1.,
Liotta L. A ., Falanga V., Kehrl J. H., and ., 1986, Transforming growth factor type beta : rapid
induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro, Proc.
Natl . Acad. Sei. U. S. A 83:4167-4171.
Roberts, L. 1., and Morrow J. D., 2000, Measurement of F(2)-isoprostanes as an index of oxidative stress
in vivo, r-ee Rad. Biol. Med. 28:505-513 .
Robinson, G. S., Pierce E. A., Rook S. L., Foley E., Webb R., and Smith L. E., 1996,
Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative
retinopathy, Proc. Nat . Acad. Sei. USA 93 :4851-4856 .
Robinson , J. M. , and Rowland R. A., 2001, Carbohydrate and carbon metabolite accumulation responses
in lea ves of ozone tolerant and ozone susceptible spinach plants after acute ozone exposure,
Photosynthesis Res. 50: 103-115 .
Rodriguez, M. M., Menendez S., Devesa E., Gomez M., Garcia J., and Carrasco M., 1993, Ozone therapy
for senile dementia, in Proceedings ofthe Xl Ozone World Congress. San Francisco, pp .M4-19-M4-
25.
Roederer, M., Staal F. J. T., Raju P. A., Ela S. W., Herzenberg Le. A., and Herzenberg L. A., 1990,
Cytokine-stimulated human immunodeficiency virus replication is inhibited by N-acetyl-L-cysteine,
Prot: Nat. Acad. Sei. USA 87 :4884-4888 .
Rokitansky, 0 .,1982, Klinik und Biochemie der Ozontherapie., Hospital is 52 :643-647.
Rokitansky, 0., Rokitansky A., Steiner J., Trubel W., Viebahn R., and Washüttl 1., 1981, Die
Ozontherapie bei peripheren, arteriellen Durchblutungs-störungen; klinik, biochemische und
blutgasanalytische Untersuchungen, in WasseI' 10A, Ozon-Weltkongress, Berlin, pp .53-75 .
Romero Valdes, A., Menendez Cepero S., Gomez Moraleda M., and Ley Pozo 1., 1993, Ozone therapy in
the advanced stages ofarteriosclerosis obliterans, Angiologia 45 :146-148 .
Romero, A., et al., 1988, La ozonoterapia en la aterosclerosis obliterante, CENIC Ciencias Biologicas
20:70-76.
Romero , A., et al. , 1993, Arteriosclerosis obliterans and ozone therapy: its administration by different
routes, Angiologie : 177-179 .
416 REFERENCES

Romero, M. J., Bosch-Morell F., Romero B., Rodrigo J. M., Serra M. A ., and Romero F. J., 1998 , Serum
malondialdehyde: possible use for the c1inical rnanagement of chronic hepatitis C patients, Fre e Rad.
Blol.. Med . 25 :993-997.
Rosa, L., Rosa E., Sarner L., and Barrett S., 1998 , A close look at therapeutic touch, .lAMA 279: 1005-
1010 .
Rosen, D. R., Siddique T., Parterson D., Figlewicz D. A., Sapp P., Hentati A. , Donaldson D., Goto J.,
O'Regan J. P., Deng H.-X., Rahmani Z., Krizus A., McKenna- Yasek D., Cayabyab A., Gaston S. M.,
Berger R., Tanzi R. E., Halperin J. J., Herzfeldt B ., Van den Bergh R., Hung W.- V., Bird T., Deng
G., Mulder D. W., Smyth c., Laing N . G., Soriano E., Pericak-Vance M. A., Haines J., Rouleau G.
A., Gusella J. S., Horvitz H. R., and Brown R. H. Jr., 1993 , Mutati ons in Cu/Zn superoxide
dismutäse gene are associated with familial amyotrophic lateral sclerosis, Nature 362 :59-62 .
Rosenberg , G. A., 1999, Ischemic brain edema, Prog. Cardiova sc. Dis . 42 :209-216.
Rosenberg. S . A ., 2001, Progress in human tumour immunology and immunotherapy, Nature 411 :380-
384 .
Rosenberg, S. A ., Lotze M. T., Muul L. M., Chang A. E., Avis F. P., Leitman S., Linehan W . M.,
Robertson C . N ., Lee R . E., Rubin J. T ., Seipp C. A., Simpson C . G., and WlJite D. E., 1987 , A
progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated
killer cells and interleukin-2 or high-dose interleukin-2 alone., N. Engl. .I. M ed. 316 :889-897 .
Ross, R., 1987, Platelet-derived growth factor, Annu. Rev. Med. 38:71-79 .
Ross , R., 1993, The pathogenesis of atherosclerosis: a perspective for the 1990s, Nature 362 :801-809.
Roth, J. A., and Cristiano R. J., 1997, Gene therapy for cancer: what have we done and where are we
going?, .!. Not . Cancer Inst . 89 :21-39 .
Rotilio, G., 2001, Risk from exposure to metals: deficits and excesses (Cu , Fe, Mn, AI, Cr, Bl, in
Nutrition and Brain (1. D. Fernstrom. R. Uauy , and P. Arroyo, Eds.), Karger AG , Basel. pp .247-262.
Rotilio, G., Carri M. T ., Rossi L., and Ciriolo M . R., 2000 , Copper-dependent oxidative srress and
neurodegeneration, IUBMB. Life 50 :309-314 .
Rotruck, J. T., Pope A. L., Ganther H. E., Swanson A. B., Hafeman D. G., and Hoekstra W . G ., 1973 ,
Selenium: biochemical role as a component of glutathione peroxidase, Science 179 :588 -590 .
Rousseau, V., Haeffner-Cavaillon N ., Poignet J. L., Meyrier A ., and Carreno M. P., 2000, In vivo
intracellular cytokine production by leukocytes during haemodialysis, Cytoklne 12:506-517.
Rowland, L. P., and Shneider N. A., 2001, Amyotrophic lateral sclerosis, N. Engl. .I. Med. 344 :1688-
1700 .
Rowland, M ., 2000, Transmission of Helicobacter pylori : is it all child's play ?, Lancet 355 :332-333 .
Rubartelli, A., Poggi A., Sitia R., and Zocchi M. R., 1999, HfV-I Tat : a polypeptide for all seasons,
Immunol. Today 19:543-545 .
Rudick, R. A ., Cohen 1. A., Weinstock-Guttman B., Kinkel R. P., and Ransoh olT R. M ., 1997.
Management ofmultiple sclerosis, N. Engl . .I. Med. 337 :1604 -1611.
Rudikoff, D., and Lebwohl M. , 1998 , Arepie dermatitis, Lancet 351:1715-1721.
Rudrnan, D., Feiler A. G., Nagraj H. S., Gergans G. A ., Lalitha P. V., Goldberg A. F., Schlenker R. A "
- Cohn L., Rudman I. W., and Mattsort D. E., 1990 , Effects of human growth hormone in men over 60
years old, N. Engl. .I. Med. 323 :1-6.
Ruggenenti, P., Schieppati A., and Remuzzi G., 200 I, Progression, remission, regression of chronic renal
diseases, Lancet 357 :1601-1608 .
Ruiz, L., Carcelain G., Martinez-Picado J., Frost S., Marfil S., Paredes R., Romeu J., Ferrer E., Morales-
Lopetegi K., Autran B., and Clotet B., 2001, HIV dynamics and T-cell immunity after three
structured treatment interruptions in chronic HfV-l infection, AIDS 15:FI9-F27 .
Ruscetli, F., Varesio L., Ochoa A ., and Ortaldo J., 1993, Pleiotropic effects of transforming growth
factor-b on cells of the immune systern., in Immunomodulating drugs. Annals 0/ The New York
Academy ofSciences, vol. 685 (V . S. Georgiev, and H. Yamaguchi, Eds .), The New York Acaderny
of Sciences, New York, pp .488-500.
Ryter, S. W ., and Tyrrell R. M. , 2000, The heme synthesis and degradation pathways: role in oxidant
sensitivity. Heme oxygenase has both pro- and antioxidant properties, Free Rad. Biol . Med. 28 :289-
309 .
Sagai, M ., and Ichinose T., 1980, Age-related changes in lipid peroxidation as measured by ethane,
ethylene, butane and pentane in respired gases ofrats, Life Sei. 27:731-738 .
 REFERENCES 417

Sagai , M., and Ichinose T., 1991 , Biochemical effects of combined gases of nitrogen dioxide and ozone.
IV. Changes of lipid peroxidation and antioxidative protective systems in rat lungs upon Iife span
exposure, Toxicology 66 :121-132.
Sagara. Y., Dargusch R., Chambers D., Davis J., Schubert D ., and Maher P., 1998, Cellular mechanisms
of resistance to chronic oxidative stress, Free Rad. Biol . Med . 24 :1375-1389.
Sali ou, c., Kitazawa M ., McLaughlin L., Yang J. P., Lodge J. K ., Tetsuka T., lwasaki K. , Cillard J.,
Okamoto T., and Packer L., 1999, Antioxidants mod ulate acute solar ultraviolet radiation-induced
NF- kappa-B activation in a human keratinocyte cell line, Free Rad. Biol. Med. 26 :174-183 .
Samanta, A., and Beardsley J., 1999, Low back pain : which is the best way forward?, Brit. Med. J.
318 :1122-1123 .
Samet, J. M., Hatch G. E., Horstman D., Steck-Scott S., Arab L., Bromberg P. A., Levine M., McDonnell
W. F., and DevIin R. B., 2001 , Effect ofantioxidant supplementation on ozone-induced lung injury
in human subjects, Am . J. Respir. Crit Care Med . 164 :819-825 .
Sarniec, P. S., Drews-Botsch C ., Flagg E. W., Kurtz J. c., Stemberg P. Jr ., Reed R. L., and Jones D. P.,
1998, Glutathione in human plasma: decline in association with aging, age-related macular
degenerati on, and diabetes, Free Rad. Hiol. Med . 24:699-704.
Sandberg. M. A., Gaudio A. R., Miller S., and Weiner A., 1994, Iris pigmentation and extent of disease in
patients with neo vascular age-related macular degeneration, Invest Ophthalmol. Vis. Sei. 35 :2734-
2740 .
Santoro, A., 1999, Quale libert ä di scelta per Je eure alternative, Il Giornale Dei Medi eo Lunedi, 21
giugno :21.
Saran, M., Beck-Speier 1., FeIlerhoff B., and Bauer G ., 1999, Phagocytic killing of microorganisms by
radical processes: consequences of the reaction of hydroxyl radicals with chloride yielding chlorine
atoms, Free Rad. Biol. Med. 26:482-490 .
Sardina, J. 0 ., et al, 1991 , Tratamiento de la giardiasis recidivante con ozono, CENIC Ciencias
Biologi cas 20:61-64.
Sarks, J. P., Sarks S. H., and Killingsworth M. C., 1988 , Evolution of geographic atrophy of the retinal
pigment epithelium, Eye 2 (Pt 5) :552-577.
Sarto , F., and Viola A., 1980, Aberrazioni cromosomiche in soggetri esposti cronicamente ad ozono, G.
Ital. Med. Lav. 2:59-61 .
Sartor, R. B., 2000, New therapeutic approaches to Crohn's disease, N. Eng!. J. Med. 342 :1664 -1666.
Sasada, T., and Yodoi 1., 1999 , Thioredoxin/adult T-cell leukemia-derived factor (ADF) as a key
regulator of redox, in Redox regulation ofeell signaling and tts clinieal applicatlon (L. Packer, and J.
Yodoi, Eds .), Marcel Dekker, Inc ., New York , pp.I-9.
Sasaki , H., Wakutani T., Oda S., and Yamasaki Y., 1967 , Application of hydrogen peroxide infusion to
maxillary cancer, Yonago . Ac;UI Med. 11: 141-149.
Sato, K. , Balla J., Otterbein L. , Smith R. N ., Brouard S., Lin Y., Csizmadia E., Sevigny J., Robson S. c.,
Vercellotri G., Choi A . M., Bach F. H., and Soares M. P., 2001 , Carbon monoxide generated by heme
oxygenase-I suppresses the rejection of mouse-to-rat cardiac transplants, .I. Immunol. 166:4185-
4194 . .
Sato , Y ., Sato K., and Suzuki Y ., 1999, Mechanisms of free radical-induced hemolysis of human
erythrocytes: comparison of calculated rate constants for hemolysis with experimental rate constants,
Arch. Biochem. Biophys . 366 :61-69 .
Schenk, H., Vogt M ., Dröge W., and Schulze-Osthoff K., 1996, Thioredoxin as a potent costimulus of
cytokine expression, J. Immunol. 156:765-771 .
Schrnid, P., Cox D., Bilbe G ., McMaster G., Morrison C ., Stähelin H ., Lüscher N., and Seiler W ., 1993,
TGF-bs and TGF-b type II receptor in human epidennis: differential expression in acute and chronic
skin wounds,.!. Patho!. 171 :191-197 .
Schmieden, U., and Wild A ., 1995, The contribution of ozone to forest decline, Physiol. Plant. 94:371-
378.
Schreck, R., Albermann K., and Baeuerle P. A, 1992, Nuclear factor kappa B : an oxidative stress-
responsive transcription factor of euk aryotic cells (a review), Free Rad. Res . Commun. 17:221-237 .
Schreck, R., Rieber P., and Baeuerle P. A., 1991 . Reactive oxygen intermediates as apparently widely
used messengers in the activation of the NF-KB transcription factor and HIV -I , EMBO J. 10:2247-
2258.
418 R EFER ENCES

