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RESEARCH

IN PHYSIOPATHOLOGY
AS BASIS OF
GUIDED CHEMOTHERAPHY
With Special Application to Cancer
EMANUEL REVICI, M.D.
Scientific Director, Institute of Applied Biology,
New York, N.Y.
Chief of Dept. Of Oncology, Trafalgar Hospital,
New York, N.Y.

Published for the American Foundation for Cancer Research, Inc., by

D . V A N N O S T R A N D C O M P A N Y, I N C .
TORONTO

PRINCETON, NEW JERSEY


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COPYRIGHT 1961, BY
EMANUEL REVICI. M.D.
Published simultaneously in Canada by
D. VAN NOSTRAND COMPANY (Canada), LTD.

CONTENTS

FOREWORD

THEORY A ND FAC TS
EW OTHER PATHOLOGICAL CONDITIONS have aroused, as cancer has, the interest of so

many scientic disciplines. Problems related to cancer have become of continuously


increasing concern in virtually every eld of medicine. In some, such as pathology, they
are a major preoccupation. But in sciences other than medicine, cancer also has been
receiving increased attention. One of the most urgent activities of synthetic chemistry
today is the search for new compounds which might possibly be effective in the control of
cancer. Physical chemistry is trying to provide new explanations about the variety of
processes present in cancer. Even mathematical studies which recently have offered an
interesting application of quantum theory to carcinogenesis, have found new applications
in cancer.
With the rapid development of physical sciences, the medical research worker has
hoped that from them might come some contribution that could help him ultimately in his
difficult task. He also appears to have been anxious to take quick advantage of the
progress of other disciplines for another reason, hoping that, through employing their
ndings and methodology, medicine in general and cancer research in particular, could be
promptly changed from the empirical discipline it has been until now into a positive
science. He has brought as many applications of other disciplines as possible into his study
and this has led to a whole series of new methods of investigation through which
interesting new information has been obtained. Yet, most of these applications have been
tried chiey because they have been at the immediate disposal of the scientist rather than
because they have represented a missing link in the development of his own ideas.
The outcome has not been rewarding. Medical knowledge appears not to be
sufficiently advanced to successfully utilize the avalanche of new, highly specialized
information offered by the investigative methods derived from other disciplines. Basic
theoretical knowledge in medicine in general, and about cancer in particular, has not yet
reached the level necessary to relate and assimilate the new data. To a large extent, basic
concepts about pathogenic problems are not even formulated as yet. When the medical
scientist has tried to transform the new data into effective therapeutic procedures, he has
failed. And the failure has made more evident how much we need basic
physiopathological knowledge before we are able to take advantage of detailed data.

Meanwhile, normal development of cancer research has been hindered, sidetracked from its logical course. While thousands of scientists with almost unlimited funds
at their disposal are presently using the most advanced methods for the acquisition of
details, almost no attempts are being made to resolve basic problems, although the cancer
investigator is continuously obliged to realize the dearth of fundamental knowledge.
If we attempt to analyze this abnormal situation further, we can nd indications
that it may have its origin also in a distortion of the proper relationship between the two
factors that, together, make for progress in researchideas and experiment.
The experimental approach provides precise information about particular
phenomena under dened conditions. The analytical method tries to investigate reality by
recognizing the proper place of the various constituents of a whole, the parts being
identied as such by the experimental ndings. On the other hand, the conceptual
method not only provides an inkling of what the completed whole will eventually look like,
but also attempts to predict the properties and the relationship of the component parts.
In dealing with a highly rened and complicated subject, the analytical method by
itself appears inadequate. For example, in atomic physics, the results of experiments are
expressed by numbers giving the values of certain physical quantities that have been
measured. In order to complete the analysis, we must simultaneously determine the
numerical values of certain quantities dening the material bodies, the objects of the
experiments. This is prohibitive so far as canonical coordinates by Heisenbergs uncertainty principles are concerned. With experimental knowledge somewhat curtailed, theory
at present must attempt explanation.
In other areas as well, experiments present only limited numerical values pertaining
to some physical quantities. Were we able to measure all quantities, we could analytically
reconstruct the entire theme of the physical reality. However, when some quantities
cannot be simultaneously determined, this direct reconstruction is not possible and
experiments merely give an indirect approach to what we regard as reality.
If the inadequacy of the analytical approach by itself is evident in the highly positive
disciplines, such as in the physical sciences, it is even more so in biology. As Bohr and
others have intimated, the conditions of uncertainty seem to be much more pronounced
in biology than in physical science. The fact that experiments in biology give only
fragmentary and unrelated results is not surprising; the need for a synthetic theoretical
method in this eld is clear.
In medicine, which is applied biology, the need for the conceptual approach is
especially profound. lt is true that this approach, as the sole approach, has shown its
inherent weakness in the past. There was a time in the development of medicine when
available data were so scarce and unreliable, and the need for ideas to provide some sort
of guidance was so great, that the worker resorted to broad imagination, using it to
replace almost entirely any other form of investigation.
Largely as a reaction to the high proportion of speculations prevalent in the early

years, the experimental approach in medicine came to be emphasized. Claude Bernard,


who almost single-handedly was responsible for this, tried to give experimentation its
rightful role. However, in ensuing decades, the relationship between theory and
experimentation has been progressively distorted. An unrestrained exaggeration of the
role of the experiment, the erroneous view that pure facts represent the aim of research,
has led to an entirely unbalanced approach. Not only have almost any data obtained by
research been considered intrinsically interesting, but obtaining them has become the sole
purpose of much research. In scientic papers today, experimental data must be reported
as such; any allusion to theoretical meaning is considered undesirable. Generations of
scientists have been schooled to believe in the intrinsic value of the experiment. As they
have applied this belief to research in biology and as they have made unlimited use of new
methods taken from other disciplines with no ideological requirement for their use, we
have had more and more data and fewer ideas. Today, with great astonishment, some
scientists are at last beginning to recognize not only that data alone do not generate ideas,
but that science cannot progress without theory.
Ideas and experiments are integral parts of all scientic research. A balance
between them is needed to assure progress. It must be understood that the function of
experimentation is to guide our thinking, to help build up new concepts, and to prove
their accuracy in accordance with reality. Certainly, fundamental concepts must not be
mere speculations. They should be accepted only after conrmation through
experimentation. Experimentation is the necessary link between mental concept and
reality. To the attempts to consider any unresolved fundamental problems in biology, one
has to try to bring a rightful balance between conceptual views and experimentation.
The exaggerated importance attributed to experimentation in biological science, its
use even as a substitute for ideas, has led recently to a massive attempt to solve the
therapeutic problem of cancer by indiscriminate screening of chemical agents. Here,
empiricism has been brought to its culmination. After tests of tens of thousands of agents,
many workers are now beginning to realize that the results are almost worthless for
cancer therapy in humans, that seemingly promising agents have an effectiveness limited
to the conditions present in the actual animal experiments. By its impressive magnitude,
the failure of indiscriminate screening, of empiricism epitomized, has begun to impel
many workers to change their ideaas to what must be done if the cancer problem is to be
solved. A rst result of this change has been a new and, this time, unbiased evaluation of
just where we stand in our assault on the cancer problem. Every day more scientists are
making the evaluation in their reports to the medical profession and to the public with a
candidness which, only a few years ago, very few would have employed.

The Present State of the Cancer Problem


Surgery in cancer can be considered to have arrived now at or near its maximum efficiency.
Thanks to progress in operative techniques, and to advances in pre- and post-operative
care, ultraradical surgery is available today. The propensity of cancer to spread far from its
original site has made such surgery obligatory in many cases if there is to be an effort to
eliminate all malignant cells. Yet ultraradical surgery has not suiciently increased the cure
rate to justify horrifying mutilations, especially when the face is involved. With few
exceptions, surgical procedures do not prevent the patient from dying of cancer sooner or
later. The so-called ve-year-cure-rate represents, to say the least, an unrealistic appraisal.
Many authors consider that even the rate of ve-year survival is not improved by surgical
procedures, and the ultimate fate of these ve-year survivors, with few exceptions, is still
disastrous. Most of the cured cases still die from cancer.
Other recently discovered facts have increased skepticism about the value of
surgery in cancer. The polycentric origin of cancer, especially incases where the lesions are
far apartconsidered by some workers to be true even in malignant melanoma, for
instancewould greatly limit the value of surgery as a means of eliminating all cancerous
cells. It is recognized that to operate on a lymphoma is useless. Furthermore, it is known
today that cancer cells are present in the circulating blood. Surgical manipulation has been
found to induce a ow of these cells into the blood even from relatively small primary
tumors.
In view of all this, cancer cannot be considered to be a condition for which surgery
is a major hope. Surgery represents only an expedientto be tried so long as nothing
better can be offered. It is probable that in the future it will be reserved, in cancer
treatment, for the correction of mechanical complications, such as intestinal or other duct
occlusion.
Unfortunately, radiation has not been much more successful in its long range
results. In order to control cancer, it is necessary that radiation destroy all the cancer cells
present in the organism while producing minimal damage to normal tissue. It appears that
such high selectivity of action cannot be obtained. The lack of it may be implicit in the
nature of the effects achieved by radiation. A study of the biological effects of radiation,
which is to be presented later in this monograph, has shown that an important part of the
action of radiation is to induce changes in certain constituents of the body, principally fatty
acids. These changes are largely responsible for the favorable effects of radiation but they
also are largely responsible for the undesired effects. It is the nature of these changes
which limits qualitatively the capacity of radiation to inuence cancerous processes, and
makes it dubious that progress in technique can ever greatly improve the qualitatively
insuficient effectiveness of radiation. Clinical results to date provide conrmation of this
pessimistic view. The recent use of extremely high voltage radiation, of radioactive cobalt,
and of other radioactive particles has not greatly improved results over those obtained
with older forms of radiation twenty years ago, except for reducing some harmful

immediate skin and systemic effects. Now, as earlier, with few exceptions, the benets of
radiation are no more than temporary. Longlasting good effects still are limited to only a
few radio-sensitive tumors. The resort to isotopes, in which the scientic world has put so
much hope and millions of dollars, also has proved greatly disappointing.
Of the thousands of cases of various kinds of cancer in which isotope therapy has
been tried, only a very limited number of cancers of the thyroid have responded. Not only
because of its continuing failures, but because of its inherent qualitative inadequacy,
radiation does not appear, any more than surgery, to represent the solution for the
problem of cancer.
With surgery and radiation therapy incapable of resolving the problem, more and
more research workers have turned their efforts in other directions. The existence of some
cases of spontaneous remission has led many investigators to believe that immunological
procedures related to cancer would be able to resolve these problems. Unfortunately the
existing knowledge in this specic eld is too meager to permit more than some tentative
investigations, usually only repetitions of similar researches made many years ago with
limited success. Fruitful development of this approach would have to follow the normal
pathway, starting with more knowledge of the complex immunological processes
intervening in cancer.
An enormous amount of cancer research in recent years has been directed toward
chemotherapy. It is a fact that many agents and groups of agents have shown the capacity
to inuence tumor evolution. However, each has had limited usefulness. Results of
treatment have been characterized by inconsistency. Even in seemingly susceptible types
of cancers, results have been good in one case, poor in another and have varied even for
the same patient at different times. The inability to explain and remedy these variations
has discouraged many workers. Although it appears evident that the source of
discrepancies resides in the patients themselves, the general tendency among researchers
has been to try to resolve the problemby nding agents able to act independently of any
differences which exist between subjects.
In despair at the lack of progress in this approach, many workers today are using
the screening enterprise mentioned above as a kind of last resort. For this project, they
have renounced the scientic concept that pharmaco-dynamic activity must serve as the
basis on which an agent is to be tried in therapy. They have fastened into a purely empiric
approach. Now, all available chemical substancesand many others which will be
synthesized especially for the purposeare to be screened indiscriminately, for their
effects on animal tumors with no reason for this test other than that the agents are, or can
be made, available. We will not dwell here on the assumption that routine technique is
more likely than imaginative brainpower to resolve the problem of cancer. The results of
this screening to date have shown it to be an invalid procedure, as expected by most
critical workers. With tens of thousands of substances already tested, the busy screeners
are obliged to recognize that the approach itself is fundamentally erroneous. Experience
has proved that an agent can be wonderfully effective against one tumor and still be en-

tirely inactive in others. Of tens ofthousands of agents tested, less than a hundred have
shown effects on tumors in animals. None appears to have signicant value when applied
in humans. These results have emphasized again the importance of factors other than the
agent itself. One factor lies in the differences which exist between various tumors. Some of
the other factors include variations between species, between individuals of the same
species, between origins of tumors, between spontaneous and transplanted tumors, and
even variations in anyone individual at different times.
Faced with this situation, some workers have concluded that not one treatment but
at least hundreds of different treatments must be found in order to cope with the huge
variety of conditions.
Taking cognizance of these considerations, it has seemed to us that a more realistic
and logical approach is to try to understand the nature ofthe existing differences and to
attempt to make the treatment adequate on the basis of that understanding. It has been
this approach which has been followed in our research.
We have studied the problem of cancer for the last thirty years from an entirely
different vantage point than that used by other workers. Attention has been focused on
the physiopathological aspect of cancer, on the basic changes that occur in the different
patients, with the ultimate aim of understanding the part played by these changes in the
response of cancer to therapeutic attempts. This emphasis on the physiopathological
aspect of cancer has been made possible by applying a more general overall idea of the
nature of the disease.
This approach is based under various new concepts. They concern,
1) The role of the organization in the pathogenesis of the conditions.
2) A dualistic systematization of the manifestations related to normal and
abnormal physiology.
3) The predominant intervention of certain constituents such as lipoids and
chemical elements in the induction of the opposite manifestations.
4) The possibility to integrate the occurring processes into a system of defense
mechanism against the noxious inuence exerted by the environment.
Many general and special problems of physiopathology, some of them concerning cancer
and other conditions, have been analyzed in this framework.
The application of this approach to therapy has resulted from a logical
development of that approach. The recognition of the intervention of a variety of
pathogenic factors, not only differing from one subject to the other, but even changing in
the same subject during the evolution of the condition has emphasized the need for
individualized therapy. As opposed to the tendency to overcome the differences existing
between individual subjects through a standard therapy, the guided therapy utilizes the
knowledge of the occurring different pathogenic particularities in order to correct them. A
high degree of exibility in the treatment has appeared necessary.
As part of this approach to therapy, has appeared the need for more complete
knowledge of the existing differences and their interpretation in terms of the pathogen-

esis of the condition. The search for adequate analytical tests has thus represented the
rst task. The development of day-by-day analysis of the condition has been possible by
choosing relatively simple but reliable procedures. The information they offered was used
to determinethe nature of the agents able to correct with a certain specicity, the
encountered pathological conditions. These two parts the recognition of the existing
condition and the adequate agents, have concretized this approach.
These considerations explain also why the new developed guided therapy cannot
be understood and correctly applied without a sufficient knowledge of its
physiopathological and pharmacological basis. These same considerations have led us to
present the research concerning this approachas a block, instead of fragmented
communications. The form of a monograph has appeared consequently the best suited. In
a further effort to achieve a cohesive presentation, we have separated from the text most
of the technical and experimental data, and presented them as notes at the end of the
text.

ACKNOWLEDGEMENT
Progress in our research has been made possible only through the day by day
cooperation of different groups of co-workers who have contributed years of assiduous
work. Many of them are mentioned in the following pages where the research in which
they took part is presented. I am deeply indebted to them.
I wish to thank all those friends whose personal efforts or, who through their
organizations, have given their material and moral support to the continuation of our
research. Special thanks go to Mrs. Sherman Pratt for her tireless efforts on our behalf.
My sincere appreciation to those who have helped me in the preparation of this
book, Mr. L. Galton, Mrs. E. F. Taskier and Mrs. H. Kennedy for the editorial work; Mrs. E. F.
Taskier for the gures; and Mrs. P. Berger, Mrs. B. Doctors and Mrs. M. Prikasky for the
secretarial work.
I wish to gratefully acknowledge Miss Fanny I-Ioltzmanns devoted friendship and
help.

CHAPTER

t s A S I CC
] O N C E P T SO F
ORCANIZATION
nr\
rNrELLEcruALMEcHANTsM
I
usedby man to acquireknowredgehas
"t
led him to recognizethe existenceo[ relationshipsbetween the various
manifestationsencounteredin nature. He has employed abstractionand
integration to build up conceprualindividualitieswhich he identified as
separateentitiesin nature. Structuralcharacteristicsand dynamic properties have appearedto be the most suitablecriteria for defining theseentities. However,curiosityhas constantlyimpelledman to attemptto extend
his knowledgeby explainingand correlatingtheseentities,and an important means has been analysesbreaking them down into their component
parts.
Out of these analyseshas come recognitionof the fundamental importanceof organization.F^ot,as entitieshave been analyzedone after the
other, it has becomeclear that the seeminglyinfinite variety of them perceived by our sensesis in reality the result of the arrangementof a relatively small number of basic units, the molecules.Moreover, analysishas
shown that only a very small numberof chemicalelementsmake up even
the most complexmolecules;that combinationsof less than one hundred
elements,in differentproportionsand relationships,
account for tens of
thousandsof compoundsand many billionsof entities.And further analysis
hasrevealedthat elementsthemselvesrepresentdifferentdynamic arrangements of only n fsw-nscording to some hypotheses,only two-fundamental corpuscles.
Upon close analysis,nature, which appearsto be so greatly varied,
turnsout, in fact, to be basedupon only a very few fundamentalconstituents and it is the manner in which theseconstituentsare bound together,
which providesvariety.
theirorganizationinto a multitudeof combinations,
I

R E S E A R c Hr N p H y s t o p A T H o L o c y

The studyof organizationobviously,then,could furnish the most valuable information about nature.And it would not appearto be too much to
expect that, if nature's seerninglyinfinite variety stems from organization
of only a few constituents,then organizationitself might also be achieved
through a few, relatively simple fundamentalpatterns.If so, seekingout
suchpatterns-systematicanalysisof organizationcomparableto the efforts
to systematizeconstituents----could
be of primary importance to better
understandingof a host of problems.
Homotropy and Heterotropy in Nature
As man attemptedto recognizeordcr in the constantchangessecn in
nature,he noted certain patternsthat appearedto indicatedefinite"laws of
nature."Someof theselaws have been observedto operateunder such a
wide variety of circumstances
that they have come to be acceptedas
"fundamentalIaws."
ln 1824, Sadi Carnot formulatedone which is known as the Second
Law of Thermodynamics.Carnot observedthat, in a given system,work
involving the transfornrationof thermal into mechanicalenergy is only
accomplishedas heat drops from high to low temperature.In more general
terms,this meansthat work accomplished
in an isolatedsystemresultsin
progressively
eliminatingdifferencesin temperature.Clausiusrecognized
this as a fundamentalprinciple and postulatedthat the amount of energy
availablefor work alwaystendstoward a maximum. This condition, called
"maximum entropy," correspondsto uniformization of temperatureand
also to homogeneousdisorganization.At first, it appearedthat the principle was in conflict with the First Law of Thermodynamicswhich expresses
the rule of conservationof energy.However, Helmholtz soon was able to
demonstrateits validity by showingthat only the secondlaw could reconcile
the first with the impossibilityof perpetualmotion.
In a more philosophicalvein, we considered,in our research,that this
Second Law of Thermodynamicsin its broadest sense could define a
fundamentaltrend toward annihilation of any existing differencesin nature, through the triumph of total uniformity. SinceClausiusused the term
"entropy" in applying Carnot's original observationto closed mechanical
systems,it has seemedpreferableto avoid confusion by utilizing another
term for this general tendency toward uniformity in its broadest sense.
Therefore, we have chosen the term "homotropy."
Despitethe theoreticallyrapid trend in the directionof absoluteuniformity, or homotropy, no such final state has yet been achieved.It must
be concluded,therefore,that someotherfactoropposedto that trend exists.

BASIC CONCEPTS OF ORcANTZATION

Wehavechosenthe term "heterotropy" for this other factor, which tends


to maintain
or produceinequalityand thus to prcservethe order that is
evident
in nature.
In order to understandthe roles of these two opposing fundamental
tendencies
in the organizationof nature in as logical a fashion and with
asmucheaseas possible,it seemedadvisableto try to study their operationfustin one of the simplestand best known natural organizations,the
atom,passinglater on to higher and lower levels of organization.
TheAtom
Therole of the two opposingtendenciesin atom organizationbecomes
clearwhen we study the relationshipbetween the forces that form this
entiry.
Each atom consistsof a positivelycbargednucleussurroundedby
negatively
charged electronsin adequatenumber to balance the nuclear
charge.
Thee.ristence
of the atom dependsupon forcesacting betweennucleus
andelectrons.One group is of coulombiannature. These are the electrostaticforcesthat account for the attraction between oppositely charged
electrons
and nuclei, and for the repulsionbetweenelectronsbearing similarcharges.
If such forcesdid not exist, electronswould wander irregularly
andwouldnot be retained around the nucleus.
Yet, if electrostaticforces were unopposed,electronswould be drawn
closer
andcloser to the nucleusand would finally fall into it, therebybringtngaboutcompleteannihilationof all charges.The fact that electronsare
notabsorbedby the nucleusindicatesthe existenceof a second,opposing
force.
This secondforce is definedby quantum mechanicsand the quantum
rheoryof fields. euantum mechanicsascribesa seriesof discreteenergy
levelsto electrons within atoms. Radiation is emitted or absorbed only
whenelectronspassfrom one stationarylevel to another.The energylevels
ue relativelystable, and a state of minimum energyexists when each of
theelectronsof the atom is as close as it can be to the nucleus on the
groundlevel.
The energy levels correspond to the orbits describedby Bohr's theory
which,although not entirely accurate,affordsa good basisfor understandingatomic properties.Bohr envisagedthe electronsrevolving around the
nucleus
in definite orbits, each orbit moving continuouslyin these states,
theatomnot emitting radiation. This differsfrom thc older theory according to which the electronscan revolve around the nucleus on any orbit.
Suchcasualorbit motions would lead to loss of energy by radiation. The

R E S E A R c Hr N p H y s r o p A T H o L o c y

electrons would come closer and closer to the nucleus and would, as
already pointed out, finally be absorbedby it. The quantum theory of
fields accountsfor the absenceof radiation and for electronsremainingin
their particular orbits. However, the concept of stationary statesfails to
explainall the propertiesof the atom, particularlyits chemicalreactivity,by
virtue of which different atoms combine to form molecules.
According to anothertenetof the quantum theory, the Pauli Exclusion
Principle,an orbit cannot be occupiedby an indefinitenumber of electrons
but, at most, by two electronsthat spin in oppositedirections.The orbits
are arrangedin shells,each shell having a definite level of energy.A shell
is completewhen it containsthe maximum number of electronscompatible
with the Pauli Principle.Completeshellsconsistof.2, 8, 18, etc., electrons.
When an inner shell has its quota of elcctrons,additional electronsmust
occupy an outer shell. Consequently,
insteadof falling into the nucleus,
the electronsin their lowest energy stateswill continue to revolve at it
considerabledistancefrom the nucleus.
As already indicated,if there were only electrostaticforces, the electronswould have long sincefalleninto their nuclei,neutralizingall electric
charges.The universewould be in a state of maximum homotropy.No
strong atomic forces would exist and no chemical reactions would take
place.The intervcntionof quantumforcesavoidsthis. It is apparent,then,
that the organizationof the atom resultsfrom the operation of two types
of forces,electrostaticand quantum, the electrostaticservingto bring and
keep nucleusand clectronstogetherto constitutethe atom, the quantum
accountingfor a motion of electronswhich preventstheir total annihilation and the neutralizationof all electricalcharges.
Homotropic and Heterotropic Forces in the Atom
We rnay now attempt to considerclectrostaticand quantum forces in
the atom in terms of homotropicand heterotropictrends.Let us hypothesize
an atomic systemin which only electrostaticforces are active and compare
it with a real systemwhich also has active quantum forces.Whereasthe
fictitioussystemwill rapidlyevolvetowardsa stateof maximum homotropy,
with annihilation of all charges,this will not occur in the real system.
When the two systemshave reachedfinal statesof equilibrium, the homotropy of the imaginary systemwill be greaterthan that of the real system.
If the quantum forces that keep the electronsaway from the nucleus in
the real atom could be withdrawn, the electrostaticforces acting alone
would bring about a state of completeannihilation,thus making available
a certain amount of energy that had previously been preservedby the

B^sIc coNcEprs

oF oRcANrzATIoN

quantrrm forces. In this sense,it is apparent that the electrostaticattraction


betweennuclei and electronsis of a homotropiccharacter,while quantum
forces are heterotropic.
Fulfillment. ol Quantum and Electrostatic Forces
There are diverse coDsequencesfrom the operation of quantum and
electrostaticforces in the atom. The partial fulfillment of the electrostatic
forces keepsthe nucleus and electronstogetherin the atom, while quantum
forces ceaseto exist with the establishmentof complete electron shells,and
have, therefore,been called "saturation forces."
In the atoms of the noble gases,quantum and electrostaticforces are
simultaneouslyfulfilled. As a result, theseatoms are inert. They have no
physical or chemical activity and their entry into the formation of molecules
is explained by the intervention of van der waal's cohesion forces. In all
other atoms, the electrostatic forces are fulfilled when the number of
orbital electronscorrespondsto the nuclear charge. However, when this
occurs, the electron shells are incomplete and consequently unfulfilled
quantum forces :ue present. when the quantum forces are fulfilled, other
electrostaticforces appear.
under these circumstances,in order to complete its outer electron
shell,i.e., to fulfill the quantum forces,an atom may borrow or lose one or
more electrons.This is achievedwith a secondatom which, by the exchange, reduces or increasesits orbital electrons to fulfrll its quantum
forces and is left with a number of electronsconsistentwith a complete
outer shell. The fulfillment of quantum forces requires changesthat involve a displacementof electronsoutside the atom itself. This exchange
of electrons,properly called "electron transfer," fulfi"lls,to be sure, the
quantum forces of the atom. However, as a result of the transfer, the
relationshipbetweeneach nucleusand its orbital electronsis changed,resulting in covalentions. Those atoms that have gainedby the transfer and
have an excessof electronsnow have a negativecharge while those that
have lost electronshave a positive charge.As a result, new electrostatic
forces appearwhich, althoughconfinedto the atoms themselves,influence
their external behavior, as evidencedby the interaction between atoms.
An antagonisticrelationship can be conceivedbetween electrostatic
and quantum forces in the sensethat the fulfi.llmentof one usually leads
to appearanceof the other.
Electron transfer representsonly one mechanismfor fulf,lling the quan. We have unwillingly resorted to this too anthropomorphic term, the use of
which has to be excusedas didactic license.

.'

R E s E A R c HI N p H y s t o p A T H o L o c y

tum forces of the atom. Two atoms which do not have sufrcient electrons
in their external shells to complete the external shells of both can fulfill
their quantum forces by sharing some of their electrons.By achieving a
complete external shell for each atom, the sharing processsatisfiesthe
quantum forcesof both atoms.This methodof quantum fulfil.lmentthrough
the sharing of electrons also can lead to the appearanceof electrostatic
forces.If the two atoms are identical,the sharedelectronshave an intermediate position and, therefore,do not influence them. As a result, the
atoms have their quantum forces fulfilled without inducing new electrostatic forces. This is the so-called"homopolar bond." If, however, two
atoms are dissimilar energetically,their shared electrons will be located
closerto one atom than to the other, the distancebeing determinedby the
competitiveinfluenceexertedby the atoms upon the sharedelectrons.At
the same time, other electronswill be influencedby the bond, and, as a
result,their orbits will be alteredto someextent.Weakerelectrostaticforces
will result and the bond will be intermediarybetweenthe ionic and the
homopolar.Both kinds of fulfillment of quantum forces-one achievedby
transfer,the other by sharing-thus lead to the appearanceof new electrostatic forces in the ions or ionoids.
We must repeathere for emphasisthat the fulfillment of quantum forces
can take place through variousavenues,either by loss or gain of elcctrons,
or by sharingwhich can range from ionic to homopolar. The plurality of
possibilitiesfor fulfillment of quantum forces is very important, making
it necessaryto considerthe resultsof such fulfillment on a statisticalbasis.
The electrostaticforces act betweenchargedions of oppositesigns,or
betweenatoms bound by sharedelectrons.Through the balanccof thesc
electrostaticforces,bound atoms appearand correspondto ncutral formations, having their electrostaticforces fulfilled. However, it is only with
the intcrvcntionof suitablequantum forcesthat the bound atoms can form
a new entity, the molecule.
Quantum and Electrostatic Forces in Molecules
Alternate operation of electrostaticand quantum forces leads to the
organizationof atomsinto molecules.The quantum forcesin the molecules
interveneto permit organizationof theseentitiesso that the constituents
are maintainedat proper distancesand positions.The result is electrostatic neutrality.The appearanceof new quantum forces that maintain the
constituents,through their organizedmovement,at certain distancesand in
but also the stabilityof
certainpositions,insuresnot only the establishment
the new formations.Besidesvibrationalmovements,other more definite

B A S I C C O N C E P T SO F O R C A N I Z A T I O N

movementscan be recognizedin the new molecule.When two or more


atomsbecomeassociatedby sharcdelectronbonds,the sharedelectronsno
longer are confined to one atom but are displacedfrom their own orbits.
Under certain conditions,electronscan travel betweentwo or more atoms,
or even surround the moleculeas a whole. These movementswhich correspond to the interventionof quantum forcesgive stability to the molecule.
The fulfillment of intramolecularquantum forces will aflect molecules
in a similar way as fulfillment of atomic quantum forces aflectsatoms. By
a processsimilarto that governingmotion of electronsin atoms,motion of
entitiesthat enter into the structureof moleculesis also controlled.The
fulfillment of quantum forces is achievedin various ways. For example,
there may be localizationof the movementof electronsin the molecule.As
the result of relative immobilizationof theseelectrons.electrostaticforces
appear in the molecule as a whole.
The relativelyimmobilizedelectronscan be consideredas being related
to the moleculeas an entity, since they cannot definitely be attributed to
any of the constituentatoms. As a result,the moleculebecomeselectrostatically active.
The positionsof electronsand evenof atomsin moleculescan be understood easilyby consideringeventsat the molecularlevel in the sameway
we consideredthose at the atom level. The molecule whose electrostatic
forces are balancedis neutral. However, it has active quantum forces
which governthe positionand mobility of the constituents
and the relative
positionsof bound atomsor of certainelectronsin the entity. Fulfillment
of molecularquantum forces is realizedthrough changesin movement
of electronswhich lead to loss or gain of one or more electrons,protons,
ions or even groups of atoms. This leads to appearanceof electrostatic
forcesand the moleculebecomesan activeentity. In the molecule,as in
the atom, quantumforcescan be fulfilled in more ways than one-although
one may representa preferred situation. For this reason, activation of
molecules,through changesin mobility of molecularelectrons,has to be
consideredon a statisticalbasis.
The electrostaticcoulombiancharacterof an activatedmoleculeis the
resultof the changesin mobility of the electrons.Positiveor negativeareas
in the moleculedevelopaccordingto the abundanceor dearth of electrons,
at thesepositions.The new electronicarrangementsin a moleculecan be
seen as representinga preparatory step for the molecule to become an
activeentity in the same way that atoms are activatedand becomeions.
The molecule loses or gains one or more electrons,(or protons, ions or
groupsof atoms) and becomeselectrostatically
active,with positiveor nega-

REsEARcH rN pHysropATHoLocy

tive charge,dependingupon the nature of the lost or gained entity, and this
is the outcome of the fulfi.llmentof the molecularquantum forces.This is
illustrated by the following examples concerning the benzene molecule,
and the carboxyl and hydroxonium radicals,in which we shall limit ourselvesto changesproducedby quantum and electrostaticforces.
In the benzenemolecule,which is electrostaticallyneutral, the electrostatic positive and negativeforces of the constituentatoms are balanced,
However,all the electronsare not in fixed positions.The ,,' electronsof the
doublebondsmove around in the molecule.Becausethe moleculeis closed,
this movementis circular, thus accountingfor the stability of the molccule,
recognizedin part by the equal reactivityof all its carbon atoms which is
encounteredunder certain conditions and results in the Kekulian forms.
The fulfillment of the quantum forces accountsfor a kind of relative
fixation of these wandering,,,electronswhich is responsiblefor the other
structuresof the benzenemoleculedifferent from the Kekulian ones. It is
this localizationof electronswith the capacityto entcr into further reactions
which results in the activation of the molecule as seen in the resulting
Dewar structureswhich in turn accountsfor active centerssuch as ortho,
meta, and para positions.These excited molecules,electrostaticallyactive,
can readily take part in chemicalreactions.Study of the mobile zrelectrons
in many other moleculesallows us to undcrstandtheir role in providing
molecularstability, while their relative localizationfavors the appearancc
of electrostaticallyactive centersin the molecule and resulting reactivity.
Here again, localizationof electronsopens up many possible avenuesto
activation.
The carboxyl and hydroxonium ions represent typical examples of
another kind of activation. Inactive carboxyl occurs when the quantum
forcescausethe electronsto wander continouslybetweenthe two oxygens
of carboxyl.Becauseof this electroniccondition,the H atom seemsno
longer to be bound to either of the o atoms,but is situatedbetweenboth:
this form correspondsto the electrostaticatly
fulfilled condition. With fulfillment of quantum forces,the wanderingelectrontakes a more fixed position at one or the other oxygen.when this occurs.the H+ ion leavcsthe
carboxyl group, and the carboxyl acquiresa negativeelectrostaticequilibrium, leadingto further combiningactivity.This fulfillment of the quantum
forcesis responsiblenot only for the appearanceof an activatedgroup of
electrostaticcharacter,but also for the existenceof two structures.each
one with anotheractive oxygen.
A similar activation takes place when a molecule acquiresan ion, as
seen for the hydroxoniumion. Water can, under ccrtain circumstances,

BASrC CoNCEPTS OF oRcANTZATtON

bind a proton resultingfrom a hydrogenatom which has lost its electron.