Schreiber , G. B., Busch M. P., Kleinman S. H., an d Korelitz J. J., 1996, The risk of rransfus ion-
tran smitted vira l infections. The Retr ovirus Epid emiology Don or Stud y, N. Eng/. .J. Med. 334 :1685-
1690.
Sch reiber, S., Hein ig T. , Thiele H. G., and Rae dler A. , 1995 , 1mmun oregul at ory role ofi nterl eukin 10 in
patients with inflammatory bo wel disease , Gastroenterology 108 :1434-1444.
Sch rop e, M., 200 0, Successes in fight to sav e ozo ne layer co uld close hole s by 2050 , Na/I/re 408 :627 .
Sch ulz, S., 1986, The roJe of ozone/oxygen in e1indamycin-ass ociated ente roco litis in the Djungar ian
ham ster (Pho dopus sungo rus sungoru s), Lab. Anim. 20:41-48.
Sch warz, K. B., 1996 , Ox idat ive stress dur ing viral infe cti on : a re view , Free Rad. Bi ol . Med. 21 :641 -64 9.
Sco tt, S . c., Amo ma O. 1. , Evan s P. J., O'Neill c., van der Vliet A., Cro ss C. E., Tritsc hler H., a nd
Halli well S ., 1994 , Lipoic and dihydr olip oic aci ds as antioxid ants . A critical e valu ation, Free Rad.
Res. 20:119 -133.
Scrollav ezza, P., Ablondi M., Pogl iacomi B., Guaresch i D. , Dall'Aglio R., Pold i R., and Pezzoli G.,
1997b , Ozone treatment in mastit is, metritis, and retent ion of fetal membr anes in the cows, in
Proceedings ofthe 2nd Inlemalional .~vmposillm onOzon e Applicalions.Ha l.all a . Cllba . p .35.
Scr olla vezza, P., Ferrari F., Martini F. M., and Isola M., 1997a , Ozone treatment and bl ood lactate
variations after thoroughbred races, in Proceedings of the Fiflh WE VA Congress. Padova, p .2.
Sechi, L. A. , Lezcano 1., Nunez N ., Espim M., Dupre 1., Pinna A., Molicotti P., Fadda G., and Zanetti S.,
2001, Antibacterial activity of ozonized sunflower oil (O leozo n), .J. Appl. Microbiol. 90 :279-284 .
Sedd on , J. M., Ajani V. A., Sperduto R. D., Hiller R ., Blair N ., Burton T. c., Farbe r M . D., Gragoudas E.
S., Haller J., and Miller D. T., 1994 , Diet ary carotenoids, vitamins A, C and E, and ad vanced ag e
related macul ar degeneration, .JAMA-.J. Am. Med. Ass n. 272 :1413-142 0.
Se eff, L. B., Lindsay K. L., Bacon B. R., Kresina T. F., and Hoofnagle J. H., 200 1, Co mple rn entary an d
alternative medi cine in chronic liver disease , Hepat olog v 34 :595 -603 .
Se eman. T. E., and Robb ins R. J., 1994 , Aging and hyp othalami c-piru itar y-adrenal respon se to cha llenge
in humans, Endocr. Rev. 15:233-260 .
Sell ey, M. L., 1998, (E)-4- hydro xy-2- no nenal ma y be invo lved in the path ogenesis of Parki nson 's dis ease,
Free Rad. Biol. Me d. 25 :169-174 .
Sen , C. K., 1995, Oxidants and anti oxidants in exercise, .I. Appt. Phvsiol. 79 :675-686.
Sen , C. K., and Packer L., 1996, Antioxi da nt and redox regul ation of gene transcripn on. FASEl / .I.
10:709-720.
Sen, C. K., Roy S., Han D., and Packer L., 1997, Regul at ion of cel lular thi ols in hum an Iymp hocyt es by
alpha- lipo ic acid: a flo w cyt ometric analys is, Free Rad. Biol. Med. 22 :1241 -1257.
Sen , C. K., Sash wati R., and Packer L., 1999, Fas medi ared ap opt os is of hum an Jurka t T-cells :
intrac ellula r events and potentiation by red ox-active alpha-lipo ic ac id, Cell Deat h. Differ . 6:481-49 1.
Sen , R., and Baltimore D., 1986 , Multiple nuele ar factors interact with the irnmun oglobu lin enh an cer
sequences, Cell 46: 705-716 .
Seo, M. S., Kwak N., Ozak i H., Yam ada H., Okam oto N ., Yam ada E., Fab bro D., Hofmann F., Woo d J.
M ., and Campoc hiaro P. A., 1999, Dram ati c inhibit ion ofret inal and cho roida l neovascularization by
oral adrninistration of a kina se inhibitor, Amer. .I. Pa/hol. 154 :174 3-1753 .
Serh an, C. N. , 1994, Lipoxin biosynthesis and its impact in inflammatory and vasc ular event s, B iochim.
Biophvs. Act a 1212 :1-25 .
Serh an, C. N ., Haeggström J. Z., and Leslie C. c., 1996 , Lipid med iat er netw orks in cell signaJing : upd ate
and impact of cytokines, FAST:B.I. 10:1147-1158.
Shah , M., Revis D., Herrick S., Baillie R., Th orgeirson S., Ferguson M ., and Robert s :\., 1999, Role of
elevated plasrna transforming gr owth facto r-beta 1 levels in wound healin g, Am. .I. Pa/hol. 154 :1115 -
1124 .
Sharma, Y. K., and Davis K. R., 1997 , The effect s of ozone on anti oxidant response s in plant s, Fre e Rad.
Biol. Med. 23 :480-4 88.
Sharma, Y. K., Le ön J., Raskin 1., and Davis K. R., 1996 , Oz one-induced responses in Arab idop sis
thal ian a: the role of salic ylic acid in the accurnul ati on of defense -rel ated tran scripts and indu ced
resistan ce, Prot: Na/. Acad. Sei. USA 93 :50 99-5104 .
Shascho va, N. M., Kachalina T. S., and Nev mjatullin A. L., 1995, Ap plicati on of ozo no rhera py in
complex treatment of inner female genital inflarnma tory disea ses, in Proceedings Ozone in Medicine.
121h World Congress of the International Ozone Association, 15111 10 111/11 MI (v 1995. Lilie F/'OI/ce
(Interna tio nal Oz one As soci atio n, Ed.J, lnstap rinr S .:\., Tours, pp .145-155.
 REFERENCES 419

Shattil , S. J., and Ginsberg M . H., 1997 , Integrin signaling in vascular biology, J. Clin. Invest 100 :S91·
S95 .
Shelton, R. c., Keller M. B., Gelenberg A ., Dunner D. L., Hirschfeld R., Thase M . E., Russell 1., Lydiard
R. B., Crits-Cristoph P., Gallop R., Todd L., Hellerstein 0 ., Goodnick P., Keitner G ., Stahl S. M.,
and Halbreich U., 200 I , Effectiveness of St John's wort in major depression : a randomized controlled
trial , JAMA 285: 1978-1986.
Shiba , M., Tadokoro K., Sawanobori M., Nakajima K ., Suzuki K. , and Juji T., 1997 , Activation of the
contact system by filtration of platelet concentrates with a negatively charged white cell -rernoval
filter and measurement of venous blood bradykinin level in patients who received filtered platelets,
Transfusion 37:457-462 .
Shimaoka, T., Kume N ., Minami M. , Hayashida K., Kataoka H., Kita T., and Yonehara S., 2000,
Molecular cloning of a novel scavenger receptor for oxidized low density lipoprotein, SR -PSOX, on
macrophages,.J. Biol . Che m. 275:40663-40666 .
Shinriki , N., Ishizaki K., Yoshizaki T., Miura K., and Ued a T., 1988, Mechanism of inactivation of
tobacco mosaic virus with ozone, Wat. Res . 22 :933-938.
Shinriki, N., Suzuki T., Takama K., Fukunaga K., Ohgiya S., Kubota K., and Miura T., 1998,
Susceptibilities of plasma antioxidants and erythrocyte constituents to low levels of ozone,
Haematologia 29:229 -239 .
Shiomori, T., Miyamoto H., and Makishima K., 2001 , Significance of airborne transmission of
methicillin-resistant Staphylococcus aureus in an otolaryngology-head and neck surgery unit, Arch.
Otolaryngol. Head Neck Surg. 127 :644-648 .
Shull , S., Heintz N . H ., Periasamy M., Manohar M ., Janssen Y. M. W., Marsh J. P., and Mossman B. T.,
1991, Differential regulation of antioxidant enzymes in response" to oxidants, .I. Biol. Chem .
266 :24398-24403 .
Siemsen, C.-H ., 1995 , Ozon-Anwendung be i akuten und chronischen Gelenkerkrankungen. in Ozon-
Handbuch. Grundlagen. Prävention. Therapie (E . G. Beck, and R. Viebahn-H änsler, Eds .), Ecomed,
Landsberg, p.V-9 .2 I-V-9 .2 14.
Sies , H., and Wendel A. , 1978, Functions ofglutathione in liver and kidney, Springer, Berlin.
Silva, D. B., Cardoso C. c ., Silva M. A. M. L., and Lucia M. T., 1999, Effect of intramammary
application of the gas mixture of ozone and oxygen in subclinical mastitis, in Proceedings of the 2°
Encontro de Pesquisadores da Mastite. BotUCOIll-SP, Brasil.
Silver, F. H ., and Glasgold A . 1., 1995, Cartilage wound healing. An overview, Otolaryngol. Clin. N.
Am e/·. 28 :847-864.
Silvestri , G ., 1997, Age-related macular degeneration: genetics and implications for detection and
treatment, Mol . Med. Today 3 :84-9 1.
Silvestri, G., Johnston P. B., and Hughes A . E., 1994, Is genetic predisposition an important risk factor in
age-related macular degeneration", Eye 8 ( Pt 5) :564 -568 .
Sirnon. R., 2000, Are placebo-controlled clin ical trials ethical or needed when alternative treatment
exists?, Ann . Intern. Med. 133 :474-475.
Simonetti, L., Agati R., Oe Santis F., and Leonardi M., 2001 , Anatomia e fisiopatologia dell 'unita
funzionale disco-sornatica, Riv . Neuroradtot. 14:7-16 .
Simonian, N . A ., and Coyle J. T., 1996, Oxidative stress in neurodegenerative diseases, Annu. Rev.
Pharmacol. Toxi col. 36 :83-106.
Simons, M ., 2001, Therapeutic coronary angiogenesis : a fronte praecipitium a tergo lupi?, Am . .I. Physiol
Heart Cire. PhysioI280:HI923-HI927 .
Slavin, 1., 1996, The role of cytokines in wound healing, .I. Path ol. 178 :5-10 .
Sionim, A. 0 ., and Singh N ., 2001, Nosocomial bloodstream infection and cost, CrU Care Med . 29 :1849 .
Slonim, A. E., Bulone L., Damore M. B., Goldberg T., Wingertzahn M. A ., and McKinley M. 1., 2000 , A
preliminary study of growth honnone therapy for Crohn's disease, N. Engl . 1. Med. 342 :1633 -1637 .
SmalI, D. L., Morley P., and Buchan A . M., 1999, Biology of ischemic cerebral cell death, Prog.
Cardiovasc. Dis . 42 :185-207.
Smith, L., 1969 , Chemonucleolysis, Clin. Ortbop. 67 :72.
Smith , L. J., Shamsuddin M., Sporn P. H., Denenberg M., and Anderson J., 1997, Reduced superoxide
dismutase in Jung cells ofpatients with asthma, Free Rad. Biol. Med. 22 :1301 -1307.
Snyder, S. H., and Baranano D. E., 2001, Heme oxygenase: a font of multiple messengers,
Neuropsychopharmacology 25 :294-298.
420 REFERENCES

Soholm, 8., 1998 , Clinical improvement of memory and other cognitive functions by Ginkgo biloba :
review ofrelevant literature, Adv. Ther. 15 :54-65 .
Spath-Schwalbe, E., Lange T., Perras B., Fehm H. L., and Bom J ., 2000 , Interferon-alpha acutely impairs
sleep in healthy humans, Cytokine 12:518-521.
Spector, A. , 1995 , Oxidative stress-induced cataract : mechanisrn of action, FASEB.I. 9:1173-1182.
Spence, V. A., Khan F., and Belch J. J., 2000, Enhanced sensitivity ofthe peripheral cholinergic vascular
response in patients with chronic fatigue syndrome, Am . .J. Med. 108 :736-739 .
Sperduto, R. D., Ferris F. L. 1., and Kurinij N ., 1990 , Do we have a nutritionaltreatment for age -related
macular degeneration?, Arch, Ophthalmol. 108 :1403 -1405 .
Spom, M. B., and Roberts A. B., 1993 , A major ad vance in the use of growth factors to enhance wound
healing, J. Clin . Invest 92 :2565-2566.
Squadrito, G. L., and Pryor W . A., 1998, Oxidative chemistry of nitric oxide: the roles of superoxide,
peroxynitrite, and carbon dioxide, F ree Rad. Biol . Med. 25 :392-403 .
Srivastava, S., Dixit B. L., Cai J., Sharrna S., Hurst H. E., Bhatnagar A., and Srivastava S . K., 2000,
Metabolism of lipid peroxidation product, 4-hydroxynonenal (HNE) in rat erythrocytes: role of
aldose reductase, Fl'ee Rad. Biol. Med. 29 :642-65 1.
Stadtman. E. R. , and Levine R. L., 2000, Protein oxidation, Ann . N. Y. Aead. Sei. 899 :191-208 .
Stadtman, E. R., and Oliver C. N., 1991, Metal -catalyzed oxidation of proteins. Physiological
consequences,.I. Biol. Chem . 266 :2005-2008.
Steidler, L., Hans W ., Schotte L., Neirynck S., Oberrneier F., Falk W ., Fiers W ., and Remaut E., 2000,
Treatment of murine colitis by Lactococcus lactis secreting interleukin- 10, Science 289 :1352-1355.
Stein, J. L., and Schwartzbrod J. K ., 1990, Experimental contamination of vegetables with helminth eggs ,
Wat. Sei. Tech . 22 :5 I-57 .
Steinberg, D., 1997, Low density lipoprotein oxidation and its pathobiological significance, .J. Biol .
Chem. 272 :20963-20966.
Steinberg, M . H., 1999, Management of sickle cell disease, N. Engl. .J. Med. 340 :1021-1030.
Steinberg, R. H., 1994, Survival factors in ret inal degenerations, Curr . Op in . Neurobtol. 4 :515-524 .
Steinbrook, R, 2001 , Providing antiretroviraltherapy for HIV infection, N. Engl. .J. Med. 344 :844-846 .
Steinhart, H., Schulz S ., and Mutters R , 1999, Evaluation of ozonated oxygen in an experimental animal
model of osteomyelitis as a further treatment option for skull -base osteomyelitis, Eur. Arch.
Otorhinolaryngol. 256 : I 53-1 57.
Stephan, F., Cheffi A ., and Bonnet F., 2001, Nosocomial infections and outcome of critically ill elderly
patients after surgery, Anesthesiology 94:407-414 .
Stephenson, J., 2000, Genital herpes vaccine shows limited promise, .lAMA 284 :1913-1914.
Stephenson, J., 2001, New therapy promising for genital herpes, .lAMA 285 :2182-21 83.
Steuer-Vogt, M . K., Bonkowsky V ., Ambrosch P., Scholz M ., Neiss A., Strutz J ., Hennig M., Lenarz T.,
and Arnold W., 2001 , The effe ct ofan adjuvant mistletoe treatrnent programme in resected head and
neck cancer patients: a randomised controlled clinicaltrial, Eur. .I. Cancer 37 :23-31 .
Stevens, W . H. M ., Conlon P. D., and O'Byme P. M ., 1995, Ozone-induced oxygen radical release from
bronchoalveolar lavage cells and airway hyper-responsiveness in dogs, .1. Physiol. 486.1 :257-265.
Stiehl, H. H., 1998, Teclmische erzeugung von ozon, in Ozon-Handbuch. Grundlagen. Präventton.
Therapie (E . G. Beck, and R. Viebahn-H änsler, Eds .), Ecomed, Landsberg, pp .I-27 .
Stocker, R., Bowry V. W ., and Frei B., 1991 , Ubiquinol-I 0 protects human low density lipoprotein more
efficiently against lipid peroxidation than does alpha-tocopherol , Proc. Na/I. Acad. Sei. U. S. A
88 :1646 -1650.
Stolarski, R S ., 1999, History of the study of atmospheric ozone, in Pro ceedings 0/ the Int . Ozone
Symposium. 21 and 22 October 1999. Basel. Switzerland (IOA - EA JG Ed .), Bauer Druck AG ,
Basel, pp.41-53 .
Stover, B. H., Shulman S. T., Brateher D. F., Brady M. T., Le vine G. L., and Jarvis W . R., 200 I ,
Nosocomial infeciion rates in US children's hospitals' neonatal and pediatric intensive care unirs, Am .
.I. Infect. Conf1'0129 :152-157.
Su, W . Y., and Gordon T., 1997, In vive exposure 10 ozone produces an increase in a 72 -kDa heat shock
protein in guinea pigs, .I. Appl. Physiol. 83 :707-711 .
Sugarman. J., and Burk L., 1998, Physicians' ethical obligations regarding alternative medicine, .lAMA
280 :1623 -1625.
Sun, J. S., Lu F. J ., Huang W . C., Hou S. M., Tsuang Y. H., and Hang Y. S., 1999 , Antioxidnut status
following acute ischemic Iimb injury: a rabbit model, Free Rad. Res. 31 :9-21 .
 REFERENCES 421

Suzuki , Y ., Ohsugi K., and Ono Y., 1996 , Oxidative stress triggers tyrosine phosphorylation in B eells
through a redox- and inflammatory cytokine-sensitive mechanism, Immunology 87 :396 -40 I.
Suzuki , Y. J., Fonnan H. J., and Sevanian A ., 1997 , Oxidants as stimulators of signal transduetion, Free
Rad. Biol. Med. 22 :269-285 .
Swain , S. L., 2001, Interleukin 18: tipping the balance towards a T helper cell 1 response, J. Exp. Med.
194 :FII-FI4.
Sweet , F., Kao M.-S ., Lee S.-C . D., Hagar W. L., and Sweet W. E., 1980, Ozone seleetively inhibits
growth ofhuman eancer cells, Science 209 :931-933 .
Symons, M. C., Rusakiewicz S., Rees R. c., and Ahmad S. 1., 2001, Hydrogen peroxide: a potent
cytotoxic agent effective in eausing eellular damage and used in the possible treatment for eertain
tumours, Med. Hypotheses 57 :56-58 .
Szatrowski, T. P., and Nathan C . F., 1991, Production of large amounts of hydrogen peroxide by human
tumor cells, Cancer Res. 51 :794 -798.
Tabaracci, G., 2001, L'ozonoterapia eon tecniea "c1assica" intramuseolo paravertebrale, Riv. Neuroradiol.
14:67-70.
Tacchini, L., Pogliaghi G., Radice L., Bernelli-Zazzera A. , and Cairo G., 1996, Post-transcriptional
control of increased hepatic catalase gene expression in response to oxidative stress, Redox Report
2:273-278 .
Taga , K., Mostowski H., and Tosato G ., 1993, Human interleukin-IO can directly inhibit T-eell growth.,
Blood 81:2964-2971 .
Tagaya, Y., Okada M., Sugie K. , Kasahara T., Kondo N ., Hamuro 1., Matsushima K ., Dinarello C . A.,
and Yodoi 1., 1988 , IL-2 receptor(p55)/Tac -inducing faetor. Purification and characterization of adult
T cell leukemia-derived factor,.I. Immunol. 140 :2614-2620.
Takahashi, N., Yu X .-Y., Sehofield B. H., Kleeberger S. R., Scott A . L., Hasegawa S., and Spannhake E.
W. , 1995, Expression of ICAM-I in airway epithelium after acute ozone exposure in the mouse, J.
Appl. Physiol. 79 :1753-1761. .
Tamura, Y ., Peng P., Liu K., Daou M., and Srivastava P. K., 1997, Immunotherapy of tumors with
autologous tumor-derived heat shock protein preparations, Seience 278: 117-120 .
Tan , D . X ., Chen L. D., Poeggeller B., Manchester L. c., and Reiter R. J., 1993 , Melatonin: a potent
endogenous hydroxyl radieal sca venger, Endo cr. .I. 1:57-62.
Tan , D. X., Manchester L. C., Reiter R. J., Plummer B. F., Limson J., Weintraub S. T., and Qi W ., 2000 ,
Melatonin direetly seavenges hydrogen peroxide: a potentially new metabolie pathway of melatonin
biotransfonnation, rree Rad . Biol. Med. 29 :1177-1185.
Tanaka, T., Nakamura H., Nishiyama A., Hosoi F., Masutani H., Wada H., and Yodoi J., 2000 , Redox
regulation by thioredoxin superfamily; protection against oxidative stress and aging, Free Rad. Res .
33 :851-855 .
Targan, S. R., Hanauer S. B., van Deventer S. J. H., Mayer L., Present D. H., Braakman T. , DeWoody K.
L., Schaible T. F., Rutgeerts P. J., and for the Crohn's Disease eA2 Study Group, 1997, A short-term
study of chimerie monoclonal antibody eA2 to tumor necrosis faetor a for Crohn's disease, N. Eng/.
.I. Med. 337 : I029-1 035 .
Tarkington, B. K., Duvall T. R., and Last 1. A ., 1994 , Ozone exposure of cultured eells and tissues, Meth .
Enzymol. 234 :257-265 .
Tate, D. J., Jr ., Mieeli M . V ., and Newsome D. A ., 1995, Phagocytosis and H202 induee catalase and
metallothionein gene expression in human retinal pigment epithelial cells, Invest Ophthalmol. Vis.
Sei. 36:1271 -1279.
Tatla, S., Woodhead V ., Foreman J. C., and Chain B. M., 1999, The role of reactive oxygen species in
trig ger ing proliferation and lL-2 secretion in T cells, Free Rad. Biol. Med. 26 :14-24 .
Taylor, R. S., Belli A. M., and Jaeob S., 1999, Distal venous arterialisation for sal vage of critically
isehaemic inoperable limbs, Lancet 354 :1962-1965.
Tazulakhova, E. B ., Parshina O . V., Guseva T. S., and Ershov F. 1., 2001, Russian experience in
screening, analysis, and elinical applieation of novel interferon inducers,.I. Interferon Cytokine Res .
21 :65-73 .
Thiele, J., and Eisner P., 2001, Oxidants and ant ioxidants in cutaneous biology, Karger, Gennany, pp .l-
194.
Thiele, 1. 1., Traber M. G., Tsang K., Cross C . E., and Packer L., 1997a, In vivo exposure to ozone
depletes vitamins C and E and induces lipid peroxidation in epidermal layers of murine skin, Free
Rad. Biol. Med. 23 :385-391.
422 REFERENCES