This bond is achieved through a valency bridge, and can be regarded
as the fulfillrnent of molecularquantum forces.Different structurescan be
consideredas resultingfrom the fixation of the hydrogenbridge in different
positions in rclation to the tetrahedralconstitutionof thc oxygen atom.
They help to give this bridge bond its high resistance.
Bonding ol Molecules
Electrostaticforces in radicalsor activatedmoleculesmay be further
balancedwhen new bonds are realizedbetweenentitieswith oppositeelectrostatic forces. Bonding of moleculeshaving electrostaticallyexcited centers may or may not be of chemical nature which is considered to
correspondto changesin the structureof the molecules.More often, only
a physical bond between moleculestakes place, in which case there are
no changesin molecular structure.Both types of bonding result in fulfillment of the electrostaticforcesthrougha balancedneutralization,but bonding alone is not sufficientto establisha new entity. A new entity, with
structural and functional individuality, apparently is realized only when
quantum forces appear and establishdefinite relationshipsbetween the
bondedconstituents'
molecules,
placingthemin ccrtainpositionsand organizing their movements.The holistic concept emphasizesthe difterence
betweenmoleculesor radicalsbound only by the fulfillmentof their electrostatic forces of gencral coulombiancharacter,and the new entities resulting from the appearanceof specificquantum forces proper to them. Here
again,then, at the molecularas at the atomic level,progressin organization
is achievedby alternateoperationof electrostaticand quantum forces.
Polymolecular Formations ;
When an electrostatically
activemoleculeor radicalbinds an electron,
ion or even a small radical,the resultingentity is still considereda simple
molecule.The group resultingfrom the bonding of severalpolyatomic
radicalsis a complex molecule.Like simple molecules,complex molecules
also can group togetherand the bonding of sevcralleads to still more
complexformations,the macromolecules.
In turn, macromolecules
also can
be grouped and the bonding of two or more producespolymolecularformation.Thus, organizationprogresses
from simplemoleculesto polymolecular formations,first through the grouping togetherof similar entities.A
' A l l t h r e e t e r m s - m a c r o m o l e c u l e s ,p o l y m o l e c u l e s
and complex molecules-are
cbosenonly for didactic convenience.

l0

REsEARcH tN pHysropATHoLocy

new entity appearswhen one of thescgroups binds a respectivesecondary


part.
Micelles
A distinctivetype of new entity resultsfrom the bonding of molecular
formationswith simplerconstituents,such as ions or ionized molecules.To
this type of entity we have appliedthe name of "micelle." I polymolecules,
macromolecules,complex moleculesor even simple moleculescan form
the principal part of thesemicellar entities.
According to the above definition,micellesare entities formed by the
binding of molecules,as principal units, to ionized molecutesor ions, as
secondaryunits. The latter originally were consideredto be "impurities"
until Duclay showed their important role in establishingspecificentities.
According to our concept,micellesare producedwhen grouped molecules
and activeimpuritiesbecomebondedas the resultof reciprocalbalanceof
electrostaticforcesand the alternatcoperationof clcctrostaticand quantum
forces,seenfor atoms and molecules,appliesagain. Fulfillment of electrostaticforcesleadsto appearanceof quantum forces,this time proper to the
micelles.The quantum forcesmaintainthe micelleconstituentsin proper
positions and govern their movements,describedas vibratory for these
entities.The operation of the quantum forces, togetherwith fulfillment of
the electrostaticforces, accountsfor the stability of micelles.
The micellarquantumforcesalsocan be fulfilled, leadingto the appearance of unequal distributionsof micelle constituents.The micclle thcn
passesfrom relativelyneutral to an electrostaticallyactive form which can
enterinto further bonds,and it is primarilyin furtherbondagethat micelles
appear in a reticular aspect.
In an overall view of the developmentof organization,from atoms to
micelles,the relatively simple pattern of alternatingoperation o[ electrostaticand quantum forcescan be recognized.The regularityof the pattern
allowsus to considerit as fundamentalto the progressof organization.We
havc tried to go further and to recognizethe existenceof this samesimple
organizationalsystemfor formations below the atom and above the miceUes.We tcntativelyconceivedof subatomicformationsbeingorganizedin
the samemanner,i.e., by alternateoperationof electrostatic
and quantum
forces.We will not go into this study here as it would lead us too far from
r l t i s t o t h i s t y p e o f s t r u c t u r et h a t w e a p p l y t h e t e r m m i c e l l e , a s d i s t i n g u i s h e d
f r o m v a r i o u so t h e r m e a n i n g sf o u n d i n t h e l i t e r a t u r e .

BASIC CONCEPTS OF ORCANIZATION

l l

the subjectof this presentation.An outline of this subject is presentedin


Note l.
Organization ol Motion as Heterotropic Achievement
An interestingaspectof the concept presentedabove is the influence
of homotropic and heterotropicforcesupon the motion of particlcswithin
the organizationalframework. At-random mobility must be consideredto
be an attributeof entitiesfrce of any constraint,and thus corresponding
to
a homotropic state.Any changetoward constraint,leading to a degreeof
immobilization, must be considereda heterotropiceffect. The systematized
mobility producedby quantum forces,which apparsto prevent annihilation of opposingcharges,accountsfor the relative immobilization.While
mobility iself is an homotropicattribute,its systematization
is heterotropic.
The correlation of mobility with homotropy, and of fixation with
heterotropy, appears basic. The processof uniformization which corresponds to homotropy appearsto be possibleonly in the presenceof a
maximum of free mobility. The heterotropicsystematizationof movement
can be seenat variouslevelsof organization.Electronsin movementin the
atom differ from electronsin movementin the environmentthrough a systematizationof their mobility, as they are constrainedto follow definite
patterns. The relative fixation of certain electrons-for instance,shared
electrons-following the fulfillment of quantum forcesalso marks a further
heterotropicinfluence.This also appliesto radicalssuch as the carboxyl.
In the formation of - C =
..----o
: 9 the movemento[ electrons,in itself, is
homotropic,while the limitation of movementbetweenthe two oxygensis
an heterotropiceffect.With the electronfixed in one position,bound to only
one oxygen,a further step in immobilizationis achievedand representsa
heterotropicfactor. In more complex molecules,such as unsaturatedfatty
acids, for example,the tendencyof the electronsto wander is related to
homotropy, while their restrictionto the moleculeor even to certain areas
of the molecule representsan heterotropic cffect. This also applies to
micelles,where the water moleculesand impuritieshave a certain degree
of mobility. This mobility must be consideredto be a vestigeof the movement of free water molecules,with a high mobility consideredas a homopolar effectat this level.The retentionof water or other moleculesin the
micelle may be consideredas a heterotropiceffect.And their further fixation, as in the activatedmicelle,is a further heterotropiceffect.
Organization,which resultsfrom the alternateoperationof electrostatic

12

xEsEARcH rN pHysropATHoLocy

coulombianhomotropic and organizationalheterotropic tendencies,leads


to the realization not merely of stableconfigurationsbut, more significantly,
to entitiescapableof reactivity,and consequentlyable to respond actively
to the various changesof the environment.The fact that fulfillment of
quantum forces causesappearanceof new electrostaticforces, which will
be further neutralized,leads to the progressof organizationto ever-higher
levels.
At each step of organization,however, another characteristiccan be
recognized.It results,in part, from the fact that progresstoward higher
entitiesis accomplishedby an increasein complexity rather than only in
size.The increasein the positivecharge of nuclei in atoms, for instance,
bringsabout a parallelincreasein the number of surroundingelectrons,but
this goes on only up to a certain point. Actually, the size of the atoms is
limited by the size of the nucleuswhich, in turn, is limited by the quantum
forcesable to insurestabilityfor the nucleus.Nuclei becomeunstablewhen
they contain too many protons.
Levels, Entities and Constituent Parts
As alreadynoted, we definedan entity through its structural and functional individuality. we used the term "level" to indicate a conceptual
groupingof entitieshavingthe samebasicconstitution,suchas, respectively,
nuclei, atoms, molecules,micelles,etc.
We have used the term "part" to define an entity when it contributes
to the formation of anotherentity. Nuclei and electronsare parts that form
an atom. Molecules are parts when they are bound through electrostatic
and quantum forces to form micellar entities.In progressiveorganization.
eachnew entity thus is composedof partswhich are entitiesfrom the level
immediatelybelow, and the new entity itself servesas a part for the immediatelysuperiorentity. we call this relationship"hierarchic," one entity
being inferior to that which it forms and superior to those which have
formedit. So conceived,
eachnew organizational
entitycan be identifiednot
only through the nature of the parts forming it and the manner in which
they are bound, but also through its level in the hierarchic succession.
we have seenthat most of the entitiesare made up of dissimilarparts.
Analysis of what happenswhen an entity is formed has shown that the
processis complex.In order for an entity to act as a part in a higher level,
it must first pass through an activatedstage.Activation opens up many
opportunities,a plurality of possibleformations.So does another process.
an immediate consequenceof activation. Almost continuously, sevcral
5imilar entitiesare seento join togetherin a kind of "common grouping,"

B^SIC CONCEPTS OF ORGANIZ^TION

13

adding further to the multiple possibilitiesof new entities.The multiplicity


of possiblities
at eachstcpin organization
explainsthe exponentialincrease
in the number and complexityof the entities,resultingfrom the hierarchic
pattern of their formation.
According to the holistic approach,an entity exists only through its
own qualities.It must have characteristics
other than thoseof its constituents. It is the relationshipbetweenthe constituents,in the new entity,
largely resultingfrom the operation of quantum forces, that characterize
the entity.
Principal and Secondary Parts
As noted, cntities at progressivelevels of organizationare formed of
dissimilarparts.Theseparts do not haveequallyimportantroles.There is
a "principal" part which is characteristic
for a given level.l'here are "secondary" parts that are nonspecific
for the level,the sameonescan serveat
ditlerentlevels.The secondarypart for a hierarchicentity often is an entity
of a far lower level. (Fig. 1)

PrincipalParts
First [ntities

Secondany
Parts
o f t h e S e c o nEdn t i t y

S e c o nEdn t i t t e s
T h i r dE n t i t i e s
F o u r t hE n t i t y

f t h e T h i r dE n t i t y
f t h e F o u r t hE n i i t y

Ftc. | . The hierarchic organization. In the organization in general, the different


e n t i t i e s a p p e a r i n t e r r e l a t e da c c o r d i n gt o a c h a r a c t e r i s t i ch i e r a r c h i cp a t t e r n . E a c h e n tity is formed by a principal and a secondarypart. An entity is hierarchically "superior" to the cntities which form its principal part and "inferior" to rhose in the
formation of which principal part it enters.

Secondaryparts have important characteristicsin common. These become especiallyevident when lower level entitiesare examined.At levels
below the atom, secondarypartsfor all entitiesare electrons.It is of interest
to observethat in higher entitiesas well, such as moleculesor micelles,all
secondary parts have a negativeelectrical charge.

L4

xESEARcHIN pHyslopATHoLocy

Also characteristicof secondaryparts is their derivation directly from


the environmentin which the entitiesof which they are components,appear.
This is clearfor many crystals,wherewater moleculesrepresentthe added
secondarypart. This water of crystallizationis not free betweenthe ions
but bound to them. Water apparsindispensable
to crystal formation, since
its loss resultsin disintegration.Water can be consideredto play a secondary part. Similarly, in some crystalssuch as gold, some electronswander
betweenthe atoms while others are concentratedin certain regions. (Brillouin) These electronsrepresenta secondarypart in these crystalsas do
water moleculesin others. Water and electronscan be related to the environment from which they derive.
Derivation of the secondarypart from the environmentappearsto be
even clearerat the micellarlevel.In the caseof the gelatinousprecipitate
obtainedby coagulatinga couoidal solution,a part of the environmentin
which the gel is precipitatedentersinto the formation of the new micelle.
For example,the micelle of colloidal copper ferrocyanatecontainspotassium ferrocyanateas a secondarypart for each principal part of copper
ferrocyanate.Micelles of fcrric hydrate, obtained by the hydrolysis of a
boiling iron perchloride solution, provide another example. Besides the
Fe2o3, moleculesfrom the Fe6Clusolution used in the preparation enter
into the formation of this hydrosol.The role of the negativelychargedconstituentsbecomesapparentwhen a part of the cationof this secondarypart
is removedfrom the intermicellarfluid and the hydrosol still persists.It is
only whcn the chloridecontentbecomestoo low that coagulationresults.
The moleculesof potassiumferrocyanateor iron perchloride,once consideredto be impurities,must be looked upon as secondaryparts of these
micelleentities,Dcrived from the environment,they enter into the formation of the micelles,especially
throughtheir negativeelectrostatic
character.
I'his concept becomeso[ even greater importance when entities are
considcredin relation to the constantlychangingenvironment.The electrostaticbalancebetweenentity and environmentrealizedat any given time
cannot be consideredto be permanentbecauseof the changeswhich occur
in the environment as it travels toward ultimate total homotropy. As a
working hypothesis,
it can be assumedthat the relationshipbetweenentity
and environmentwould changeas the lattermovestowardtotal homotropy.
It can be assumed,too, that as hierarchicorganizationdevclopsin time,
secondaryparts from the environmentwould diffe.rfor differententities.
Changesin environmentwould providcevidencethat thcsesecondaryparts
are relatedmore closelyto the environmentas it existedin the past when

BASTC CONCEpTS OF ORCANTZATTON

15

these entities are assumedto have appeared.we will see below how important this is for the more complexentitiesof higher levels.
In the role played by secondaryparts in progressiveorganization,the
changesin their mobility are of specialsignificance.we have already seen
that in hierarchicentitiesthe secondaryparts are simpler units than the
principalparts,a factor which facilitatestheir mobility. The mobility can
be related to the fact that these secondaryparts are derived from the
environmentwhere they arc mobile, with their motion not systematized.
The interventionof quantumand quantum-likeforces,which help to create
new entities,can be seenas a kind of organizationof the relativemobility
of the secondaryparts. It is the systematization
of their movementwhich
preventscompleteannihilationof electrostaticforces present.The relationship between secondaryparts and environmentthus explains the
characterof the mobility encounteredthroughouthierarchicorganization.
It must be emphasizedhere that, becauseof the electrostaticnature of
the bond betrveenprincipal part and secondaryparts, a principal part is
capableof entering into the formation of more than one specifictype of
entity. similarly, fulfillmentof quantumforcescan lead to more than one
typc of structure,However, of the many possiblenew entities,or structures,only a few will fulfill the requirements
for developinga still higher
organization,i.e. will be capablcof acting as principalpart in a new cntity. some rcmain at their original level without progressing,
even after
being bound to other entities.Even many of thosewhich have somecapability for higher organizationcan go only one or two steps.Only a very
few will continueall the way up. [n other terms,only a few will be able
to utilizc new quantumforcesin order to realizencw entities.While various bondsand structuresoffer a largevarietyof possiblenew entities,it is
thc entity with a capacityto adcquatelyresistthe cffectsof the changing
environmentwhich will take part in progressiveorganization.
In considering the forces which intervene in progressivehierarchic
organization,one has to considerthe free cnergyavailablein the environment. The immenseamount of energyreceivedfrom the sun representsa
typc of heterotropicenergy which can intervenein organization.We will
see that this is easilyrecognizedfor higherentities.The ability of certain
entitiesto develop may be relatedto their peculiarability to utilize heterotropic forces, most of them of solar origin. The lesssuccessfuldisappearor
remain at lower levels.

16

xESEARcHIN PHYSIoPATHoLocY

The Organized Boundary


We have seen that an entity achievedthrough systematizationof the
movementsof its componentsacquiresa boundary betweenitself and the
environmentas a result of this restrictedmovement.The boundary does
much more than delimit the entity and constitutea barrier betweenit and
the environment.The electronsof the outermostshell form the boundary
of the atom, for example.It is through them that the atom realizesits relationship with the environment.Chemicalreactionis largely limited to this
boundary. In the atom, where the nucleusis the principal part, and the
electronsare the secondarypart, it is evidentthat it is the organizedmovement of the electronsthat providesthe boundary.The form and organization of electronicshells,and specificallyof the boundary shell of an atom,
are resultsof quantum forces.The environmentalnature of the secondary
parts and their buffering role make them of great importancein complex
boundary formation. Theoretically,hierarchicprogressmay be considered
to dependupon the developmentof secondaryparts which allow increasingly complex boundary formations. This explains the importance we
attachto the study of boundaryformation in higher entities.
To summarizethe above conceptof organization,the different entities
can be integratedinto a hierarchicorganizationalpattern which depends
upon alternate operation of the two fundamentalforccs, electrostaticof
coulombiannature, and quantum of organizationalnature. Entities can be
identifiedby the nature of the inferior entitiesthat act as constituentparts
and the relationshipbetweenconstituentsas principal and secondaryparts.
While the principal part is formed by an hierarchicallydevelopedentity,
the secondarypart is a secondentity from the environment.The incorporation of a part of the environmentinto a new entity correspondsto a systematizationof its motion. And it is through the organizedmotion that
appearsa boundary formation which marks the realizationof a new hierarchic entity.
This concept of organizationhas made it possibleto understandthe
relationshipof the seriesof entitiesthat composethe biological realm.

CHAPTER

BIOLOCICAL ENTITIES

\xr

VVrt" rHE coNcEpr of hierarchicorganization,it becomespossibleto


gain a new insight into, and understandingof, the biologicalrealm.
In the classicalview, just as simple substancesin nature are conceived
of as being formed by moleculesand atoms, biologicaUycomplex organisms are consideredto be composedof cells as fundamentalentities. In
arriving at complex organisms,however, it is granted that organization
has followed a definitepattern.At first glance,it is apparentthat cells are
grouped in morphologicallyregular ways to form tissues.Similarly, tissues
are groupd to form organs and thesein turn composethe organism.In
this classicalsystematization,
a complex individual would appear to be the
result of a grouping of cells, tissuesand organs,bound together in what
has been describedas an harmoniousmorphologicalrelationship.
In our study of organizationof the biologicalrealm, we have emphasized the individualization of both conceptualand material entities. In
some cases,entitieshave been simple to identify becausethey are easily
separablemorphologically.Where morphologicalseparationhas not been
immediately evident, other criteria-such as structural and functional
properties-have been used for identification.Besidesplaying its part in
organization,each entity has its own individuality,and consequentlycan be
recognizedholisticallyas a well-definedunity. Startingwith chromomeres,
the entity status is easily acceptedbecause,in addition to clear morphological and functional properties,there is a degree of independentindividuality. Following up, chromonemata,chromosomes,nuclei are other
entities.
The study of the organizationof biological entities has shown, however, that in all casesthere is a specificpatternof interrelationshipwhich is
more complex than the classicallyacceptedpattern. In the simplestmicrot1

18

R E S E A R c Hr N p H y s I o p A T H o L o c y

scopicallyidentifiedentities,it could be seenthat a seriesof chromomeres


are bound together through a specialfibrillar formation, which stainsdifferently from the chromomeres,
to producethe chromonemata(I,2), which
can be consideredholisticallyas a new entity. Two or four chromonematas
( 3 ), togetherwith the chromosomalsap,form the chromosomeas a new entiry. Simi.larly,severalchromosomestogetherwith another part-this time
representedby the nuclear sap and the proper nuclear membrane-form a
nucleus.In turn, the nucleus,plus protoplasmicformations,cytoplasmand
the cellular membrane,form the ccll. Even superficialanalysisindicatesa
common fundamentalpattern in the organizationalchangestaking place
for entities ranging from chromomeresto cells. Becauseof this pattern,
these entitics can be consideredto be "hierarchically" interrelated.One
entity is hierarchically"superior" to the entitieswhich form it and "inferior" to those it will itself help form. Two immediatelyinterrelatedentities thus are hierarchicallysuperior and inferior, respectively,just as in
organizationin other realms.(FiS. 1)
Analysisof the progressionof organizationfrom simple to more complex entitiespermitsus to recognizeother characteristics
of the fundamental
pattern.In order to form a hierarchicallysuperiorentity, severalsimilar entities first join to form a group. It is the group which then will bind
other constituentsto bring into cxistencea new, hierarchicallysuperior,biologicalentity. Thus, the chromomeresas a group join with a fibrillar formation to producechromonemata;
join with chromosomalsap
chromonemata
to form chromosomes.Groups of chromosomesplus nuclear sap form the
nuclei.It has appearedevidentthat the partswhich are bound to form each
hierarchicentity do not play equal roles. In each case, the principal part
is the one which is composedof similar entitiesacting as a group; the
other part is the secondary.Figure 2 offers a graphic representationof
hierarchicorganizationfrom chromomeresto cells.
In all entities,mentionedabove,thereis the samerelationshipbetween
principal and secondaryparts. The secondarysurroundsthe principal part.
The moqphological
relationshiphas helpedus to apply to thoseentitiesthe
hypothesisdiscussedpreviouslyconcerningthe mechanismthrough which
hierarchicprogressionhas taken placein nature.According to the hypothesis, severalsimilar entitieswould first associateand form a group. In a
secondstep, the goup would tend to maintain around it a small portion
of its immediateenvironment.From this portion o[ the environmentrvould
come the secondarypart for the next superior hierarchic entity. With a
boundary formation, separatingthis minute part from the rest of the environment,the new entity would be established.Such a processcould oc-

BroLocrcAL ENTtTIES

19

cur, although rarely, with a singlebiological entity serving as principal part.


Usually, severalentities grouped togetherwould be needed.This pattern
explainswhy the secondarypart can be conceivedof as a part of the environment retained around the principal part, and rvhy the establishmentof
a new and higher entity can be consideredto occur only when this secondary part is detachedfrom the rest of the environment and separated
from it through the interventionof a boundary formation,

Entities.

AddedSecondary

Format
ions
Chromomeres
Chromonemata

Fibrillar material
Chromosomial
sap

Chromosomes

lfucleus
CelI

Nuclearsap
Cytoplasma

Ftc.2. The hierarchic relationship in the organizationof morphological entities below


c e l l s . F o r e a c h e n t i t y i t s p r i n c i p a l p a r t i s r e c o g n i z e da s b e i n g m a d e b y a g r o u p i n g o f
entities hierarchically inferior to it. The secondarypart which correspondsto a kind
of environment for the principal part usually surrounds the principal part.

Having recognizedthis pattern of organizationfor lower entities, we


lr'ent on to determinewhetherit remainsthe same for higher entities.It
could be seenthat groups of cells,along with interstitialformationsand
fluids around them servingas secondarypart, createthe tissueas a new
hierarchicallysuperiorentity. Indeed,a proper boundaryformation morphologicallylimits and conceptuallydefinesthe new entity. The interstitial
fluids are separatedby a continuousendotheliumlimiting the lymphatic
spaccsas a systemclosedtoward the intercellularspaces.Under thesecircumstances,the lymphaticendotheliumservesas the correspondingboundary formation that limits the tissueentity. Severaltissuesgrouped together,
playing the role of the principal part, bind the lymph, as secondarypart,
to form the organ, as a new hierarchic entity. Lymphatic vesselsand
connective tissues represent the organ's boundary formations. Further-

; : : i '
. r ' :
I ]!a:; trr:4trr:itl'riitir{)

Nl

20

RESEARcH rN pHysropATHoLocy

more, organs grouped together, with blood as secondary part, form the
entity called the organism.(Fig. 3,)
Does the samepattern apply for entitieshierarchicallyinferior to chromomeres?For these lower entities, morphologicalinformation to define
the relationshipbetweenprincipal and secondaryparts is unavailablefor

Entitv

Added
Secondary
Formations

_ _ I n t e r s t i t i a lf o r m a t i o n s
Tissue--Lymphat
ic fornations
0rgans

0rganism

- -8lood f ormat
ions

Ftc. 3. The oganization abot'e the cells. The hierarchic pattern of the organization
in general is recognized also in the organization above the cells. Tissues, organs.
organism are seento representhierarchic entities,each one being made by a principal
p a r t f o r m e d b y t h e g r o u p i n go f e n t i t i e sh i e r a r c h i c a l l yi m m e d i a t e l yi n f e r i o r t o i t . T h e
principal part is bound to a secondarypart characteristicfor each entity. This secondary part would correspond to the proper environment in which the entities forming the principal part, have evolved.

the moment. Until electron microscopyand other means provide such


data,wc are obligcdto find other criteriato indicatewhich, in thesehierarchic entities,is principal and which secondarypart. We have considered
electricalcharacteristics
of the constituents
as criteria for identifyingtheir
role in the hierarchicorganizationof biologicalentitiesbelow chromomeres.
Biological Realm
Before going further in trying to analyze submorphologichierarchic
entities,one problem was to establishthe limits of the biological realm

BroLocrcAL ENTITtES

2l

itself. The questionwas,how far below the morphologicalformationscould


we go and still have entitieswhich can be regardedas biological.With the
progressof science,criteria previouslyusedto definelife have becomeoutmoded. A separationline betweenthe animate and inanimate no longer
cain be drawn. with the study of the propertiesof viruses, it appeared
impossibleto maintain the last vestigeof the old vitalisticconcept.According to most of the classicalcriteria, viruseswould representanimate entities sincethey are able to multiply and conservea strict identity. However,
they also form salts,are crystallized,broken down, and then reconstructed
in the same or another order. If the virusesare acceptedas ,,borderline"
entities,as proposed,then the concept of animate and inanimatecan no
longer be sustained.
Although the animateand inanimatecannotbe distinguishedin terms of
a specificproperty, we cannot totally overlook the fact that an important
group of entitiesappear quite different from others in nature. As animals
and plants are difterent from stones,even without any vitalistic concept,
we are obliged not only to recognizethe difference,but also to try to
establishwhere the differencelies. To conform to reality, we have applied
the term "biological" to a group of entities,but we have given the term a
new meaning.
Just as organic chemistryis considered-whether correctly or not-to
comprisecertain combinationsof carbon,we considerthe biological realm
to compriseentitieshierarchicallydevelopedfrom a specificchemicalradical. This basic radical includesnitrogen and carbon atoms bound together
to form the N-c-N-c group. According to this concept,starting from the
basic nitrogen-carbonformation, an entire seriesof entities has been developed through hierarchicorganization.Togetherthey form the realm to
which the term "biological" can be applied.
The N-c-N-c group, through combinationwith hydrogen,would result
in radicals with a strong alkaline property; that is, with strong positive
electricalcharacter.Someof theseN-C-N-C groupstake part in the formation of nitrogenousbases,pyrimidinesand purines,while some,by acting
as principalpart and bindingvariousamino acid radicalswould, with the
necessary
electrons,
build up a new group of entities,arginineand histidine
as alkalineamino acids.Thesecan be considered,
hierarchically,
to be the
immediatesuperiorsof the N{-N-C goup and thus to representthe first
biological molecules.The alkaline positivecharacterof these moleculesis
noteworthy.Following the hierarchicpatterndescribedabove,severalsuch
alkalineamino acidslinked togetherto a seriesof entitiesof the samelevel
(simple amino acids) will form new groupsthat are still electricallypositive:

22

nESEARcHrN PHYSToPATHoLocY

will act as principalpart


In a newstep,thesehistones
thealkalinehistones.
(Fie.a)
to produce
nuclearentities.
parts,can form not one
Principalparts,by bindingdifferentsecondary
but manynew and differententities.More than one hierarchicline can be
identified.An especially
importantline resultswhenhistonesbind one or
more entitiesof the nucleicacid group,to form nucleo-proteins.
Other
histonescan bind variousother secondary
partssuchas carbohydrates
or
lipids and, in so doing,form differentbiologicalentities.Someof these

Princtpal

Added
Secondarv
Format
ions

il-C-l{-CqrouD'

Alkaline-ami!o

Aminoacid group

Histones

aminoacids

Deoxycibo-, \
nutleoproteins

Deoxyribonucleic
acld

Genes

Nucleooroteins
and
o t h e ls^e c .e n t i t i e s

Ftc. 4. The organizalion ol the submorphological entities of the biological realm.


T h e s a m eh i e r a r c h i cg e n e r a lp a t t e r n .w i t h p r i n c i p a la n d s e c o n d a r yp a r t s i s r e c o g n i z e d .
S t a r t i n gw i t h t h e p o s i t i v cC N C N g r o u p , e a c h e n t i t y h a s i t s p r i n c i p a l p a r t m a d e b y t h e
grouping of immediately inferior entities bound to secondary parts, generally more
electronegative.

entitiescan continue their hierarchicdevelopment.Nucleo-proteinscan


have riboseor deoxyriboseand consequcntly
form more complexnucleoproteins.Through all hierarchicachievement,
it can be secnthat new lines
are formedwhen groupsof hierarchically
lower entities,actingas principal
organizedpart, bind diflerent secondaryparts, all more negativcthan the
principalpart. Theoretically,this would rcsult in a sericsof entitiesall at
the samelevel,i.e. entitieswith similar principalparts but havingdifferent
sccondaryparts.Somewould go on to developsuperiorhierarchicentities,
otherswould evolve no further.
Differententitiesof the the samelevelcan be groupedtogetherin various ways to form a variety of principalparts. Always the group must be
madeup of entitiesof the samelevel.This requirementhas been seento
can exist betweenthe
be generalat all hierarchiclevels.Sincedifferences

B t o L o c r c A L E N T T T T E S/

23

constituents
forming the principal part for a new entity, the predominance
of one or another constituentwill make entities at the same level differ.
This mechanismof differentiationthrough the constitutionof the principal
part has been extremelyimportant throughoutthe biological realm.
Nucleolus
The concept that plural groupingscan enter into the principal part of
the nucleusputs the role of the nucleolusin a new light. It was accepted
for a long time that the nucleolus representsonly the reserve material
necessaryfor metabolism of the nucleus. The strong positive electrical
characterof the nucleus,as recognizedthrough its rather alkaline reaction,
would give it roles more important than that of the other constituents.
According to a work hypothesiswhich we advance,successivenuclcolar
formationswould representthe principal parts of hierarchic organization
below the nucleuslevel. In chromonemata,chromosomeand nucleus,the
partscorrespondingto the nucleoluscan be recognized.These formations
are grouped together with genesto form the principal part of the chromonemata.Similarly, chromatineformationsrepresentingentitiesof the
samelevel as the respectivenucleolar formations,will form togcther the
groupscharacterizingthe principal part of chromosomes.In the nucleus,
the nucleolusis joining the other formationsto form is principal part.
Protop lasmatic F ormations
In the cell, a hierarchicallysuperiorentity, a similar condition also
appearsto persist.The protoplasmaticformationswith ribo-nucleicacid
can be conceivedas representingentitiesof a nuclearlevel, that is, a level
similar to that of the nucleus.Togetherwith the nucleusthey would form
the group correspondingto the cell. This kind of evolution of entities in
relatively separateparallel lines, with their further grouping together to
form principal parts for new entities,is part of the typical pattern of
organizationespeciallyevidentin the biologicalrealm.
Boundary Formalions
We have mentionedthat groupingsof severalentitieswould not be
sufficientto form a new entity so long as the secondaryenvironmentalpart
is not isolatedfrom the medium from which it originates.Consequently,
the new entity appearsonly when a distinctboundaryformation is formed.
Progressivehierarchic developmentis dependentupon the appearanceof
suchboundary formations.For the first biologicalentities,the radicals,the
boundary semsto be more an energeticproperty than a morphologically

24

nESEARcH rN PHYStoPATHoLooY

organizedformation. For the molecules,it can be consideredto consist of


molecular surface forces, recognizedas the van der Waals cohesionforces.
A similar but more apparent boundary formation can be found in higher
molecularcomplexes,especiallythe micelles.The molecular arrangementat
the surface of micelles separatesthem from their environment and consequently insurestheir identity. In the caseof morphologicallyidentifiableentities,of course,boundary formations can be easilyrecognized.Chromomeres
are welldefined and separatedfrom the chromosomal sap. The chromosomes,in turn, show a real membranejust as the nucleusand the ceil do.
The next higber entity, the tissue, is bounded by the endothelial cellular
layer, separatingthe interstitial formations from the lymphatic spaces.Usually the boundary of organs which have tissuesas principzrl and lymph as
secondaryparts is representedby organizedblood vessels.As far as the
organism is concerned,the mucous membranesand skin are boundary
formations.(Fig.5)
H i erarchi c I nterrelationship
Viewed as a heterotropiceffect, hierarchic organizationcan be consideredto be a method of conservingexistingentitiesas such, in spite of
changesoccurringin the environment.TeleologicaUyspeaking,by entering
into the formation of a new and superior entity tfuough the system of
hierarchicorganization,each entity, in fact, protectsis own individuality.
The hierarchic organizationmakes it possiblefor each entity to continue
to live in a medium which correspondsto its own environment.The constituentsof the secondarypart in the new entity are chosento correspond
to the environmentin which the principal part of the entity has existed.
The successive
secondaryparts, added during the hierarchicdevelopment,
act as multiple protectivebuffers for the first entities,thus insuring their
unaltcredconservationin spite of continuouschangesin the environment
brought about by increasinghomotropy.
PhylogeneticDevelopment
Hierarchic organization,when relatedto time, would appear to correspond to evolution. The conceptof ontogenesisreproducingphylogenesis,
appearsin a new light when analyzedin accordancewith hierarchic organization. The parallelism between actual hierarchic organization and
hierarchicphylogeneticand ontogeneticdevelopmentgreatly helps to increaseunderstandingof many principal problemsof biology.
In accordancewith the conceptof hierarchicorganization,when a new
level is realizedthrough the binding of entitiesfrom a lower level, as prin-

BroLocrcALENTrrrEs

25

cipal part, to differentsecondaryparts, severaloutcomesare possible.


Someof the new entitiesare unableto continueto existand disappear.
Of
the others,somearerelatively
wellbalanced
entitiesandconsequently
can

Enti ty

ChromomeresChromonemala/-Chromosomes.Nucleus- -

formation
Boundary

boundary
Chromomere
boundary
Chromonemata
membranE
Chromosomial
-- Nuclearmembrane
-.Cellularmembrane

- Lymphat
ium
ic endothel

C eIIs

Tissues-J-

0roans
- F-- I
\

B l o o dv e s s e l s
S k i na n dn u c o u s
menbranes

\
\
\

0rganism

Fto. J. The boundary lormation in the hierarchicorganizationof the biological realnr.