Thiele, 1. J., Traber M. T., Podda M., Tsang K., Cross C. E., and Packer L., I997b, Ozone depletes
tocopherols and tocotrienols topically applied to rnurine skin, FEBS Len. 40] :167-170.
Thomas, D. D., Liu X., Kantrow S. P., and Lancaster J. R., Jr., 2001, The biological lifetime of nitric
oxide: implications for the perivascular dynamics of NO and 02, Proc. Natl. Acad. Sei. U. S. A
98 :355-360.
Thomas. J. A., Darby T. D., Wallin R. F., Garvin P. J., and Martis L., 1978, A review of the biological
effects of di-(2-ethylhexyl) phthalate, Toxicol. Appl. Pharmacol. 45 :I-?
Thomas, M. A., Grand M . G., Williams D. F., Lee C. M ., Pes in SR., and Lowe M. A., 1992, Surgical
management of subfoveal choroidal neovascularization, Ophthalmology 99:952-968.
Thomas, T., Thomas G., McLendon C., Sutton T., and Muliart M., 1996, Beta-Amyleid-mediated
vasoactivityand vascular endothelial damage, Nature 380:168-171.
Thompson, A. M., Wille J. C., Hudson R. D., Guo H., Herman J . R., and Fujiwara M., 2001 , Tropical
tropospheric ozone and biomass buming, Science 291:2128-2132.
Thorin, E., and Shreeve S. M., 1998, Heterogeneity of vascular endothelial cells in normal and disease
states, Pharmacol. Ther. 78:155-166.
Tibbles, P. M., and Edelsberg J. S ., 1996, Hyperbaric-oxygen therapy, N. Eng/. .J. Med. 334:1642-1648 .
Tissieres, A., Mitchell H. K., and Tracy U. M ., 1974, Protein synthesis in salivary glands of Drosophila
rnelanogaster: relation to chromosome puffs,.J. Mol . Biol. 84:389-398.
Titheradge, M. A ., 1999, Nitric oxide in septic shock, Biochim. Biophys. Acta 1411 :437-455.
Tombola, F., Morbiato L., Dei Giudice G., Rappuoli R., Zoralti M ., and Papini E., 2001, The
Helicobacter pylori VacA toxin is a urea permease that promotes urea diffusion ac ross epithelia, .I.
Clin. Invest 108:929-937.
Tommasini, G., Lavaroni A., Petralia B., and Fabris G., 1998 , Ozono-terapia intradiscale, in Lombalgle e
lombosciatalgie (F. Ceccherelli, and A. Ricciardi, Eds .), Edizioni Libreria Cortina, Torino , pp.351-
359 .
Torres, A., Utell M . 1., Morow P. E., Voter K. Z., Whitin 1. c., Cox C ., Looney R. J., Speers D. M., Tsai
Y., and Frampron M. W ., 1997, Airway inflanunation in smokers and nonsmokers with varying
responsiveness to ozone, Am . J. Respir. Crit Care Med. ]56 :728-736.
Torri, G ., Della Grazia A., and Casadei C., 1999 , C1inical experience in the treatment of lurnbar disk
disease, with a cycle of lumbar muscle injections of an oxygen + ozone mixture, Int. .I. Med. Biol.
Environ . 27:177-183.
Towbin, H., Schmitz A ., Jakschies D., von Wussow P., and Horisberger M . A., 1992 , A whole blood
irnmunoassay for the interferon-inducible human Mx protein.,.!. InterferonRes. 12:67-74.
Toyokuni , S ., 1996, Iron-induced carcinogenesis : the role of redox regulation, Free Rad. Biol. Med .
20 :553-566.
Toyokuni, S ., Okamoto K., Yodoi J., and Hiai H., 1995, Persistent oxidarive stress in cancer, FERS Lett ,
358 :1-3.
Toze, S., 1999, PCR and the detection of microbial pathogens in water and wastewater, Wal. Res.
33:3545-3556.
Trippel, S . B., 1995, Growth factor actions on articular cartilage,.J. Rheumatot. 43 :129-132 .
Trussell, J., 1999, Pills or placebos", Science 284:913.
Tsuchihashi, H., Kigoshi M., Iwatsuki M ., and Niki E., 1995, Action of b-carotene as an antioxidant
against lipid peroxidation, Arch. Biochem. Biophys. 323 :137-147.
Uchida, K., 2000, Role of reactive aldehyde in cardiovascular diseases, Free Rad. Biol. Med. 28:1685 -
1696.
Ueno, 1., Hoshino M ., Miura T., and Shinriki N ., 1998, Ozone exposure generates free radicals in the
blood samples in vitro. Detection by the ESR spin-trapping technique, Free Rad. Res. 29 :127-135.
Uppu , R. M., Cueto R., Squadrito G . L., and Pryor W . A ., 1995, What does ozone react with at the
air/Jung interface? Model studies using human red blood cell membranes, Arch . Biorhem. Biophvs.
3] 9:257-266.
Urschel, H. c., 1967 , Cardiovascular effects of hydrogen peroxide: current status, Dis. Chest 51: 180-192 .
Valacchi, G., and Bocci V., 1999, Studies on the biological effects of ozone: 10. Release of factors frorn
ozonated human platelets, Mediat. Inflamm. 8:205-209.
Valacchi, G., and Bocci V., 2000, Studies on the biological effects of ozone: 11. Release of factors front
human endothelial cells, Mediat. Inflamm. 9:271-276.
 REFERENCES 423

Valen , G., Erl W ., Eriksson P., Wuttge D., Paulsson G., and Hansson G. K., 1999, Hydrogen peroxide
induces mRNA for tumour necrosis factor alpha in human endothelial cells, Free Rad. Res. 31 :503-
512 .
Valen, G., Starkopf J., Takeshima S., Kullisaar T., Vihalemm T., Kengsepp A. T., Lowbeer c., Vaage J.,
and Zilmer M., 1998, Preconditioning with hydrogen peroxide (H202) or ischemia in H202-
induced cardiac dysfunction , Free Rad. Res. 29 :235-245 .
Valer i, C. R., Contreas T. J., Feingold H., Shebley R. H., and Jaeger R. J., 1973, Accumulation 01' di-2-
ethylhexyl phthalate (DEHP) in whole blood , platelet concentrates and platelet-poor plasma. I: effect
01'DEHP on platelet survival and function , Environ. Health Perspect. 3:103-?
Valeri, C. R., and Hirsch N . M., 1969, Restoration in vivo of erythrocyte adenosine triphosphate, 2,3-
diphosphoglycerate, potassium ion , and sodium ion concentrations following the transfusion of acid-
citrate-dextrose-stored human red blood cells,.J. Lab Clin. Med. 73 :722-733 .
van der Vliet , A. , O'Neill C. A., Eiserich J. P., and Cross C. E., 1995, Oxidative damage to extracellular
fluids by ozone and possible protective effects of thiols , Arch . Biochern . Biophys. 321 :43-50.
Van der Zee, J., van Beek E., Dubbelman T. M. A. R., and Van Steveninck J., 1987, Toxic effects of
ozone on murine L929 fibroblasts, Biochem. .J. 247 :69-72 .
van Eden, W ., van der Z. R., Paul A. G., Prakken B. J., Wendling U., Anderton S. M., and Wauben M.
H., 1998, Do heat sho ck proteins control the balance 01'T-cell regulation in inflammatory diseases?,
Immunol. Today 19:303-307.
van Hoof, H. J. M., Zij1stra F. J., Voss H.-P., Garrelds I. M., Donnans J. A. M. A., Van Bree L., and Bast
A.. 1997, The effect of ozone exposure on the release 01' eicosanoids in guinea-pig BAL fluid in
relation to cellular damage and inflammation, Mediat. Inflamm. 6 :355-361 .
van Parijs, L., and Abbas A. K., 1998, Homeostasis and self-tolerance in the immune system: turning
Iymphocytes off, Science 280 :243-248.
Vanchieri , c., 2000 , Alternative therapies getting notice through Best Case Series Program. .I. Nat/.
Cancer Inst . 92 :1558-1560 .
Vandenbroucke, J. P., 1997, Homoeopathy trials : going nowhere, Lancet 350 :824.
Varro, J., 1966, Über das Krebsproblem und seine Therapie, Ztschr. Int. Med. Ges. F. Blut U.
Geschwulstkrankheiten 4:5-6.
Varro , J., 1974, Die krebsbehandlung mit ozon, Erfahrungsheilkunde 23:178-181.
Varro, J., 1983, Ozone applications in cancer cases, in Medical Applications ofOzone (J. LaRaus, Ed.),
International Ozone Association, Pan American Cornmittee, Norwalk.Conn., pp .94-95 .
Vasiliou, V., Pappa A., and Petersen D. R., 2000, Role 01'aldehyde dehydrogenases in endogenous and
xenobiotic rnetabolism, Chern. Biol . Intera ct. 129:1-19.
Vaughn , J. M., Chen Y. S., Novotny J. F., and Strout D., 1990, Effects 01' ozone treatment on the
infectivity ofhepatitis A virus, Can. ./. Microbiol. 36 :557-560 .
Vaupel , P., and Hockel M., 2000, Blood supply, oxygenation status and metabolie micromilieu ofbreast
cancers : characterization and therapeutic relevance, Int . .J. Oncol. 17 :869-879 .
Verga , c., 1989, Nuovo approccio terapeutico alle ernie e protrusioni discali lornbari , Riv. Neuroradiol.
2:148.
Verrna, A., Hirsch D. J., Glatt C. E., Ronnett G. V., and Snyder S. H., 1993, Carbon monoxide: a putative
neural messenger, Science 259 :381-384 .
Verma, I. M., and Stevenson J., 1997, IkappaB kinase : beginning, not the end, Pro c. Natl. Acad. Sei. U. S.
A 94 :11758-11760 .
Verrazzo, G., Coppola L., Luongo c., Sammartino A., Giunta R., Grassia A., Ragone R., and Tirelli A.,
1995, Hyperbaric oxygen, oxygen-ozone therapy, and rheologie parameters of blood in patients with
peripheral occlusive arterial disease, Undersea Hyperbar. Med . 22:17-22 .
Verweij , H., Christianse K., and Van Steveninck J., 1982, Ozone-induced fonnation of O,O'-dityrosine
cross-linked in proteins, Bioch im. Bi ophys. Acta 701:180-184 .
Victorin, K., 1992, Review ofthe genotoxicity 01'ozone, MI/tat. Res. 277:221 -238.
Victorin, K., 1994, Review of the genotoxicity 01' nitrogen oxides, Mutat , Res. 317:43 -55 .
Viebahn -H änsler, R., 1999b , Einfluss auf den erythrozytenstoffwechsel, in Ozon -Handbuch. Grundlagen.
Prävention. Therapie (E. G. Beck , and R. Viebahn-Hänsler, Eds.), Ecomed, Landsberg, p.l- 15
Viebahn-Hänsler, R., Lell B., and Kremsner P. G., 2001 , The effect of ozone on plasmodium falciparum-
infected red blood cells , in Proceedings of the }5th Ozone WOI'ld Congress, London, UK. l Ith-I 5th
Sept ember 2001. Medi cal Therapy Conference (lOA 2001, Ed.), Speedprint MacMedia Ltd, Ealing,
London, UK, pp .26-39 .
424 REFERENCES

Viebahn, R., 1975, Phisikalisch-chernische, grundlagen der ozontherapie, Erfahrungshetlkunde 5: 129.

Viebahn, R., 1999a, The use ofozone in medicine , ODRE1 Publishers, Iffezheim, pp .I-148.
Vingerling, J. R., Dielernans 1., Bots M. L., Hofman A , Grobbee D. E., and de Jong P. T., 1995, Age -
related macular degeneration is associated with atherosclerosis. The Rotterdam Study. Am. .1.
Epidemiol. 142:404-409.
Vingerling,1. R., Hofman A., Grobbee D. E., and de Jong P. T., 1996, Age-related macular degeneration
and smoking. The Rotterdarn Study, Arch. Ophthalrnol. 114 : 1193-1196.
Wagner, J. R., Hu C. c., and Ames B. N. , 1992, Endogenous oxidative damage of deoxycytidine in DN.'\,
Proc. Nml. Acad. Sei. U. S. A 89 :3380-3384.
Wahl, C., Liptay S ., Adler G., and Schmid R. M ., 1998 , Sulfasalazine: a potent and specific inhibitor of
nuclear factor kappa B, .I. Clin. Invest. 101 :1163 -1174.
Walker, C. M., and Levy 1. A., 1989, A diffusible Iymphokine produced by CD8 ' T lymphocytes
suppresses HIV replication., Immunology 66 :628-630.
Wallner, F., 1998, Photometrische verfahren (UV und VIS), in Ozon-Handbuch. Grundlagen . Prävention.
Therapie (E . G. Beck, and R. Viebahn-H änsler, Eds.), Ecomed, Landsberg, pp .I-5 .
Wang, G. L., and Semenza G. L., 1996, Oxygen sensing and response to hypoxia by mammalian cells,
Redox Report 2 :89-96 .
Wang, J ., Huang C. J., and Chow C. K., 1996, Red cell vitamin E and oxidative darnage . a dual role of
reducing agents, F/'ee Rad. Res . 24 :291-298 .
Wang, P., Chen H., Qin H., Sankarapandi S., Becher M . W ., Wong P. C; and Zweier J. L. , 1998 ,
Overexpression of human copper, zinc-superoxide dismut äse (SOD I) prevents posrischemic injury,
Proc . Natl . Acad. Sei. U. S. A 95 :4556-4560.
Wang, P., Wu P., Siegel M. 1., Egan R. W ., and Billah M. M., 1995, Interleukin (lL)-lO inhibits nuclear
factor kappa B (NF kappa B) activation in human monocytes. IL-IO and lL -4 suppress cytokine
synthesis by different mechanisms,.J. Biol. ehern. 270 :9558-9563 .
Wang, W ., and Ballarori N ., 1998, Endogenous glutathione conjugates: occurrence and biological
functions, Pharmacol. Rev. 50 :335-356.
Ware, J. A, and Heistad D. D., 1993, Platelet- endothelium interactions, N. Engl . .1. Med. 328:628-635 .
Warren, D. L., and Last J . A., 1987, Synergistic interaction of ozone and respirable aerosols on rat lungs.
111. Ozone and sulfuric acid aerosol, Toxicol. Appl. Pharmacol. 88 :203-216.
Warren , J. B., and Higenboltam T., 1996, Caution with use ofinhaled nirric oxide, Lancet 348:629 -630 .
Washiittl , 1., and Viebahn R., 1986, Immunologische Aspekte bei chronischen Erkrankungen unter Ozon-
Sauerstoff-Therapie, Ozotvachrichten 5 :11-15.
Washiittl, J., Viebahn R., and Steiner 1., 1988, Immunologische Untersuchungen bei chronischen
Erkrankungen unter Verabreichung von Ozon-Sauerst off-Gemischen, Ozo lvachrichten 7 :30-36.
Washiittl , J., Viebahn R., and Steiner 1., 1990, The influence of ozone on tumor tissue in comparison with
healthy tissue (in vitro)., Ozone-Sci. Eng. 12:65-72 .
Wasser, G. H., 1995a, Behandlung von Verletzungen mit ozoniertem Wasser, in Ozon-Handbuch.
Grundlagen. Prävention. Therapie (E . G . Beck, and R. Viebahn-H änsler, Eds .), Ecomed, Landsberg.
p.V-7.4 I -V -7.4 8.
Wasser, G. H., 1995b, Zerebrale Durchblutungsstörungen, in Ozon-Handbuch. Grundlagen. Prävention.
Therapie (E . G. Beck, and R. Viebahn -H änsler, Eds .), Ecomed, Landsberg. p.V-6.3 1-V-6.3 12.
Wayner, D. D . M., Burton G. W ., Ingold K. U., Barclay L. R. c., and Locke S. J., 1987 , The relative
contributions of vitamin E, urate, ascorbate and proteins to the total peroxyl radical-trapping
antioxidant activity of human blood plasma, Biochim. Biophys. Acta 924:408 -419 .
Wayner, D. D. M ., Burton G. W ., 1ngold K. U., and Locke S ., 1985 , Quantitative measurement of the
total , peroxyl radical -trapping antioxidant capability of human blood plasma by controlled
peroxidation, FEBS Leu. 187 :33-37.
Weber, W ., 2000, New ozone exposure thresholds signal step forward for health , Lancet 356: 1419 .
Weber, W ., and Butcher J., 2001, Doubts over cell therapy for Parkinson's disease, Lanret 357 :859 .
Webster, G. 1., Hallett R., Whal1ey S . A., Meltzer M ., Balogun K., Brown D., Parrington C. P., Shanna
S., Hamilton G ., Farrow S. c., Ramsay M . E., Teo C. G., and Dusheiko G. M., 2000, Molecular
epidemiology ofa large out break of hepatitis B Iinked to autohaemotherapy, LanceI356:379-384 .
Weck, P. K., Buddin D. A., and Whisnant 1. K., 1988 , Interferons in the treatment of genital human
papillomavirus infections,Amer. .I. Med. 85 :159-164.
Weeks, D. L., Eskandari S., Scott D. R., and Sachs G., 2000, A H+-gated urea channel : the link between
Helicobacter pylori urease and gastric colonization, Science 287 :482-485.
 REFERENCES 425

Wefers, H., and Sies H., 1988, The protection by ascorbate and glutathione against microsomal lipid
peroxidation is dependent on vitamin E, EIII·. .I. Biochem. 174:353-357 .
Wehrli, F., and Steinbart H., 1954, Erfahrungen mit der Haematogenen Oxydations - Therapie (HOT),
Ars Mediä 10:44-51.
Weinblatt , M. E., Kremer 1. M., Bankhurst A. D., Bulpitt K. J., Fleischmann R. M., Fox R. 1., Jackson C.
G., Lange M., and Burge D. 1., 1999, A trial of etanercept, a recombinant tumor necrosis factor
receptor : Fe fusion protein, in patients with rheumatoid arthritis receiving methotrexate, N. Eng/. .I.
Med. 340 :253-259 .
Welch , W. J., 1992, Mammalian stress response: cell physiology, structure/function ofstress proteins, and
implications for medicine and disease, Physiol Rev. 72:1063-1081.
Weleber, R. G., 1996, The Cuban experience. False hope for a cure for retinitis pigmentosa, Arch.
Ophthalmo/. 114:606-607 .
Wells, A. D., and Malkovsky M., 2000, Heat shock proteins, tumor immunogenicity and antigen
presentation: an integrated view, Immunol. Today 21 : 129-132.
Wells, K. H., Latino J., Gavalchin J., and Poiesz B. 1., 1991, Inactivation of human immunodeficiency
virus type I by ozone in vitro , Blood 78 :1882-1890 .
Wenning , F., 1956, Veränderungen der Blutelemente durch UV.Bestrahlung in Sauerstoffatmosphäre,
Wiener Mediz Wochensehr. 51152:I067-1080 .
Wenzel , R. P., and Edmond M. B., 1999, The evolving technology of venous access, N. Engl..1. Med ,
340:48-50.
Wenzel , R. P., and Edmond M. B., 2001, The impact of hospital -acquired bloodstream infections, Emerg.
Infect. Dis. 7 :174-177.
Werkme ister, H., 1995, Dekubitalgeschwüre und die Behandlung mit der Ozon-Unterdruckbegasung, in
Ozon-Handbuch. Grundlagen . Prä vent ion. Therapie (E. G. Beck, and R. Viebahn-H änsler, Eds .),
Ecomed , Landsberg, p.V-7.1 I-V -7.1 22.
Wessely , S., 2001, Chronic fatigue : symptom and syndrome, Ann.Tntern. Med. 134:838-843.
West, S., Vitale S., Hallfrisch J., Munoz B., Muller D., Bressler S., and Bressler N. M., 1994, Are
antioxidants or supplements protective for age related macular degeneration?, Arch . Ophthalmol.
112:222-227 .
Westend orp, M. 0 ., Shatr ov V. A. , Schulze-Osthoff K., Frank R., Kraft M., Los M., Krammer P. H.,
Droge W., and Lehmann V., 1995, HIV-I Tat potentiates TNF-induced NF-kappa B activation and
cytotoxicity by altering the cellular redox state, EMBO.J. 14:546-554.
Westermann, J., and Bode U., 1999, Distribution of activated T cells migrating through the body : a matter
of life and death, Immunol. Today 20 :302-306 .
Westermann, J., and Pabst R., 1992, Distribution of Iymphocyte subsets and natural killer cells in the
human body ., Clin . Investigator 70 :539-544.
Whitley, R. J., and Roizman B., 2001, Herpes simplex virus infections, Lancet357:1513-1518.
Whysner, J., Conaway C. c., Vema L., and Williams G. M., 1996, Vinyl chloride mechanistic data and
risk assessment : DNA reactivity and cross-species quantitative risk extrapolation, Pharmacol. Ther.
71:7-28 .
Wigley , F. M., Wise R. A., Seibold J. R., McCloskey D. A., Kujala G., Medsger T. A., Jr ., Steen V. D.,
Varga J., Jimenez S., Mayes M., and ., 1994, Intravenous iloprost infusion in patients with Raynaud
phenomenon secondary to systernic sclerosis. A multi center , placebo-controlled, double-blind study,
Ann . Intern. Med. 120:199-206.
Williamson, L. M., 2000, Leucocyte depletion of the blood supply - how will patients benefit?, BI' . .I.
Haematol. 110 :256-272 .
Willis, W. D. 1., 1995,11 sistema somatosensoriale, in Fisiologia (R. M. Beme , and M. N. Levy, Eds.),
Casa Editrice Ambrosiana, Milano , pp.130-151.
Wink, D. A., Grisham M. B., Mitchell J. B., and Ford P. c., 1996, Direct and indirect effects of nitric
oxide in chemical reactions relevant to biology, Methods Enzymo/. 2611:12-31.
Wink , D. A., and Mitchell J. B., 1998, Chemical biology of nitric oxide : insights into regulatory,
cytotoxic, and cytoprotective mechani sms ofnitric oxide , Free Rad. Bio/. Med. 25 :434-456.
Winterboum, C. c., 1995, Toxicity of iron and hydrogen peroxide: the Fenton reaction, Toxicol. Lett. 82-
83 :969-974 .
Wiseman , H., and Halliwell B., 1996, Damage to DNA by reactive oxygen and nitrogen species : role in
inflammatory disease and progression to cancer, Biochem. .I. 313 :17-29.
426 REFERENCES