A proper boundory lormation delimits each entity, insuring lhus its individuality. It
is by separatingcach time the secondarypart from the rest of the environment that
the boundary formations have pcrmitted the progressivedevelopment of the hierarchic organization. The boundary formation governs the rclationship bctwecn the
entity and its environment.

persist.But with further changesin the environment,some of theseentities


disappear.Even among those that remain, some do not representfully
satisfactorysolutions and further hierarchic developmentis necessaryto
insure tbeir persistence.They must evolve further to correspondto the

26

REsEARcH rN pHysropATHoLocy

new unfavorableexternal conditions.This has led to the developmentof


the complex entities presenttoday in nature.
The multiple entity possibilitiesat each level have resultedin a wide
variety not only of entities which, with further evolution, could produce
higher complexes,but also of others which have ended their evolution at
Iower levels. So the existenceof many varied entities apDearsto be the
result of the existenceof multiple solutionsfor the same problems which
have been part of the mechanismof hierarchicevolution.
From this viewpoint,existingindependententitiescan be recognizedas
correspondingto the different levels of hierarchic organization.Viruses
can be consideredto be at the same level as genesor even the entities
immediatelybelow genes; the microbes at the same level as the nuclei:
monocellularorganismsat the level of cells, etc. Furthermore,in each
independentorganism, from simple to most complicated,the same progressionin organizationof successive
hierarchicentitiescan be seen,starting with the simplest biological entities and continuing until the level at
which the entity has actually stoppedits hierarchicevolution.
In a further step, having arrived at thc concept that the secondary
part of each entity correspondsto the kind of environmentin which the
entity found itself at the time of its phylogeneticappearance,we tried to
seewhat information about theseenvironmentscould be obtainedthrough
study of the secondaryparts.
The first biological entities,the alkaline amino acids, could have appearedin an atmosphererich in ammonia,water and methane.Experimentally,electricaldischargesthrough mixtures of these materialshave
induced the appearanceof amino acids.It appearsplausiblethat such an
environmentcould have existedaroundvolcanoesin times when the earth's
atmospherewas formed by ammonia. with steam formed by the heat of
the volcano, with methaneresultingfrom the interactionsof erupted metallic hydrocarbonswith watcr, and with lightning so frequent around
volcanoesin eruption, the necessaryconditionsfor synthesisof amino acids
may have been present.The simple amino acid moleculeswhich would
haveresultedcould haveconstituteda group neededfor hierarchicdevelopment. Out of a seriesof such amino acids,some could have bound the
group N-c-N-c which also could have been synthesizedunder the influenceexertedby radioactiveelementsor radiation.(Note I )
Additional information concerningthe constituentsof secondaryparts
which enter into the developmentof all the subnuclearentitiesis mcager.
Simple amino acids or urea are presentin the chromosomialand nuclear
sap which are practicallyfree from K and Na. We could thus consider

BfoLoorcAL ENTtTTES /

27

arumonium as the predominantcation for all hierarchicentities up to the


nucleus. This would accord with geologicaldata concerning a primitive
atmospherein which ammonia was predominantat the time when the first
biological entitieswould have appeared.we can then, tentatively,in view
of this cation common to all, classify the hierarchic entities below the
nucleusin what we will call "the nuclearcompartment."
In the same way, we analyzedthe compositionof cytoplasm,with the
thoughtthat it could provide informationabout the constitutionof a second
environmentin the evolution of biologicalentities.The principal cation of
cytoplasm is potassiumwhich also representsthe principal cation of the
earth'scrust. Curiously enough,potassiumand the other constituentscould
oe found in the same relative proportions in the earth's crust as in cytoplasm, (Note 2) a f.actwhich seemsto confirm the hypothesisthat mud,
humid earthcrust,represented
the environmentin which entitiesat nuclear
levels lived. The cytoplasmconservedthis constitution,with potassiumas
principal cation, when it was separatedfrom the environmentto become
the secondarypart which, with the nucleus,formed the cell as the next
superiorhierarchicentity. The cell by itself, representsa new compartment
with potassiumas principal cation.
It is only for the animal cell that the environment seems to have
changedagain.This time the new environmentwas the sea.when several
cells joined togetherto organizetissueas a new entity, they had to maintain their environment,now representedby the sea.The hierarchicentities
above the animal cell show sodium as principal cation in their secondary
parts, thus indicating that when they were organized the sea was their
environment.This characteristic
allows us to group togetheranimal hierarchic entitiesabove cells to form a new compartment,the metazoic,with
sodium as the principal cation.
With passagefrom marine to terrestriallife, air is found as the new
environment.Not integratedas a new secondarypart, without a separating
boundary formation, the part of the environmentkept in the respiratory
apparatusdoesnot enter however,into the formation of a new entity. Only
the presenceof air in the bonesof birds can be seenas such integration.
A certain fundamentalfurther development,in the same direction can
be seenin animalsas well as in humans,in the areaof sociallife. (Note 3)
Divisionof complexhierarchicorganization
into compartmentsappears
to be relativelysimple. There are changesin the principal cation from
compartmentto compartmentwhich correspondto similar fundamental
changesin the environmentthrough which the organismpassedduring its
phylogeneticevolutiol-f1e61 volcano to mud, to sea, to the surface of

28

REsEARcH rN pHysropATHoLocy

the earth. We tried similarly, to correlateother elementsin the periodic


chart with hierarchiccompartments.The resultswill be presentedin detail
later. For the moment, it can be stated that elementscorrelated to different environments are also found in the difterent compartments correspondingto these environments.For example, Mg like Na representan
elementof the metazoiccompartmentand of the sea where this compartment was phylogenetically
organized.Fe, Cr, Ni, Zn, Ca, Mn, Co, As, and
Se which fall into the cellular compartment, represent characteristic constituentsof the earth'scrust.
Constants
The conceptthat matterin generalis the expression
of the heterotropic
trend permits us to explain further some of its characteristicswhich have
been of great importance in biological development.Heterotropy can be
seenworking to maintain existingentitiesas long as possible,to conserve
their characteristicproperties in spite of changesin the environment. Heterotropy could result in unchangedvalues which would appear as constants of an entity and would indeed identify the entity. It is hierarchic
organizationwhich would tend to permit conservationof constants.
Each biological entity-just as any entity in nature----canultirnately
be defined by the series of characteristicproperties which it is able to
conserve.Constancyis the criterion which permits us to judge the importance of a property to an entity. The longer a property is kept constant
despiteenvironmentalchanges,the greaterits fundamentalimportanceto
the entity. The progressiveaddition of secondaryparts through hierarchic
organizationrepresentsan effectivemeans of preservingconstantsof the
lower entities. New properties, added with the formation of each new
hierarchicentity, representednew constants.This explainswhy considering
the constants,it can be seen that those incorporatedin the lower entities
are the best conserved.The higher a constantin the organizationalhierarchy,the lesswell-preserved
it will be.
The idea that constantscorrespondto the character of the environments through which the individual has phylogeneticallypassed,and that
they are conservedthrough hierarchicorganization,permits us to try to
extend our understandingof conditionspresent in past environments.
Cationsand even anions would representonly one type (apparentlythe
most important) of the constantsmaintainedthrough hierarchicalorganization. Other constants,correctly interpreted,would indicate in what
direction we must search for conditions which prevailed in the environ-

BroLocrcAL

ENTtTTES

29

mentwhen the respectivehierarchicentitiesthat make up a given organismwere establishedduring phylogeneticdevelopment.


As examples,let us considerthe conservationof salhity and temperatureas constants.Values for salinity of the metazoiccompartmentand of
theblood and valuesfor temperaturehavc been seento be constantscharacterizingspecies.Differencesbetweentheseconstantsin different species
showa succession
conforming with paleontologicaldata. In the interpretation given by Rene Quinton, constantswould indicate the times when
various speciesoriginally appearedin nature.
According to our concept of hierarchicorganization,these constants
may be interpretedotherwise.They would not indicate the moment when
the lowest entities of the respectivespecieswere formed, but rather the
time in the developmentof thesespecieswhen the hierarchicentitiescapable of conservingthe respectiveconstantsappeared.In other words, they
would not indicate the earliestmomentsof appearanceof the first entities
which later developedto form the respectivespecies,but would indicate
a relatively Iate moment in the creation of the metazoicentity which has
appearedable to conserve,as its own constant,this specificattribute of the
environment.In the caseof salinity,this would correspondto the constitution of the metazoicentity itself which has retainedthe compositionof the
early seain its intercellularfluids. As far as tempratureis concerned,the
entity which would appear able to conserveit has to be regardedas much
more complex and even to be related to the appearanceof systemsof
organswhich are sufficientlysensitiveto changesin temperatureand which
also possessthe means of insuring constancyfor temperature.
The conservationof different elementsin different compartmentsappears to be characteristic.
In order to maintainits constantsfor elements
the entity has to opposetheir uncontrolledcirculation.The role of hierarchic entitiesin conservingancestralconditionswould explain why an
entity would have to opposeparticularlythe penetrationof the constituents
which characterize
the succeeding
environments.
This has appearedevident
for the cations.The boundary formationswhich have to ptay the principal
role in the creation of each entity must also insure its identity by barring
uncontrolled penetrationof elementscharacterizingthe new environments.
Invasion by such elementswould correspondto an abnormal event which
must be corrected. If the invasion progressedbeyond a certain limit, it
would createa conditionincompatiblewith further existenceof the entity.

30

nESEARcH rN pHystopATHoLocy

|later Circulation
The passageof complex organizedanimals into the new environment
of terrestrial life brings to the fore the problem of the place of water in
hierarchic systematization.It appears to us an acceptable concept that
water doesnot circulatefreely in the organism.Its appearancein hierarchic
entitiescan be understoodif, as we did for the other constituents,we relate
water to its place in the environmentin which phylogeneticdevelopment
has taken place, In the first near-volcanoenvironments,which applied to
the subnuclearentities,water was relativelyscarce,which explainsthe high
concentrationof the constituentsin the nucleus.The mud of the earth's
crust is richer in water, which explainsthe difterencebetweenthe nucleus
and the cytoplasm, with the latter richer in water. The sea was the
environmentfor the metazoiccompartment,which explainsthe richnessof
water in this compartment.The so-called"internal sea" consequentlycan
be seen only in the metazoic compartment.With the passageinto the
terrestrialenvironmentwith its air medium,the water againbecomesscarce
and has to be conserved.The circulationof water betweencompartments
is governedby osmotic forces which are determinedby the original richness in water of the respectiveenvironments.The importance of water
circulation appearsevidentwhen an abnormalityin its distribution occurs.
Water arriving in a compartment-alone or with a cation-in an amount
abovethatcorresponding
tothe constantfor that compartment,isseparated
from the constituentsof the entity in order to reestablishthe characteristic
constant value. Such separationof abnormal amounts is accomplished
accordingto the compartment,through the appearanceof vacuoles,edema,
exudatesor diuresis.
Animals and Plants
The conceptof hierarchicorganizationin which each entity canconserve
its own environment allows us to consider in a new light various other
problems of living organisms.One concernsthe fundamentaldifferences
betweenanimalsand plants.Analysisof the constituentsof the metazoic
secondarypart providesa new criterion for distinguishingbetweenanimals
and plants and gives logical meaningto its distinction.Animals can be
characterized
as having sodium as the cation of thcir metazoiccompartment; from the cell level on, they have had the sea as their temporary or
even permanentenvironment.Plants,on the other hand, have potassium
as the principal cation for their metazoic compartment, indicating that,
from the cell level on, they have had the earth'scrust as their environment,

B r o L o c r c A LE N T r r r p s /

lt

passingthus directly from mud to air. By their actual attachmentto the


soil, plants continuethis relationshipto the mud. Their relative immobility
is in accord with continuation of the terrestrial-airenvironment in their
development.The mobility of animals, on thc contrary, can be seen to
have its origin in the fact that they have had the sea as thcir environment
at least for a period of time, r.e.,from the cell period until the appearance
of those animalswhich left the sea. We can interpretthe appearanceof
celluloseand lignin as part of the plant-sustainingmeans which would
bring to plants indispensableexternal protection against the hardnessof
the soil environment.Cellulose and lignin are not necessaryfor animals
which experienced
much of their evolutionin the sea.
Multiplication
The hierarchic organization of organisms,with the conservationof
successive
entities,puts the problem of cntity multiplicationin a new light.
According to the hierarchicorganizationconcept, the multiplication of a
complex entity means the reproductionof the entire seriesof hierarchic
entities forming it. In this process,the intervention of each hierarchic
entity appears highly individualized.And this applies not only for the
morphologicallyidentifiableentities,but even for the most primitive entities.The difterencebetweenthe role playedby the principal and secondary
parts becomescapital for theseprocesses.
While for each entity the principal part has to be built as such, the parts correspondingto the secondary
Parts are taken from the immediateenvironment,The quantitativedisproportion betweensome principal and secondaryparts makes the role of the
first diftcult to be recognized.The complex entity, through changesthat
are the reverseof thoseof ontogeneticand phylogenctichierarchicdevelopment, separatesthe successive
principalparts which characterizeit. With
the replication which takes place the division occurssuccessivclyfor these
hierarchic entities. In scissiparitythe division morphologicallyoccurs at
the cell level; in karyokinesis,it can be identifiedat the chromomerelevel
and certainlytakesplacemuch lower in the hierarchicentities.In replication in general,different constituentsavailableare adequatelychangedby
the respective principal part to form the necessarysecondary parts.
Through these changesthe same processesare reproducedwhich originally occurredwhen the entity had been phylogcnetically
organized.
individualization
With the
of the low hierarchic entities the problem
of replication is simplified.Once replicationoccurs,the sameprocesstakes
place successivelyfor the progressivelyhigher levels.Above chromomeres
this appears very clear in karyokinesis.

32

REsEARctf IN

PHYSIoPATHoLocY

After the chromomeresdivide, two or four chromonemataappear.The


processgoes on within the chromosome,nucleus and cell. In order to
protect its individuality each hierarchicentity is protectedduring its division. The chromosomialmembrane,the cellular cytoplasmand the cellular
membranecontinue to protect the respectiveentities as they divide. The
cell itself divides only when the two nuclei have had their protecting membranes rebuilt.
In the division and multiplication of a complex entity, the return to
entitiesas low as subnucleicparts indicatesthe relativeimportancefor the
characterizationof the complex entity and for the conservationof its particular properties of the parts added during the hierarchic development.
The entity must, in fact, rid itself of these added parts which, although
they have other importance,have a secondaryrole even in the processes
of multiplication.
It is interestingto note that a similar return to more primitive component entitiesalso occurs,althoughit is lesspronounced,when an entity,
tissue,organ or organismhas to fight a noxious intervention.The defense
is passedprogressivelyfrom the organ to the tissuesand from theseto the
cells.In effect,there is a renunciationof addedparts during thesemoments
of crisis.Even at the cell level, a similar processis seen.The added parts,
represented
formations,disappcar.The almostnonby the protoplasmatic
differentiatedcell fights the noxiousfactor at the lowest levelsof its organization.
Lile and Death
We have seenthat the term "biological realm" can be applied to hierarchic developmentstartingfrom the N-C-N-C radicals.We have employed
this term for didacticconveniencealthoughit is unrelatedto the commonly
acceptedconcept of life. The study of hierarchicorganizationalso led us
to considera conceptof life and death which, while retainingsome of the
common meaning, also accordswith the phenomenaof hierarchicorganization.
In the complex entity, each lower level entity lives and dies with
relative independence.An organ can be dead and yet have living cells
in it for a time. There are always dead cells to be found in living tissues
of the different hierarchicentiand organs.It is the relative independence
ties making up a complex entity that explainstheseseemingpeculiarities.
Our concept of life and death stemsfrom considerationof the nature
of hierarchic entities. We have seen that all matter in nature. from the
simplestto the most complex entity, is a result of heterotropy.The per-

B I O L O c T C A LE N T I T t E S

33

sistenceof constantsproper to each entity is distinctly opposed to homotropy. Life in its broadestsense,correspondsto the capacity of an entity
to maintain heterotropyby conservingits characteristicconstants.The life
of any entity aPpearsto be synonymouswith conservationof its constants.
An entity dies when it has permanently-that is, irremediably-lost its
capacityto conservethe constantswhich characterizeit. Death then representsexhaustionof heterotropyfor the specificentity.
The fact that, in essence,life appearsto be synonymouswith the conservation of constants and is heterotropic,relates it, and especiallyits
origin, to one of the important sourcesof heterotropicforce, solar energy.
A distinction has to be made betwcen heterotropyas one of the fundamental laws of nature and the meansby which it is exerted.Solar energy,
with all of its quantas,would greatly increasethe effectsof heterotropic
forces in nature. It would not createsuch forcesbut would simplify them
and extend their applications.The origin of matter and, as we have seen
above, of entities, biological or nonbiological,is in the final analysisthe
result of heterotropicforces.External conditionsqualitatively and quantitatively influenceoperation of heterotropicforces.
The sun's heterotropiccontributionshave to be consideredunder this
aspect.Throughthe quantasit disposes
of, solarenergyhasnot createdlife,
as conceivedabove, but by permitting more and more entities to appear,
has greatly facilitated their extension.Its effect, although certainly not
limited to any group of entities,seemsto be especiallyimportant to those
forming the biological realm. Similarly, the effect, of a special type of
energ.y,radiation,also must be considered.Radiation appearsto be related
to the elements,and will be discussedin a later chapter devoted to the
elements.
Sincelife itself is related to changesdirectly aimed at conservingconstants,in this broadestsenseit is no longer limited to the specificgroup of
cntitiesfound in the "biological" realm. Life has the samemeaningfor an
atom, crystal or micelle, as for a cell, organ or organism.It is for this
reason that knowledge of the mechanismthrough which constancy is
achievedbecomesof great importancein the study of all matter and especially of the biologicalrealm.
Maintenattce ol Constants
To study the mechanismused to maintain constants,we must define
exactlywhat constancymeans.According to Cannon'sprinciple of homeostasis,
constantshave beenconsidered
to correspondto a dynamicbalance
that results from the continuousoperation of two opposingfactors. And

34

R E S E A R c Hr N p H y s r o p A T H o L o c y

it was conceivedthat by actingconcomitantlyas coupled antagonists,these


factorsor groupsof factors insureconstants.The interventionof thesetwo
opposite factors becomesapparentonly if an exterior cause upsets their
balance.
However, our study of the processesthrough which dynamic balance
is maintainedhas permittedus to recognzea differentmechanismthan the
one which is commonly accepted.
A value consideredto be a constantfor an entity is not fixed or static.
It represents,rather, a statisticalvalue, the result of a seriesof dynamic
changeswhich must also be consideredin terms of time. Consequently,a
constanthas to be seennot only as the averagevalue of a seriesof organized changes,but also must be identifiedby the characteristicsof the variations. An averagevalue around which variationsoccur thus representsthe
first attribute of a constant. The second attribute is the existenceof a
rhythm in the variations,the third involvesintensityof variations.For
instance,when we say that human body temperatureis constant,we mean
that 37'C is averagevalue for oral temperature,and also that body temperature presentscharacteristicvariations having a 24-hour rhythm and
also that the occurringchangesconsistof variationsof a few tenthsof a
degreeabove and below the averagevalue.
The two antagonisticinterveningfactors do not operateconcomitantly
to maintain a constantvalue, but rather act alternately,each being predominantfor a period of time.The resultis not a continuouslysteadyvalue
for the constant,but an oscillatorymovementwith successive
passages
from one sideto the other of the averagevalue.This oscillatorymovement
appearsto be the generalrule throughoutnature, prevailingin everything
from the wavesin the smallestsubatomicparticlesto the pulsationof the
universe. The rhythm periods appear to correspond to environmental
rhythms.A rhythm relatedto the day, for instance,is seenfor temperature.
In other constantswe recognizea l2-hour rhythm which could correspond
to that of the oceantides.Other rhythms,with periodsrangingfrom two
hours to a few minutes are seen for severalchangesoccurring in blood.
Therc are also some in which the influcnceof thc moon is evident; for example,the hypophysis-ovarian
cycles;and for others,the influenceof the
seasonsis apparent.
Teleologicallyspeaking,balancerepresentsa very effectivemethod for
maintainingconstants.Any deviationin any direction as a result of an
externalinterventionwill be counteracted
by the opposingphaseof the oscillatory balance.This occursbecauseof the existenceof two phasesof thc

BIOLOCIC^L ENTITIES

35

oscillatorymovementitself. Such would not be the caseif there wcre fixed


valuesfor constants.
Related to the pattern of the organizationo[ matter in gcnera.t,this
oscillatorymovementcan be consideredto bc anotherinstancein which the
two oppositefundamentalforcesof heterotropyand homotropy, which are
basicto progressivehierarchicdevclopmentitself, also operate.This oscillatorybalancecan be relatedultimatelyto the alternatesuccessive
interventionof thc heterotropicand homotropictrendsin the organizationand the
manifestations
of entitiesexistingin nature.
Dualism
The concept of dynamic oscillatorybalanceis of great importance in
the study of biological phenomena.coupled facrors with opposite propertiescharacterizeall constantsand are involved both in the processes
throughwhich constantsare maintainedand in their manifestations
as well.
Recognition
of this dualismin all biologicalphenomenahas been of great
valuein the investigationof normal and abnormal physiology.
According to our concept,dualismresultsfrom the alternate.not concomitant,operationof two opposingfactors.And, as we have seen,these
factorsultimately can be related to the two fundamentalforces in nature,
homotropicand heterotropic.Thus, in a unifiedconcept,in every phenomenon in which dualism appars,one force will be homotropicthe other will
be heterotropic.Homotropy is relatedto fulfillment of clectrostaticforces,
andhasgeneralcoulombianelectriccharacter.Heterotropyis quantum-like
and organizational.Homotropy would keep entities simple. Hcterotropy
would lead to more organizedbonds and to more complex synthesis.In
everyphenomenonstudied,thesecharacteristics
of the two fundamental
forceshave permitteddualism to be recognizedand interpreted.The dualisticview has becomeour basicapproachfor all of the problemsrelatedto
matterand, more specifically,to biologicalentities.
The dualistic conceptof interveningforcesbrings an entirely new light
in any analysis in which a graphicalrepresentation
is different from a
straightline. From the curvesof spectralanalysisof constituentsto those
of complex phenomena,the existenceof oscillationsrevealsthe intervention of opposed forces and offers a valuable mean to study them. This
broad approach has a specialfield of applicationin biology.
Dualism can be further recognizedeasilyin the manifestations
of the
in
biologicalentities, their function and in the substances
composingthem.
In the case of the elements,such a dualismcan be relatedto atomic structure and the propertiesof the electronicshells,as will be seen below. In

36

nEsEARcH rN pHysIopATHoLocy

complex molecules, a simple form of dualism can be seen in acidity and


alkalinity, electrophily and nucleophily or, furthermore, in positive and
negativeelectrical characteristics.We will seelater, how important dualism
is for the different groups of constituentsand how, without this dualistic
concept, it would be di-fficultto understandthe roles of most of theseconstituentsin biologt.
In part, as a consequenceof the separationof the constituentsinto
two groups,dualism can be observedeasilyin the hierarchic organization
of higher entities. Dualism appearsin the relationship between primary
and secondaryparts, the fust having a more positive character than the
second.The study of cancermanifestationsunder this dualistic aspecthas
beenhighly rewarding and is the subjectof the following chapters.
In a more concretestep, the dualisticconcepthas provided new understandingof abnormality.
Normal and Abnormal
A normal entity can be conceivedof as one which is able to maintain
its constantswith their characteristicvalues, rhythms and intensitiesby
meansof the alternateoperationof homotropic and heterotropicforces.A
normal entity, thus, can be definedas one havingconstantswithin the limits
that statisticallycharacterizethis particular kind of entity. we can define
the abnormal entity as one in which a constant'scharacteristics-average
value,rhythm, intensity-are altered.It is alteration,without completeloss
of the characteristicsof constants,that differentiatesabnormality from
death.In death the constantsthemselvesare irremediablylost. This definition also distinguishesabnormal from physiologicalmanifestations.In the
physiologicalmanifestation,oscillatorymovementpersistsand only its intensity is influenced,usually becomingexaggerated.
As expectedfrom the dualistic concept, abnormal changescan take
place in either of two opposite directions and this is a significant fact of
abnormality.The two possibilitiesare inherent in the oscillatory balance
characterizingthe constant itself. It is the offbalance,resulting from the
predominanceof one of the coupledfactorsover its antagonist,
exaggerated
which leads to the abnormal. Persistentpredominanceof one factor abnormally affects,and even suppresses,
normal oscillatoryrhythm.
For each normal condition, then, two oppositeabnormalitiesare possible.By relating the abnormalcondition to one or more alteredconcepts,
and the alterationin each constantto one of the dual changespossible,a
new systematizedanalysisof the abnormal becomesfeasible.The large
number of constantswhich composeeach entity and which can become

BtoLOctc^L ENTTTTES /

37

abnormalhelp not only to explain the great varieg of abnormalitiesbut


also offer a meansof obtaininganalyticalpicturesof disease.
It is with this approachthat wc havetried to study pathologicalconditions. with specialemphasison cancer.This study is presentedin the following pages.

CHAPTER

CANCERAS AN ORGANIZED
CONDITION

il

.llN rue coNcEpr now most widely accepted,cancer is consideredto be


the result of abnormal changeswithin cells. Although it is admitted that the
diseasemay have different etiologies,it is the cell which is regarded as the
pathogenic entity. A group of specific changesin the cells is believed to
rcpresent the fundamental abnormality.
In today'sprevailingoutlook, differencesbetweentumors are attributed
to the multiple secondarycharacteristics
presentin the diseasedcells along
with a primary specificanomaly. The complex clinical manifestationsof
cancerare further explainedin termsof the relationshipbetweencancerous
cells, as pathogenicentities,and the whole organism. Clinical evolution,
from local innocuous process to lethal disease is related to anatomical
spread of cancerous cells from their original site. Abnormal metabolic
changcs seen in the organism are believed to result from the influence
exertcd by functional abnormalitiesof the cancerouscells. In a still narrower view, canceris consideredto be the result of abnormalityof a single
s-1-cificfunction of the cell-its gro\rnh. Qualitatively and quantitatively,
abnormalgrowth has becn consideredto be the capital factor in the pathogcncsisof the discase.(292)
In contrast to this classicalview, our studies have lc-d us to regard
ccnc!'r as somethingother than an abnormality limited to the cell alone.
As we have seen,the organismis a conrplcxhierarchicorganizationof
different biological entities.We soueht to detc'rminen'here cancer fits in
this complexorganization.
Can cancerrvith is manifc'stations
and is evolution be L*tter understoodif s1'stematized
in ;.rccordirnce
s'ith the hierarchical
organizationof the organism?Cm both nunifcst.rti(rnsilnd r'r'olution be
rclutednot alone to a cellular abnormaliry"
but rathc.rto :l pR)gressive
par38

CANCER ^S

^N

ORGANIZEDCONDITION

39

ticipation in the diseaseof the different hierarchiclevels of the organism?


we have found that suchparticipationcannotbe analyzedreadily in the
advancedcanceroussubject with so many and such varied manifestations
of the diseasealready present.Similarly, incipient caseswith a paucity of
clinical manifestationsare not ideal for the purpose.It was only by following the successiveappearanceof manifestationsduring the evolution of
cancerthat their relationshipto the level of hierarchicorganizationinvolved
could be clearly seen.
Identificationof the level involved at each point in the developmentof
cancerwas greatly facilitatedby conceptuallyseparatingthe clinical evolution of the diseaseinto a seriesof successivephasesand identifying the
changeswhich characterizethe passagefrom one phase to the next.
we have chosento call thesephasesprecanccrous,
noninvasive,invasive, painful, preterminal and terminal. we will briefly identify them and
their salient features here.
Precancerous Phase
In the precancerousphase, the diseaseis not clinically apparent.Yet
this phasehas beenrecognizedas pathogenicin experimentalcarcinogenesis
and its characteristicchangesalso have been identifiedin human subjects.
Morphologicalchanges-abnormalitiesin size and form--<an be observed
in the chromosomes.These changesare not identifiedas related to cancer
in human subjectsexceptwhere multiple centersof cancerizationare found
(as in the stomach,for instance). (Note 1) Thesechromosomalabnormalities can be considered to be precancerouslesions, since experimental
carcinogenesis
has indicatedthat thesechangespreccdethc appearance
of
cancerous
cells.In termsof hierarchicorganization,
then, the precancerous
phasecan be consideredto be limited to the subnuclearlevels.
Noninvasive Phase
In the noninvasivephase,also known as "cancer-in-situ,"abnormal
intra-epithelialcells are present.The abnormality involves two changes.
One, morphological,affectsthe nucleus;the other affectsarrangementof
the cells in the epithelium. The abnormal changesin the nucleus in this
phasehavebecn widely studiedin exfoliativccytology.(Note 2)
Abnormalityin this phaseappearsto be limited entirelyto the nucleus.
The cells continue to have an almost normally differentiatedcytoplasm,a
fact which originally led to the descriptionof this phaseas "cancer of differentiatedcells." Besidesthe nuclear changes,cells in this phase show,
histologically,an anarchicarrangementdifferent from the regular disposi-

40

REsEARcHrN pHysrop^THoLocy

tion which is one of the basic characteristicof the epithelium. Since the
regularrelationshipbetweenthe cells forming epitheliumcan be attributed
to dipolarity, the anarchicdispositionseenin this phaseof cancer can be
ascribedto loss of cellular dipolarity.
cancer-in-situ, in terms of hierarchic organization,would appear to
involve the level of the nuclei, and the noninvasiveness.
characteristicof
this phase,persistsas long as the "cancerous"abnormatityremainslimited
to this level, that is, as long as the cytoplasmof cells remains apparently
unaffected.
Invasive Phose
This phaseis characterizedby irregularproliferationof cells and penetration into neighboringtissues.To the anarchicarrangementsnoted in the
noninvasivephase, now has been added exaggeratedgrowth. And the
changeof a noninvasivecancer into an invasiveone can be consideredto
result solely from the addition of the new factor of abnormal growth. The
invadingcells will persistonly if, concurrently,there is a loss of the defense
mechanismof the invaded tissues,as will be seen later.
Studiesof invading cells have revealed,in this phase,an anomaly no
longer limited to the nucleusbut now encompassing
the cytoplasmas well.
Exfoliative cytology has shown an abnormal and rapidly disintegrating
cytoplasmand this has servedas an important diagnosticcriterion. From
the point of view of organization,it can be said that, with the participation
of the cytoplasm,the diseasehas progressedfrom the nuclear to the cellular level.
Painlul Phase
Pain is the principal clinical manifestationcharacterizingthe next phase
of the disease.As we shall explain in greaterdetail later, pain arisesfrom
changesin the pH of the intercellularfluid that bathessensorialnerve endings.For the moment, we can remark that biochemicalchangesnow occur
outsidethe cells, and, with the participationof interstitial formations,the
diseasehas progressedto the tissularlevel.
Preterminal and Terminal Phases
In the next stage,the preterminal,biochemicalchangesaffect the function of variousorganswhich may or may not in themselvescontain cancerous cells. while some changesin function may be seen even before this
preterminalphase,now, there is manifestimpairment.And, while the invasionof an organ by cancerousmassesis a factor precipitatingthe func-

c A N c E R A S A N

O R C A N I Z E DC O N D T T I O N

4 l

tionalchanges,
invasionis not indispensable.
Abnormatbiochemicalchanges
leadingto seriousfunctionalimpairmentsare seenin organsentirelyexempt
from tumor masses.
with further progress of cancer, metabolic functions that are systemicallyimportant becomeabnormal,Later, we will analyzr,in detail these
changeswhich affect the whole organismprofoundly.For the moment we
want only to note that, with thesechangescancerpassesfrom the clinically
preterminal to the terminal phose.
In the light of this systematization,cancer then appears to progress
clinically in organized fashion as it passesfrom the relatively innocuous
nuclearnoninvasivecancer-in-situto a lethal systemicdisease,the progress
being marked by the successiveparticipationof difterent hierarchic levels
of the organization. Table I sums this up.
T,fSLP

Organizational
Level
Subnuclear
Nuclear
Cellular
Tissular
Organic
Systemic

PhysiopathologicalChanges
Gene and chromosomeanomalies
Nuclearanomaliesand atypical
cellular arrangemenls
Atypical growth
Local pH changes
Organic metabolicchanges
Systemicmetabolicchanges

Ctinical phase
precancerous
Noninvasivecancer
Invasivecancer
Painful cancer
Preterminalcancer
Terminal cancer

By extrapolation,a similar progressiveparticipationof hierarchic entities can be conceivedof below morphologicallyrecognizablelevels.This


would permit us, as a working hypothesis,to attribute the pathogenesis
of
cancerto abnormalitiesin nucleo-proteinsor, even lower in the scale,to
abnormalitiesin histonesor alkaline amino acids.(Note 3)
This concept----ofprogressiveparticipation of successivehierarchic
levels in cancer-<ontrasts sharply with the view generally held today
which placesthe entire burden of anomalyon the cancerouscell itself. The
classicalconcepthas led to the currently prevailingaU-or-nothingapproach
in which therapeuticattemptsare directedto the cancerouscells as the only
avenuefor controlling the diseaseat any moment of its evolution. Under
our hierarchicconcept,therapeuticpossibilitiescan be extendedbeyond
the cancerouscell.
These considerationsraise the questionof the relative importance of
themultiplechangeswhichoccurin cancer.Subnuclear
and nuclearchanges
are of relatively little importanceas long as there is no progressof disease
beyondtheselevels.Corroborationfor this can be found in the great num-

42

RESEARcH rN pHysropATHoLocy

ber of casesin which cancer-in-situ


cellsare noted in an organ,yet clinical
cancerdoesnot follow. In our concept,the changeswhich occur at levels
abovethe nucleararc critical in the evolutionof the diseaseand, as such,
are the important pathogenicfactors. On the other hand, as we shall see
later,changesat higherlevclssimilar to thoseencounteredin cancermay
occur independentlyof cancer,and without a sequenceof changesat lower
levels.It is only when changesat higher levelsappearin proper sequence,
affectingalready abnormal entitiesof the lower levels,that clinical cancer
resultsand the malignancymovesrelentlesslyfrom the noninvasivecancerin-situ to the terminal phase.
This concept,then, focusesattentionon all changesoccurring at difterent hierarchiclevels of the organizationrather than on those in the cell
alone. It emphasizesthe importanceof the relative independencewhich
existsbetweenthe different hierarchiclevels,an independencewhich governstheir participationin the complexconditionwhich is cancer.
From the therapeuticstandpoint,then,it seemslogicalto supposethat,
if the progressiveparticipation of successivelevels can be interrupted,
many if not all of the noxiousmanifestationsand the courseof cancercan
be favorablyinfluenced.In view of this, it has beenessential,first, to obtain
more information about the cancermanifestationswhich are added as the
diseasetakes its hierarchicallyprogressivecourse and about the mechanisms that account for thesemanifestations.

CHAPTER

DUALISIU

'fhe
/ \\s wE HAVEotssERvED
ABOVE,
dualismprevailsin nature.
concept
of an oscillatorydynamic balance-the result of alternateopcration of opposedforccs-has beenof specialvalue in the study of most of the physiological phenomcna.Over the yearswe have alsoconstantlyobservedthat in
most physiopathological
manifestations,
dualisticpatternscan be recognized.
This dualisticpathogenicconccpthas helpcd to guide our study of disease.
In cancer, it has permitted bettcr understandingof many processesand
manifestations.It has also servedas a basis for our attemptsto influence
cancer and other conditionstherapeutically.
It was in the study of pain
that, initially, we found clear evidenceof pathogcnicdualism.