Witko -Sarsat, V ., Friedlander M., Nguyen K. T., Capeillere-Blandin c., Nguyen A T., Canteloup S.,
Dayer J. M., Jungers P., Drueke T ., and Descarnps-Latscha B., 1998 , Advanced oxidation protein
products as novel mediators of inflammation and monocyte activation in chronic renal failure, .I.
Immunol. 161 :2524 -2532 .
Witko-Sarsat, V ., Rieu P., Descamps-Latscha B., Lesavre P., and Halbwachs-Mecarelli L., 2000,
Neutrophils: molecules, functions and pathophysiological aspects, Lab Invest 80 :6 I7-653.
Wits chi , H., Espiritu 1., Pinkerton K. E., Murphy K., and Maronpot R. R., 1999, Ozone carcinogenesis
revisited, Toxicol. Sei. 52 :162-167 .
Witztum, J. L., 1994 , The oxidation hypotheses of atheroscJerosis, Lancet 344 :793 -795 .
Wolff, H. H., 1974, Die Behandlung per ipherer Durchblutungsstörungen mit Ozon, Erfahr. ttk. 23: 18 1-
184 .
Wolff, H. H., 1979 , Das medizinische Ozon . Theo retis che Grundlagen, Therapeutisch e Anwendungen,
Verlag für Medizin, Heidelberg.
Wolff, S., 1996 , Aspects of the adaptive response to very low doses of radiation and other agents, Mittat .
Res. 358:135 -142 .
Wong, R., Menendez S., Castaüer J., Perez P., and Abreu A , 1995 , Ozonetherapy in ischemic
cardiopathy, in Proceedings Ozone in Medi ctne /21h World Congress of the Internattonal Ozone
Association. l ith 10 l Sth Mav /995. Lille france (International Ozone Association, Ed .), Insraprint,
Tours, pp .73-77 .
Wood, M . J., Johnson R. W ., McKendrick M. W., Taylor J., Mandal B. K., and Crooks 1., 1994 , A
randomized trial of acycJovir for 7 days or 21 days with and without prednisolone for treatment of
acute herpes zoster, N. Engl. .J. Med. 330 :896-900.
World Health Organization, and International Federarien of Red Cross and Red Crescent Societies, 2000,
Safe b100d starts with me, World Health Organization, Geneva, Switzerland, p. I2.
Xie, c., Lovell M. A, and Markesbery W. R., 1998, Glutathione rransferase protects neuronal culrures
against four hydroxynonenal toxicity, Free Rad. Biol. Med. 25 :979 -988 .
Xu , G. Z., Li W . W., and Tso M . 0 ., 1996 , Apoptosis in human retin al degenerations, Trans. Am .
Ophthalmol. Soc . 94 :411-430 .
Yakes , F. M., and Van Houten 8., 1997 , Mitochondrial DNA darnage is more extensive and persists
longer than nucJear DNA darnage in human cells following oxidative stress, 1'/'OL'. Natl. Acad. Sä. 11.
S. A 94:514 -519 .
Yakovleva, Ye . 1., 1995, The influence ofthe efferent ozonotherapy on pulmonary functions in norm and
pathology in experimental animals, in Proceedings Ozone in Medicine. /21h World Cong ress ofthe
International Ozone Associatton, 15/h /0 l Sth May /995, Lilie, Fran ce (International Ozone
Association, Ed .), Instaprint S.A. , Tours, pp .31 -40 .
Yamamoto, H., Yamamoto Y., Yamagami K., Kume M ., Kimoto S ., Toyokuni S. , Uchida K., Fukumoto
M ., and Yarnaoka Y., 2000, Heat -shock preconditioning reduces oxidative protein denaturation and
amelierares liver injury by carbon tetrachloride in rats , Res. Exp. Med. (Red) 199 :309-318.
Yamamoto, M., et .al., 1996, The effect of ozone on treatment of 4 patients suffering from Heparins C,
Bulletin ofJapan Research Associatton forthe Medical Use ofOzone 3:1-2 .
Yarnamoto, Y., 2000, Fate oflipid hydroperoxides in blood plasrna, Free Rad. Res. 33 :795-800 .
Yan copoulos, G. D ., Davis S., Gale N . W ., Rudge 1. S., Wiegand S. J., and Holash J., 2000, Vascular-
specific growth factors and blood vessel formation , Nature 407 :242-248 .
Yang, E. Y., and Moses H. L., 1990, Transforming growth factor beta I-induced changes in cell
migration, proliferation, and angiogenesis in the chicken chorioaJlantoic rnembrane, .I. Cell Biol.
111:731 -741.
Yoritaka, A., Hattori N., Uchida K., Tanaka M., Stadtman E. R., and Mizuno Y., 1996 ,
Irnmunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinsen disease, Prot:
Nat, Acad. Sei. USA 93 :2696-2701.
Young, R. W ., 1987 , Pathophysiology of age-related macular degeneration, Surv . Ophthalmol. 31:291-
306 .
Youngman , L. D., Park J. Y., and Ames B. N ., 1992 , Protein oxidation associated with aging is reduced
by dietary restriction of protein or calories, 1'/'01:. Natl. Acad. Sei. U. S. A 89 :9112 -91 16.
Younossi, Z. M., MuJlen K. D., Zakko W ., Hodnick S ., Brand E., Bames D. S., Ca rey W. D.,
McCuJlough A. C., Easley K., Boparai N ., and Gramlieh T. , 2001, A randomized, double-blind
controJled trial of interferon alpha-2b and ribavirin vs. interferon alpha-Zb and amantadlne for
treatrnent of chronic hepatitis C non-responder to interferon monotherapy,.I. Hepatol. 34 : 128- I33.
 REFERENCES 427

Yu, B. P., 1994, Cellular defenses against damage from reactive oxygen species, Physiol. Rev. 74 :139-
162.
Yu, B. P., 1996, Aging and oxidative stre ss : modulation by dietary restrietion, Free Rad. Biol. Med. 21
:65 1-668.
Zabel, W ., 1960, Ganzheitsbehandlung der Geschwulsterkrankungen, Hippokrates 31 :75 1-760.
Zagury, D., Lachgar A ., Chams V ., Fall L. S., Bemard J., Zagury J. F., Bizz ini B., Gringeri A ,
Santagostino E., Rappaport J., Feldman M., Burny A, and Gallo R. C., 1998, Interferon alpha and
Tat involvement in the immunosuppression of uninfected T cells and C-C chemokine dec1ine in
AIDS, Proc. Natl. Acad. Sei. U. S. A 95 :3851-3856 .
Zagury, D., Lachgar A , Chams V ., Fall L. S., Bemard J., Zagury 1. F., Bizzini B., Gringeri A.,
Santagostino E., Rappaport J., Feldman M., O'Brien S. J., Burny A ., and Gallo R. C., 1998, C-C
chemokines, pivotal in protection against HIV type I infection, Proc. Natl. Acad. Sei. U. S. A
95 :3857-3861.
Zajicek, G., 1995, The placebo effect is the healing force of nature, CancerJ. 8:44-45.
Zanker, K. S., and Kroczek R., 1990 , In vitro synergistic activity of 5-fluorouracil with low-dose ozone
against a chemoresistant tumor celliine and fresh human tumor cells, Chemotherapy 36 :147-154.
Zeuzem, S., Feinman S. V ., Rasenack J., Heathcote E. J., Lai M. Y ., Gane E., O'Grady J., Reichen J.,
Diago M., Lin A., Hoffman 1., and Brunda M. J., 2000, Peginterferon alfa-2a in patients with chronic
hepatitis C, N. Engl . .1. Med. 343 :1666-1672.
Zhong, H., De Marzo A . M., Laughner E., Lim M., Hilton D . A, Zagzag D ., Buechler P., Isaacs W . B.,
Semenza G. L., and Simons J . W., 1999, Overexpression of hypoxia-inducible factor lalpha in
common human cancers and their meta stases, Cancer Res. 59 :5830-5835.
Zhulina, N. 1., et al. , 1993, Ozonetherapy efficiency in the treatment of patients with atherosc1erosis of
coronary and cerebral vessels, in Ozone in Med icine: Proceedings ofthe 11th Ozone World Congress
(P. A C . International Ozone Association, Ed.), Stamford, Conn., pp .M-2-9-11.
Ziegler, D., and Gries F. A ., 1997, Alpha-hpoie acid in the treatment of diabetic peripheral and cardiac
autonomie neuropathy, Diabetes 46 Suppl 2:S62 -S66.
Zimmermann, P. H., 1999, Tracing the sources of tropospheric ozone, in Proceedings of the Int. Ozone
Symposium. 21 and 22 October 1999. Basel. Switzerland (IOA - EAlG Ed .), Bauer Druck AG,
Basel, pp.157 -160 .
Zino , 5., Skeaff M., Williams 5 ., and Mann J., 1997, Randomised controlled trial of effect of fruit and
vegetable consumption on plasma concentrations of lipids and antioxidants, Brit. Med. .1. 314 :1787-
1791 .
Zollman, C; and Vickers A ., 1999, What is complementary medicine?, Brit. Med. J. 319 :693-696 .
 INDEX

Acetylcholine: 162
Acid hydrolases: 152
Acidosis : 162
Acquired immune defieieney syndrome (AIDS) : 13, 174
Activatorprotein I (AP-I): 199
Aeupuneture: 367
Aeute care : 363
Acute cerebral isehemia : 197
Aeute oxidative stress : 76, 107,210,223
Aeute phase reaetants (APR) : 229
Aeute respiratory distress syndrome (ARDS): 75, 139,297
Adaptation to ehronie oxidative stress (COS) : 213,233 ,309
Adenosine diphosphate (ADP) : 124,137,152
Adenosine monophosphate (AMP ): 124, 126
Adenosine triphosphate (ATP) : 112,124,125 ,126,137,152,180
Adenosine : 162
Adeny1ate cyclase (AC) : 112
Adrenoeortieotrophie hormone (ACTH) : 196,229,254
Advanced glyeation end produets (AGE) : 343
Age related macular degeneration (ARMD): 102, 125, 128,211 ,280,286
Ageing : 235
Alanine aminotransferase (SGPT): 207
Albumin : 87, 1I1
Aldehydes: 60, 73
Alkalosis : 162
Alkoperoxyl radical (Raa'): 61,62
Alkoxyl radieal (Ra'): 68
Allogenie blood transfusion : 185
Alpha.vantitrypsin : 229
Alphaj-antiplasmin: 152
Alpha-macroglobuhn: 152
Alpha-linolenic acid : 71
Alpha-lipoic acid (Thioetie acid) (LA): 93
Alpha-tocopherol (vitamin E) (EH) : 42,62,82, 89, 115,238
Alpha-tocopheryl radieal (E): 105
Alternative medieine : 57
Aluminium : 43
Amaurosis fugax : 229
American College of Rheumatology (ACR) : 323
Amputation : 278
Anal abseesses : 213
Angina abdominis (Aa) : 280
Angiogcnes is: 243
Angiostatin : 80,304
Angiotensin Il (Agil): 162
Angiotensin-converting enzyme (ACE) : 228, 230
Anion superoxide (0 2 ") : 64,65,68, 162
Anion-exchange protein (AE 1): 168
Ankle-brachial index (ABI) : 277
Antibodies : 132,273
Antibody-depcndent eellular citotoxieity (ADCC) : 150

429
430 INDEX

Antigen presenting cells (APC): IR7

Antigens: 132
Antioxidant system : 79 , 109, 171
Antioxidanttherapy and suppleruentation (AT): 237
Apnoea: 173, 178
Apoptosis : 233
Arachidonic acid (AA): 65 ,71 , R7
Archeus : 372
Arginine: 67, 165, 166
Arterial gas cmbolism : 361
Arterial pO l: 180, 204
Ascorbic acid (AH ') : 12, 42, R2.113 , R7. 105, 111, 2311 . 352
Aspartate aminotransferase (SGOT): 207
Asthma: 230, 297
Atmospheric chemistry: 2
Atopic dennatitis (AD): 296
Auricular route : 173
Aulacoids: 184,210,236,245,247,371
Autohaemotherapy (AHT): 267
Autohomologous immunotherapy (AHIT): 305
Autoimmune diseases: 239 , 271
Aulovaccines : 1117
Azidoth ymidine (zidovudine) (AlT) : 253
2,2' -azinobis-(3ethyl-benzothiazoline-6-sulphonic acid) (ABTS): 110

Bacillus anthracis: 2
Back -Ache: 314,315
Bacteria: 57 , 66 , 79,99, 146. 160, 179, 220 . 221 , 233 , 244, 24 7, 2411, 272. 309 , 331, 347, 3411, 349 , 351 ,
354
Bacterial flora : 214 , 220 , 221 , 275
Banked blood : 351
Basic fibroblast growth factor (bFGF): 152,IRO
Basic Iife support (BLS): 364
Beta carotene: 91
Beta thromboglobulin (ß-TBG): 152
Beta- Mieroglobulin (ß1M): 255
Bilirubin : 62 , 112, 94 , 106, 170
Bioflavonoids : 96
Blood : 113
Blood letting : 16
Blood mononuclear cells (BMC) : 9, \35
Blood pressure: 203
Blood substitutes : 353
Bod y ozonc exposure (BOEX): 199,256, 262 , 2113 , 296, 3011, 360 , 371
Body weight: 203
Bohr effect : 127
Bone marrow : \30
Bradykinin : 162
5-bromo-2 ' -dcoxyuridine (BrdU): 376
Bronchial-associated lymphoid tissue (BALT) : 146
Bronchoalveolar lavage fluid (BALF): 132
Bums : 212 ,324,361