PAIN
Many years ago, during expcrimentswith an alcoholic cxtract of human placentaas a therapeuticagentin terminalcancercases,a curiouseffect was observed.In somepatientswith painful lesions,administrationof
the preparationresultedin a decreasein the intensityof pain and even in its
disappearance
within a few minutes,with rclicf usuallylastingfor hours. In
other cases,however,there was an oppositeeffect;pain increasedin intensity within a few minutesafter an injcction.In somesubjects,the exacerbation was so great and pain becamcso unbearablcthat the experimental
treatmenthad to be discontinuedquickly. In severalcasesin which the
preparationwas used in progressivelylarger doscs,another notcworthy effect was observed: after the first injections,pain decreasedand even disappearedfor severaldays, only to have a new pain arise as treatment
continued.This new pain becamemore intcnseaftcr each injcctionso that
the therapyhad to be discontinued.
Patientsclearly recognizedthe differpains.
encebetween
The new one frequentlyhad a burningcharacter.
43

44

REsEARcH rN pHysropATHoLocy

Thus, it became apparent that one substancecould increasepain in


some subjectsand alleviateit in others,and could even alter the nature of
the pain in the same subject. Pain, as demonstratedby antagonisticresponsesto a single agent, thus appearedto have a dual nature. Our immediateproblem was to investigatethis and its significance.
Physiological and Pathological Pain
In discussingthe sensationof pain,mostauthorshave found it necessary
to distinguishbetweendifferenttypesof pain. Somehave classifiedpain as:
l) spontaneous,
or 2) provoked,accordingto its modeof induction.others
have definedpain accordingto its site of origin and quality as: r) superficial or cutaneous,and 2) deep visceralor somatic.Superficialpain from
skin and mucousmembranesnear body orificeshas been describedas bright
or burning in quality, while deep visceral or somatic pain arising from
mesenchymalstructures,certain mucous membranesand viscerahas been
describedas diffuse and aching in quality. In many respects,attemptsto
define and classifypain in theseterms have servedto confuse rather than
to clarify the problem.
That differenttypesof painsdo exist is an observationbasedupon common experience.For example,when a stimulusof sufficientintensityis applied to the skin for an adequateperiod of time, a sensationof pain is
induced.This pain disappearsrapidly when the stimulusis removed.But
if the stimulushas been of such intensityand duration as to producetissue damage,an after-pain may recur spontaneouslysome time after the
stimulushas ceased.The originalpain servesas a warningthat the tissues
are endangered.The after-pain,however,cannot be consideredas a direct
effect of the applicationof an externalstimulus,but is rather a manifestation of true tissuedamage.
As a first step, two categoriesof pain-one induced in normal tissue
by external intervention,the other appearingas a pathologicalmanifestation of an existinglesion-were established.we called the first, which is a
normal sensorialsensation,"physiological"or "sensorial" pain. The second, a symptomof an abnormallocal condition, was called "pathological"
or "symptomatic"pain. This separationhelpedto eliminatediscrepancies
otherwiseencountered
in the studyof pain,discrepancies
which resultwhen
two entirelydifferentmanifestations,
one sensorialand the other symptomatic, are studied under the same heading and investigatedby the same
methods.
Pain may be induced in damagedtissuesby various stimuli not of
sufficientintensityto arousepain sensations
in normal tissues.This sensi-

DUALTSM

45

tivity constitutesan abnormal responseof tissuesthat have undergone


pathological
changes.Inflammatory,traumatic,circulatory,neoplasticor
otherpathologicalchangessimilarly may bring about either spontaneous
painor an abnormaldegreeof sensitivityof the inyolved tissuesto external
stimuli.
There are, therefore,two gcneraltypes of pain which are biologically
different.Thc first is a direct responseof normal tissuesto external stimuli
whichservesas a warning of danger.The organismreacts to this type of
painby seekingto run or to fight. The secondtype of pain arisesas a consequence
of tissuedamage or diseaseto which the body respondsby attemptingto put the injured area at rest. This secondtype of pain, whether
spontaneous
or provoked,superficialor deep,is biologicallydifterent from
the first pain experiencedfollowing the applicationof sufficientlyintense
externalstimuli to normal tissues.
For purposesof further study, it would appear to be advantageousto
distinguish
betweenphysiological
or sensorialpain which is the responseof
normaltissuesto noxiousexternalstimuli,and pathological
or symptomatic
painwhich is a manifestationof abnormaltissues.In the study of pain in
all its aspects,it is necessaryto keep this distinctionin mind. While various
investigativemethods have furnished data concerningphysiologicalpain,
the information thus obtainedis of very limited value when applied to the
problemof pathologicalpain. However,it is pathologicalpain which constitutesthe vital clinical problem, physiologicalpain being of concern in
me'dicine
primarily in the field of anaesthesia.
(Note I )
Dualism in PathologicalPain
It wasonly in pathologicalpain that a dual characterwas encountered.
For
onething, it was noted that in some patientswith chronic pain-associated
*ith tumors, arthritis or other conditions-the pain intensitywas not constant.In many of thesepatients,variationsin pain intensitycould be seen
to follow a pattern. Although the variations usually are referred to as
"spontaneous,"we could show that they were related to the time of day.
Furthermore,the variations were not the same for all patients. In one
group, pain was severein the morning and diminished toward evening,
whilein anothergroup, little or no pain was felt in the morning and exacerbationsoccurredin the evening.
The intake of food also had a dual influence.In some patients with
tumorsfar removedfrom the gastro-intestinal
tract, pain was increasedby
catingwhile in otherspain decreased.Patientsthemselvesoften recognized
this relationship and many whose pain was increasedwith the intake of

46

nEsEARcH rN pHyslopATHoLocy

food refusedto eat for fear of aggravatingtheir suffering,while thoseof the


other group wanted to eat wheneverpain was scverein order to reduceits
intensity.
These observationson the influenceof time of day and intake of food
pain
on
led to the study of the acid-basebalance of the body since this
balanceis known to be influencedby the same two factors-time of day
and food intake.
In a preliminary study we considereda specialaspectof the acid-base
balanceof the body, that is, the severalmechanismsinvolved and their
possibleinterventionin the changeof pain intensity.A study was made of
blood pH, titrimetric alkalinity,CO2 combiningpower, rclative chloride
distribution betweenerythrocytesand plasma, as well as urinary pH for
the indicationsthey furnish concerningthe acid-basebalance.The blood is
highly buffcred in order to avoid damage,through abnormal pH values,
to cells in generaland especiallyto those of the nervous system.Consequently, the variationsin the blood pH are as limited as possible.Alkaline
reserveand the chloridesrepartitionrepresentonly part systemsin the general acid-basebalance.Titrimetric alkalinity, correspondingto the sum of
ionized and nonionizcd constituents,furnish information of the broadest
scale of the acid-basebalance. Consequentlyit appears to be a higily
significantmeasurement.
Through the non-ionizedconstituents,it can vary
greatly without influencingblood pH. It reflectsthus otherwischidden
changesin the acid-basebalance.We could show that alone, among all the
variablefactorsof the blood acid-basebalance,total titrimetric alkalinity of
blood varies in parallel with the urinary pH. (Nore 2)
As an immediateresult of this research,it was possibleto utilize the
changesin the urinary pH as an indicationof the most importantvariations
occurringin the systemicacid-basebalance.This makes it possibleto use
changesin the urinary pH as an indicator of the relationship between
acid-basebalanceand variationsin pain intensity.
Pain and Acid-Base Balance Changes
When changesin pathologicalpain intcnsitywerc studiedin relationto
changesof the urinary pH, a correlationcould be establishedin the majority of cases.Two oppositekinds of relationshipwere observedwhen curves
of the variationsin pain intensitywerecomparedwith thoseof the urinary
pH.
with chronic pathological
Patientswho had experiencedpain associated
lesionsover prolongedperiodsof time were instructedto record carefully
the relativeintensityof their pain at regularintervals,such as every hour.

DUALTSM

47

No analgesicswcre administeredfor at least six hours before or during the


testperiod which was continued as long as possible,even for twenry-four
hours.Patientswere instructedto concentrateon a singlepainful area and
to estimate the degreeof pain intensity.They were told to consider an
averagedegreeof pain during each hour rather than momentarypeaks
during the observationperiod or the pain at the moment of recording. The
PTIN
Unbaroble
Very*vere
Severe
Moderole
Sl,9ht
None

pH
8.O
Unne
7.5
7.O
6.5
6.O
5.5
5.O

to

tz

'6
,.1,!

tB

22

24

Flc. 6. A pain pa!lern is recognizedby comparing the concomitant changes present


in the curves of pain intensity with those of the urinary pH, The parallel variations
of the two curves indicate an olkaline patter,t with the pain more intensive when thc
urine is more alkaline, as seen in a case of carcinoma of the coloq with painful abdominal mass.

degreewas recordedin relativeterms of no pain, slight, moderate,severe,


very severe and unbearable,or as figuresfrom 0 to 10.
Urine specimenswere obtainedeach hour immediately after the pain
intensityobservationswere recordedand the pH was determinedelectrometrically. Two curves-for the hourly variations in pain intensity and
for urine pH-were then plotted.
Two distinct types of correlationswere found. In the first, the two
curvesparalleled each other, the pain being more intensewhen the urine
pH was higher, and less severewhen the pH was lower. (Figs. 6 and 7)
Becausethe maximal pain of this type of correlationis associatedwith a
changetoward alkalinity, this was caUedan alkaline pattern of pain,

48

RESEARCH IN

PHYSIOPATHOLOGY

PAIN
Unbcorobh
Vcry Scvcn
Scwrc
Moduote
S,,9ht
l{ona
0H
Utina

6.2
6.O
58
5.6
5

5.2
5.
l'lour

Fto. 7. The alkaline paltern ol pain in which the concomitant variations in the curves
of pain intensity and urinary pH are parallel, seen iD a case of arthritis.

In the secondrype of correlation,the two curvesvaried inversely,pain


being most marked when the urine was most acid, and least so when the
urine was most alkaline.(figs. 8 and 9) This secondtype was called acid
pattern of pain becauseof its associationwith a changetoward acidity.
Consideringthe highly subjectivenatureof pain, the inconsistencies
which
occur are minor. Fig. l0 showsthesecurvesfollowedduring days.
The correlationbetweenthe pain intensityand urine pH curves arc
relativerather than absolute.The generallevel of urine pH apparently
m,A/
Ltibcorobfe
Very *ere
Scvcrc
Itlodatota
Sl,ght
Nona

pH
7.5
Uttna
7-O
6.5
6.O
5.5
5.O

Hour

Ftc. 8. An ocid pain patlern is seenin a caseof carcinoma of the prostate with meta.
s t a t i c b o n e l e s i o n s ,i n w h i c h t h e c o n c o m i t a n tv a r i a t i o n so f t h e c u r v e s o f p a i n i n t e n 5ity and urinary pH are divergent.

DU^LrsM

49

dependsupon other factors. Consequently,only the fluctuations of the


hydrogenion concentration,rather than the absolutelevels,are considered
in this relationshipto pain.
Changesin pain intensitywere found to have a similar dualisticcorrelationwith other factors as well as the acid-base!3l3nss-v/ith potassium
contentof blood serum, for example.Studieswere made to compare the
concomitantchangesin pain intensityand in potassiumcontent of blood
serurn.In severalcasesthe acid and alkaline patternsof thesepains were
determinedthrough the relationshipto urinary pH variations.At different
PAIN
U^Eorablc
Very fucn
Sevtre
Hoocrcle
Sl,ght
l{ona

_N_
Utlte

8.O
7.5
7.O
6.5
6.O
5.5
llou I

Ftc. 9. An acid pattern ol pain recognizedby the divergent variations of the curves
o f p a i n i n t e n s i t y a n d u r i n a r y p H i s s e e ni n a c a s eo f p h a n t o m l i m b .

moments,especiallywhen the pain was markedly different in intensity,


blood was obtained by finger puncture,and collectedin glass capillaries.
After clotting, the serum was immediatelyseparatedand the potassium
contentmeasuredusing a flame photometer.Curvesof the valuesof pain
intensityat these moments, establishedby the method previously rnentioned,and of the concomitantK+ contentwerecompared.Figs. I I and 12
showthe two curves in two casesin which, the two pain patterns-acid
andalkaline, were primarily recognized.The caseswith high serum potassiumvalues and with parallel changesbetweenthe two curves-pain intensityand K+ content-were seento correspondto alkalinepain pattern;
the other cases with less serum potassiumand opposite variations of the
two curyes were seento correspondto the acid pain pattern.
We extended the study of the pain pattern, as revealedby concomitant

50

nESEARcHrN pHysropATHoLocy

variationsof pain intensityandbody acid-base


balance,from cancercases,
in which a frank dualismhad beenseen,to other painful conditions.It
wasinteresting
pain can haveone or the
to note that,in manyconditions,
otherpatternbut thereare someconditionsin which only one patternis
consistentlyfound. Pain followingtraumaof any kind-the pain of post-

C o r c r n o m oo f R e c t u m
|9 4 3

+++ +
+ + +
c
++
o
o+

8.2
74
--

6'2
54
46
C o u r t e s yo f D r .M o r r oR o g n o n i

Fto. 10. The pain pattern can be recognizedalso through the characteristicopposite
variations of the curves of pain intensity and urinary pH, followed during successive
days-instead of hours-as seen in the above curves of a case of cancer of the rectum. (Courtesy of Dr. Rognoni)

operative and accidental wounds, burns and fractures, for example-always has an alkaline pattern. This is also true for the pain of gallbladder
colic. In other conditions,eitherpatternmay be presentand must be determined by analysis,For instance,the pain of neuritis and simple headachehas an acid pattern in somecases,alkalinein others.
In rheumatoidarthritis,an alkalinepain is almost constantlyfound.
ln osteoarthritis,the pain is of an acid type. In arthritic patientsin whom

DUALISM

5I

this relationshipdid not seemto hold, it was possibleto recognizenot only


the existenceof both rheumatoidand osteoarthritisbut also to note that the
pain pattern, as shown by test, was relatedto the more painful condition.
we utilized the diagnosisof the type of pain presentas an indication of
the nature of painful processes.We will seelater how this correlationhas
been confirmed by therapeutic trials.
lo
I

Pain
i n t e n ist y

6
4
2

(a)
K+ in serum
in mEq
.2
.0
1

H o u r s
Frc. I l. Pain pailern and potassium in blood serum. The comparison of the concomitant changesin the curves of pain intensity and those of the amount of potassium
in blood serum shows parallel variations in a case with an alkaline pattern.

Two types of pain associatedwith two different conditionspresent in


the same individual have been found to occur more frequently than expected,althougbusuallynot simultaneouslyactive.
In most patientswith two or more anatomicallyseparatedpainful foci,
parallelvariationsoccurredbetweenthe curvesof the differentpains. Only
in occasionalcaseswere the rwo painsfound to vary simultaneouslybut in
oppositefashion, Their oppositepatternswere well describedby patients
who observed"the two pains act as if they were part of a balance;when
one goesup, the other goesdown, and the opposite."In Figure 13, the pain
curye of lesion A is seento vary inverselywith the curve of urinary pH,
while the pain curve of lesion B is parallel with the urinary pH curve. Thus,

52

RESEARCH IN

PHYSIOPATHOLOGY

t0

Pain
Intensity

1
7
o

(b)
1.0

K+ tn serun 58

i n dq

16
54
32
3

Hours
F t o . 1 2 . D i v e r g e n t v a r i a t i o n sb e t w e e np a i n i n t e n s i t yc h a n g e sa n d t h o s e o f t h e b l o o d
serum potassium in a case of acid pain pattern.

PAIN LESION A
YaTt
Sevlrl
'.odarota
Sllghl

Nonr
MIN LESION8
Vr"t S.v.r.'l
Stvrrr
I
Hod.rlt
I
srbhf
{
I
ttolr

n!__UntlE ?.o
6.5
60
3.5

Frc. 13. Pains. Acid and alkaline pains can co-exist on different lesions,as seen in a
patient with multiple osseous metastasesfrom breast carcinoma. Lesion A, which
c o r r e s p o n d st o a n a c i d p a t t e r n . s h o w s d i v e r g e n t v a r i a t i o n s b e t w e e n t h e c u r v e o f
u r i n a r y p H a n d t h a t o f i t s p a i n i n t e n s i t y ,w h i l e f o r l e s i o n B . w i t h a n a l k a l i n e p a t t e r n ,
t h e v a r i a t i o n so f t h e c u r v e so f p a i n i n t e n s i t ya n d o f u r i n a r y p H a r e p a r a l l e l .

I _H..1"*_

:" .

_,

l,lil{Si"Ss\jilJ,1,$s-\i1,,i"r".+r,,"

DU^LrsM /

53

the pain of lesionA is of an acid patternwhile that of lesionB is of alkaline pattern.


Also interestingto note is the persistence
of the samepatternfor pains
associated
with chronic conditions.We have headachepatients,for example,in whom, during the 20 yearssincewe first determinedthe patternof
pain, there has been no changesof pattern. In others,on the contrary,
PAIN
UnDmmbla
Itry Scverc
Slvero
Modctolc
Slghl
lVorp
pH
ul tnQ

HrPOr

7.O
6 .o
5 0
4.O
8

9
t
Houl

F t c . 1 4 . T h e c h a n g e si n d u c e d i n t h e p a i n i n t e n s i t yb y t h e a d m i n i s t r a t i o no f a n a c i d i fying agent indicate the pattern present. Pain with an acid pauern is intensified foll o w i n go r a l a d m i n i s t r a t i o no f 1 . 5 c c . o f . a 5 o % s o l . o f p h o s p h o r i ca c i d . U r i n a r y p H
changesreflect the induced systemic acidification.

changes
occur rapidly. In a caseof sciatica.we have seenrapid and frequentchangesin pattern,especiallyin responseto therapeuticmeasures.
Worthy of being noted is the correlationfound betweenvariationsin
painintensityand changesin the acid-basebalanceof the body even in
casesin which nerves are directly involved in lesionsand in which a
mechanicalpathogenesis
usuallyhas been accepted.This would indicate
thatthe chemicalfactor mentionedabovehas a role in the pathogenesis
of
paineven in thesecases.
Acidilying and Alkalizing Agents
We next demonstratedthe cause-effect
correlationbetweenacid-base
changes
and variationsin the intensityof pain. Administrationof acidifying or alkalizing substancescould induce the same changesin pain
intensityas those causedby spontaneous
variationsin the acid-basebalanceof the body.
The relationshipbetweenacid-basebalancechangcsand pain intensity
was thus investigatedby administeringstrong acidifying and alkalizing

54

RESEARcH rN PHYsropATHoLocy

agentsto patientsafter the type of pain-urinarypH correlationhad been


determined.Administeredorally, strong acidifyingsubstances,
such as
phosphoricacid, ammonium chloride or mono ammonium phosphate,
increasedthe severityof pain with an acid pattern,(Fig. 14) and reduced
the severityof one with an alkalinepattern.(FiS. 15)At the sametime, it
causeda loweringof the urine pH.

PAIN
Severe

Poo
JHt

Moderote
Slight
None

pH
Urine

7.O
6.5
6,O
5.5

Hours
Flc. 15. Pain with an alkaline pattern is relieved by oral administration of two doses
of 1.5 cc phosphoric acid (50Vo sol.). Urinary pH changes reflect the systemic acidification.

Sodium bicarbonate or ammonium acetate in quantities that alkalinized the urine increasedintensityof pain with an alkaline (Fig. I6) ^nd
diminishedintensityof pain with an acid pattern.(Fig. 17)
These changes of the systemic acid-basebalance induced by administration of strong acidifying or alkalizing agents explains how similar
changes,when they occur spontaneously,affect pain intensity. The effect

DUALISM
PAIN
Unbrorcble
Very Scvere

lVoHCOr

Sevcre
Moderote
S1 , 9 h ,
NonG
pH
Uttt'

/ s s

80

7 0
6 0
5.O
9
Houl

F t c . 1 6 . P a i n w i t h a n o l k a l i n ep a l t e r n i s i n t c n s i f i e db y o r a l a d n r i n i s t r a t i o no f 5 g r a m s
of sodium bicarbonate.urinary pH changesreflect the systcmic alkalinization.

may be to increaseor decreasethe intensity,dependingupon the pattcrn


of the existing pain. It is intercstingto mention that similar changesof the
systemicacid-basebalance,spontaneousor induccd by the administration
of acidifying or alkalizing agents,do not influenceeither the thresholdor
intensity of physiologicalpain. we have often utilized this rcsponseto
alkalizing or acidifying substancesas a method of recognizingthe acid or
alkalinepattern of pain. (Fig. I8 bis)
Dualism in Local pH Measurements
Since it had been observedthat a definite correlation exists between
the variations in pain intensity of abnormal foci and changesin the gen-

fllA/
U^bP'oroble
Wy Severe
Sevcrc
Modercle

NoHCOs

Slighl
None
PH
Unne

8.O
7O
6.O
a n
I

a
Houl

F t c , 1 7 . P a i n w i t h a n a c i d p a t t e r n i s r e l i e v e dh y o r a l a d m i n i s t r a t i o no f 5 g r a m s o f
s o d i u mb i c a r b o n a t e .U r i n a r y p H c h a n g e sr e f l e c tt h e s y s t c m i ca t k a l i n i z a t i o n .

56

xESE^RcHrN pHysropATHoLocy

eral reaction of the body, it was desirableto ascertainwhat changeswere


taking place within the abnormalfoci themselvesat the same time.
Patientswith easily accessiblesuperficiallesions,especiallytumors, in
which painful areascould be well localized,were employedin theseexperiments. The pattern of pain, acid or alkaline, was first determinedin the
manner previously described.Local pH determinationswere then perCo of Utcrut
45
55 05

Hours 35

unbccroblc
vcry rcvtra
t cv0ra
modorolo
rlight
nona

Co of Prortolr
f5

25

35

45

1 . 5g m r . A m o n i u m

55

05

15

25

9m3A
. monium
occloll

ocr tot.

r.5eT
A m o n i u mo c c l o l c

Go of U l c r u r
fo
20 30 4 0

oo

oo ro
l

unbcoroblc O . 5g m t . A m o n i u mC h l o r i d r
varyr

Co of 8r.ort
20
30 40

50

l g m . A m o n i u mC h l o r l d r

taYcra
nodcroh
I light
nona

Co of Ulcrur

Co of Uteruc

30

YaTy scvarc
stvcrS

40

50

00

ro

to

? o r c 4 p 5 0

3O drope phorphoric ocid


off. sol.
I
I
r7

modorolc
rllght
nonc
Ftc. l8bis. The responseof the pain of a lesion to an agent permits to identify the
acid or alkaline pattern present.The effect of an acidifying and alkalizing agent correspondsto an increaseor a decreasein pain intensity, according to the pain pattern
present.First row, left side-acid pattern, right side-alkaline; second row, left sideacid, right side-alkaline; third row. right side-alkaline, left side-acid pattern.

DUALTSM

57

formed. Specialglasselectrodest were used for this purpose and determinations were carried out employing a sensitivepH meter. The tip of the
electrodewas placed on the surface of the area to be testedif ulcerated,or
was introduced into the tissue to be tested through a small incision. In
reality this givesa measurementof the pH of the local interstitialfluid.
Urine pH and local pH determinationswere then performed at difterent
times correspondingwith spontaneousvariationsin the pain intensityexperiencedby the patient. Simultaneously,the pH values of normal tissue
areas and, when possible,of nonpainful tumor areas were determined.
Similar studieswere carried out after administrationof strong acidifying
and alkalizingagents.
Many difficultieswere encounteredboth in the choice of suitableclinical subjectsand in techniques.Neoplasmsproved to be the simplesttype
of painful abnormalprocessto employ.The neoplasmhad to be locatedin
a readily accessibleregion so that the electrodescould be introducedinto
an ulceratedarea or through small incisions.The patient had to be able
to very accuratelylocalize the area of pain since considerabledifferences
in pH valueswere found to exist in difterentparts of the same lesion.The
pain had to be superficiallylocalizedbecauseaccuratedeterminationsin
the depths were not possible.Finally, the complete cooperation of the
patientwas essential.
The data obtained for a patient with an ulcerated,profusely draining
carcinomaof the breast is recordedin TnsLe II. Pain was most intense
T,rsl.e II
Onse.nvrtroNsIN A Crse wlrn Alx.,rllxe P,rlx P,rrrenx

Treatment (oral)

Phosphoricacid
S O V o . 2c c .
Sodium bicarbonate
5 grams

Pain Intensity pH Urine

pH Normal
Tissues

pH Tumor

None
Moderate
Very severe

5.4
6.2
7.1

7.3
7.4

7.6
8.1
8.5

Slight

5.5

7.3

7.9

Unbearable

7.8

1 A
I .t+

8.8

when the acid-basebalance of the blood, as reflected in the urine pH


changes,was relatively most alkaline, and was less intensewhen the balancwas more acid. The pH valuesof the painful areasof the tumor in
* S u p p l i e db y H a r t m a n a n d B r a u n , P a r i s .

58

R E S E A R c Ht N p H y s r o p A T H o L o c y

this case showed considerablelability under the influence of spontaneous


changesin the general acid-basebalanceof the body, reaching a high of
8.5. At this time, the pain was very severe.The pain becameunbearable
following oral administrationof 5 grams of sodium bicarbonate,and the
pH within the sametumor area reached8.8. The pH of the normal tissues
in this case,even after the administrationof strong alkalizingagents,never
exceeded7.4, while the tumor pH was neverbelow 7.6.
The findings in a casewith acid pain are recordedin TenlE III. This
T,rsr-EIII
OnseRv.rrroNs
rN A C,r,se
wrrH Acro prrN prrrrnN
a=_

pH TUMOR
Treatment(oral)

Pain
Intensity
None
Moderate
Severe

Phosphoricacid
5 0 V o, 1 . 5 c c .
Unbearable
Sodiumbicarbonate
5 grams
None

p H r pH Normal Painful
Urine
Tissues i area

7.0 l

7.4

i 66 . 3r

Nonpainful area

5.8
5.3

t.)

5.8

7.2
7.1
6.9

5.0

t.-)

5.5

6.8

6.6

7.2

l.+

patient had an extensivesarcomaof the face which was not ulceratedbut


involved the skin. Pain was most intensewhen the blood titrimetric alkalinity was relatively low and less severewhen the alkalinity was higher.
The pH values in the tissuesof a painful area of the tumor were always
more acid than the values found in another nonpainful area. The pH of
the nonpainful area was slightly below that within normal tissues.Spontaneouschangesin the acid-basebalance of the body brought about a
reductionof the pH of the painful tumor area to 5.8, at which time the
patient reported severepain. The nonpainful tumor area had a pH of 6.9
at the same time. Following administrationof L5 cc. of 5o% phosphoric
acid, pain becameunbearablcand the pH within the tumor tissuesof the
painful area fell at oncc to 5.5. At the same time, the nonpainful tumor
areapH was 6.8 and the normaltissuepH was 7.3.
Similar results were obtained in other casesand lcd to the following
conclusions:
The pH valuesof the interstitialfluid of painful lesionsstudied
in vivo differ from thoseof normal tissues,the hydrogenion concentration
being eithcr higher or lower than that of normal tissuesof the same individual. Thc pH of painful abnormal tissuesis much more labile and

DU^LrsM

59

extremelysensitiveto generalbody pH changes.Spontancousor induced


variations in the acid-basebalance of the body produce slight changesor
none at all in the pH of the interstitialfluid in normal tissues,but give rise
to more pronouncedchangesof the pH within painful pathologicaltissucs.
Through this research,we have thus been able to conncct pathological
pain to the pH of interstitial fluid of painful lesions.A further connection
could be made with the richnessof the interstitialfluids in potassium.In
the alkaline pattern of pain, more potassiumwas found in the interstitial
fluids. The presencein thesefluids of potassium-the cation of the cytoplasma-in higber amountsthan in normal conditionswas scen to induce
pain. The subcutaneousadministrationof potassiumcompoundswas secn
to be painful, while similar saltsof sodium werc well tolcrated.As a local
acidosiswas found in lesionshaving an acid pain pattern, a local alkalosis
and an increasein potassiumcontcnt appcarcdin those with an alkaline
pain pattern. The intensity of pain in this case was found to be proportionate to the degrec of abnormal deviation of thc local pH and to the
abnormalamountsof potassiumin the interstitialfluids. In addition to
variationsin the acid-basebalanceof the body, variationsin the amount
of serum potassiumappear able to alter pain intensityin caseswith an
alkalinepattern.This correlationis further explainedbelow by the place
of potassiumin the organization.
O.rido-red uction Potential
Differencesindicatingthe samedualismwere also found in other manifestations
in painfullesions.The oxido-reduction
potentill was measuredin
tumorswith pain. Patientschosenwere thosewith easily accessiblesuperficial tumors in wbich painful areas could be localized. Platinum needle
electrodeswere introducedin the painful areasthrough small incisionsor,
if the lesionswere ulceratedin the lesionsthemselves.
The measurements
of thepotentialpresentweremadeusinga BeckmanpH meter.As had been
thecasefor the pH, it appeared
importantthat thc patientbe ableto indicate
clearlythe painful areas.Becauseexactlocationof theseareasin the depths
was almost impossible,superficiallesionsusually were chosen for these
determinations.In general,in lesionswith an alkaline pattern, the measurementsshowedhigh values (such as from +100 to +350 millivolts)
whilein lesionswith pain of the acid pattern,the valueswere low (such as
-2 to -15 millivolts).

60

REsEARcHrN pHysropATHoLocy

Abnormal Substances
changes in the local pH, like changesin acid and alkaline patterns,
could be related to the appearanceand subsequentaccumulationof difterent substancesin the lesion. Increasedconcentrationof lactic acid was
found in the interstitialfluids of acid pattern lesions,while increasedconcentrationsof sodium and especiallypotassiumions were found in the
interstitialfluid of lesionswith an alkalinepattern.
Processesleading to a local acidosisare known and ascribed to the
well-known anoxybiotic metabolismof carbohydrates.They result from
lack of the "respiratory" oxybiotic phase of carbohydrate metabolism,
with the consequentconversionof pyruvic acid into lactic acid. only part
of the lactic acid is changedinto glycogenthrough the pasteur-Myerhoff
reaction,and an accumulationof lactic acid results.While the presenceof
lactic acid is well known, its presencehas not previously been related to
pain or other manifestations.
We could showthis correlationin somecases.
(Note 3 )
The scientificliteratureofreredno information concerningthe appearance of alkaline compounds.We were able to establishthat the presence
of sodium ions coincideswith another anomaly found in theselesions-a
high fixationof chloridesin the lesionsthemselves.
Valuesof 1600 mgr. of
cl., or even higher, per 100 gms. of wet tissuewere found in theselesions
insteadof about 400 mgr./ 100 gms. measuredin normal tissues.The local
alkalosisand the resultingalkaline pain pattern could thus be correlated
with an abnormal sodiumchloride metabolismat the tissularlevel in which
with chloride ions fixed by the cells, sodium ions remain free to combine
with carbonate anions and form alkaline compounds. If the abnormal
NaCl metabolismoccurs in the interstitial fluid, the subsequentalka.losis
inducespain. We will seelater how abnormal NaCl metabolismalso takes
place at other levelsof the organization.In thesecasesof alkaline pattern
of pain, the fact that abnormalamountsof sodium ions still enter the cells
will result in a loss by thesecells of potassiumwhich will accumulatein
the pericellularfluids,and form alkalinecompounds.
An immediateconclusionthat could be drawn from these studieswas
that there is a definitedualismin pathogenesis
of pain originatingin abnormal tissuesand that the two pain patternsevidencedin lesionswith acidosis
or alkalosisof the interstitialfluids indicatethat processesof two opposite
naturesgo on at the tissularlevel.

DU^LtsM

OTHER ACID AND ALKALINE

6l

SYMPTOMS

using the same method of investigationas in the study of pain, a relationship betweenvariationsin intensityof certain other symptomsand
variationsin urinary pH could be found. Acid and alkaline patternsof
itching could be recognized.The samepatternscould be found for vertigo
and impairedhearing.Among psychiatricmanifestations,
manicdepressive
statesshowedchangesthat could be relatedto acid-base
variations.
The correlationbetweendyspncaand acid-base
variationswas contrary
to what we expected.classically,dyspneais believedto be associated
only
with an increaseof acidityof blood. However,both acid and alkalinepatterns of dyspnea were observed,and could be related more directly to
changesat the tissuelevel.We will rcviewherethe pathogenesis
of itching,
vertigo,dyspnea,and other conditions,undcr this dualisticaspect.
Itching
The problem of itchingis interestingfrom other than the therapeuticpoint
of view, sincelittle is known regardingthe natureand causeof this condition. Various hypotheses
have been offeredbut have failed to explain its
pathogenesis.
our interestin itching originatedduring the study of pain
and led to an hypothesiswhich allowsus to approachthe problemfrom a
new angle. By analogy with pain, we separateditching into two types:
physiological,
as the responseof normal tissuesto externalstimuli; and
pathological.as a sensationarising within diseasedor damagedtissues.
Ticklingthe normalskin or evenstimulatingit throughheat.colcl,etc.,may
causa sensationof itchingand lead to scratching,Certainmucousmembranes,such as thoseof the nose,and the skin aroundnaturalorifices,arc
especially
sensitiveto such stimulation.This type of itching,as a response
of normal tissuesis phy,siological
or sensoriul.
Under pathologicalconditionshowever,the skin, mucousmembranes
andother formationsmay itch without externalstimulationor in response
to stimuli which ordinarilydo not producethis sensation.
The itching then
can be consideredas a manifestation
of diseasedor damagedtissuesand,
as such can be describedas pathological.Just as does pathologicalpain.
pathologicalitching representsa symptom rclated to abnormal changes
afreadypresent.(Note 4)
In spite of their relative independence,
the fact that similar fundamentalmechanismsare involved in the productionof itching and pain
explainscertain characteristics
they have in common. Like pathological

62

R E S E A R c Hr N p H y s I o p A T H o L o c y

pain, pathologicalitching varies in intensitywith the time of day. patients


with chronic pruritusare awareof this,some have more itching in the morning, others experienceexacerbationsat night. The same dualism is seen
with intake of food. For thesereasons,the relationshipbetweenchanges
in acid-basebalanceof the body, as reflectedin the urinary pH, and variations in intensityof itching was investigated.
Patientswith long standingpruritus associated
with a variety of chronic
skin conditionswere studied.They were askedto note over periods of six
to twelve hours the changesin itching intensity.Evaluation of the changes
was made by the patientsthemselves,using a seriesof qualificationssuch
as none, slight, moderate,severe,very severeand unbearable,or a scale
rangingfrom 0 to 10. They were instructedto considerthe averageinten-

lTCHING

Very Severe
Seyere
Moderote
Slight
None
PH
Urine

5.5

6.O
5.5
5.O
t

4
Hours

Ftc. 20. Thc alkaline puttern ol itching is recognized i n a c a s e o f s e n i l e v u l v a r


p r u r i t u s ,t h r o u g h p a r a l l e l v a r i a t i o n so f t h e c u r v e s o f t h e intensity of the itching and
of tbe urinary pH.

DUALTSM

63

sity of their itching for each hour, rather than to indicate the maximum
intensity at the exact time of recording. voided urine specimenswere
obtained at the end of each hour. The pH of the urine specimenswas determined electrometrically.A graph was plottcd to compare thc variations
in the subjectivedata furnishedby the paticntwith the concomitanthourly
changesin the pH of the urine. It was usuallynecessaryto repeat the test
severaltimes before the patient appearedable to satisfactorilyevaluatethe
changesin the intensity of the symptomfor hour-long periods rather than

ITCH IN G

VerySeyere
Severe
Moderote
Slight
IVone

fi_

Urine

7.O
6.5
6.O
5.5

Hour
FIc. 21. An acid patlern ol itching is recognizedin a caseof pruritus ani through the
divergencein the concomitant variations of the curves of itching intensity and urinary pH.

for just the moment of recording. (we also tried to judge the intensity of
itching through the frequency,intensityand duration of scratching,as
notedby an observer,but without success.)Fiftecn patientswere studied
and, becauseof the limited number, the resultsare presentedas merely
preliminary.

64

x E S E A R c Ht N p H y s l o p A T H o L o c y

In four cases,the curvesof itching and urinary pH did not show any
definitecorrelationeven after repeatedtests.of the remainingI I cases,7
showed a distinct parallelismof the two curves, Md in the other 4, an
inverserelationshipbetweencurveswas apparent.The graphs obtained in
two characteristiccasesare presentedhere. (Frgs. 20 and 2l )

Severe
lvloderole
Slf9ht

HsPOe

None

pH
Urine

7.O
6.5
6.O
5.5
5.O

Hours
F t c . 2 2 . A d m i n i s t r a t i o no f p h o s p h o r i ca c i d - 1 . 5 c c p h o s p h o r i ca c i d , s o l . 5 0 c Z - i n d u c e s t o g e t h e rw i t h a n a c i d i f i c a t i o no f t h e u r i n e , a n i n c r e a s ei n t h e i n t e n s i t vo f t h e
itching with an acid pattern.

There is a distinctparallelismbetweenthe two curves in Figure 20.


indicatingthat itchingwas more intensewhen the urine was relativelymore
alkaline,and slightor absentwhen the urine was more acid. We have consideredthis as an "alkaline pattern" of itching in accordanccwith the
designationfor pain, An inverserelationshipbetweenthe two curves is
seenin Figure 21. ln this case,itching was more intensewhen the urine

DU^LtsM

65

\\'asmore acid, and less severewhen the urine was more alkaline.This
representsan acid pattern.
The effect of a strong acidifying agent,phosphoricacid, in caseswith
acid and alkalineitching,is illustratedin Figures22 and 23. The intensity
of the alkalineitchingin the first casewas reducedby the acidifyingaction
of phosphoricacid, while the acid itching of the secondcasewas intensified. In Figure 24, the responseof a patient with alkaline itching to the
administrationof sodium bicarbonateis shown. The intensityof itching
wasgreatly increasedafter the alkalizingagent was given.

ITCHING
Seyere

IH'Po'

Moderote
Slight
None

pH
Urine

7.O
5.5
6.O
5.5
4
Hours

F l c . 2 3 . l t c h i n g w i t h a n a l k a l i n ep a t t e r n i s r e d u c e di n i n t e n s i t yb y t h e a d m i n i s t r a t i o n
o f a n a c i d i f y i n g a g e n t . I n t h e a b o v e e x a m p l e ,t w o d o s e s ,e a c h o f t . 5 c c p h o s p h o r i c
a c i d( 5 0 % ) , w e r e n e c e s s a r iyn o r d e r t o o b t a i n t h i s e f f e c t .

The fact that both pathologicalpain and pathologicalitching undergo


thesamechangesin intensityrelatedto the generalacid-basebalancewould
indicatethat a similar mechanismmay be involvedin the pathogenesis
of
bothsymptoms.It can be conceivedthat a slight local pH changeconfined
to the skin or mucousmembranecould act on the itching end organsand

66

R E S E A R c Hr N p H y s r o p A T H o L o c y

evokethe sensation
of itching.More intensepH wouldresultin pain.The
fact that itching,one of the principalsymptomsof dermatological
conditions,can be relatedto local acid and alkalinechangeswithin the skin
wouldpermit the integrationof skin pathologyin a more generalphysiopathological
mechanism.
The conceptof the interventionof two difterent

ITCHING

Seyere
lvloderote
Slight
None

pH
Urine

7.O
6.5
6,O
5.5
6
Hour

F r c . 2 4 . T h e a d m i n i s t r a t i o no f 4 g r a m s o f s o d i u m b i c a r b o n a t et o a s u b j e c tw i t h a n
alkaline patteril of. itching induces together with the alkalinization o f t h e u r i n e , e x acerbalion of the symptom.

one rcsultingin acid substancesand the other in alkaabnormalprocesses,


line compounds,reprcsentsa new approachto the study of many skin
conditions.The therapeuticapplicationof this concepthas producedinterestingresults.(See ChapterXIV)

DUALTSM

67

Vertigo
vertigo has been of specialinterest in our research.while it can be
inducedby various etiologicalfactors, a basic dualism in its pathogenesis
is evident.
Many patientsrvith vertigo have been found to experiencewide variations in the intensity of the symptom. Some have exacerbationsin the
morning,others in the evening.This observationsuggesteda possibledual
patternand we investigatedvertigo by the samemethod usedfor the study
of pathological pain.
In patients with vertigo, the intensitywas determinedat hourly intervalsby using either a scaleranging from 0 to 10, or a seriesof qualifica-

x
@
g

(u

5
U'
o
OJ
g
N
N

8.7

7.
H
!

7o
o 5.6
L

o
c
L

=
,'D

46

Noon

pm.

midnightatn.

Frc. 25. Shows the curves of the intensity of lerligo and of the urinary pH in a case
w i t h a n a l k a l i n e p a t l e r n , t h e v a r i a t i o n so f t h e t w o c u r v e s b e i n g p a r a l l e l . ( 8 . W e l t ,
A M A A r c b i v e s o f O t o l a r y n g o l o g y ,5 8 : 2 7 3 - 3 0 0 ,1 9 5 3 . )

68

REsEARcHrN pHysropATHoLocy

tions, such as none, slight, moderate,severe,very severeand unbearable.


At the same intervals,urine sampleswere collectedand their pH values
measured.Data were plotted in curveshaving time as common abscissa.
Here again, as for pain, two different correlationscould be observed.In
somecases,the two curves--{ne for intensityof vertigo, and the other for
urinary pH level-showed parallelvariations.(Fig. 25) The vertigobecame
more intensewhen the pH was high, and this was consideredto be vertigo

:\
v,

c
o
c
o

CD
L

(u

o
(_
ftt
E
L

Hours
Ftc. 26. Vertigo with an acid pottern. The symptom is more intensive when the
urinary pH values are lower. (B. Welt A M A A r c h i v e s o f O t o l a r y n g o l o g y ,5 8 : 2 7 3 300,1953.)

DUALtSM

69

of an alkaline pattern. In other cases-of an acid pattern-vertigo was


m o r ei n t e n s ew i t h l o w e r v a l u e so f u r i n a r y p H . ( F i g . 2 6 )
The acid-basepathogenesi.s
of vertigo was further confirmed by the
responseto acidifying and alkalizing agenrs.B. welt has widely investigatedvertigo by this method. He also has used the responseto therapcutic agentsas a criterion for the pattern present.The administrationof
[glenel, heptanol or unsaturatedfatty alcohols was seen to induce an
increasein the intensity of vertigo of acid pattern and a decreasein the

500cycles
--- 1000cycles
-2000 cycles
o2o
C)

'3a

o
o

,'=u4j)

an9^-to-tl-t2-/^;-Z-J

zt
Elq
L

Eto
L

I
n.n

t2

HouFs

t
e^1.

Fto. 27. The analysis of the concomitant changesin thc curvc of the intensity of thc
auditive acuity and of the urinary pH shows opposite variations iu a subject with
impairedhearing. The opposite variations arc especiallymanifest for some velucr2000cycles in this case. This rclationship corrcspondsto an acid pottern. (Courtesy
of Dr. B. Welt.)

70

*EsEARcH rN pHysropATHoLocy

alkaline type. Agents such as sodium thiosulfatehave an opposite eftect.


The great similarity in the responseof pain and vertigo to the same
agentshas establishedthe role of acid-basechangesin the pathogenesis
of
vertigo as well as the possibilityof influencingthese changesin order to
relievethe symptom.Thesestudieshave shown that, in spite of the variety
of etiologicalfactors which can induce vertigo, the condition can be considered,from the point of view of therapy,in terms of its dualistic pathogenesis.This has simplifiedthe therapeuticapproach,limiting it to a choice
betweenonly two groups of agents.We will see later how successfulthis
approachhas been in Welt's hands.

:b

.at

1to

{A

tO

&

.t-

r-.

a6

a-

F. f{.
In i t i a l
PostPlacebo
P o s tA c i d i f i c a t l o n

u,
q,

o
o
o,
C

.n
C'
-t

ttiohtear A.C.

\.

\
F r c . 2 8 . T h e a d m i n i s t r a t i o no f a n a c i d i f y i n g a g c n t - 2 g r a m s o f m o n o a m m o n i u m
phosphate-induces a marked increaseof the heoring acuity if. the abnormality presenl correspondsto an alkaline pattern. Only minimal or no changesare seen to occur
if a few drops of acetic acid are used as placebo. (Courtesy of Dr. B. Welt.)

7l

F. |l.
- - - lnltial
"'-" PostPlacebo
-Post Acidification

o
o,
D

(,
c,

o
c

ti
an
C'
J

L e f t e a rA . C .

Fto. 29. Alkaline pattern of hearing impairment correspondsto an increasedhearing


a c u i t yf o l l o w i n g t h e a d m i n i s t r a t i o no f 2 g r a m so f a m m o n i u m p h o s p h a t e .

.*i*r#d'ltW$l$*iliffik,s;l.ii$i,,i#:

72

RESEARCH IN

PHYSIOPATHOLOGY

o
A. l.

--- Initral
F o s tA c i d r f r c l i r o n

o
tf,
20

zf
?t
(y
cl
IJ

&
,t

o,

c ,o
rt
an
o

af

to

Left car
to

30. Alkalinc pattern of hcaring impairment.

DUALISM

73

Impaired Hearing
In studying thc various otologicalprobrems,B. welt and I noted
thatmany patientswith impaired hearingexperiencedvariationsin auditive
acuityat different times of the day, or even in conjunction with food intake.This led us to investigateimpairedhearingby the samemethod used
for pain. (Fis. 27) we studied the influenceof acidifying and alkalizing
agentsupon auditive acuity in casesof impaired hearing.
complete audiograms were obtained, employing differences of only
two decibelsbetweenmeasurements
and using all of the acceptedfrequenciesfor air and bone conduction.Audiograms were obtained in subjects
beforeand after administrationof acidifying agents,such as ammonium
fe

7fo

t6o

K.H.
- -- lnitial
' ..' ....
PostPlacebo
P o s tA c i d i f i c a t i c n
o
o
D
(J

c,
o
c
t^

o
o

'at-e'a

-'-

-./'/-./u"
'"
a-.

L e f t E a rA . C .

F r c . 3 t . T h e a d m i n i s t r a t i o n o f 2 g r a m s o f m o n o a m m o n i u m p h o s p h a t ei n d u c e s a
decreascin hearing acuity if the abnormal pattern is acid.

74

R E s E A R c Hr N p H y s r o p A r H o L o c y

chloride and ammonium monophosphate,and alkalizing agents such as


sodiumbicarbonate.In normal subjects,the audiogramsshowedlittle or no
change.In subjectswith impaired hearing,three types of responseswere
noted for any one agent.The audiogramwas either not changedat all or
an increaseor decreasein acuitywas seen.If a manifestincreasein acuity
2ooo

K .H .
----|nitial
- * - - P o s tP l a c e b o
-f65[
Acidification

U'

o,

-r/--t'

II

-'1

I
(u
o

t
"t""'-

o
U'
o

llight Ear8 . C .

ta

2ooo

4ooo

K.H.

at

o
o

--- lnitial
' - - - - PostPlacebo
-Post Acidification

(,

o
ct

C
v,
U'
C'
-J

L e f t E a r8 . C .
Ftc. 32. Acid pattern of hearingimpairment.

was obtained with one group of agents,an opposite effect was obtained
when an agentof the oppositegroup was administered.
changes of at least 6 decibelswere required before they were considered to be induced by an agent.when changesof such intensity were obtainedwith an agent,it was invariablytrue that oppositechangeswould be
obtainedwith an oppositeagent.It was also true that the same responses

DUALTSM

75

couldbe obtainedin the samepatient in repeatedtests.This appearsto be


highlysignificant,indicating that the responsewas, in fact, correlatedwith
changes
induced by the administeredagent,changessimilar to those seen
in thecaseof acid-basesymptoms.It was thus possibleto integratehearing
impairmentin the group of acid-basesymptoms and to recognize two
well-definedtypes, one correspondingto an acid pattern, the other to an
alkaline.Figures 28 through 32 illustrateseveralcasestaken from Welt's
observation.It must be noted that changesunder the influence of the
agentsare seenat almost all frequenciesin some casesbut at only certain
frequencies
in others.
It must be noted also that not all casesof impaired hearing could be
placedin one or the other category.while this could be done almost without excePtionfor young subjectsor for thosewith still evolvingconditions,
it could not be done for subjectswith old, fully evoluted impairment. It
aPpearsthat once the pathologicalprocesseshave arrived at a terminal
point-and an inactive scleroticscar is present-a responseto acidifying
or alkalizingagentsis no longer to be expected.

Manic-depressiveCondition
We have studiedthe relationshipbetweenintensityof manifestations
and
systemicacid-basebalanceof the body in another group of subjectswith
variousmental disorders.of all the casesstudied, the only condition in
whicha clear relationshipcould be shown was in manic-depressive
subjects.From patients presentingchangesduring the day, passing from
periodsof high excitation to calm, from deep depressionto calm, we obtainedhourly urine specimens.At the same time, observationsof their
mentalstate were made. The evaluationsof mental condition were made
by trained observersor membersof the family using a conventionalscale
whichpermitted translationinto graph. The pH of the urine sampleswas
measured
electrometrically.Curvesof the pH and of the mental conditions
wereplotted having the common time as abscissa.A striking correlation
betweenthe two curves was found in the first investigationsin manic patients.with the pH of the urine at higher values, the patient was calm,
whilethe more acid urine correspondedto periodsof intensiveagitation.
Figure33 showsan example.
This correlationalsocould be demonstrated
by administering
acidifying
andalkalizingsubstances
to manic patients.Acidificationthrough the administrationof phosphoric acid was followed by a manifest increasein
agitationwhile administrationof an alkalizingagent-sodium bicarbonate

76

REsEARcHrN pHysropATHoLocy

-was followed by a pcriod of calm. An oppositebut less evidentcorrelation was seen for severaldepressedpatients.we must mention that the
usual difficulty of judging accuratelythe degreeof depressionfrom one
hour to another can explain this lessercorrelation.Manic manifestations
were much more readilv evaluated.

o
(o
q)

o_

Io

(u

c 6o
L

Hours
Fro. 33. Tbe variations of the curve of the ogitotion of a lO-year-old boy are oppositc to that of the urinary pH, indicating an acid pattern.

DUALTSM

77

Dyspnea
Certain characteristicsof dyspnea suggestedthat this symptom might be
exploredby the srme meansemployedin studying pain associatedwith pathological tissue changes.A regular pattern is observed in certain types of
dyspnearelated to the time of day. Cardiac asthma or paroxysmal cardiac
dyspneaoccur mainly at night after patients have gone to sleep. However,
this is not due to the recumbentposition since patients may assumethis
position during the day without any ill effect. Harrison's "evening dyspnea"
(20) is characteristicallyabsent in the morning, but develops slowly during the course of the day, reaching a maximum in the evening.
Just as for pain, the degreeof intensity of dyspneawas studied in relation to hourly acid-basebalancechangesas indicated by changesin the pH
of the urine. Patientshaving dyspneaof prolongedduration as a result of
variouspathologicalconditions,with no treatmentof any kind for at least
six hours before or during the period of observation,were the subjectsof
this investigation.The degreeof intensityof the dyspneawas estimatedby
trainedobserverswho were in constantaftendance.Dyspneawas recorded
by the observersas absent,slight, moderate,severe,or very severe,estimationsbeing basedupon rate depth and evidentdifficulty in respiration.
At the time of these observations,hourly urine specimenswere obtained from patients with as little disturbanceas possible. No patient
showedevidenceof renal disease.The pH of the urine specimenwas determined potentiometrically.The curves showing hourly fluctuations in
the intensity of the dyspneaand the changesin the pH of the hourly urine
specimenswere then plotted and compared.Acidifying and alkalizingsubstanceswere administeredto patients during the course of some tests in
order to observe the influence of induced changesin the acid-basebalance
uponthe degreeof dyspnea.Phosphoricacid and sodium bicarbonatewere
usedfor this purpose.
Fourteen patients with different pathologicalconditions were studied.
In ten cases, a distinct correlation betweenthe intensity of the symptom
and acid-base variations was found.
Four patients had pulmonary edema associatedwith the symptom of
dyspnea.One patient had edemadue to congestiveheart failure; another
had pulmonary edema and lung metastases
from a carcinomaof the pancreas.Two other patients with cancer of the breast metastaticto the bone
andskin also had pulmonaryedema.In one of these,the pulmonaryedema
appearedto be a result of the accidentalintroduction of a fatty acid in oil
preparationinto the blood stream following an intramuscularinjection. In

78

n E S E A R C Hr N p H y s r o p A T H o L o c y

all four of the caseswith pulmonaryedema,the intensityof dyspnea


was
found to be increased
whenthe urinarypH showedchangestowardmore
alkalinevaluesand was diminished
whenthe pH changes
wereopposite.
In thesecases,the intensityof the dyspnea
wasrelievedfollowingadmin-

DYSPNEA
Seyere
Moderote
Stight
None

pH
Urine

6.5
6.O
5.5
5.O
4

Hour
Ftc. 34. The parallel variations between the curves of the intensity of dyspnea and
o f t h e v a l u e o f t h e u r i n a r y p H i n a c a s e o f p u l m o n a r y c o n g e s t i o nf o l l o w i n g a n
a c c i d e n t a li n t r a v e n o u si n j e c t i o n o f a f a t t y a c i d p r e p a r a t i o n ,i n d i c a t e s a n a l k a l i n e
patlern.

istrationof phosphoricacid, while sodiumbicarbonateincreasedthe dyspnea. By analogywith pain, we called this correlationan alkalinepattern.
(Fie.34)
Six casesshowedan oppositetype of correlationbetwecnthe intensity
of dyspneaand the acid-basechangesin the pH of the urine, All of these

DUALISM

79

caseshad mediastinalor pulmonary massesand failed to show signs of


pulmonaryedema. In these cases,the maximum degreeof dyspneawas
associatedwith a relatively more acid urine, and the dyspnea was less
intensewhen the urine was more alkaline.In thesecases,phosphoricacid
increased
the degreeof dyspnea,while converselysodium bicarbonatedec r e a s eidt . T h i s w o u l dc o r r e s p o n tdo a n a c i d p a t t e r no f d y s p n e a(.F i g s . 3 5 ,
36)
Thesefindings,althoughobtainedin only a limited number of patients,
stronglysuggesta similaritybetweenthe fundamentalorigin of both pain
anddyspnea.As in pain, the two patternsof dyspneawere associatedwith
a relative alkalosisand a relative acidosis.

Severe
Moderote
Slighl
None

pH
Urine

6.5
6.O
5.5
5.O
3

Hour
F r a . 3 5 , T h c o p p o s i t ec o n c o m i t a n tvariations betwcen the intensity of dyspnea and
urinary pH in a case of mcdiastinal metastasesof a hypernephroma indicatc an acid
paltefn,

80

REsEARcH tN pHysropATHoLoGy

certain differencesexist betweenthe investigations


of pain and dyspnea. In studyingpain, it was necessary
to dependentirelyupon the observations
of the patientas to the relativeintensityof the pain experienced
from hour to hour. In dyspnea,the patient'sown observations
werefound

oYSPNEA

NoHCOs

Seyerc

Moderotc
Slight
None
7.5

70
6.5
6.O

5.5
f

Hour
Flc. 36. The administration of sodium bicarbonatc increases the intensity of the
dyspneain a casc of pulmonary metastases
of cancer of the gall-bladder,indicatin3 an
alkaline pattcrn.

to be less reliable due to the emotional factors associatedwith dyspnea.


It was, however, possibleto depend upon trained observerswho could
estimateaccuratelyenough the degreeof dyspnea on the basis of rate,
depth and apparentdifficulty of respirarion.It was found that the greater
the experienceof the observer,the closer was the correlation betweenthe
curyesof intensity of dyspneaand the changesof the urinary pH values.

DUALtSM

8l

since it appearedevident that acid-basechangesplay a significantrole


in influencingthe degree of dyspneacxperiencedby the patient in two
oppositedirections,it was important to considerthis influencein relation
to the physiologyof respirationwhich is known not only to be affectedby,
but even dependenton acid-basechanges.
concerning the respiratorycenter, two different mechanismsof direct
chemicalstimulationhave beensuggested
as being responsiblefor dyspnea.
One is lack of oxygen, but considerableevidenceexists to indicate that
simpleanoxemiais not a true causeof dyspnea
. (zl, 22) on the other
hand,an acidosisof the respiratorycenter is known to causedyspnea,but
it has been establishedthat only a very profound generalacidosis,such as
that connectedwith diabetes,acid poisoningor emphysemacan create a
changein blood pH sufficient to affect the respiratory center. This is also
true for the chemoreceptorcenterslocatedin the carotid sinus, which are
still lesssensitiveto the generallack of oxygenor acidosisthan the respiratory center. while general acid-basechangessecm to play an important
role in dyspnea,thesechemicalchangeswould not appear to act directly
upon the respiratorycenter since the actual blood pH is not sufficiently
changed.
other factors-involving direct local, rather than systemicinfluencesmaybe considered.One suchdirect action would be relatedto reflex stimulation which is generally accepredas having an important role in the
controlof respirationand even dyspnea.This reflex control of respiration
is connected with nerve endings within the lung parenchyma which are
stimulatedwhen the walls of the alveoli are stretchedin the respiratory
phase.The stretch reflex inducesimpulses,carried by way of the vagus,
which acting upon the respiratorycenter, stop the inspiratory phase and
bring about expiration. It may be assumedthat the nerve endingswithin
theparenchymacan be stimulatednot only by mechanicalchangeswithin
theparenchymabut also by local chemicalchangestoo. Under abnormal
conditionssuch as pulmonary congestion,there may be a change in the
tissuereaction, as has been recognizedby a higher pH of the edema fluid
itself.Through this reflex mechanism,these pulmonary tissue reaction
changes
may producedyspnea.The generalacid-basefluctuationsinfluencingthe local alkalosiswould, as seenin pain, indirectlyinfluencethe degree
of dyspnea.such a mechanismmay accountfor dyspneawith an alkaline
patternfound in pulmonary congestion.
A simi.larlocal factor also could be seen for the acid pattern. It was
observedthat all patients with an acid pattern of dyspnea had tumors
locatedwithin the mediastinumor in the lung parenchymaitself. From

82

R E s E A R c Ht N p H y s r o p A T H o L o c y

the study of pain, we know that abnormal degreesof acidosiscan occur


in tumors.pH changestoward acidosiswithin a tumor tissuein the vicinity
of chemoreceptorsmay produce impulse dischargesin these centers especially sensitiveto changestoward acidosis.They may alter the character
of respirationand result in dyspnea.As in other conditions,these local
changesoccur even with reducedchangesin the systemicacid-basebalance.Through this mechanism,variationsin the intensity of dyspneacan
occur without the intensivechangein the generalacid-basebalancewhich
is considerednecessaryto affect directly the respiratory center itself.
The fact that relatively small acid-basebalance changesaffect the intensityof dyspneain two oppositedirectionscan thus be explainedby this
indirect influenceexertedupon a local process,Ieadingto alkalosisin abnormal conditions affectingthe pulmonary parenchymaand to acidosisin
lesionspresentin the neighborhoodof the chemoreceptivecenters.These
two mechanismsproposedas part of this working hypothesis,appear able
to explain the paradoxicalexpcrimentalfindingsin clinical studiesof this
symptom, where opposite responsesupon dyspneaare seen for the same
acidifying and alkalizingagents.
A more completestudy of dyspneaunder this aspectwill be published
separately.
Dualistic Patterns at Other Levels
Cellular Level
Cytologicalstudieshave revealedthat some characteristicsof the cell,
other than those typifying the cancerousanomaly itself, exhibit dual patterns.For the cellular level, they could ultimately be related to changesin
the aging processes.In tumoral foci having an acid pain pattern, cytological
characteristicsindicate a prolonged cellular youth. A round aspect of the
nucleus,with a fine texture of chromatin and well-separated
nucleolus,
and a basophylcytoplasmrepresentmajor characteristics
of cellular youth.
In lesionswith an alkalinepain pattern,the cells show rapid early aging.
The tendencyto lobulationof the nucleus,to separationof chromatinand
formation of clumps, to cytoplasmaticoxyphily and the appearanceof
azurophylgranulaecharacterizesuch aging.Rapid aging was seento lead
to prematurecellu.lardeath through piknosis and karyorrhexis.Opposite
cellular aging processescould be further related to differencesseen in evolution of tumors. Rapid aging of cells,associated
with alkalinepain patterns, results in necrotic tumors and ulceration of superficial lesions.
Frequently,it could be noted that a changefrom acid pattern to alkaline

DU^LtsM

g3

occursand is accompaniedby a melting away of massivetumors and their


replacementusually by ulceration.
TissularLevel
As seen above,pain, dyspnea,vertigo and itching can be considered
tissuelevel manifestationswhich show dualism. Additionally, the nasal
pH (Note 5/ deviateseithertoward acid or alkalinevaluesand thesedeviationscan be correlatedwith similar acid-base
changesat the tissularlevel.
Thesemeasurementsare useful as a diagnosticcriterion for tissue level
changes.
Organic Level
The same dualismobservedat the cellular and tissuclevcls also was
foundin signsand symptomsinvolvingthe organ level.Dual patternswere
foundfor dysfunctionsof variousorgans.Insomniaand somnolence,
diarrheaand constipation,oliguriaand polyuria,tachycardiaand bradycardia,
all representexamplesof dualism at the organic level.
The dualismevidentfor the lengthof persistence
of a wheal (Note 6)
inducedby intracutaneous
injectionof a salinesolutionwas also related
to the organ levelwith the skin considered
to be an organ.In normal subjects,the resorptionof the wheal is completedin about ls-zo minutes.
In onegroup the resorptiontime is short,evenreducedto a few minutes.In
othergroups, on the contrary, the resorption time is greatly prolonged,
thewheal sometimesbeing presentevcn after more than 90 minutes.
Svstemic
Level
Studiesof dualism at this level covered temperaturevariationsand
changes
in variousblood and urine values.
Temperalure
Two patternsof temperaturechangeswere found in cancer patients.
For oral temperature,37oc (98.6"F) was consideredas the reference
va.lue.
Temperaturesmeasuredseveraltimes during the day showedthat,
for many patients, the values were fixed either above or below this referenceline. Normal individualsexhibit daily variationsof temperature;the
curvenot only crossesthe referenceline but also shows broad changes.
In contrast,variationsusuallyare smallerin abnormalcases,and the curve
remains
on one or the othersideof the referenceline.Figures37, 38 and 39
showexamplesof such curves.The two patternshave been found to be

Flc" 37, The curve of oral temperutureof a casewith gcneralizedmctastaticmelanoma is pcrsistentlyabovc the 98.6'F (37'C) line, which corrcspondsto the averagevaluc of normal individuals.

Fre. 38. The curve of the oral tempcratureof a paticnt wilh gcneralizcdmctastases
of the breastshowsvaluesconstantlybelnx'lhe 9t.6"F averase
of adcnocarcinetma
line.

DUALTsM
r{i r1,..

R.
6-lo

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IT

* 1 "

rr .,r,a rrrk

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/3

/2

/ 8 5

t,g

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rl

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lt

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t

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Ftc. 39. Curve of the oral temperature of a patient witb bronchogenic cancer with
abdominal metastassshows values constantly helnw g8.6"F (37'C) avcrage line.

independentof type or site of origin of tumors. we will discussthe significaoce of tempefature patterns later.
.Syrtenrc Analyses:
Changesin urine or blood valueswere followed in hundredsof subjects
for long periods,even years. It appearedessentialto plot the data graphically. An average value, obtained from a significant number of normal
subjects,was riepresentedin graphs as a referenceline. In normal subjects,
as a general rule, relatively wide oscillations omur around the average
value line. By contrast, in diseasestates,there is a fixation of the curve on
one or the other side of the averagevalue, the curvesexhibiting only slight,
or even no, variations.Two opposingpatternsare thus evident in disease
sialesfor each type of analysisof blood and urine. The dualism indicates
once again the existencein systemicmetabolismof two kinds of abnormal
changeswith antagonistic characteristics.It is important to note that the
advancedcancerouscondition is characterizedby a marked dualistic pattcrn at the systemic level.

86

REsEARcHrN pHysropATHoLocy

Blood
In blood analyses,the concentrations
of potassiumand calcium, the
presenceof C reactiveproteins,the number of leucocytesand of circulating eosinophiles,(Nole 7) and the red cell sedimentationrate, have been
studied,usingclassicalmethods.They all show the samedualism.Figs. 40
to 43 show someof thesecurveswith the averagevaluesas referencelines.
with the intention of obtaining information concerning the amount of
potassiumpresent in cells, we investigatedthe content of this cation of
the red cells.(Note 8)
Urine
In urine studies, measurementswere made for pH, specific gravity,
surfacetension,oxido-reduction
potential,excretionof sodium,potassium,
calcium, chlorides, phosphates,sulfates, sulfhydryl, indoxyl, grucuronic
acid, peroxides, etc. Most studies were carried out by routine test techniques.For some analyses,however,conventionaltechniqueswere found
to be inadequateand new testsdevised.
For the urinary excretionof sulfhydryl,a new techniquewas devisedby
M. Bier and P. Teitelbaum in our laboratories.Using the warburg
micromanometer,
thc nitrogenliberatedfrom sodium azide in a buffered
solution in the presenceof free iodine was found to be directly related to
the amountof sulfhydrylpresent.The amountof nitrogenfreed at a determined momcnt-13 minutes-was most indicative.(Note 9)
For the information which we neededconcerningthe amount of calcium in urine a very simplemethodwas devised.(Note I0)
For measurementof urinary surface tension, we devised a new
technique.We used a capillary so calibratedas to give the surfacetension
in dyne/cm for a fluid with a specificgravity of 1,015. In this method,
severalarrestsor slowdownsof the descendingcolumn are noted and make
it possibleto obtain informationabout an extremelyimportant factor which
is usuallynot consideredin the measurement
of surfacetensionwith other
methods.It is known that urine is formed of differentconstituents.
some
of them with the tendencyto move toward the surfacewhile others tend
to move toward the bulk of the fluid. Changesin the distributionof these
constituentstake place. They induce changesin the value of the surface
tensionof the urine which occur even during the time measurements
are
taken. The descendingcolumn in a capillary will indicatethesechanges
which are important for precisemeasurement
of surfacetensionof urine.
(Note I I )

D U A L I S M

87

lz

!'
a1 a

Ftc. 40. Curve of the value of the K + in blood serunt in a case with periarteritis
nodosa. The values remain above 4.5 mEq, which represent the average value obtained from seriesof normals.

H
Y

5
c
o

a/,

F I c . 4 1 . C u r v e o f t h e v a l u e so f K + i n t h e b l o o d s e r u m o f a s u b j e c tw i t h c a r c i n o m a
of thc breast, liver metastasesand jaundice. The values remain almost constantly below the 4.5 mEq line.

,:{#:i;;!lil:tt;iiil:tiii'i1l,l1$.'*ii
{llii*ri'+

88

R E S E A R C H

I N

P H Y S I O P A T H O L O C Y

U'
O.J
t)
o

IJ
f

Days
F I o . 4 2 . T h e c u r v e o f t h c n u m b e r o f b l o o d l e u c o c y t e so f a c a s e o f b r e a s t a < J e n o carcinoma shows constantly values abovc 7.0O0,considercdas the average value for
normals.

9ao
C

7
(e
3n
(l,
(J

f,

(J

5
o,

-J

0ays
Ftc. 43. The curve of thc number of blood leucocytesof a casc of brcast carcinoma
showspcrsistentlyvalues below the averageline of 2.000.

DUALTSM

89

Two methods were employed to rneasurethe oxido-reduction potential


of urine. In one, the measurementwas made with a potentiometerusing a
platinum electrode,fust at the pH of the urine and once again after bringing the pH to 7 by adding the necessaryamount of NaoH or HCl. (Note
l2) rn the secondtechnique,difrerencesin potentialwere measuredthrough
the time necssaryto discolor a solution of toluidine blue in an acid
medium at l00oc. The value was determinedin seconds,the concentration of the dye having been chosen so that discoloration in 100 seconds
represented
an averagevalue for normal subjects.More rapid discoloration
indicated higher potential, while a low potential correspondedto slow or
evenno discoloration.(Note I3)
The presenceand relativeamountsof oxydizingsubstancesin the urine
were also investigatedby meansof two reactions:in one, through the passageof indoxyl into indigotine and indigorubineunder the influence of
sulfuric acid (Note 14); in the other, through liberation of iodine from
iodides in the acid medium.
It is interestingto mention at this moment, the variationsencountered
in the metabolismof nitrogen and the form under which it is excreted
through the kidney under normal and pathologicalconditions. we could
showthat in generalthe form of excretionis determinedby the amount of
wateralso excretedby the organism.High amountsof water will thus induce
the excretionof nitrogenprincipallyas ammonia,low amountsas uric acid.
This relationshipwas explainedby data found in comparativephysiology.
The availability of water in the environmentof variousanimalswas seento
determinethe form under which theseanimalsexcretenitrogen.The occurrenceof similnl conditionsconcerningthe excretionof water in pathological
states,furnishesan interestingexplanationfor the form under which it is excretedin thesepathologicalconditions.In Note l5 this problem is discussed
in more details.
Iu all tests requiring quantitative measurements,an important problem
arose.It appearedpractically impossibleto obtain 24 hour urines routinely
for periods of months in large groups of individuals in order to note the
continuouschangesin excretionof the differentsubstancesstudied.Measurements of various substanceseliminated in urine could be made under
thesecircumstancesonly by using isolatedsamples,The values so obtained
expressthe concentrationsof the substances
in the sampleand consequently
weredirectly related not only to the amount eliminatedby the kidney but
alsoto the amount of water excretedat the same time. The values varied
greatly from one specimento the next, according to the amount of water
excreted.Therefore thesedata could only be of relative usefulness,Since

90

xESE^RcH tN pHysropATHoLocy

specificgravity is also a direct functionof the amountof water excretedin


a urine sample,we related the concentrationof a substanceto the specific
gravity of the same sample.This ratio provided a new value which is independentof the amount of water in the sample and is more closely relatedto the amountof the othersubstances
which vary much lessin amount
than water. From the physiopathological
point of view, the data obtained
were seen to correspondto the degreeof active reabsorptionof a given
substance
by the kidney.While the ratio of the concentrationover specific
gravity would vary directly with the excretionof the substance,the inverse
ratio would rcpresentan index of retention,which increaseswith the retention of the substancein the body. Such indiceswere routinely applied for
the differentsubstancestestedin urine to obtain more reliable valuesthan
could be obtainedfrom the concentration
data alone.(Note t5)
Urinary Patterns
various urinalyseswere performedduring sufficientlengthsof timc on
a larger number of subjectsconsideredto be normal. For each test, an
averagevalue was thus obtained.This averagevalue servedas a referencc
line for the curvestraced with the data obtainedfrom the subjects.The
AcC u3i

Sagiibr,

| l ? o ? 2

?r
0" 2 1
.o 15
9

AI

ERS

Flc. 44. Curves of. diflerent anall'sesof daily urine samples of a normol 4O-yearo l d m a l e . F o r e a c h k i n d o f a n a l y s i s t, h e c o r r e s p o n d i n ga v e r a g cv a l u e w a s c a l c u t a t e d
from the measurementsobtained in more than one hundrcd normal subjects. The
curves are seen to pass from one to the other side of the lines correspondingto thc
r e s p e c t i v ea v e r a g ev a l u e s .A c e r t a i n c o r r e s p o n d e n c ies s e e n f o r t h e c h a n g e sw h i c h
take place at the same time in these curves. Parallel variations are seen between spccific gravity and chloride index, while opposite to thosc for the pH and surface
tension,

9l

D U A L I S M

study of thesecurves has permitted us to recognizeseveralcharacteristic


patterns.Under conditionsconsidered
normal,the curvcswere seento pass
from one side to the other on this averageline with relativelywide variations. When an abnormalityexisted,the variousurine analyseswere characterizedby a curve with only small variations,fixed on one or the other
side of the averageline. For each test, two such characteristicabnormal
palternsare encountered.Fig. 44 showsthe curvesof differenttestspassing
trl
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50
PH

6
I
6

Frc. 45. Curves of the valuesof the urinary pff followed during 50 days in five
s u b j c c t s .T h e c u r v e ( a ) c o r r e s p o n d st o a n o r m a l i n d i v i d u a l w h i l e t h e o t h e r f o u r
( b , c , d , e ) t o s u b j e c t sw i t b d i f f e r e n t c a n c e r o u sl e s i o n s .F o r t h e n o r m a l c a s e , t h e
values pass above and below the 6.2 value which is consideredas the average computed values obtained from normals, For the abnormal cases,the curves which show
l i t t l e v a r i a t i o n s a r e f i x e d a t o n e o r t h e o t h e r s i d e o f t h e a v e r a g el i n e , F o r c a s e ( b ) ,
an adenocarcinoma of the ovary' and case (c), a bronchogenic cancer, the curves
are fixed below the averageline. For case (d), with a bronchogeniccancer. and case
( e ) . a c a n c e r o f t h e b r e a s t w i t h g e n e r a l i z e dm e t a s t a s e st ,h e c u r v e s a r e a b o v e t h i s
averageline.

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D U A L I S M

/ e 3

July

June

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Ftc. 47. Curve of the values of the urinary specific gravity in a case of cancer
of thc breast, with the values fixed above 1016 which is considered as an avcrage
value for the specificgravity as computed from a seriesof measurementsin normals.

from one side to the other of the respectiveaveragelines as corresponding


to a normalsubject.Figures45 to 5l showseveralexamplesof two opposite
patternsfor the various testsusedsuch as urinary pH, specificgravity, surface tension,chlorideindex and sulfhydrylindex.
F undatnental Ofi balances
When the curvesof variousanalyses
obtainedat the sametime for thc
samesubjectwere checked,an interesting
relationshipwas found.
In patientswith small localizedtumors, only some of the analyses
showedabnormalpatterns.However,with the evolutionof cancertoward
the terminalstage,abnormalpatternsbecameapparentfor more and more
analyses.In advancedcases,most of the analysesshowedabnormal patterns.