CI inhibitor 152
Ca l > adenosine triphosphataso (Ca 1' -ATPase): 102
Calcium lcvels: 11 , 140, 152
Cancer: rs, 19,303
 INDEX 431

Candidiasis : 270
Carbon dioxide (CO"): 215
Carbon monoxide (CO) : 3, 21, 33,162,170,359,361 ,371
Carboxyhaemoglobin (HbCO) : 127
Cardiae angiosteno sis : 197,244
Carotenoids : 90
Catalase (CAT) : 99, 138,236
Cathepsins: 152, 272
CD4 >Th-I response : 221,254,272,296
CD4+Th-2 response : 221,254,272, 296
Cell antiviral faetor (CAF): 254
Cell-mediated immunity: 143,257
Central nervous system (CNS) : 364
Cerebral ischaemia : 276
Cerebrospinal fluid (CSF): 107
Ceruloplasmin : 98 ,111
Chelation therapy: 367
Chemieal aeupuneture : 228,319,356
Chemilumineseenee: 63
Chlorine (CI): 32,66
Chlorofluoroearbons (CFCs) : 31
Cholesterol : 171,180 ,196,284,294
Cholitis : 213
Chronie fatigue syndrome (CFS) : 211,322,361
Chronie hepat itis: 197,211
Chronie obstruetive pulmonary disease (CO PD): 297
Chronie Oxidative Stress (COS) : 76,371
Cilostazol : 277
Citrate-phosphate dextrose (ePD): 122
Citrie acid-citrate, dcxtrose solution (ACD) : 153, 155
Clostridial myonecrosis : 361
Collagen I!III: 180
Common cold : 269
Complementary medieine : 57,365
Coneanavalin A (ConA) : 132
Conjugated dienes : 61, 73
Contraetion faetor I (EDCF -I): 162
Copper (Cu) : 98
Copper/Zinc superoxide dismutase (Cu/Zn-SOD): 98, 236
Cortieotrophie releasing honnone (CRH) : 196, 229
Cortisol : 196, 229, 254
Cosmetolog y : 244,333
C-reaetive protein (CRP) : 149,180,229
Creatinine : 207
Crohn 's disease : 139,213 ,274
Cryptosporidium infeetion: 213, 252, 270,347
Cutaneous infeetions : 178
Cutaneous route : 173
Cyclic adenosine 3' -5'-monophosphate (cAMP) : 112,151 ,277
Cyclooxygenases : 153
Cyelopcroxides : 61,73
Cysteine nitrosothiols (Cys-NO): 167
Cytokine indueers : 132
Cvtokines: 180, 271
Cytotoxic T lymphocytes (CD8 +) : 257
Cytotoxic T lymphocytes (CTL) : 257
432 INDEX

Decompression sickness : 361

Degenerative diseases : 235 ,239,300
Dehydroascorbic acid (DHA): ll4
Dehydroepiandrosterone (DHEA): 196,229,256,343
Demcntias: 213
Dentistry : 244 ,331
Deoxyribonucleic acid (DNA): 72,102
Dermatology : 244 ,296
Desfcrrioxamine: 9ll
Di(2ethylcsil) phthalate (DEHP): 376
Diacylglycerol (DAG): 112, 140
Diffused noxious inhibitory control (DNIC): 321
Dihydrolipoate (DHLA): 93
2,3-Diphosphoglycerate (2,3-DPG): 124,126 , 127, 12ll, iso, 27ll, 352
2,3-Diphosphoglycerate mutase (2,3-DPGM): 127
2,3-Diphosphoglycerate phosphatase (2,3-DPGP): 127
Disseminated intravascular coagulation (DIC): 324
Dopaminc: 229
Doping: 339 ,341
Duct-associated lymphoid tissue (DAL T) : 146

Eicosanoids: 73
Eicosapentanoic acid (EPA): 7
Electron paramagnetic resonance spin trapping technique (EPR): 31ll
Emergency surgcry : 324
Emphyema: 55
Emphysema: 297
Endorphins : 196,229
Endothelial cells (Ecs): 161, IllO
Endothelial-derived relaxing faclor (EDRF): 162
Endothelin-I (ET -I): 162, 166, 16ll
Endothelium-derived contracting factor (EDCF): 162
Endothelium-derived hyperpolarizing factor (EDHF): 162, 16ll
Endotoxins : 132
Energy charge (EC) : 124, 125
Enzymaue system: 9ll
Enzyme-Linked lmmunosorbent Assay (ELlSA) : 72, 202
Epidermal growth factor (EGF): 152, IllU
Epinephrine : 162
Erythrocyte sedimcntation rate (ESR): IllO
Erythrocytes - Tc 99 uptake: 130, 131
Erythrocytcs : 109, 121, iso, 207
Erythropoietin (EPO): 341
E-selectin : 166
Estrogen : 343
Ethane: 72, 73
Euphoria: 344
European Phannacopea (EP) : 376
Extracorporcal blood eireulation against OZ-Ol (EBOO): 129, 179,lll9, 2ll3, sos, 360, 371

Fj-isoprostanes (Fz-lsoPs): 71, 73

Faetor VIII antigen: 152
Fatigue : 306,310
Ferric rcduciug/antioxidant power (FRAP): llll
Ferritin: 97
Fctal ealf scrum (FCS): 376
Fibrinogen : IllO,196, 294
 INDEX 433

Fibroblast growth factor (FGF) : 152

Fibroblasts : 180
Fibromyalgia : 322
Fibronectin : 152, 180, 181
Fistula: 55.173 ,213
Fontaine's c1assification: 277
Food and Drug Administration (FDA) : 11,175 ,277,348,370
Food processing : 348
Free radical: 58, 75
Free Radicals in Biology and Medicine (FRBM): 21,22
Fresh frozen plasma (FFP) : 351, 356
Fungi: 179,347

Galactose - oxidase : 132

Gamma-glutamyl transpeptidase (GGT) : 259, 260
Gastroenterology: 244
Gerontology: 244
Giardiasis: 252,270,347
Glueose-6 phosphate dehydrogenase (G-6PD) : 103,124,126,230,236,284
Glucose-regulated proteins (GRP) : 84
Glutahione reduced form (GSH): 12, 84, 10 I, 102, 105, 124, 154,352
Glutathione disulfide (GSSG) : 84, 101, 105
Glutathione peroxidases (GSH-Px): 100,236, 284
Glutathion e reductase (GSSGR) : 84, 124,236
Glutathione transferase (GSHT) : 101
Gluthatione nitrothiols (GS-NO): 167
Glycation-related aldehydes : 73
Glycemia : 171
Glyceraldehyde 3-phosphate dehydrogenasc (GAPDH) : 124
Glycolysis : 124,180
Graf! versus host diseasc (GVDH): 185
Granulocyte-monocyte Colony Stimulating Factor (GM-CSF): 11, 132
Granuloc ytes: 109
Growth factor release : 151
Growth hormone (GH) : 229, 256, 273, 343
Guanylate cyclasc : 170
Gut-associated lymphoid tissue (GALT): 146,221
Gynaecology : 244

Haematoerit: 122,207
Haematological : diseases: 299
Haeme-oxygenase I (HSP 32) (HO-I): 170,236
Haemoglobin (Hb) : 97.110,127
Haemoglobin sickle eell (Hbs) : 299
Haemolysis: 61,114,122
Haemostasis : 151
Half-life (TV,): 38, 53, 54, 68
Haptoglobin : 149,229
Heart ischaemia : 276
Heat shoek proteins (HSPs) : 233,234
Heat, ozone and ultraviol et light (H-O-V) : 4,254
Hclieobacter pylori (H.p.): 54, 252
Helminth eggs : 347
Heparin : 122, 155
Hepatitis A virus (HAV): 257
Hepatitis B virus (HBV) : 257,261
Hepatitis C virus (HCV) : 75, 197,257
434 INDE X

Hepatit is delt a virus (HDV) : 25 7

Hepatitis : 213 , 25 7
Hepatocyte growth factor (HGF): 152
Herbalism : 57,367
Herpes Zostcr (HZ): 264
Herpetic infections: 264
Hexek inase (HK): 124
High pres sure liquid chromatography (HPLC) : 376
High-dens ity lipoprotein (HD L): 171,196
Highly active anti-retroviral therap y (HAA RT) : 2 13, 243, 256, 334
Hind-Iimb ischaemi a: 276
Histam ine: 162
Histo rieal aspects : I, 7
Homeopathy: 57
Honnesis : 234
Housc of Lords S.C.Se .Teeh. : 367
Human immunodeficiene y virus (HIV) : 12, 75, 235, 253, 361
Human vascular endothelial cell s (HUVECs ): 161 ,1 63,297
Humoral immun ity : 143
Hyaluron ic acid: 180
Hydrogen pero xide (H20 2) : 59, 60, 62, 63, 68, 73,99,1 24,13 2,137,1 38 ,13 9.15 2,1 53,1 64.1 65,3 54
Hydroperoxide (ROO H): 60
Hydrop eroxy radical (H0 2): 6 1
Hydroxy eth yl starc h (HES) : 353
5-hydro xy-2'-d eoxycyt idine (5-0H-dCyd) : 72
8-hydroxy- 2'deoxyg uanosine (8-0 hdG) : 72
4-hydro xy-2 ,3-trans-nonen al (4-HN E): 6 1, 69, I U2, 120
Hydroxye icosat etraenoic acid (HETE) : 162
8-hydro xyguan ine (8-0 HG): 72
Hydro xyl radical (OH"): 33, 59, 61 , 68,71,75,85
Hyperbaric oxyg en therap y (HOT): 26,359
Hypertension : 283
Hyperthyroidi sm : 230
Hypoclorou s acid (HOCI): 66. 68
Hypox ia inducible Iactor-I (HIF- I): 30 6, 308

lIopros t: 277
Immune sys tcm: 132
Immunoglob ulin A (lgA): 221
Immuno globulin E (lgE): 143. 296
Immun oglobu lin G (lgG) : 143
Immunesuppressive thcrap y: 272, 274 , 275
Inducers : 7, 132. 143
Infectious disea se (Idis): 239,24 6
Initiat ion : 59
Inositol -l ,4, 5-trisphosphate (lP 3) : 112, 140
Intens ive therap y: 244
Interferons (l FNs): 7,11 ,1 32, 261
Interleukin (lL ): 11,13 2.133,134.1 35,142,1 58,1 66.205
Internat ional Medi cal Ozonc Socic ty (IMOS): 5.28
International Ozone Association (IOA) : 29
Intraarter ial (lA) : 173, 176, 278
Intraarticular (la t): 173
lntr abladder route : 173
Intrad isc (10) : 173
Intraforaminal (l F): 173
Intralesional (lies): 173
 INDEX 435

Intramuseular (IM): 173 ,176

Intraperitoneal (lpe): 173 ,176
Intrapleurie (IPL): 173 ,176
Intravenous (IV) : 12, 173
Iodide: 2
Iodometrie method: 47,411
Iron (Fe 2> ~FeH) : 59 ,65,85 ,97,353
Isehaemie diseases : 276

Keratinoeyte growth faetor (KGF): IlW

Keratinoeytes: IllO

Laetie dehydrogenase (LDH): 169

Laetobaeillus (Lb): 220
Legionella: 347,349
Leukoeyte depletion (LD): 185,256,351 ,356
Leukoeytes: 132, iso, 205
Leukotriene B. (LTB.): 162
Limb ischaemia: 173, 196
Linoleic acid : 71
Lipid emulsion (LE): 356
Lipid oxidation products (LOPs): 56 ,76,121 ,130,144,218,236,355
Lipid peroxidation : 60 ,61 , 115
Lipodystrophies : 197,212,333
Lipopolysaccharides (LPS): 146
Lipoproteins: 109,171 ,
Liver: 130
Low Density Lipoproteins-tl.Dl.): 111, 171, 196, 2114
Low Molecular Weight Antioxidants (LMWA): 86
Lungs : 130
Lymph nodes: 130
Lyrnphocytes: 109.142
Lymphokine acti vated killer cells (LAK): 304