Moy

ro

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25

25
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Potienf-Mrs FS.
F r c . 4 8 . T h e v a l u e so f t h e u r i n a r y s p e c i f i cg r a v i t y i n a c a s eo f c a n c e r o f t h e b r e a s t .
are the whole time below thc averatc value of 1016.

94

RESEARCH IN

PHYSIOPATHOLOCY

Ftc. 49. The urinary surloce tension remains constantly fixed abot,e the average
value of 68 for more than a year in a case of cancer of the urethra.

2oo
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F t c . 5 0 . T h e u r i n a r y s u l f h y d r y l i n d e x r e m a i n s b e l o w t h e a v e r a g ev a l u e o f
c a s co f c a n c e ro f t h e b r e a s t ,w i t h b o n e m e t a s t a s e s .

DUALISM

95

It was especia.lly
in advancedstagesthat definitegroupingsof patterns
with opposite characteristicscould be recognized.They correspondedto
two fundamentaloffbalanceswhich we have called "Type A" and ,.Type
D." 1"4" for anoxybiosis,"D" for dysoxybiosiswhich representthe principal manifestationsof oxygenmetabolismin a phaseof theseoffbalances.
)
The correspondencebetwecn the different abnormal patterns defining
the offbalanceA or D, is seenin Figures52 to 60. The abnormalpattern of

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Frc. 5 1 . T h e u r i n a r y s u l f h y d r y l i n d e x r e m a i n sa b o v e t h e a v e r a g ev a l u e o f r . 5 i n a
caso f h y p e r n e p h r o m aw i t h l u n g m e t a s t a s e s .

the low urinary specificgravity appearstogether with a pattern of high


valuesfor the pH, both corresponding
to the offbalancerype A. (Fig. 52)
A high urinary pH and low chlorideindex patterncorrespondto the offbalancetype A, as seenin Fig. 53. The analysesof a casewith low specific
gravity,high pH, low chlorideindex and high surfacetension,as present
in offbalancetype A, is shown in Fig. 54. An oppositecase,offbalancetype
D, with high specificgravity, low pH, high chloride index and low surface
tensionis shownin Fig. 55. A similarcaseof offbalancetype D is shownin
Fig. 56 with high urinary specificgravity,low pH, low surfacetensionand
low blood leucocytenumber.In Fig. 57, the low pH, high sulfhydrylindex
and low surfacetensionshow an offbalanceof the type D.
of the levelsmay explainwhy, in the samesubject,
The independence

e 6 /

RESEARCH IN

t94l
Moy
30

PHYSIOPATHOLOGY

Ju n e

ro

r5

20

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62
54
4.6
Polrenl- Mrs Von

Frc. 52. A correspondenceis seen between the patterns of. urinary specific grat,ity
and that of the urinary pH in a cancer of the colon correspondingto tbe type A offbalance.The changesstill present in the curves are opposite.

not all the analyticalpatternsobligatoryconcord at all times in the same


subject.Especiallywhen defensereactionsintervene,the offbalanceat one
levelcan be differentfrom that at otherlevels.Fig. 58 showssuchexamples.
Usually,as the diseaseprogresses,
many of thesedifferencesdisappear,the
manifestations-analyticaland clinical at different levels-entering in the
sametype of offbalance.Fig. 59 showssuch an example.

ZC
62
4 6

cl
I tiaar

2.5

F r c . 5 3 . I n a c a s e o f s a r c o m ao f t h e l e g w i t h l u n g m e t a s t a s etsh c c u r v e o f t h e a r i nury pH is fixed above the averageline (6.2) while the curve of chloride index Wl o w t h e a v e r a g el i n e o f 2 . 5 . T h i s r e l a t i o n s h i pindicates a type A of the offbalance.

D U A L I S M
Fab.n,t
5
r

/ e 7

-22

lo 't .2
18r

is: {-- 1
4CJ

323
Oro

t = ^
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F t c . - 5 4 .T h e a n a l y s i so f a t e r m i n a l c a s eo f c a n c e ro f t h e b r e a s t ,s h o w i n g t h e
fundanrcntaloflbolance, low specific gravity, high pH, low chloride index and high surface tension, correspondingthus to the offbalance A.

The passageof a subjectfrom one offbalanceinto the oppositeone is seen


to occurduring the evolutionof the conditionmost often inducedby therapcuticattempts.Fig. 60 showssuchan example.
The two oppositeoffbalances,identifiedfirst through the urine analyses,
couldbe recognizedto exist for all the manifestations
taking place at the
diffc'rentlevelsof the organization.The manifestationsseenat theselevels
could thus be interpreted as correspondingto one of the two opposite
fundamental
offbalances,
type A and D. Table Iv showsthis coordination
of the different manifestationsaccordingto the two oppositeoffbalances.
on the basisof all thesedata, clinical manifestationsand analyses,the
dualisticpathogenicconceptappearedto be well establishedand the dual-

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60

t'/

r/\r

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Ftc. 55. A terminal case of cancer of the breast shows all the analyses fixed in
opposite position to the case of Fig. 54, i.e., high specific gravity, low pH, high
chloride index and low surface tension, corresponding to an offbalance of the type D.

RESE^RcH IN

Leucocyt
es
o

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'8'
o

PHYSIOPATHOLOCY

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Ftc. 56. offbalance type D, shows high urine specific gravity, tow pH, low surface
tension and low blood leucocyte number.

66
64
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6.o
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-

a{,

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/3'

io'

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56

Ftc. 57. The 3 curves, PH, sulfhydryl index and surface tension show an offbalance
type D.

DUALtSM

gg

isticphysiopathological
mechanism
studiedin complexconditions
in gcneral
andin cancerin particular.
r5
5?

so G. t 6

o
9H

7d
62
46

Cl

2 5

sl

74
68
@
Potranr Alr

F t c . 5 8 . T h e u r i n e a n a l y s e so f a p a t i e n tw i r h c a n c e ro f t h e o v a r y . s h o w i n g a d i s c o r d a n c e b e t w e e nt h e p a t t e r n sp r e s e n t .W h i l e a l l t h e a n a l y s e ss h o w p a t t e r n so f t h e
t y p e D , t h e c h l o r i d e i n d e x r e m a i n s t h r o u g h o u t t h e e n t i r e o b s c r v a t i o nc o n s t a n t l yo f
type A.

Place ol Dualism in Cancer Physiopathology


After recognizinga dual pattern in most manifcstationsof cancer,the
problem was to considerthe relationshipbetweenthis dualismand cancer
itself.
One critical observationwas that a pattern in a given patient may
change.During the evolution of the condition, seldom is the changefrom
abnormal to normal, but usually from one abnormal to the opposite abnormal pattern. This fact has a specialimportance as will be seen later. In
some cases,even the immediate,direct causcof such changcscould be
established.
The following case is illustrative.
Mr. S. L., a patientwith cancerof the prostateand metastaticdestruction of half of the sacrum,was in very severepain. Study of variationsin
pain intensity and in urinary pH, as well as the responseto acidifying and
alkalinizing substances,indicated that the pain was of a typical acid pattern.The patientstartedradiotherapyand pain decreased
with almostevery
treatment,until after a few sessionsit had disappearedcompletely.Out of
bed and feelingwell, he continuedthe treatment.Howevcr, at about the
nvelfth session,pain again appearedand thereafterincreasedwith each
treatment.After five more sessions,
he was back in bed and sufteringsuch
unbearablepain that radiotherapyhad to be discontinued.
At that time, a
new analysisof the patternof pain showedthat it had changedfrom thc
that this changemight be thc
originalacid to alkaline.It was hypothesized

- U 3 c i <n

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V

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DUALISM

l0l

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d16
a

Ftc. 60. A condition can pass from one offbalanceto the opposite one. The passage
ol the analyses lrom the oflbalance lype D into the oflbalance trpe ,,1 is seen in a
caseof caocer of the lung. The analyseswhich first show high specific gravity, Iow
pH and high chloride index, pass subsequentlyto the opposite side, with low specific
gravity, high pH and low chloride index. This new oflbalance remains unchanged
for a long time as shown in the second part of the curve.

resultof irradiation. Study of other cases,after irradiation, has confirmed


the hypothesis.
Changesof pain from an acid to an alkalinepattern also were observed
patients
in
who had sufferedshock or seriouspyrogenic infections.viral
infection such as flu, or even smallpox vaccination,seemedto induce an
opposite change-from alkaline to acid. Changesobtained under the influence of therapeutic agents were frequently observed and will be discussedlater.
The dual pattern seen for most cancer manifestationswas integrated
into the concept of the diseaseas a complex organized condition that developsthrough progressiveparticipation of hierarchically superior levels of
the organization. From a clinical point of view, such participation correspondsto the successiveaddition of new manifestationsto a previously less
complexcondition. According to their levels,these"Added Factors" correspondto prolonged youth or rapid cellular aging,to acid or alkaline pain,
to blood and urinary analytical data fixed in one or another of two opposite
patterns.Dualism is the principal characteristicsharedin common by all
theseprogressively added manifestations.
In trying to understandthis dualism and its significance,we had to

102

*ESEARcH IN

PHYsroPATHoLocy

consideragainthe placeof dualismin the generalorganizationof nature.


The studyof reactivityin naturehas led to the recognitionthat a basic
dualismexists-that the forcesoperatingin naturecan be separatedinto
two oppositegroupsfor almostall the interveningfactors.Forceswhich
would tend to lead toward annihilationof differencesand producea state
of maximumentropyor homotropyor the antagonistic
forceswhich tend
TrsLe IV
M,tNtreSrrTIONS
kvel

Offbalance"A"

Offbalance"D"

Cellular Prolonged cellular youth

Rapid cellularaging
Less difterentiatedcells, especially More differentiated cells, espeof connectivetissues
cially of connective tissues
lncreased amount of connective Decreasedamount of connective
tissue
tissue

Tissular Increasedlymphatic tissue

Organic

Low
oxido.reduction poten
processes
Low chloride content
Local acidosis
Acid pattern symptoms
(Pain, dyspnea,itching,etc.)

Decreasedlymphatic tissue
High oxido-reduction potential
processes
High chloride content
Local alkalosis
Alkaline pattern symptorns
(Pain, dyspnea,itching, etc.)

Somnolence
Constipation
Polyuria
Exophtalmia
NasalpH below6.5
Slow absorbtionof skin wheal

Insomnia
Diarrhea
Oliguria
Enophtalmia
Nasal pH above6.5
Rapid absorbtionof skin wheal

Systemic Blood
Leucocytosis
Eosinophilia
High color index
Low potassiumcontent
Low R.C. sed.rate
No C reactiveprotein
Urine
Low specificgravity
High pH
High Cl, Na excretion
Low SH, Ca, phosphate,sulfate
excretion
High surfacetension
Death in coma

Blood
Leucopenia
Eosinopenia
Low color index
High potassiumcontent
High R.C. sed.rate
High C reactiveprotein
Urine
High specificgravity
Low pH
Low Cl, Na excretion
High SH, Ca, phosphate,sulfate
excretion
Low surfacetension
Presenceof oxidizing substances
Death whi.leconscious

DU^LrsM /

103

to maintainand increasedifferencesand lead to more complex organization


-and are thus cataloguedas ncgatively+ntropic,
ectropicor heterotropic
-would thus appear with each new added factor when a higher hierarchic entity is realized.For this reason,they will appearespeciallymanifest for the same added factors intervcningunder abnormal conditions.
Dualism consequentlyconcernsthe manifestationsprogressivelyadded.
The result is seenin the alterationof the oscillatorymovementwith alternatepredominanccof one and then the other of the antagonisticforcesfor
theseadded factors.This fact would explain the dualism seen especially
in abnormalities.It also explains the interventionof the dual patterns
mentionedabove.
Dualism becomeseven rnore important when it can be seento play a
capital role in the mechanismthrough which agcntsact upon abnormal
conditions.Originally,the dualisticactionsof agcntscould be ascertained
through the changesinduced in pain and various other patterns. It has
consequentlybeen possibleto classifythe effcct of various therapeutic
agentsaccording to their influenceupon these patterns and, through this
more readily measurableinfluence,on the fundamentaloffbalancesthemselves.
At the sametime, the study of the influenceexertedby variousagents
upon these patterns, which is presentedlater, has posed the problem of
preciselywhat effectsupon thesepatternsare producedby the substances
which are body constituents.And this led us to a third basic conceptdealing with the importantrole playedby body constituents
and, in particular,
by the lipids.

CHAPTER

TH E CONSTITUENTS

E-lr
.lL lltsnencHrc oRcANrzArroNand dualism have opened the way for

a
study of the body constituentsin an attempt to systematizethem and their
functioningin accordancewith thesetwo concepts.In this research,we
consideredbesidesthe constituents
separatedin groupsas lipids, proteins,
carbohydrates
and electrolytesalso the elementsas a sourceof interesting
information.
With most of the manifestations
integratedin dualistic patterns,rvc
plannedto test the differentconstituents
by noting their influenceon these
patterns.It was to be expectedthat some might have selectiveactivity at
certain levelsof organizationand the manifestationsrelatedto theselevels.
This selectivitydid becomeevidcntbut it also turned out that any agent,
in sufficientamount, cxertcd an effect at any level. The problem was to
selectthe manifestationwhich would respondmost readily to the greatest
number of agents.This would make comparisonsbetweenagentseasier
and serveas a practicalcriterionfor the start of classification
on this dual
basis.
The measurementof the influenceexertedby various agentsupon the
secondday wound crust pH (s.d.c.pH) proved to be particularlyrewarding and was employedin the first part of the investigation.
The s.d.c.pH
providedan indication not only of acidifyingand alka.lizingeffectsbut also
comparative values for these effects. Later, the influence exerted upon
many other manifestations
was studiedfor corroboration.Details of the
s.d.c.pH techniqueand thc resultsobtainedare in Note 1.
In studyingthe elements,we chose,first, simplecombinationsin which
they appear.Each anion was investigated
by studyingit as it occurredin
thc respective acid and in compounds in combinations with diffcrent
cations;each cation was studiedin its combinationswith differentanions.
104

THE CONSTITUENTS

105

ln this way, we obtained a seriesof data which enabled us to pinpoint the


influenceof each element.
TheElements
Using this method, we could determinethat elementssuch as Li, K,
Na,Fe, Ni, Zn, Hg, Bi, B, F, Cl, Br, I-in sufficientamount-produced in
the s.d.c. pH an acidifying effect. The opposite eftect-alkalization-was
seenfor Mg, Ca, Sr, Ba, Cu, Pb, S and Se. It must be emphasizedthat
someelements-such as K, Fe, Zn, Hg, Cl in the acidifying $oup and Ca,
Cu, S and Se in the other-produced an intense effect while others had
a weak though still clear, action. We must add that Ni and Cr showed a
relatively weak acidifying effect. This separationof elementson the basis
of acidifying or alkalizing effect agreed with almost all data available
about antagonismbetween elements-for example, the known antagonism
betweenK and Ca, Mg and Cu, and betweenMo, Zn and Cu.
As a secondstep,we relatedthe elements,through their influenceupon
the s.d.c.pH, to one of the two fundamentaloffbalances.
Those inducing
acidificationwere thus classifiedas an "inducing offbalancetype A," or
"anti offbalance type D" or "anti D," while those producing alkalization
wereca.lled"inducing D," or "anti offbalancetype A," or "anti A." Going
anotherstep, the acidifying elementswere consideredto have a tendency
towardincreasingheterotropy;the alkalizing,a tendencytoward increasing
homotropy.This led us to attribute to the first, the acidifying group, the
qualification"hetero" type, and to the second,thc "homo" type. We did
not give these designationsany other meaningthan that indicated above,
usingthem for didactic facility.
TheSeries
After classifying elementsinto hetero and homo groups, we studied
thesegroupings in terms of the place of the elementsin the periodic chart.
We could quickly see that when two or more elementsare part of the same
series,they also belong to the same group. For example, all elementsin
the I A and the VII A seriesare A inducing,or "hetero." The members
of the II A and VI A seriesare D inducing or homo. At this point, we
tentatively extended these hetero or homo characters to an entire series
after one or more elementsin it had been recognizedas such. The I A,
Il B, III A, V A, VII A seriesand the Fe subseriesof VIII were classified
ashertero or A inducing, while the II A, I B, VI A seriesand the Co subseriesof VIII were labelled homo or D inducing.
This hetero or homo grouping of the various series permitted us to

106 /

xESEARcHrN pHysropATHoLocy

make other correlations.We could seethat among all the seriesdesignated


as A in the periodic chart, those numberedoddly are hetero type, while
thosewith even numbersare homo type. Among the B series,the opposite
is true; those with odd numbersare homo type, while thosewith even are
hetero type. Extrapolating, we could classify all the series according to
this criterion. This view was confirmed by the hetero character seen in
Cr and especiallyMo, and the homo characterfor Mn.
We could go farther and correlatethe aboveclassification,made on the
basisof biologicalproperties,with the electronicconfigurationsof the elements. For the members of the A series,those with an odd number of
electronsin the valency shell were hetero type, while those with an even
number were homo type. Among the B series,this criterion did not hold
true. Wc found, howevcr,that a similarcorrelationexistedif consideration
was given not to the valencyshell alone but to the sum of the two external
shells,the valency and the shell beneathit. This accordswith the fact that
in the B serieselements,the two shellshave insufficientelectronsto frrlfill
the quantum numbers. we saw thus that those membersof the B series
with an odd number for thc sum of elcctronsof the two shellshave a homo
character,while those with an even number havc a hetero. This criterion
appliesto all membersof the B group, includingthose in the I B and II B
series,which have their full quantumquota of electronsin the shell beneath the valency shell. This samecriterion was used to classifythe three
subseriesof seriesVIII shown in the chart. The Fe subseriesand Ni subserieswere consideredhetero type, the Co homo type. This antagonism
was seento be in accordwith experimental
findings.
These considerationsalso permitted us to classifythe membersof the
Lanthanium and Actinium Series.Characteristically,all show nonfulfillment of three of their electronshells-the valencyand the two shellsbeneath.We established
for theseelementsa separateseriesdesignation,C.
Using thc sum of the electronsof the three shells,we separatedthe elementsof the C seriesinto hetero and homo categories.Here, the criterion
was the opposite of that used for the B scries.Thc members with odd
numbersof electronswere consideredhetero, those with even numbers
homo.
We will discusslater the biologicalsignificanceof this separationof
elementsinto A, B and C serieswith their respectiveone, two and three
unfulfllledshells.For the moment,we will only remark that if we consider
an even number of electronsas corresponding
to a kind of partial fulfillment of quantumforces,especiallyas comparedto an odd number, such
partial fulfi.llmentis seen,among all the A series,in those with even num-

rHE

CoNSTTTUENTS /

107

bers (II, IV, VI); amongall the B series,in thosewith odd numbers(I B,
III B, V B, VII B); and, amongthe C series,in thosewith alternatenumbers which can be consideredto correspondto even numbers. All these
series-with partial fulfillment for the sum of their shells-have a D inducing or anti A, character, and thus a homotropic tendency.
Periods
Coming back to the influenceexertedby the elementson test manifestations, we found that elementsof the same seriesshow some similar
propertieswhen acting at a certain level of organization,but some differencesappearwhen they are acting at difrerentlevels.Someof thesedifferences are important. Magnesium,calcium and strontium act similarly on
the s.d.c. pH, and againstconvulsionsas well. However, magnesiumhas
been found to induce somnolenceor even deep sleepin test animals,while
calcium immediately wakes them from magnesium-inducedsomnolence.
This type of antagonisticaction among membersof the sameseriessometimes apparsespeciallypronouncedbetweentwo consecutivemembersin
the series-for example,betweensodiumand potassium,magnesiumand
calcium,oxygenand sulfur, and sulfur and selenium.
Study of this "antagonism" has permitted us to recognizea specific
characteristic.When two elementsof the same seriesact upon the same
entity, one may substitutefor the other.Sodiummay replacepotassiumin
cells.Magnesiumand calcium.oxygenand sulfur,and sulfur and selenium
can replaceeachother in this kind of reciprocalactivity.There is no truly
antagonisticaction betweenthem. This explainsthc fact that two elements
of the same series,if in sufficientamount can have similar activity at a
given level-that of the tissular,for instance,where the changesof the
s.d.c. pH take place.
Further analysisof the activityof membersof the sameserieshas revealedanotherimportantcharacteristic
which has permittcdfurther classification. Differencesin activity of membersof the same seriescould be
related to the organizationalcompartmentsinvolved.This becameclear
when activity of members of the I A series was analyzed according to
whether these elementsform constituentsof the metazoic,nuclear or
subcellularcompartments.
Sodiumis the predominantcation of the metazoiccompartment,which consistsof the interstitialfluids,lymph and blood.
Potassiumis the principalcationof the cellularcompartment.Ammonium,
which corresponds in most of its propertiesto rubidium, representsthe
cation at the nuclearlevel.It could be seenthat the developmentof hier-

108

nEsEARcH rN PHYSToPATHoLocY

archic organizationhas involved elementswith progressivelysmaller atomic


weights.
Study of constituentsin compartmentsand in the environment has
further permitted us-as seen above-to correlate the metazoic compartment with the sea, the cellular compartmentwith the crust of the earth,
and the nuclear and subnuclearcompartmentswith the formations in
which their constituentswere found in the vicinity of volcanoes.
This correlation of the metazoiccompartment to the environmentof
the sea becameespeciallyinterestingwhen we could recognizein its constituentsnot only sodium,but, curiouslyenough,the other membersforming the sameperiod in the periodicchart. Chlorine, magnesiumand sulfur,
predominantin the sea and also found in the metazoiccompartment,are
in the same period in the chart as sodium.
We have thus tried to extend the conceptof a correlationbetweenthe
periods of the chart and the different compartmentsof hierarchic organization. Tentatively we correlated the second period of the chart to the
total organism as an entity. Oxygen, carbon and nitrogen-principal elements in air----enterinto direct contact with the organism as such. The
third period contains sodium, magnesium,sulfur and chlorine, which are
found in the sea and can be correlatedwith the metazoic compartment.
The fourth period containspotassium,calcium, iron, nickel, copper,selenium and bromine-all common to the earth's g1u5t-3nd, according to
our tentative systematization,correlated with the cellular compartment.
Following the same plan, we could relate the fifth period-<ontaining
rubidium, molybdenum,silver, tellurium and iodine-to the nuclear compartment.
As a possiblebasis for a working hypothesis,we could consider the
sixth period-with cesium,barium, gold, mercury, lead, bismuth-and the
lanthanium series to belong to a subnuclearor, rather, submorphologic
compartment.The seventhperiod includesthe radium and actinium series,
characterizedby radioactivity. This period could be related to the lowest
level of the biological organization,the primary one, probably even the
submolecularlevel. This would relate the interventionof radioactivityfrom cosmic rays and especiallyfrom the earth's radioactiveslsrnsnt5-ts
the beginningof the biological realm. Such radioactiveinterventioncould
have brought togetherC and N to form N-C-N-C, which we consideredin
our hypothesis to be the fust entity in the biological realm. This view
represents,at least,a new basisfor an interestingworking hypothesis.
Thus we have the conceptof hierarchiccompartmentsrelatedto changing environments.We also can correlate,further, the environmentsto the

THE CoNSTtruENrs

lo9

periods in the periodic chart to which their principal constituentsbelong.


It is difficult to accept as purely accidentalthe correlationof the changes
in environments with the progressivedisplacementof their constituents
toward periods in the chart with memberseach time having lower atomic
weighs. It is in this progressionthat we can see homotropy developing
toward its maximum, complete value. This view, which will be discussed
in more detail later, again relates evolution of the biological realm to
progress of homotropy, with the cnvironment representingthe concrete
realizationof homotropic evolution.
.
The entire chart can be consideredin terms of hetero and homo series
and of periods that correspondto hierarchiccompartments,as shown in
Tenle V.
For the moment, the possibility of relating an element, tfuough its
membershipin a series,to the hetero or homotropic trend and, through
its place in a period, to an organizationalhierarchiccompartment,helps
to explain many of the peculiaritiesseen in the biological distribution of
the elementsand especiallyin the role played by them at the "proper"
levels to which they belong.
As a generalrule, the presenceof an elementat the level to which it
belongsis directly correlatedto quantitativeoptimum valuescorresponding
to the constantsof the level and to the qualitativerole which it performs.
lts presenceat levelsother than its own must be interpretedin connection
with its activity at its own proper level. Increaseor decreasein the amount
o[ an elementhas a different meaningaccordingto the level at which the
variation occurs. If it occurs at the specificproper level to which the element belongs,it would indicate a direct quantitativeor qualitativechange
in the activity of the element.At other levels,this is not true. If the activity
of an element is qualitatively impaired at its own level, the amount of
the elementat the immediatesuperiorlevelwill increase.The increaseat the
superior level can be interpreted as taking place in order to keep at the
disposal of the impaired level a sufficient amount of the element for possible later use. On the other hand, an abnormally intensive activity of an
element at its own level will reduce the amount of it present at the level
immediately superior. The decreaseat the upper level can be interpreted
as a defensiveattempt to reduce the abnormal activity by limiting the sup-

plv.
The generalrule, which appearsto governthe variationsin distribution
of an elementwithin the organism,makesit important to know the proper
level of an element. Some exampleswill illustratewhat we mean, An increaseof copper is seen in the blood serum of cancer patients,althougb

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THE CoNSTrruENTs

lll

a manifest reduction in catalaseas well as in copper content is seen in


the tumor cells themselvesand in the liver cells. According to the view
presentedabove, these findings can be interpreted to reflect a primary
insufficiency
of copper at its specificlevel, that of the cell. copper is quantitativelydeficientat the levelof the cell not becauseof its low availability,
but becauseit cannot be utilized well enoughqualitativelyto form catalase.
The qualitativeimpairmentin copper'suse at its proper level would lead
to an increasedamount of copper in the immediatelysuperiorcompartment,that of the blood serum.
The knowledgethat copper belongsto the cellular levcl, becauseof
theperiod to which its belongs,could explainthis peculiarity.The organismdoesnot have too much copper althoughthe amount of it in the bloo<J
is incrcased.
Neitherdoesit have too little copperat the proper level.The
abnormalityresidesin a qualitativelyimpairedcapacityof abnormalcells
to utilizecopper.In tentativctherapeuticapplication,we haveto try neither
to increase
nor decrease
the amountof copperbut to obtainits proper utilizationby the abnormalcells.
This view appliesalsoto potassium.
An increaseof potassiumin blood
serumis seenin subjectswith type D offbalance.
with potassiumbelonging
to thecellularlevel,the primary abnormalityhas to be soughtat this level.
In fact such a primary anomaly of potassiummetabolismis seen in the
cellsfor, in oflbalanceD, the cellsare poor in potassium,possiblybecause
thecationmovesout of the cellsas the resultof beingdisplacedby sodium.
The increasein the amount of potassiumfound in the circulatingblood
thuscan be interpretedas secondary,designedto offer the cells a sufficient
amountof potassiumto be utilizedin attemptsto overcomcthis offbalance.
on the other hand, in abnormal offbalanceA, when quantitiesof potassiumare found presentin proliferatingcells,an abnormallyIow amount of
potassium
is found in blood (as low as 3.0 m Eq or less). As potassium
is stillexcretedthrough the kidney, this low blood potassiumis not to be
interpreted
as a quantitativedeficiencybut rather as a teleologicalresponse
to the abnormallyhigh utilizationat the cellularlevel.
A study of potassium,presentedunder this aspect,is thc subjcct of
Note2.
Thc relationshipbetweenelcmcnts,pcriodsand levelsof the organizationexplainsa curious distributionof elementsas seen in the following
e x p e r i m e nlt/.1 0 m o l a r s o l u t i o n sw i t h p H , o f d i b a s i cp h o s p h a t eosf l i t h tum,sodium, potassium,ammoniumand rubidium were prepared.Each
solution
was injectedintravenouslyinto mice, l/4 cc. per minute, until
theanimaldied. The organs,and especiallythe brains,werc immediately

ll2

RESEARcH rN PHYsroPATHoLooy

fixed in Bouin solution and studied histologically. For ammonium and


rubidium, vacuoleswere seen presentin cells and especiallyin nuclei; for
potassium,the vacuoles were present in the cytoplasm, while for sodium,
only a pericellular edema was noted. No such changeswere observed for
lithium. Considering the diiension of the atoms, an opposite occurrence
would have been expectedwith lithium penetratingmost into the cells and
rubidium the least. The fact that the heavier elements correspond to the
lower levels of the organization,accordingto the concept presentedabove,
explains the occurrence.
This could also be seenfor the distributionof seleniumand tellurium.
We could show that while seleniumaccumulatesin the cytoplasm,tellurium
-which is the next heavier elementof the VI series-is fixed preferentially
in the nuclei.
It must be recognized that many problems result from exaggeratedor
reduced amounts of elementsat compartmentswhere they do not belong
as characteristicconstituents.The therapeutic effort, until now, has been
to try to eliminale an excessor make up for a deficiency at any level. According to the view presentedabove, the main effort should be to try to
correct the anomaly in the metabolism of the element at its proper Ievel
for this will lead to correction at other levels. we will consider, later, some
examplesof such eftort.
The concept of dualism and of the place of elementsin hierarchic organization has opened a new way to study the influence exerted by these
elementsin abnormal conditions as will be presentedbelow.
The same type of analysis used for elements can be applied to the
other body constituents.we will start with those which we believe to be
the most important for the problem of the offbalances,the lipids,

CHAPTER

LIPIDS AND LIPOIDS

1r
pATHwAys,one theoreticaland the other experimental,
lLrvo DTFFERENT
have led us to considerlipids as possiblythe most important constituents
involved in thc dualisticpatternsof physiopathological
manifestations.The
study of all the constituentsof the organism--electrolytes,
proteins,carbohydrates and lipids-has shown that for each of them, a rough division
into two classeswith antagonisticreactivitycan be made accordingto the
positive or negativeelectrostaticcharacterof their polar groups-nucleophilic or electrophilicfor some,anionic or cationicfor others.However,
these fundamentaldifferenceswhich can explain thcir interventionin
processesin which dualism is apparent,do not representthe reason for
their rolc in the inductionof patterns.
The reactionsin which someof theseconstituents
take part are carried
out as rapid changeswhile others are complet.,ed
only slowly. It is these
slow rcactions,once accomplished,
which tend to be stablefor long periods
of time. Sinccsuchstabilityis characteristic
of clinicaland analyticalmanifestationswhich have dual patterns,it appearedlogical to considerthe
with slow reactivitywhich are relatedto thesemanifestations.
constituents
Becauseof their hydrosolubility,
and the rapidityof the reactionsin which
part,
proteins
take
most
they
electrolytes,
and evencarbohydrates
probably
play a lesserrole in theselong lastingprocesscs.
The lipids, on the contrary,seemto be especiallysuitedfor this role.
Many of the reactionsin which the lipids participateare slow. As we will
seelater,this is primarily becauseof their insolubilityin water.They form
in the organisma group "apart" from all the water solubleconstituents,
a fact which permits them to function through proper reactions largely
without continuous interferencefrom thc other constituents.For these
reasons,the lipids appearedto be the most likely of all constituentsto be
lt3

114 /

R E S E A R c HI N P H Y S t o P A T H o L o c Y

of major importance in physiopathologicalmanifestationswith long-lasting


patterns.The study of the lipids has substantiatedthis.
However, before discussingthese substancesand their properties, a
nosologicalproblem must be considered:What are lipids? How can they
be defined?
DEFINITION

OF LIPIDS

The literature fails to furnish an adequatedefinition for the group of


that show thosepropertieswhich biochemistryand experimental
substances
biology attributeto the lipids, A definitionon a chemicalbasis,such as one
which considerslipids to be fatty acids and fatty acid derivatives,appears
to be insufficient. It excludes substancessuch as those forming insaponifiable fractions which not only have properties attributed to lipids, but
continuouslyintervenein the processesrelated to them.
such as "greasiness"and solubility come nearer
Physicalcharacteristics
to the real situation without providing a satisfactorydefinition. Bloor's
definition (Note /J, widely acccptedtoday in spite of having been found
inadequate,has introduced-in addition to the important solubility characteristics-certain less acceptable criteria such as the origin of these
substances
and the direct relationshipto fatty acids.Without thesecriteria,
lipids would have to include the group of hydrocarbonswhich have the
same solubility property but usually are not encounteredin organisms.
However, with these criteria, Bloor's definition, besideslimiting the field
too much through the requirementfor a relationshipto fatty acids, excludes the entire important group of synthetic agents with similar properties.To be complete,a definition would have to include these artificial
substances.
Thus confronted by the need for a satisfactorygeneral definition, we
proposedone in 1940 (23) which has since been of great help to us in
all our research: a Iipoid is a polar-nonpolar substancein which the nonpolar part is predominant. It is thus lormed by one or more polar groups
bound to one or more nonpolar groups, the last being energeticallypredominant. In terms of intervening forces, this definition considers the
cohesionforces of the nonpolar part, and especiallythose related to its
surfaceand known as the constant"b" of van der waals forces, which in
lipoids are predominant upon the electrostaticforces of the polar part.
The definitionhas provided the key for the study of the multiple problems
in which these substancesappear to be involved. This definition appears
acceptablesinceit explainsall the known proprtiesof the lipids. Further-

LTPTDSAND

LTPOTDS

ll5

more,the study of the specificrelationshipbetweenthe forces involved


couldevenpredictnew properties,
as will be shownlater.
The distinctionbetweennaturaland syntheticsubstances
as a basisfor
a definifioahasbeenobsoletein biochemistryfor a long time. In our study
however,
it appearedto be didacticallyusefulto indicatewhetheror not
a substanceis encounterednaturally in the organism.Therefore,while
adheringto our generaldefinition,we haveemployedthe term "lipoids"
for the entiregroup of polar-nonpolarsubstances
with a predominance
of
thenonpoliupan, and have conservedthe term "lipids" to designatethe
naturallyoccurringmembers.With this separation,
we havealso avoided

Hydro
id
Borderl
ine
Lipoid

FN\\\
FI\\\\\\\\\

eot.rGroup

Group
Sl llon-Polar

Ftc.6lbis. Schematic representationof the predominant relationship of the polar


and nonpolar parts in hydroids, borderline substancesand lipoids.

a certain apprehensionfelt by many workers about incorporating indistinctly, in the same group of agents,substanceswith vastly different chemical constitutions,which until now have not been associatedwith lipids.
The fact that, beyond physicochemicalconstitution, biological properties
characterizingthe lipids are common to the entire group of lipoids, will
in time, we hope, help to reduce the importance of this separation betweenlipoids and lipids.
The structure of the lipoids-with a large variety of polar and nonpolar
Sroupsbut always with the same characteristicenergeticrelationshipbetweenthem-has led to a logical systematizationof thesesubstances,using
thenature of the polar and nonpolar goups as criteria.
CLASSIFICATION OF LIPOIDS

Lipoids may be subdividedaccordingto differentcriteria.


l. According to the polar group.
A. Lipoids classifiedaccording to the nature ol their polar group.
( -COOH)
1. Lipo-carboxylic acids
(-SH)
2. Lipo-thiols
(_sofi)
3. Lipo-sulfonic acids

l16

nESEARcH rN pHysropATHoLocy

4.
5.
6.
7.
8.
9.
10.

Lipeamines
Lipo-amides
Lipo-alcohols
Lipo-aldehydes
Lipo-ketones
Lipo-halogens
Lipo-metals
etc.

( -NH.)

-coNH,)
-oH)
-cHo)

(:CO)
(-Cl, etc.)
( -Na, etc.)

B. Lipoids classified according to the predominantelement ol the polar


group.
l. Lipo-sulfur compounds
a. Lipo'thiols
b. Lipo-sulfonicacids
c. Lipo-sulfides
d. Lipo-sulfoxides
e. Lipo-sulfones
f. Lipo-sulfites
etc.

(-SH)

( -so"H)
(:S)

(-so)
(:SO,)
(:SO")

2. Lipo-nitrogenderivatives
a.
b.
c.
d.
e.

Lipo-amines
Lipo-amides
Lipo-nitriles
Lipe.isocyanides
Lipo-nitro derivatives
etc.

(-NH,)
(-CONH,)
( -CN)
(-NC)
( -NO,)

C. Lipoids classified according to the energetic character ol their polar


8roup.
A. Lipoids with negativepolar groups
l. Lipoacids
a. Lipo-carboxylicacids
b. Lipo-thiols
c. Lipo-sulfonicacids,etc.
2. Lipo'atdehydes
B. Lipoids with positivepolar groups
3. Lipobases
a. Lipo-amines
b. Lipo,guanidines
c. Lipo-imines,etc.
4. Lipo-alcohols
ll. According to the nonpolar group.
A. Lipoids classifiedaccording to the structure ol their hydrocarbon chain.
l. Aliphatic
2. Alicyclic
3. Aromatic
4. Heterocyclic,etc,

L t P t D s A N D L r P o t D S

l 1 7

accordingto theircarhttnbond.s.
B. Lipoidscla.ssified
l. Saturated
2. Unsaturated
a. Ethenic(mono-,di-,poly-)
b. Ethynic
Someaspectsof this classification
requirediscussion.
The genericterm lipoacidhas beenemployedto describesimplelipoids
having polar groupswith acid functions.While the principallipoacidsare
the fatry acids,other membershavc other acid polar groups,such as SO3,
SH, NOr, etc. The significanccof this groupingtogethcrof lipoids with
negativepolar groupshas becomeevidentespeciallyin studyingthe similaritiesin the biologicaleflectsof thcsesubstanccs.
In certain aspectsof
our research,this correlationhas pcrmittedus to substituteone category
of lipoids (lipo-thiols or lipo-aldehydes) for another (lipo-carboxylic
acids), therebyavoidingcertain undesirableeffectsof the latter group of
substances.
Lipoids havingpolar groupsenergetically
oppositeto thoseof the acids
have been grouped togcther.O[ thcse,the memberswith a polar group
with alkalinefunctionshave becn classifiedas lipobascs.The tcrm baseis
generallyappliedto ionizablecompoundswhich influcncethe pH of solutions and combine readily with acids by losing an OH and gaining a
proton. Another group is formedby the lipoidicalcohols.Recentevidence
indicatesthat, in miiny circumstances,
the differencesin the reactionsof
alcoholsand common basesare quantitativeratherthan qualitative.Quite
often the reactionof an alcohol with an acid is analogousto the reaction
befweenan acid and sodium hydroxide,thc H' of the acid combiningwith
the OH- of the alcohol.The differcnces
betweenreactionsare considered
to be mattersof time-rate.Whereasthe reactionof the baseis almost instantaneous,
that of alcohol is a slow reactionand is less complete.This
behavior of alcoholic substancesis particularly clear when the hydroxyl
group of an alcohol is replacedby a halogento preparethe alkyl halides.
For instancc,accordingto Karrer (24): "This can be done by the action
halogcnacidson the alcohol:
of the concentrated
CH3OH + HCI -----> CHrCI + H2O
The reaction correspondssuperficiallyto the formation of a salt from an
acid and a base:
NaOH + HCI ------>NaCl + H:rO
There is, however,a differencebetweenthe two processes.
Basesand acids
are largelydissociated;when thcy come together,hydrogenions and hy-

ll8

,/

R E S E A R c HI N P H Y S t o P A T H o L o c Y

droxyl ions combine almost at once to give the very little electrostatically
dissociatedwater, so that the reactionwhich really occurs is:
Nal * OH- + H+ + Cl- -----> Na+ * Cl- + HrO
Ionic reactionsalwaysoccur instantaneously.
Reactionbetweenalcohol
and hydrogen halides is governed by other laws. Alcohol is only very
slightly ionized. For the removal of the hydroxyl group a certain time is
required.The reactionbetweenalcoholand acid with eliminationof water,
known as esterification.
is thereforea time reaction."No essentialdifference exists between the reaction of lipo-alcoholsor lipo-amines,for instance,with organicacids.
Theseconsiderationswould have been sufficientto allow lipobasesand
lipo-alcoholsto be grouped together.There are othcr considerationsas
well. Their common biologicalactivity and mutual interchangeability
also
justify
appcar to
grouping them together.Furthermore,the recognitionof
the existence
of a generalmutual antagonismbetwecnlipoacidson the one
hand and lipobasesand lipo--alcohols
on the other hand, chemically,physically and biologically,has proven of considerablevalue in explaininga
variety of experimentallyobservedfacts in many aspectsof our research.
Following this through, we have found it advantageousto define the
two groupsof lipoids by a more generalcharacter,the electricalaspectof
the polar part, negativcfor the lipoacidsand lipo-aldchydes
and positive
for the group of lipo-alcoholsand lipobases.The terms,"positive and negative lipoids,"servealso to emphasizethe natureof their antagonism.
The structureof thc nonpolargroup as it confersphysical,chemical
and biological propertieson the lipoids permits further subdivisions.Lipoids may be classifiedon the basisof thc a.liphatic,alicyclic.aromaticor
heterocycliccharactcrof the nonpolar group. While the negativelipids
are principally formed by fatty acids,the positive are made up principally
of sterols.The prcsenceor absenceof doublebondsdefiningsaturatedand
unsaturated
carbon chainshas becn one subjectof our study and considerablebiologicalimportancehas bcen found to be relatedto this character
as well as to the positionalrclationshipof the double bond to the polar
group and the polarity inducedby the doublebond.
The study of lipoids has shownthat, bcsidespropcrticscontributedby
the elementsand groups which compose thcm, they have additional
physico-chemical
and cven biologicalpropertieswhich are characteristic.
We have termedthcse"lipoidic propcrties"to indicatethat they are consideredto resultdirectlyfrom the particularconstitutionof the lipoids.

LIPIDS AND

LIPOIDS

II9

Physical Lipoidic Properties


Solubility
Solubility representsthe first and most important of thcselipoidic prop
erties.Characteristically,a lipoid has a greatersolubility in neutral solvents
than in water.This is explainedby the fact that the two constituentgroups,
polar and nonpolar,inducediffercntsolubilitypropertics,
to a frec movementbetwcen
As is well known, solubilitycorresponds
moleculesof the solventand the solute.(25) Solubilityis greaterwhen
the physicalpropertiesof the groupsformingthe solventand thoseforming
Lhesolutearc similar;it is impairedwhcn thcy are different.Consequently,
polar groupsin a solutewill tend to favor solubilityin solvcntswith polar
groups, such as water. At the same time, thcy will oppose solubility in
neutral solventsformed by nonpolargroups.On thc othcr hand, nonpolar
groupsin a substancewill favor solubilityin nonpolarneutralsolventsbut
will opposeit in polar solventssuch as watcr. Polar groups thus are hydrophilic and lipophobic,while nonpolarare lipophilicand hydrophobic.(26)
While the solubility characteristics
of substances
composedonly of
polar or nonpolargroupsare rcadily apparent,thc problcm is more complex when a substancecontainsboth polar and nonpolar groups. Since
groupswith antagonistic
such a compoundpossesses
solubilitytendencies,
"in
toto" will dcpendupon thc relationshipbctweenthe opits solubility
posing forces. For a borderline group of polar-nonpolarsubstanceswith
approximatelyequalforces,therc will be cqual solubilityin polar and nonpolar solvents.For other substances,
thc predonrinancc
of one or the other
group will determinesolubilitycharacteristics.
If thc electricalforces of
the polar group predominatc,the substanccwill be hydrosolublebut insolubleor only partly solublcin nonpolarsolvent.s.
If, on thc contrary,the
cohesion-i.e., the van der Waals forccs-of the nonpolar group predominate, the substancewill be solublein nonpolarsolventsand less,or even
not at all, solublein water. (Tnnn VI)
Polar and nonpolarforcescan be calculatedand thcir study can indicate the place of a substancein this systematization.
The importanceof
solubility for defining and systematizing
lipoids became apparent in a
physicomathematical
study of thescsubstances
carried out by Jean Mariani
in our laboratories.(Note 2) We havc defined as "hydroids" those substanceswith predominant polar groups which are morc soluble in polar
with no predominance
solventssuch as water.The "borderlinesubstances,"
of either group, show the same solubilityin polar and neutral solvents.

120 /

R E S E A R , C HI N

PHYSIOPATHOLOGY

TrsLe VI
CursstntcerroNoF Cueulc,rl Coupouxos
Composition

Predominance

Name

Polargroupsonly
Polar-nonpolar
groups

Nonpolar groups
only

Example
Water

Polar group
predominant

Hydroides

Glycerin

No predominance Borderline
substances

n-Propyl alcohol

Nonpolargroup
predominant

Oleic acid
n-Butyl alcohol

Lipoides

Paraffin

The "lipoids," in which the nonpolar groups predominate,are more soluble in neutral solventsthan in water.
As we have mentionedabove, from a practical point of view, a substancecould be judged to be a hydroid, borderlinesubstance,or lipoid by
consideringthe differencesin its solubility in water and in a nonpoliu solvent, such as petroleumether, which correspondsto a mixture of the first
aliphatic saturatedhydrocarbonsliquid at normal temperatureand pressure. ,{ polar-nonpolar substancemore soluble in water than in neutral
solvent is considered a hydroid; one equally soluble in both solvents is
classifiedas a borderline substance;w,hilea substancemore soluble in the
neutral solvent than in water is a lipoid.
Difterent polar groups such as COOH, OH, NH2, CO, SOz, SH, etc.,
enter into the constitutionof various lipoids. They differ considerablyin
their electrostaticforces. As a result, the forces of the nonpolar goups
required for predominance,if a lipoid is to be formed, also will difter. A
different nonpolar group thus is necessaryfor each different polar group.
For aliphatic molecules,it is principally the length of the chain which
determinescohesionforces and a differentnumber of carbons in the nonpolar group appearsto be necessary,dependingupon the polar group, in
order to form a lipoid. The study of homologousseries from this point of
view is interesting.
Sincethe value of the electrostaticforcesvariesgreatly from one polar
group to another, the first members of the various homologous series.
which are also lipoids, will differ from seriesto series,dependingupon

L T P T D S^ N D L t P o t D S

l2l

the nature of the polar group. The lengh of the carbon chain of the nonpolar group will thus indicate in what member of a series the lipoidic
characterappears.By comparingmathematicallythe value of the electrostatic forces of each polar group and the cohesionforces of the nonpolar
group in the respectiveseries,it is possibleto determinewhich member of
each homologousseriesof substanceswill first show the properties of the
lipoids. This a.lsocan be determinedexperimentally,as seen above, using
the solubility characteristicsof the lipoids. For the different membersof
the series,degreesof solubility in a polar solvent such as water, and in a
nonpolar solvent such as petroleum ether, were determined.The first member of an homologous series to be considereda lipoid was the one found
to be more soluble in the nonpolar than in the polar solvent.All members
with a large number of carbon atoms show lipoidic properties;those with
fewer carbon atoms lack those properties.
Thus, lipoidic properties first become manifest, among the carboxylic
acid series,in valeric acid,i.e., the five-carbonmember.The shortercarbon
chain membersare soluble to an equal or greaterdegreein water than in
petroleum ether, while those having a carbon chain longer than four show
a higher degreeof solubilityin the nonpolarsolventsthan in water. (Tnnr-E

vlr)
TesLe VII
Sor,usrLlrresor CrnsoxyLtc Aclo HovolocuEs

ility in

Substance'

Common Name

Methanoic acid
Ethanoic acid
Propanoic acid
Butanoic acid
Pentanoic acid
Hexanoic acid
Heptanoic acid
Octanoic acid

Formic acid
Acetic acid
Propanoic acid
Butyric acid
Valeric acid
Caproic acid
Enanthic acid
Caprylic acid

Polar
Solvent
( W a t e ra t 2 0 " )
I
2
3
4
5
6
7
8

Nonpolar
Solvent
( Petroleum
Ether)
insol.

cO

3 . 7( a t 1 6 " )
0.4
o.24

0 . 2 5( a t 1 0 0 " )

' Namesapproved
by International
Unionof Chemistry.
The sameis true for the trJkylalcohols.n-Propyl alcohol and the members below it are either miscible with both water and petroleum ether or
more soluble in water, indicatingthat the nonpolar forcesdo not predomi-

?.:.]l,:'j

\jii;

122

RESEARcH rN pHysropATHoLocy

natein their molecules.


Therefore,they are not lipoids.n-Butyl alcohol,
moresolublein neutralsolventthan in water,thus is the first lipoidic member of this homologousseries.However,this is not true for all its isomers.
The primary,secondaryand iso butanolare the first in their respective
seriesto possessthe solubility propertiescharacteristicof lipoids. In the
tertiaryalcoholseries,however,the four+arbonmember,the tert.-butanol,
doesnot showthe samesolubilityproperties.Tert.-butanolis misciblewith
water and neutralsolventand as such,is not a tipoid. For this tertiary
alcoholseries,it is the five-carbonmember,the tert.-amylalcohol,which
first showsthe solubilitypropertiesof a lipoid,being only 12.57osoluble
in waterandinfinitelysolublein petroleum
ether.Thus,of the four isomers
of butyl alcohol,three are lipoids,while one, tert.-butylalcohol,is not.
( Trn l n V III)

Trnle VIII
Sor-uslI-rrresoF THE Ar-xyr- Alconor-s
7o of solubilitvin

Substance'

Conrmon Name

Methanol
Ethanol
I -Propanol
2-Propanol
l-Butanol
2-Butanol
2-Methyl, 2-propanol
2-Methyl, I -propanol
l-Pentanol
2-Pentanol
3-Pentanol
2-Methyl, 2-butanol
2-Methyl, l-butanol
3-Methyl, 2-butanol
l-Hexanol
2-Hexanol
3-Hexanol
l-Heptanol
l-Octanol

Methyl alcohol
Ethyl alcohol
Propyl alcohol
Isopropyl alcohol
n-Butyl alcohol
sec.-Butylalcohol
tert.-Butyl alcohol
Isobutyl alcohol
n-Amyl alcohol
sec.act. Amyl alcohol
Diethyl carbinol
tert.-Amyl alcohol
n-act. Amyl alcohol
Isoamyl sec. alcohol
n-Hexyl alcohol
sec.-Hexyl alcohol
Ethyl propyl alcohol
n-Heptyl alcohol
n-Octyl alcohol

No. of
Carbon
Atoms

Polar
Solvent
(Water
at 20")

Nonpolar
Solvent
(Petroleum
Ether)

I
2

4
4
4
5
5
5
5
5
5
6
6
6
8

r Names approved by International Union of Chemistry.

7.9
t2.5
@

9.5
2.7
5.3
insol.
t2.5
insol.
sl. sol.
very sl. sol.
very sl. sol.
0.9
insol.
insol.

co
oO
co
oO
@

co

&

*
t

*
cc

r
co

LrPrDsAND LrPorDs

123

The same methods were used to recognizethe first Iipoidic membcrs


of various alkane derivativesstudied.-fnnle IX shows the first lipoid
membersof severalhomologousseries.
T,rgls IX
Flnsr Lrpololc MevneRs lN Vrnrous AlxeNr' Denrv,rrrve
Hovor-ocous SE'Rrr.s
S u b s t a n c eI

Conrnron Name

N{ethanethiol
Propanal
Propylcarbylamine
l-Butanol
2-Butanone
Butanamide
2-Methyl, 2-butanol
Pentanoicacid (n)
H e x y l a m i n e( n )
l, I Octandiol

Methyl mercaptan
Propionaldehyde
Propyl isocyanide
n-Butyl alcohol
Butyl ketone
Butylanride
tert.-Amyl alcohol
Valeric acid
Hexylamine
l. 8 Octandiol

Polar
Group
-SH
-CHO
-NC

-oH
:CO
-CONH.

-oH
-COOH
-NH'

-oH

i
]

No. of
Carbon Atoms
l
J
J
A

,
A
a

5
5
6
8

t N a m e sa p p r o v e db y I n t e r n a t i o n a l
Union of Chcmistrv.

Molecular l^oyer Formation


Other fundamentalcharacteristics
result from the different solubility
propertiesof the two parts, polar and nonpolar, forming a lipoid. Introducedin a diphasicmediumin which one phaseis watcr and the other oil
or even air, the polar group, with the tendencyto be solublcin water, will
penetratethe water. Since the nonpolar group, which is hydrophobic, is
predominant,not only will it not cnter the water but it will also prevent
theentire moleculefrom moving frecly in water.Conscquently,
the lipoid
moleculewill remain at the surfaceof the water with only its polar group
penetrating.
Becauseof this, the moleculewill assumean oricnted position
towardthe surfaceof water. If the secondphaseis a neutral solvent,the
lipoidmolecules,which will accumulateat the interphasewith the polar
group in water, will have the nonpolar group penetrating the ncutral
solvent.
In both cases,the moleculesform oricntedmolccurarlayerswhich, if
presentat the limit betweentwo phases,would appearas organizedformations.This property which appearsas a direct consequence
of the characteristic
constitutionof the lipoids has further consequcnces.
In such a
layer,the polar groups penetratingin water will influencethe properties
of its surface,and thus reduceits surfacetension.

124 /

REsEARcHrN pHystopATHoLocy

Through the coulombiancharacterof their electrostaticforces,the polar


groups will thus confer, accordingto their nature. a positive or negative
electricalcharacterto the layer.
In a mixture of water and oil, the presenceof a lipoid layer will lower
the intersurfacetension and will favor the breaking down of the phases,
facilitatingthe formation of an emulsion.The presenceof the same electrical chargeat the surfaceof theseresultingemulsiondroplets will act as
a repeUentforce betweenthem and increasethe stability of the emulsion.
(FiS.62) This is anotherimportantcharacteristic
of lipoids which results
from their peculiar constitution.

F t c . 6 2 . T h e p r e s e n c eo f t h e s a m e e l e c t r i c a lc h a r g e a t t h e s u r f a c eo f d r o p l e t so f a n
emulsion insures,througb the repellent forces, the stability of the emulsion.

Chemical Properties
Lipoids have two groups of chemicalpropertieswhich can be related to
their two principal parts, polar and nonpolar. The polar groups with their
electrostaticforces give to the lipoids one group of characteristicreactivities. A carboxylic lipoid will act like any other organic acid, while the
lipo-'alcoholswill act like other alcohols,a thiolipoid like a mercaptan,
and so on. A characteristicof chemical reactionsinduced by the polar
groupsis that, while they are occurringin a water medium, they are largely
limited to the site where the lipoid is localizeddue to the insolubility of

LtPtDS AND LtPOtDs

125

the entire molecule in water. Through this localization, the polar reactivity
of the lipoids becomeslargely a "surface reactivity." It is interestingthat
even minute amountsof lipids are able, through this localizationat separating surfaces,to induce important changes.
A second group of reactionstake place at the nonpolar group and
especiallyat the different formations presentin it, such as double bonds,
cycles, etc. The hydrophobic and lipophilic character of the nonpolar
groups confers a specialcharacteron thesereactions.Most of them take
place in nonionic nonpolar media. Many occur at the semipolar double
bonds with nucleophilicor electrophiliccarbonswhich appear to be especially suitablefor this reactivity. This would explain the fact thar nondissociatedmoleculesmay take part in thesereactions.Most of thesereactions
are relatively slow. This double reactivity,ionic through the polar group
and rather nonionic through the nonpolar group, makesthe study of these
Iipoids one of great interestand it will be discussedbelow in more detail.
Biological Properties
The biological properties of lipoids in general also can be related directly to their physiochemicalcharacteristicsand, thus, to their peculiar
constitution.
Lipidic System
The relative insolubility of the lipids in water and their solubility in
neutral solventshas permitted us to separatethesesubstancesas a group
from the other constituentsof organisms.For more than just didactic purposes,we considerlipids to constitutea separatesystemin the organism.
The part played by lipids in the organizationand the functioningof various entities supports this concept.For example,when a lipoid is introduced into the organism, it will be selectivelydissolved in, circulated
through, retainedby and metabolizedas part of the lipidic system.overtone's"Index of Repartition" of anestheticsin the organism can be seen
to be a direct corollary of the existenceof such a systemalthoughthe anestheticagent can be a lipoid or a nonpolar substance.
A great degreeof independenceof this system is morphologically evident as in adipous cells, when fats circulateas chylomicronsor when
they form oriented layers. We have seen above how the orientation of
lipoids at the surface of water results from the relationshipbetweenthe
solubilitiesof the two constituentgroups,polar and nonpolar. Along with

126 /

s,EsEARcH
rN pHysropATHoLocy

their insolubility in water, the orientation of lipoids has allowed them to


play a very important role in biology.
The very existence of biological entities appears to depend upon the
ability of lipids to build up boundary formations separating and thus assuring the individuality of biological entities.
Through peculiar, reciprocallyopposedorientations,two or more layers
of lipids can form a membrane with two polar faces which has the ability
to separatetwo aqueousmedia. In its simplestform, such a membrane
appears in mitochondria. (Note 3/ Similar boundary formations identify
nuclei and cells and appear in higher entities, as in the membranes and
intercellular cements of lymphatic and blood vessel endothelia. It is this
peculiar orientation which allows lipids to establishthe necessaryboundary
formations resulting in complex hierarchic organisms.The existenceof
biological entities, at least from the chromosome level up (and probably
even below that level), can be seento result directly from the intervention
of lipids as a separatesystem,particularlyin the formation of the dipolar
lipidic boundaries.
However, boundary formations which separatethe biological entities
would not have been efficient if they did not fulfill another capital role:
that of allowing selective passageof metabolites. A totally impermeable
membranewould isolate the respectiveentities and result in their death.
On the other hand, a totally permeablemembranewould have no usefulness.The boundaryformation has to act selectively,pcrmitting the passage
of some,but not all. substances.
But even this does not seemto be sufficient to insure an efficientboundary. Most important, such a membrane
must be able to alter its permeability,quantitativelyand qualitatively,according to variationsin circumstances.
Such capacityfor altering perrneability can be relatedto the presenceof the two groups of lipids, fafty acids
and sterols,with their antagonisticpropertiesrelating to permeability.
The fatty acids appear to induce permeabilityin the membranethey
form, especiallypermeabilityfor anions.The perpendicularposition to the
surfaceof water assumedby the nonpolar aliphatic groups when the fatty
acids form this boundary membraneappearsto be favorable for the passageof a substancethrough the membrane.The fatty acid moleculesthus
can be separated,permitting other moleculesto pass between them; that
is, to passthrough the membraneformed by the fatty acids.The negative
electricalcharacterof the polar groups of these fatty acids explainswhy
they representa kind of barrier to the free passageof cations.Thesecations
are attractedand retainedby the acid polar group. This would explain the
manifestchangesin permeabilityunder the influenceof calcium ion. The

LtPTDS AND

LTPOTDS

127

removalo[ calciumfrom cellularmembranes,


throughtrcatmentwith oxalates, increasespermeability,while trcatnrentwith calcium salts reduces
permeability.The bivalentcalcium ion, when it binds the polar groups
of two adjacentfatty acid moleculcsin the membrane,prcventsthe passage
of other moleculesbetweenthcse parts o[ thc membrane,a fact which
explainsthe manifestdecrease
in permeabilityinducedby this cation.
The other group of lipids, the sterols,have an effect on permcability
opposite to that of fatty acids. This can be related in part to the bond
rvhich these sterols make with the fatty acids. Consequently,they block
any passagethrough the part of the membraneformed by fatty acids.The
impermeabilityis due, to someextcnt, to a peculiarityof the layersformed
by thesesterolsthemselves,
The polycyclicmolcculesof sterolsdo not take
the same perpendicularposition toward the surfaceof water as fatty acids
d o . ( 2 7 ) ( F i S . 6 J l S i n c es t e r o lm o l e c u l eass s u m ea p o s i t i o na l m o s tp a r a l l e l
(a)

-ll+llllll
ooooooooc'

Fattyacidorientedlayr

ratgr

(b)

o
/z///,///,.

rater

oooooooo

Sterol orientedlayer

l.-

polar group
rrcn-polargroup

Ftc. 63. Schenraticuspcct ol orientcd interluce lu1'ers.The perpendicularposition to


t h e s u r f a c eo f w a t e r o f f a t t y a c i d m o l e c u l e s( a ) f a v o r s t h e p a s s a g eo f o t h e r m o l e c u l e s
t h r o u g h t h e s e p a r a t i n gm e m b r a n e st h e y f o r m . O p p o s i t e l y t, h e a l m o s t p a r a l l e l p o s i t i o n
t o t h e s u r f a c eo f w a t e r o f t h e p o l y c y c l i cm o l e c u l c so f s t c r o l s ( b ) p r e v e n t st h e p a s s a g e o f o t h e r m o l e c u l e st h r o u g h t h e m e m b r a n et h e y f o r m ,

to this surface,the layer which they form exhibits no permeabilityproperties.It evenopposesany passage
throughit. It seemsthat fatty acidsand
sterolsmake separate"spots" in the cellularmembranesso that, through
their quantitativerelationships,
they confer differentdegreesof permeability to differentregionsof the mcmbrane.The changesin permeability
which resultfrom the antagonistic
interventionof the two groupsof lipids
seemto play an important role in norrnaland abnormal physiology.
With fatty acids inducing permeability,and sterols opposing it, the
fundamentalcharacter of their biological relationshipcan be recognized.
It would seemthat part of the function of sterolsis to opposethe activity
of fatty acids.Conceptually,sterolswould appear,in this specificactivity,
to be "anti-fatty" acid agents,with a capacityto control the activity of fatty
acids rather than be active by themselves.
Partly for this reasonas well as
for greater general understanding,it is necessaryfirst to investigatefatty
acid activity.
'EllElt;tlf

[Ln[it8.AIf

128 /

nESEARcHrN pHystop^THoLocy

FATTY ACIDS
Besides their constructive role in cstablishingboundary formations,
fatty acids appear to serve various othcr purposesin the organism.They
can be used as caloric metabolites,and they play an active functional role
in a biological change. while all fatty acids may exhibit these three
activities---<aloric,constructiveand functional-there are important individual difterences.with the carboxyl as common polar group, the differencesbetween the various fatty acids can be related to the nonpolar
groups.we will discussthis aspectof fatty acids, emphasizingonly what
can be consideredto be new contributionsto understandingthe biological
role of the substances.
Rancidity
The srudyof changeswhich take placein virro, on lipids and especially
on fatty acids after they have been separatedfrom the organisms,led us
to consider a possible parallelism between them and the changes which
take placein the organism.We tried thus to utilize especiallythe knowledge
furnishedby the study of the chemicaldeteriorationof natural fats generallyknown as rancidity(28), to betterunderstandand alsoto systematize
many of the processes
occurringin vivo.
Three typesof rancidityare describcd.In onc-hydrolytic rancidityfats are separatedin free fatty acids and glycerol (or mono- or di-glycerides), through thc intervcntion of lipolytic enzymes. These are often
producedby molds (Penicillium,Aspergillus,ctc.) or by microbesrich in
suchlipolyticenzymesor cven thc lipasepresentin the tissuesfrom which
the lipids are obtained.]-he characreristic
of rhis typc of rancidityis the
interventionof enzymesand the appearance
of frce fatty acidsas a result.
In a secondtyp. of rancidity,also occurringunder the interventionof
enzymes,an oxidativeprocessis involved.Thc characteristic
of this typc
of rancidityis that it affectsaJmost,if not cxclusively,saturatedfatty acids,
convertingthem into methyl-ketones
by a bcta oxidation process.This
"perfume rancidity" called so becauscof the odor of the methyl-ketones
with seven,nine or cleven carbonswhich rcsult-takcs place apparently
through the interventionof a peroxidaseprescntin certain molds (such
as penicillium glaucum) , one of its characteristicsis that it occurs
e s p c c i a l loyn s a t u r a t e fda t t y a c i d sw i t h a l o w n u m b c ro f c a r b o n s( 8 t o l 2 ) .
The third type of ranciditygroupstogethcrthe oxidativechangeswhich
take placeat the unsaturatednonpolargroup of the lipids. As they result
from the interventionof double bonds, the reactionsdiffer accordinsto

L T P T D SA N D L t P O t D s

Lzg

the energeticcenterpresent.In one which occursat room temperatureonly


for the conjugatedfatty acids,such as eleostearicacid, and at 100"c only
to some extent for oleic, linoleic and linolenic acid, the oxidation leads to
thc appearance
of peroxides.(29) In anotherform of this oxidation,taking
place for oleic. linoleic and linolenicfatty acids at room temperatureor
below 50"c, hydroperoxidesresult, as it has been shown by Farmer and
corvorkers,
first for rubber (30) and afterfor fats (31). Another important
fact seen in rancidity changesis that the atmosphcricoxidation of polyethenoidfatty acidscan resultin a displacement
of the double bondswith
the appearance
of conjugatedisomcrs.(32)
The study of natural rancidityhas represented
the basicguide for our
study and systematization
of the processes
encounteredin normal and abnormal physiology. we searchedand found this similarity not only in
generaloutlines,but also for most of their details.By referringto the processesfound in rancidity, we were able to identify, besidesenzymaticlipolysis and enzymaticKnoop beta oxidation,known to occur in the organism,
also the interventionof hydroperoxides,peroxidesand the conjugationof
double bonds. Not only the processesthemselvesbut a.lsothe conditions
under which they take place and their inter-relationship
have been found
p
a
r
a
l
l
e
l
to
i n v i v o t h o s ew h i c h c a n b e s e e ni n v i t r o .
We rvill seeall along in the studyof fatty acidshow far the biological
interventionof this parallelismgoes.
Caloric Metabolism
Although all the fatty acidsarc ulrimatelyusedby differentorganisms
as caloric metabolites,the saturatedand monocthenicmembersare most
importantfrom this point of view. Among the saturatedand monoethenic
fatty acids,the memberswith long chainsappcar to be thosc which are
kept in reservefor caloric purposes.we could show that the principal form
of caloric desmolysis,
the Knoop beta oxidarion,takes place directly, almost exclusively,on memberswith relativelyshort chains,tlat is, with a
maximumof l0 or 72 carbons.
While fatty acids with short chainstake part directly in thesecaloric
metabolicchanges,thosewith longercarbonchainsmust undergopreliminary changesbefore entering into caloric metabolism.A desaturation,
changinga saturatedfatty acid into a monoethenic,appearsto be a first
stepin caloricmetabolismof the long chain members.The monoethenoids
thus can be seento be intermediaryforms bctwcenthe saturatedreserve
and the short-chain,easilymetabolized
fatty acids.
The double bond in these monoethenicacids would rhus appear to

130

RESEARcH IN

PHYSIoPATHoLocY

have two uses: one, to reducethe melting point below body temperature
and thus permit easymobilization,and two, to induce changeswhich lead
to the breaking up of the long moleculeinto two shorter ones which can
be metabolizedthrough the Knoop oxidation.
All the data indicate that this fission would not take place at the double
bond but through a more complex process.A first change consists of
oxygen fixation at the carbon near the double bond. This leads to the appearanceof a hydroperoxidegroup. It is only in a subsequentstep that
the moleculebreaks at a place betweenthis carbon near the double bond
and the double bond itself, resulting in the appearan of short chains
which have an even number of carbonscapableof being directly metabolized through the beta oxidation.(Note 4)
The positionof the doublebond in the naturally occurringmonoethenic
fany acids, separatingalmost always a group of nine carbons toward the
carboxyl or the mcthyl end, (Note 5/ acquiresa special significancefor
the breakingdown of the moleculesfor caloric purposes.
The desaturationof the saturatedfatty acids,which would representa
first step toward allowing them to participate in metabolic caloric changes,
would usually takc place in the liver, apparentlythrough the same processesby which polyunsaturatedfatty acidsare partially saturated.(Note 6)
An interestingpart of the caloric metabolismof the saturatedand
monoethenicfatty acids, which will be shown below, is their combination
with glycerol to form triglycerides.
Constitutional Role
Although saturatedand monoethenoidfatty acids enter into the formation of boundary membranes,the di-, tri- and tetraenicmembersseem to
havea particularlyimportant role in the constructivefunction of fatty acids.
Some of them entcr directly into the formation of the membrane;some
forrn complex lipoids such as lecithinewith the glycerophosphoricradical
and nitrogen containing bases.As a rule, these last representa lipoidic
substratewhich would act as a neutral natural solvent present in membranes,and as such, intervenein the realizationof a diphasic medium at
the level of the boundary formation.This medium would largely insure
the orientationof the fatty acidsat the separationsurfaceand the formation of permeablelipidic layers,
FunctionalRole
The third role of fatty acids is as functional agentstaking part in certain reactions.This activity appearsto be strongly related to two factors:

LTPIDSAND LTPOTDS /

l3l

the presenceof an uncombinedcarboxyl $oup and the energeticintervention of the double bonds of the nonpolar part of the polyunsaturated
members.
Free fatty acids appear to be functionally active while the combined
ones usually are inactive.The activity is relatedonly partially to the direct
capacity of the carboxyl to realize new combinations.It resultsfrom the
induction exertedby the carboxyl upon the nonpolar group. The so-called
free fatty acids of the organism are probably bound in a labile form to
proteins, but this bond will not influencethe induction effect exerted upon
the nonpolar group. The intensive positive carbon of the carboxyl, together with the zig-zagdispositionof the fatty acid molecule,causesthe
inductive effect to charge the successive
carbons of the chain. They will
thus show alternativesigns.The even carbonsshow a negativecharacter,
while the odd ones are positive.The fact that oxygencombineswith positive carbonsexplainsnot only why, as in Knoop oxidation,this bond occurs
at C3, which is strongly positive,but also explainsthe so.calledalternate
oxidation (33) where the other following odd carbonsare binding oxygens.
Through the influenceexerted by the carboxyl, the double bond shows a
specialactivity which has been worth studying.
Double Bonds
There has been some tendencyto regardthe double bond as a weak,
easily broken point of the molecule.Actually, it emergesas an important
center of activity. With its capaciry to become a semipolar center, and
consequently,to bind or lose radicals,the double bond is an energetic
center in the molecule.Is importantcharacteristic
is the ability to effect
such changeswithout altcringthe chain of the moleculeitself. Since this
type of reactionis reversibleand can be repeatedfor the same molecule,
the double bond appearsto representa functional entity. Becausethe reaction principally involvesnonmetallicelements,the unsaturatcdfatty acid
takesan activepart in the metabolismin which theseelementsappear.
The study of rancidity has helpedus, by analogy,to systematizeoxidaas they take placein vivo. In addition to Knoop beta, several
tion processes
other g'pes of oxidation could be recognizedin which double bonds intervene more directly. The double bond, with its semipolarcharacter,influences nearby carbons, rendering them highly reactive. In one form of
oxidation, a molecularoxygen is bound to a nearby carbon to produce a
formation,as was shownto occur in vitro by Farmer. (31)
hydroperoxide
this oxygenfixationbecomesreversible.
When,under certaincircumstances,
the fatty acid will liberate the oxygen. It appearshighly probable that in

132 /

R E s E A R C Hr N p H y s r o p A T H o L o c y

such a process,the oxygen is libcratedas a free radical, the entire process


thus corresponding
to an activationof oxygen.The changeof a molecular
oxygcn into a frec radical would representthe physiologicalrole of unsaturatcdfatty acidsin oxidationprocesses.
The presenceof two double bondsin non-parallelposition,contmon to
most of the naturallyoccurringpolyunsaturatcd
fatty acids,is even morc
important; the two double bonds exert a parlicularly strong influcnce on
the specialcarbonwhich is in thc intermediarypositionbetweenthem. Becauseof the alternateinduction producedby the strongJypositive carbon
of the carboxyl,the carbonsof the chainhave alternatecharacters,
positive
and negative.When an intermediarycarbonalso has a strongpositivecharacter, it appcarsto be cspeciallyable to fix oxygen.This strong.lypositive
intermediarycarbon,occurringin naturalpolyunsaturated
fatty acidswith
more than two double bonds, may be the rcason for the important role
playedby theseacids whcn they act as essentialfatty acids in the organism. (Note 7)
The study of rancidity has further shown that, while the in vitro
oxidationof an unsaturated
fatty acid under mild conditionssuch as room
temperatureleads to the appearanceof hydroperoxidcs,oxidation at a
higher temperaturewill result in anothcrfixation of oxygen,this time at
the doublebond itself.Epoxidcsor peroxidcswill appearaccordingto the
ionic or molecularcharactcr of the oxygen. This extremely important
processalso occurs in rancidity under the inl'luenceof an enzyme. It is
highly probablc that a similar proccsstakes placc in vivo in those pathologica.l
conditionsin which pcroxidesappearin the urine.Radiation,certain
inflammations(especiallythosc due to streptococci),administrationof
seleniumpreparations
or of highlypolyunsaturated
fatty acidsare followed
by the appearanceof theseoxidizingsubstances
in urine. As mentioned
above, when these substancesappear, there also arc increasesin indoxyl
and glucuronicacid, which can be considered,up to a certain point, to
resultfrom abnormallyintensiveoxidationtakingplaccon tryptophaneand
glucose.(See below.) while acrivationof oxygenis a physiologicalprocess,peroxidesappearunder abnormalconditions.