Macrophage inflammatory protein 10. (MIP-l 0.): 254

Macrophage inflammatory protein Iß (MIP-Iß): 254
Major histocompatibility complex (MHC): 137,257
Malonyldialdehyde (MDA): 61 , 69 , 71 ,115
Manganese (Mn): 43
Manganese-superoxide dismutase (Mn-SOD): 98 , 139
Melatonin : 96 ,343
Messenger RNA (MRNA): 137
Metalloproteinases: 272
Metastas is: 13U, 173, 197,211 ,239
Methaemoglobin (MHb): 122
Methaemoglobin reductase: 124
Methane (CH.): 214
Methylene blue (MB): 352
I million units (MegaU): 262
Monocyte chernotactic protein 1 (MCP-IIJE): 274
Monocytes: 109,142
Multi -Drug-Resistant-Mycobacterium Tubercolosis (MDR-MT): 1117
Multiple sclerosis : 139,271,275,361
Mus cularis mucosae (MM): 217 ,218
Mutagenicity: 223
Mxprotein (IFN marker) (Mx): 148
Myeloperoxidase (MPO): 205 , 2011
436 INDEX

NalK ATPase : 168

N-acetyl-cystcine (NAC): 42.104,238
Nasal route : 173
Natural Killer (NK): 141, 150.257
Necrotizing fasciitis : 248 , 361
NG-nitro-L-arginine methyl ester (Nos inhibitor) (L-NAME): 164
Nickel (Ni) : 43
Nicotinamide adenine dinucleotide phosphate, oxidised form (NADP): 102, 103, 105, 106, 124, 125, 126
Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH): 103. 105, 106, 124. 125. 126
Nicotinamide adenine dinucleotide, oxidiscd form (NAD): 64 , 124
Nicotinalllide adenine dinucleotide, reduced form (NADH): 124
Nitric oxide (NO'): 21,33,67,68,151 ,162,168,171, 180,297,371
Nitric oxide synthase (NO,) : 67
Nitrite: 164, 165
Nitrogen (N l) : 43 , 175
Nitrogen dioxide (NO'l): 33
Nitrogen oxides (NO ,): 3,43
Nonsteroidal anti-inflammatory drugs (NSAID): 274
Nosocomial infections : 349
Nuclear factor Kappa B (NFKB) : 136, 137,272,297

Oedematous-fibro-sclerotic panniculitis (OFSP): 334

Oleic acid : 71
Oncology: 244
Onychomycosis: 270
Oral route : 173
Orosomucoid : 149,229
Orthodox medicine : 57.365
Orthopaedics : 244,314
Osmotic fragility: 123
Osteomyelitis : 55, 248, 361
Osteo-radionecrosis: 361
Oxidative preconditioning : 234 ,271
Oxidative shock proteins (OS Ps): 234
Oxidative Stress Proteins (OSP): 234
Oxidative stre ss : 71,75,107,149.233
Oxidized low-density lipoproteins (OxLDLs): 171
Oxygen ro,» 38 ,44,57,68 ,215
Oxygen availability: 180
Oxyhaernoglobin (Hb0 2) : 124, 127,359
Ozone (0 3) : 3, 31, 37,79. 109, 132, 137, 199,347.349.351
Ozone concentrations: 34 , 44 , 45, 47 . 49. 52, 119. 159,219,320
O zone destructor: 43 , 44, 51 .. .
Ozone dose: 45, 225
Ozone generator: 43
Ozone in Science and Engineering (OSE) : 29
Ozone tolerance: 234
Ozonetherapy: 7, 10,46,57
Ozonides : 55,60
Ozonized major autohaemotherapy (OrAHT): 4, 8, I L 34 , 39. 130, 147. 179, 182, 238 , 239 , 262 , 278 .
283 ,308,353,359,360,369,375 ,380
Ozonized minor autohaemotherapy (01 -AHT minor): 8. 182.186
Ozonized oil : 51,55,249,280
Ozonized water: 51 , 249 , 269. 280

Palladium (Pd) : 43
 INDEX 437

Papillomavirus infections (HPV) : 268 .

Parts per billion volume (Ppbv) : 32
Parts per million volume (Ppmv) : 34, 41
Pentane : 72
Periodate : 132
Peripheral occlusive arterial disease (POAD) : 277
Peroxynitrite (ONOO"): 67,68
pH : 207,215,216
Phorbol esther : 132
Phosphatase (Ppase) : 137
Phosphofructokinase (PFK) : 124,126
6-phosphogluconate dehydrogenase (6PGD) : 103
Phospholipase A 2 (PLA 2) : 153,272
Phospholipase C (PLC) : 112
Photometrie determination: 49
Phthalates: 183,228,259,375
PhysiologicalIFN(cytokine) response : 146
Phytohaemagglutinin (PHA) : 132
Piruvate kinase (PK) : 124, 126
Placebo effect: 184,242,327,369
Plants: 233
Plasma proteins : 86,97,109
Plasma : 113, 180,351
Plastic bags : 183,375
Plastic particles : 378
Platelet activating factor (PAF) : 73, 151
Platelet factor 4 (PF4): 152
Platelet-dcrived growth factor (PDGF) : 152,158,180
Platelet-rich plasma (PRP) : 153
Platelets : 109, 151, 153, 155, 180,207
Pneumolog y : 244,297
Pokeweed mitogen (PWM) : 132
Poliethylenglycol-Intcrferon CI. (PEG-IFNa): 261
Polyethvlenglycol-superoxide dismutase (PEG-SOD): 99
Polyunsaturated fatty acids (PUFAs): 60, 71,112,252,356
Polyvinyl chloride (PVC) : 43,259,375
Post-herpetic neuralgia (PHN) : 264
Pregnancy : 230
Primary root carious lesions (PRCLs): 331
Probiotics : 273
Proctitis : 213
Proliferation index (PI): 376, 379
Propagation : 59
Prostacyclin (PGIz): 151,162,168,297
Prostagiandin H2 (PGH 2) : 162
Prostaglandins (PGs) : 65, 151, 180,277
Proteasome : 137
Protein bloeking NFKB activity (IKB) : 136, 137
Protein carbon yl content: 73
Protein G (G): 112
Protein Kinase C (PKC): 112
Protein kinase phosphorylating IKB (IKK-a): 137
Protein kinase phosphorylating IKB (IKK -ß) : 137
Protein thiol groups (PTG) : 110,114,154,157,164,194,195.206,216,217,357
Proteinases: 132
Psolaren S-59 UVA (S-59-UVA): 352
Psoriasis: 296
438 INDEX

PVC-di(2ethylesil)phthalate (PVC-DEHP): 375 ,377

Pyruvate kinase (PK): 124

Radiation damage: 361

Randomised clinical trials (RCTs): 20 ,239,242 ,262,299,306,371
Raynauds phenomenon : 276
Reactive oxygen spccies (ROS): 56, 76, 111,218,236,297
Recommended dietary allowances (RDA): 83, 90
Rectal abscesses : 213
Rectal insufflat ion (RI) : 125, 128 , 173, 213 , 256 , 262 , 283, 360
Redox balance: 235, 236
Regulated upon activation, normal T-cell expressed and secreted (Chemokine) (RA NT ES) : 254
Reju venating agent: 343 ,361
Renal diseases: 29R
Rcspiratory diseases: 239
Respiratory tract lining fluids (RTLFs) : 34 ,41
RetieuJo endothelial system (RES): 146, 172
Retinal pigment epithelium (RPE): 286, 288 , 290
Retinitis pigmentosa: 287
Retinoic acid (RA) : 91
Retinol (Vitamin A) : 90 , 106
Retinol bind ing protein (RBP): 91
Rhagases : 213
Rheumatoid arthritis (RA): 139,271 ,273 ,274

Sauna: 207 ,209

Scanning electron microscopy (SEM ): 153,156
Scavenger receptor A (SRA): 75
Sch önbein: 1,27,29
Sclerodermia: 212
Selenium : 238
Semidehydroascorbate radicalanion (A-): 84
Scnil ity : 361
Serotorune (5-hydroxytryptaminc) (5-HT): 151. 152, 162,229
Serum amyloid A (SAA): 229
Sickte cell anaemia (SCA) : 299
Side efTects: 227
Siemens 'tube: 3
Singlet oxygen (' 0 ,): 66 .68
Sister chromatid exchange (SC E): 224
Sj ögrens syndrome: 271
Skin: 199,200,209,212
Skin-associated lymphoid tissue (SA LT) : 146
S-nitrosothiols (RSNO): 167
Sodium thiosulphate (Na,S,Oj): 48
Solvent-detergent (S-D): 352
Somalostatin : 229
Spleen : 130
Staphylococcal Enterotoxin B (SEB): 9 ,132
Stratosphere: 32
Stress tolerance: 233
Subcutaneous (SC) : 173,176
Substartee P: 162
Sulphate dermatan : 180
Sulphidric acid (H1S) : 214
Supergifted erythrocytes: 126, 129,308
Superoxide dismutases (SOOs): 9R, 255
 INDEX 439

Surgery: 56, 244

Systemic lupus erythematosus (SLE): 271
Systemic sclerosis : 271

Technetium 99 (Te 99 ) : 131

Temperature: 203
Termination: 59
Testost erone: 343
Therapeutic " shock" : 76, 107,245,301,371
Therapcutie response to COS : 234
Therapeutic window: 119, 141
Thiobarbiturie acid-reactive substanees (Marker ofperoxidation) (TBARS): 71,88,110,114,115,154,
15~ 164, 194,195,206,216,217,357
Thioredoxin (TrX): 95
Thioredoxin peroxidase (TrX Px): 95
Thioredoxin reduetase (TrXR): 1\4,95
Thiyl radieal (RS'): 90
Thiyl /sulphenyl radicals (RS'/RSO'): 90
Thrichloro acetie acid (TCA): 110
Thrombomodulin: 152
Thrombospondin (TSP-I): 152
Thromboxane A z (active form) (TxA z): 151,153,162
Thromboxane B~ (stable form) (TxB~) : 151\
Total (peroxyl) Radioal-trapping Antioxidant Parameter (TRAP): 1\7
Total Antioxidant Status (TAS): 1\6,87,110,113 ,114,154,157,206,357
Toxicity : 21,25 ,33 ,41,54,139,223,369
Trans -activator of transcription (HIV protein) (Tat) : 253
Transaminases : 207 , 258, 259
Transferrin : 97, 111
Transforming Growth factor alpha (TGFu): 258
Transforming Growth faetor beta (TGFß): 132,152,158 , 159,180,181
Transfusion-telated acute lung injury (TRAU): 185
Transient isehem ic atlacks (TlAs) : 285
Transmission eleetron microscopy (TEM): 153, 156
Transplantation: 324
Trauma: 324
Tri-(2 etylesil trismellitate) (TEHT): 377
Triglyccrides: 171
Triolein triozonide: 56
Troposphere: 32
Tubal route : 173
Tumor Necrosis Faetor alpha (TNFu): 11, 132
Tumour infiltrating lymphocytes (TIL): 304
Tumours : 130,303

Ubiqu inol (QHz): 92

Ubiquinone (QlO): 90 ,92, 106
Ultravioletlight (UV) : 31
Urethral infections : 178
Urethral route : 173
Urie acid: 65. 81, 87,111
Urology : 244

Vaginal infeet ions : 178

Vaginal route : 173
Vascular endothelial Growth Factor (VEGF): 152, 169, 180, 21\8
Vascular system : 109, 130, 161
440 INDEX

Vaseulitis . 271
Vas culopathies : 211,239, 244
Vasopressin : 162
Venous C O 2 (pvC0 2): 204,215,216
Venous O 2 (PV02): 180,104 ,215,216
Venous stas is : 276,28 I
Very low density lipoprotein (VLDL): 171
Vetcrinary mcdicine: 337
Viral discases: 211
Visual acuity (V A): 293
Visual analogue scalc (VAS): 266
von Willebrand factor (vWF): 152

Watcr disinfectant: 347

Work site concentration (WSC): 34
World Health Organisation (WHO): 34,270,356
World Medical Association (WMA): 328
WOllnd healing : 159, 181,361
Wound hormone: 159

Xantine dehydrogenase (XDH): 64

Xantine oxidase (XO): 64

Yin-Yang: 80