ABNORN{AL FATTY ACIDS


T h e s t u d yo f t h e r e l a t i o n s h i bp e t r v c c na b n o r m a cl o n d i t i o n sa n d l i p i d s h a s
progrcssively
led us to considerthe existence
of clualitative
changesin these
l i p i d s ,b e s i d e st h e c l u a n t i t a t i voen e s .T h e c x i s t c n c eo f a b n o r m a ln r e t a b o l i c
processes,
and espcciallythe fact that such abnormalitiesare often of long

L T P t D SA N D L t P o t D s

133

duration,could hardly be attributedto variationsin the quantityof the interveninglipids alone. More probably they would result from changesin
the nature of the interveninglipids themselves.
We have investigated
this
aspectof the fatty acids presentunder abnormal conditions.
As a guide for the direction to be followed in theseinvestigations,we
used the information furnishedby the study of rancidity. We believedrancidity would be ableto indicatebroadly the natureof the qualitativechanges
which the lipids may undergounder abnormalconditions.Conceptually,the
abnormal can be consideredto result from a loss of the capacity of the
organism to sufficiently control occurring processesand keep them in
the frame of the constantswhich characterizethe entity. Due to this lack of
effectivecontrol, the in vivo occurringchangesunder abnormal conditions
would closelyapproachthose which take place in vitro where such a control doesnot exist,Theseconsiderations
led us to searchfor changessimilar
to those seenin rancidity,or occurringin vivo in lipids, under abnormal
conditions.
As mentioned above, in rancidity a first group of changesconcerns
the polar group. Someof them result in the appearanceof free fatty acids,
others correspondto changesin the carboxylsthemselves,while still others
are representedby processes
of oxidationwhich occur in the chain near the
polar group. A secondgroup of rancidity changesconcernsthe nonpolar
group and especiallythe energeticcenterspresentin it, the double bonds.
The study of this last group of changesled us to consider,the changes
appearingin vitro under the direct influenceof heat and oxygen.As part of
thesechanges,we consideredof specialimportancethe conjugationof the
double bonds seen to occur in vitro as a step in the oxidation of polyunsaturatedfatty acids.This conjugationcorrespondsto a characteristicdisplacementin the moleculeof two or more of the double bonds present,so
as to result in parallel reciprocalpositions.
While in the simple bond two tetrahedriccarbons are bound through
their peaks,in the double bond they are bound by one edge, and in the
triple bond by a surface.In the conjugatedformation, the common edges
of two doublebonds,being separatedby one simple bond, are consequently
parallel.The planesin which the electronsof thesedouble bondsare moving
for eachdouble bond and which are perpendicularto that of the bond itself,
becomeparallel. Through the resultingreciprocalinduction their energetic
value is enhanced.
We have studied systematicallythe different qualitative abnormalities
concerning the fatty acids, guided mainly by the information obtained
throughthe study of rancidity.

.^

134 /

nESEARcHrN pHysropATHoLocy

Methods ol Investigation Used


Followingthis line,we firstinvestigated
the forms underwhich the lipids
in gcneralare presentin the organism.We utilizedthe differences
in solubility betweenthesedifferentforms, separatingthem into free lipids, lipids
kept in a labile bond with other constituents,
as in cenapse,lipids bound
throughtheir polar group as in fats,or in the still strongerform as lipids in
combinationsso firm that thcy cannot be separatedexcept through saponification.The methoddevisedfor this study and someexamplesare in Note
8A. This researchshowedthat under abnormalconditions,very important
variationsoccur in the amountsof the difterent forms. This study pointed
out that the free lipids are greatlyresponsible
for the importantnranifestations in which lipids appearas activeagents.
In the study concerningthe abnormal metabolismof the carboxyl and
nearby carbons, we investigatedthe appearanceof fatty aldehydesor
ketonesin blood, urine and in the cells.
Onc of the major problemsencountered
was the appearance
in vivo o[
conjugatedfatty acids,as abnormalfatty acids.In order to ascertaintheir
presenceand to measuretheir amounts,we had utilized three differcnt
methodsof investigation:spectralanalysisin ultra-violetand in the first
portion of the visible spectrum(Note 8Bl; the study of the place of thc
doublebond in the fatty acidsmoleculethroughthe fissionof thesemoleculesand the analysesof the resultingfractions.(SeeNote I, Chapter 101
More recentlywe have tried the vapor fractionationmethod (gas chromatography) (Note 8C).
The first, and especiallythe secondmethod,gave us valuabledata permitting us to recognizethe interventionof conjugatedfatty acids in ab'l'hese
normal conditions.
studiesrevealedthe appearanceof conjugated
fatty acids,especiallyas trienes,the increaseof their amountwith the progressof the conditionsand especiallythe fact that deathoccurswhen their
concentration
in the bodieshasreacheda criticalvalue.This has markedthe
importanceof thesesubstances
in physiopathology.
The fact that gaschromatographydid not revealthe presence
of conjugatedfatty acidsappearsdue
to the conditionsunder which the methodactuallyworks.
Later, we will frequcntlyreturn to the variousproblemsrelatedto interventionof conjugatedfatty acids.We could thus directly correlatethe
interventionof theseabnormalfatty acidswith the pathogenesis
of the manifestationsof many conditionssucb as trauma,shock,adrenalectomy,
and
especiallywith the noxiousmanifestations
following irradiation.(Chapter
I0)

LIPIDS AND

LIPOIDS

,/

I35

In abnormal metabolic changes,an important factor is the intervention of abnormal fatty acids in the metabolismof chloride ions, producing
an especiallystrongfixation of the chlorideion to the carbonsat the double
bonds. The conjugateddouble bonds in a fatty acid moleculeappearto be
especially suitable for this since an abnormal, irreversible fixation of
chlorides occurs in two steps. First, the halogen is fixed at the extreme
carbons of conjugatedformationswith a displacementof the double bond
in the intermediaryposition. In the secondphase,the fixation takes place
in the intermediarycarbons,too. (Nole 8D)
Functionally,fatty acids induce activationof oxygenas a norrnal process, but the appearanceof peroxidesor irreversiblefixation of ctrloride
ions is an abnormal event.
It is the abnormal fixation of chloridesby the conjugatedfatty acids
which leads to a more complex group of processcsinvolving sodium
chloride metabolism.with the chloride ion fixed. the sodium ion of sodium
chlorideremainsfree to enterinto othercombinations,
especially
with a carbonateion, producingstronglyalkalinecompounds.This processexplains
the appearanceof local alkalosisas a result of thc intcrvention of conjugated abnormalfatty acids,corresponding
to the chloride phaseof ',D."
The division of fatty acids into four groups-l ) saturatedand monounsaturated,2) di-, tri- and possiblyalso tetra-unsaturated,
3) tetra- and
higher polyunsaturated,and 4) conjugated---{orresponds
schematicallyto
the four principal roles---caloric,organizationa.l,functional and pathogenic
-which fatty acids play in the organism. These roles are seen to be
dictated both by the differentstructuresof the fatty acids and the different
substancesto which they are preferentiallybound. The fate of a fatty acid
in the organism seemsto be greatly influenced by its bond to other substances.As already noted, we have called these other substances"antifattv acids."
THE

ANTI.FATTY

ACIDS

Gl;'cerol and Glyc'erophosphoricAcid


It is classically acceptedthat the intestinal absorption and circulation
of fatty acids is made through bonding to varioussubstances.
The analysis
of this absorption shows, however,that different fatty acids have preferential bonds. For saturatedand monothenicfatty acids,the bond is principally with glycerol. Although mono- and di-glyceridescan be identified
in the cells of the intestinalmucosa,these fatty acids leave the intestine
as triglycerides,forming the largest part of the chylomicrons.They are

135

nESEARcH rN PHYStoPATHoLocY

also found in reserye in adipous cells as triglycerides. The di-, tri- and
even tetraenic fatty acids usually enter the circulation as phospholipids,
that is, in direct combination with glyccrophosphoric ions. The polyunsaturated acids are bound to sterols when they enter the blood, circulate
and are stored. While the structures of the various fatty acids determine
their difterent roles in the organism, it is the anti-fatty-acid constituents
to which they are bound which enhancethcseroles.The study of the antifatty acids has shown that these substancescan even dictate, by themselves,difterentfates for the variousfatty acids they bind.
The combination of glycerol with any fatty acid seemsto establisha
caloric metabolic character. This is true for the very different fatty acids
found in plants and animalsas triglycerides.Even the ricinoleic triglyceride,
if fresh, is used as comestibleoil, castor oil. The same is true of the oil
of triglyceridesof polyunsaturated
fatty acids found in marine animal oils.
In the seeds,all the triglyceridesof fatty acids,even the conjugatedones
such as eleostearicand parinaric,representenergeticsources.It seemsthat
it is their combination with glycerol which has given all these fatty acids
value as caloric metabolites.The same is true for the bond to glycerophosphoric ion.
Combination with glycerophosphoricacid endows various fatty acids
with the abi.lityto participatein the constructionof membranes.The bond
to sterols,on the contrary, inducesan ultimate functional activity provided
the fatty acid iself is so constitutedas to be able to fulfill this function.
The influenceexcrtedby anti-fattyacidscan be understoodin terms of
the changesthey induce in the activity of the fatty acids.Sincethe activity
of the last is largely relatedto their presenceas free substances,
it is principally through their combinationwith fatty acids that the anti-fatty acids
intervene.By inactivatingthose free fatty acids which form a membrane
and insure its permeability,an anti-fatty-acidagcnt can causethe membrane to changeits permeabilityand evento becomecompletelyimpermeable. Similarly, an anti-fatty acid, by combining with a polyunsaturated
fatty acid, can reduce or even suppressits functional activity. It is to be
notedthat,by both changingpermeabilityand suppressing
functionalactivity, the anti-fatty acids exert their influenceultimately by altering oxygen
metabolism.From this point of view, metabolismbecomespredominantly
anoxybioticin contrast to normal oxybiotic metabolism.For glucose.for
instance,suppression
of the oxidativephasearrestsmetabolismat pyruvic
acid which passesinto lactic acid. The appearanceof acid substancesas a
biological effect of the action of anti-fafty acids results,in fact. from the

r.rPrDs^ND LrPotDS

137

rc'ductionof the fatty acid's activity, aflectingoxidative processesdirectly,


or indirectly through reductionof membraneFrcrmeability.
while one group of anti-fatty acidscan be directly relatedto hydroids,
and especiallyto glycerolor to glycerolbound to phosphoricacid as in the
ion, a secondgroup is represented
by lipoids, principally
-elycerophosphoric
formed by derivativesof a characteristicring system,the cyclopentanophenanthrene.As anti-fatty acid lipoids, thesc compounds,the steroids,
were of special intcrest in lipoid research.only some aspectsof the biologicalactivity of steroids-mainly, thosewhich reprcsentnew views in the
studyof thesesubstances-willbe discussed
here.

Steroids
A fundamentalrole of thesesubstances
in biology is determinedby the
'l'his
fact that they are polycyclic.
leadsus to considerthe role of the ring
iself in reactivity, as shown by a study of the steroidsin oppositionto the
fany acids.In thc fatty acids.the bondsbetwecncarbonsas presentin the
aliphatic chain, insure a high reciprocalmobility between these carbons.
As a result, the entirealiphaticchain is highly llexible.on the other hand,
ri-uidityis characteristicfor all the rings,and is increascdby the polycycling
of the nrolcculcs.The constitucnts
of the moleculesare kept in fixed reciprocalpositions.while, in the fatty acids,the flexibility of the chain permits
the energeticcentersto take different relative positionsamong themselves
towardother nrolecule-s,
the rigidity o[ thc p<llycyclicmoleculcsmaintains
theenergeticcentersof the cycle,or thoseattachedto it, in the samerelative position. This fundamentalcharacteristic
of the cyclic moleculesappearsto be an important factor in determiningthe biological role of the
variousagentswhich have such cyclesin their molecules.
In the case of steroids,this attributcacquiresspecialimportance.An
understanding
of the differentbrologicalactiviticsof steroidscan be obtainedby an ana.lysisof the forces resultingfrom this characteristiccomposition.Besides the energeticcentersor formations attachcd to it, two
energeticcenters appear as part of the steroid nucleus itself. One is at
Cr and the other center is rcpresentedby the cyclopentanicgroup. The
factthat these centersare maintaincdin fixed relativeposition through the
rigidityof this polycyclicnucleushas resultedin an importantpropertyof
thenucleus itself which becomestranslatedinto a dipolarity of the molecule.The study of thesetwo energeticcentershas advancedour knowledge
of the role of steroids.
The study of the polar groupsbound to C,, of the polycycleskeletonof

138

nESEARcH rN pHysropATHoLocy

steroidshas permitted us to recognizethe conditions which induce stronger


activity for these polar groups, conditions which are usually fulfilled in the
naturally occurringmembers.It could thus be seenthat the reactivityof an
oxygen bound to Cs is increasedif another double bond present in the
cycle is parallel to the double bond through which the oxygen is bound to
Cr.A doublebond betweenC1 and Cr,,as shownin Figure 64 (a), fulfills

(a)

(b)

(L'

F l c . 6 4 . I n f l u e n c ee x e r t e du p o n t h e o x y g e n b o n d a t C ' b y t h e p o s i t i o n o f t h e d o u h l e
bond in the cycles I and 2 of the cyclopentanephenanthrene
molecule. A parallelism
b e t w e e nt h e d o u b l e b o n d o f o x y g e na n d t h a t p r e s e n tb e t w e e nC , a n d C , i n c r e a s e st h e
e n e r g e t i cc h a r a c t e ro f t h e c a r b o n y l ( a ) . A s i m i l a r i n f l u e n c eb u t l e s s a c t i v e , i s e x e r t e d b y t h e d o u b l e b o n d b e t w c e nG a n d C , . A d o u b l e b o n d a d d e d b e t w e e nC , a n d
C ' ( c ) i n c r e a s e st h e a c t i v i t y . S t i l l m o r e a c t i v i t y w o u l d r e s u l t f r o m a t b i r d d o u b l e
b o n d a d d e d b e t w e e nC c a n d C , ( d ) .

such a condition.A similar influenceis exertedindirectly by a double bond


betweenC6 zrndCz (b) which, through induction,will influencethe parallel
Cr and C5 bond and further the double bond of the oxygen.This explains
the influenceexertedby the double bond presentbetweenC, and C, (c).
as in the synthetic,prednisolone.
Further enhancement
of reactivitywould
be obtainedwith a third doublebond addedbetweenC6 and C7. The parallelism bctwecn three double bonds (d ) would produce an increased
reactivity.
For the hydroxyl, a similar enhancedreactivity is induced by double
bonding of the carbon to which the hydroxyl is attachedwith a double
bond for the Cs - Ca or C, - C., as shown in Figure 65 (a and b). A
similar condition is fulfillcd if a doublc bond is prescntin the moleculc
parallelto any of thesebonds,as seenin Figure 65 (c) where the double

r*

,\/-t-r*oAA
(a)

rff

-k

.*

Ho,/W

*o&
(b)

(c)

6)

F t c . 6 5 . T h e i n f l u c n c ee x e r t e du p o n t h e h y d r o x y l b o n d a t C . b y a d o u b l e b o n d i n
t h e c y c l e I a n d 2 o f t h e p h e n a n t h r e n ei s i n c r e a s e di f t h e d o u b l e b o n d i s a d j a c e n to r
parallelto the bonds of C., bearingthe hydroxyl.

LTPtDSAND LrPorDS

139

bond is betweenC5 and Co. An enhancedreactivityof thesecompounds


would be obtainedwith one double bond betweenC3 and C. and another
betweenCr and C,, (d ).
We will come back to this important intervention of the double bonds
in cyclic molecules.
The energeticproperty of the cyclopentanegroup apparsto be correlated with its odd number of carbons.The a.lternatesuccessionof carbons
with positive and negativecharactersresultingfrom the induction effect
causestwo carbonsof this cycle to have the sarnesign. This "twin formation" inducesa specialmolccular reactivity related to the pentanic cycle
of the steroidmolecule.(Note 9)
The special reactivity seen for c3 of thc cyclopentanophcnanthrene
molecule can be explained through a hypothesiscovering the origin of
these substances.
Although the origin of a cholesterolmoleculethrough a
cyclizationof squalene(35) appearsplausible,this seemsless probable
for the corticoids.We havetried to connecttheir origin to arachidonicacid.
Severalconsiderationssuch as the high levelsof arachidonicacid and
corticoids in the adrcnals,and the reduction of the former when an important amount of the latter is excreted(Note I0), seem to establisha
correlation between these substances.According to our hypothesis,the
steroidswith a two carbon chain at Cr;, as seenpresentin the corticoids
and luteoids,would result from a cyclization of the arachidonicmolecule.
(Note 11l This would explain the specialreactivityof C., which woutd
correspond to ce of the arachidonicacid bound in this molecule by a
double bond.
A study of the different steroidsunder this energeticaspecthas permitted us to understandtheir physiologicpropcrties.
with the c3 having a hydroxyl or an oxygen as polar group in almost
all the steroids,the variety of the biologicalproperrieswould be relatedto
the different conditionsat the other extremity of the molecule,principally
at Crz, which result from the specia.lenergeticconditionsprevalentat this
regionof the molecule.The simpleststeroidsare thosehavinga polar group
representedby an OH or O fixcd at C17.Such naturally occurring steroids
have propertiesrelated to secondarysex characteristics.We will discuss
them briefly here.
Sex flormones
This group of steroidshas two polar groups, one at C.qand one at C17.
The energeticcenter at C3 can have negativeor positive polar characters,
accordingto the presenceof oxygen or hydroxyl. The energeticcenter at

140 /

R E S E A R c Hr N p H y s r o p A T H o L o c y

Crz also can have an oxygen or hydroxyl group and thus be negative or
positive. An important factor for the properties of the substanceis the
relationship between the two polar groups in the same molecule. It is apparent that the polarity of the molecule will vary according to what polar
groups are present at C3 and C17.[n a very simplified concept, which we
consider only partially accurate, we have tried to associate female and
male hormonal charact,eristicswith this polarity.
In the simple steroid molecules,a folliculinoid or estrogenicbiological
property seems to be conferred if the two polar groups-at Cs and Crz
-are formed by hydroxyls. The molecule appears to have a dipositive
polarity. It seemsto be important that the two hydroxyls be kept in the
relatively fixed reciprocal positions---+orrespondingto C3 and C,7-as
part of the solid skeleton of the steroids.Estrogenic properties are present
in various steroids that fulfill this condition. Furtherrnore, substancesfar
removed from the steroids have folliculinoid properties if they have this
relationship between the two hydroxyls. As shown in Figure 66 diethylstilbestrol,which has its two hydroxyls maintained in a fixed relative position similar to that of steroid estrogens, also shows potent estrogenic
activity.
OH

Estradiol

D i e t hIvs ti l b e s t r o l

Fro. 66. The lolliculinoid activity appears to be related to the existence of ru,o
hydroxyls kept at lhe same relative position. as it appearsin estradiol and in diethylstilbestrol.

ln the same way, we tried to correlate testoid activity with the presencc

of positive-negative
polarity; that is, with two polar centersenergetically
different, one corresponding to an oxygen and the other to a hydroxyl,
maintained in the same fixed relative position. The importance of this
relative steric position of the two polar groups for testoid activity becomes
evidentwhen it is found in substances
other than testosterone,
the principal
male hormone,with an oxygenat C3 and a hydroxyl at Cri. Testoid activity is presentin androsterone,which has an oxygenand an hydroxyl maintained in the samereciprocalpositions,althoughhere the oxygen is at C17

LIPTDS AND

LtPotDS

l4l

and the hydroxyl at C3, the reverseof testosterone.In both substances,


testosteroneand androsterone,the two conditions for testoid activity, positive-negative polarity and the same relative position between the polar
groups, are fulfi.lled.(Fis. 67)The differenceswhich exist betweenthese
substancesin their specifichormonal activity can be explained through the
difterent influence exerted upon the two polar groups in these substances
by the rest of the molecules.

Testosterone

Androsterone

Ftc. 67. The testoid activity seemsto be related to the presenceof a hydroxyt and a
carbonyl in the same fixed relative position which is insured by tbe rigidity of the
steroid molecule. The same positional reciprocal relationship is seen to exist between
these two polar groups in testosteroneand androsterone.

The testoid activity secn for cortisone, hydrocortisone and other hormones also can be explained by the presencein these moleculesof oxygen
and hydroxyl at C3 and C17,and maintenanceof the fixed position between
thesetwo polar groups.
Conceprually,the antagonismbetweenestrogenicand testoid biological
activitiescan be consideredto be ultimately related to the differencesin
polarity, which in one form or anothercan be found in other factors differing for the sexes.we will men[ion here only that a similar differencebetween male and femalecharacteris seenin the sexualchromosomes,where
the female characteris related to the XX chromosome,and the male to
an X and a Y chromosome.As we will see below, a relationshipexists
betweenlipids in generaland sex.
Besidesthe sex hormones,fatty acids appear to be connectedwith male
sex characteristics,
while femalecharacteristics
are relatedto anotherg.oup
of steroids,the sterols.
Sterols
Characteristic of the structure of this group of steroids is the presencc
of a hydroxyl at Cs and a long chain bond at C,z. Through the hydroxyl,

:-ttr I

t.t

t;

142 /

xESEARcH rN pHysropATHoLocy

the center at Cg has a nucleophilic character. This is reinforced by the


presenceof a double bond botweenC5 and C6 which, by paralleling the
bond betweenC3 and C1, increasesits ionic character and consequently
the reactivity of the hydroxyl bond to C3. Through this hydroxyl, sterols
combine in general with substanceshaving a negative polar group to form
esters.
Besidesthe capacityto combine with fatty acids in general,one of the
most important characteristicsof the principal sterol of animals, cholesterol, is its selectiveaffinity for certainfatty acid membcrs,the polyunsaturated.We tried to explain the specificityof this bond through an interesting
processwhich could be called "steric coupling."
In this process,two molecules,usually lipoids, are kept together not
only by the combination of their polar groups but also through a bond
betweentheir nonpolar parts. The two moleculesare reciprocallyattracted
through the multiple forces presentin the nonpolar groups. Some are related to attached centers, while some, such as those correspondingto
cohesionforces,are relatedto the rings themselves.An important factor is
the rigidiry of the sterol moleculewhich permits another molecule,if it is
flexible,to make the steric coupling.The rigid skelctonnot only kecpsthe
energeticcentersof one moleculein a fixed positionbut permits the flexible
aliphatic chain to cover over the polycyclic molecule and thus bring the
energeticcenters of one molecule in contact with those of the other.
Through this, steric coupling completesthe bonding of the polar groups.
The greaterthe concordanccbetwcencnergcticcentcrsin both molecules,
the more perfect the coupling is, for the more complete is the reciprocal
neutralizationof the energeticcentersof the two molecules.Stericcoupling
explains why, of all the fatfy acids presentin the organism, cholesterol
seemsto prefer to bind thosewith polyunsaturatedchains.It is thesefatty
acidswhich have severalenergeticcentersin thc nonpolar group as representedby double bonds. The long chains of these fatty acid molecules,
havinga certaindegreeof flexibility,will then completcthe stericcoupling.
(Note I2 )
Stericcoupling, in addition to its generalimportancein biology, where
it representsa kind of molecular reactivity,seemsto cxplain the antagonistic influence exercisedby different constituents,especiallythe sterols
and polyunsaturatedfatty acids.Through stericcoupling.cholesterolcould
influencethe activity of thesefatty acids more dircctly related to the nonpolar group. It has to be emphasized,however, that the neutralization
resultingfrom steric coupling is not irreversible,On thc contrary, through
the interventionof various factors,such as the breakingdown of the bond

LTPTDS AND

LTPOTDS

143

betweenthe polar groups,the two coupledmoleculescan regain their independence.This would explain the relativelability of the combinationsberween fatty acids and sterols. The antagonism between fatty acids and
sterols is an important aspectof biological dualism which will be discussed
in more detail later when these substancesare studied in terms of their
influence at the difterent levels of organization.

Steroids with a Two-Carbon Chain


Among the most important steroids are those having a two-carbon chain
fixed at C17.
Two groups, the Iuteoids and corticoids, appear directly related to
allopregnanehydrocarbon,the steroidpolycyclewith a two-carbonlateral
chain fixed at Cr;. As we have alreadyseenin the hypothesisconcerning
the origin of the steroids(Note 11l, this hydrocarboncould have been directly derived from arachidonicacid, thc two-carbonlateral chain correspondingto the tail chain of this acid, a tail which remainsafter cvclization.
The Luteoids
The prototype of the luteoids is progesterone.Two polar groups C : O
are present,one at C3 of the polycycleand the other at C:roof thc tail chain.
A paralleldouble bond betweenCa and C; completesthe formula. Energetically,progesteronepresentsa first center at C3 which appearsstrongly
nucleophilicfor two reasons:first, becauseit correspondsto the potent
electronegative
Ca and second,becauseit is reinforcedby a double bond
presentbetweenC1 and C5,and whichis henceparallelwith the doublebond
of the carbonyl.The second : O is attachedto the Cs,yof thc tail chain.
This alsoappearsreinforced,the doublebond of this carbonylbeingparatlel
to the bond betweenthe Crr and C17,which in the cyclopentane,
accordhypothesis
of
ing to the
twin carbons,binds two negativechargedcarbons.
Through its constitutionprogcsterone
is also a lipoid, thc complexhydrocarbon group being predominant over the polar groups. With its polar
nucleophilicccnters,progesterone
has the fundamentalcharacterof acid
luteoid activitycorresponds
lipoids. Progesterone's
to the presenceof two
relativelystrong neutrophiliccenterskept in the characteristicpositions,
one at C3 and the other at C2s.
We can see that any disturbancein this energeticpicture, any change
from the dinucleophilicat any center, decreasesthe luteoid propertiesof
the substance.With more profound changes,the luteoid activity is even
(Note 13)
suppressed.

144 /

nESEARcHtN pHystopATHoLocy

Corticoids
The corticoidsrepresentthe group of hormonesupon which the attention of scientistsrecentlyhas beenintensivelyfocusedbecauseof their new
therapeutic applications.
chemically, they appear to be the same as luteoids,derivativesof the
s:rmeparent hydrocarbon, allopregnane.Structurally, all these adrenocorticoid hormoneshave: a) a c3 bindingan o group; b) a double bond betweencr and cc in the first cycle;c) a two-carbontail chain with an o
attachedin ketone form to Czui d) an oH as primary alcohol presentat
c2t. This structure,common to all corticoids,seemsto be responsible
for
the principal propertiesof thesesubstances.
Corticoidshave been separated
into subgroupsbasedupon the presenceof aftachedgroups oH or _--o at
Crr or OH at Ctz. The presenceor absenceof attachedradicalsat C,,
appearsto be most important. Corticoidswithout attachedradicals at the
C1l have a major influenceon the metabolismof electrolytes.The second
group of corticoids,having the radical, are known as neoglucogeniccorticoids,the nameindicatingtheirprincipalbiologicalcharacteristics.
Energetically,the corticoids present a nucleophilic center at c3,
reinforcedby the presenceof the double bond in the cycle betweenCr and
cr. The doublebond is parallelto the doublebond of the carboxyl,and thus
inductivelyincreasesthe ionic characterof the latter.
A secondenergeticgroup of the tail chain appearsin toto as a strong
tripolarcenterwith a nucleophiliccenterat C.:,of this chain and an electrophilic center at c31. (Note 14) To this basic pattern is added, in the
neoglucogenic
corticoid,a separateenergeticcenter at C11.which can be
either clectrophilic,formed by a hydroxyl,or nucleophilic,formed by an
oxygen.
Corticoidsappeil, in general,to act as positivelipoids.(Note t5)
Becauseof their importancein relation to anti-fatty acid activity, we
will discussfirst the neoglucogeniccorticoids,the members with a polar
group also at C11.Accordingto our hypothesis,
thesesteroidshave a special biological activity, a role in the processof synthesisin the organism.
The part of the molecule between C11 and Crr constitutesan energetic
formation with a peculiar property. It representsa kind of energeticmold
or template,in which each carbon has its specificenergeticcharacter.Different radicalswould be attractedby the energeticcentersof this template
formation accordingto their own energeticnature.Kept in their respective
positions,they would be induced to bind together in order to fornr new
substances.In this manner this template formation would promote new

L T P T D SA N D L T P O T D S /

145

syntheses.[n difterent corticoids the constitution of the Crr : Czr formation


will differ and this will determine which substanceis to be synthesizedby
the respectivemold or templateformation.(Note I6)
Using the template hypothesis, we studied an entire series of body
constituentsforming the "gluco group." Glucose,galactose,glucosamine
and galactosamine,with their respectiveacids, as well as ascorbic acid,
:ue rmong thesessubstances.According to our hypothesis,thes neoglucogenic corticoids would have the important role of producing, possibly
along with other mechanisms,the entire seriesof "gluco" constituents.The
existence of difterent template formations would result in a variety of
synthesizedconstituents.
The intervention of the template formation in synthesis can occur
again and again without aftecting the molecule of the corticoid as such. It
is interesting to note here a structural curiosity which could be interpreted
as being related to template activity. In this template,the group of successivec,r, c,r, c13 and crz ?re part of the rigid skeletonof the cyclic
molecule,while C:o and Czr are forming the lateral chain attachedto C17.
This can be regarded as conferring a certain propr mobility to this lateral
chain as related to the polycycle.It is conceivablethat this lateral chain
would becomea closedformation when synthesistakes place.A movement
of the chain at Crz would permit the mold to open and thus liberate the
synthesizedmolecule. It is interesting to note here the importance of the
structure of the template for the constitution of the substancessynthesized.
Besidesthe polar group at c17, that at Crr is also important for neogluco
genic activity since it insuresa six-carbonchain in the synthesizedmolecules. A hydroxyl or carboxyl at the Co of the synthesizedsubstancewill
appar, according to the nature of the polar group at Cn of the steroid.
The respectivecharactersand positionsof Czr and C12 will permit thc
appearanceof a cycle formed by five carbons and an oxygen, characteristic
for the pyranic form of newly synthesized
substances.
An interesting confirmation of the template hypothesis was obtained
when glucosamine which, according to the hypothesis,is synthesizedby
the cortisonemolecule,was found to inducein patientsmany of the clinical
changeswhich are obtainedby treatmentwith cortisone.We will consider
these resultslater in our discussionof therapy.The capital role played by
glucosamine,galactosamineand the respectiveuronic acids in the constitution of the connective tissue representsthe "missing link" for the
explanation of the relationship betweencortisone,the other neoglucogenic
corticoids, and this tissue. Some part of the therapeutic eftect obtained

146 /

R E s E A R c Hr N p H y s r o p A T H o L o c y

with theseneoglucogeniccorticoidsin diseasesof the connectivetissuehas


to be attributed to the intervention of the amino sugars.
In the study of anti-fatty acid activity, glycerol and glycerophosphoric
ion werefound to control the absorptionand circulationof saturatedmono-,
di-, or tri-unsaturatedfree fatty acids.The sterolsappearto counterbalance
the normal polyunsaturatedmemberswhile adrenal corticoids, and especially the neoglucogeniccorticoids, counteract the toxicity of fatty acids in
general and of the abnormal conjugated members in particular. Research
done in our laboratoriesby E. F. Taskier indicatesthat the adrenalsintervenein the defensemechanismagainstfatty acids,and especiallyagainst
the conjugated members which appear to be related to abnormal conditions. (Note 17)
The part of our researchconcernedwith the role of lipoids in normal
and abnormal physiologyhas been almost entirely guided by the concept
of an antagonismbetween the two groups, one with a positive and the
other with a negativepolar character.This specificaspecthas led us to
study, together with the fatty acids and the anti-fatty acids, other substancesrelated to this antagonism.In the group of Iipoacidsor acidic lip
oids, as obtained from tissues,organs or organisms,we rccognizedthe
group of porphyrinic acids,relatedto various hemespresentin the organisms. In the group of anti-fatty acids obtained from the same sources,
differentconstituentsform the insaponifiablefractions.
As related to this dualistic aspectwe have studicd another group of
substances,
which appearto act in the organismagainstthe anti-fatty acids
themselves.These other substanceswould representa kind of biological
brake to counteract an exaggeratedintervcntion of anti-fatty acid constituents,
we have made a specialinvestigationof two substancesof this group,
glucuronic and sulfuric acid anions, which charactcristicallyseem to opposecertain anti-fatty acid substances.
Thesesubstances
appearas a result
of an exaggerated
oxidationof normalmetabolites.
Under abnormalconditions, the oxygen resultingfrom the interventionof peroxidemay be fixed
to carbohydrateseven bcfore they have undergonethe preliminary fermentativetransformationsseenin normal metabolism.with the aldehyde
group bound to phosphoricacid, the oxidationtakesplaceat c6, the second
most reactivecarbon h the molecule.This direct oxidation would represent, accordingto our view, one of the sourcesof glucuronic acid. Similarly the sulfuric anion would result from the oxidation of sulfur present
in the organism.They correspondto the oxygenphascof offba.lance
D.

LIPIDS AND

LIPOIDS

147

Glucuronic and Sulfuric Anions


urine specimensthat contain abnormal oxidizing substancesshow significant amounts of glucuronic and sulfuric acid compounds.(Nore Ig)
The analysisof the conditions under which these two substancesexert
anti-toxic activity permits a better understandingof their role in general
biology. A certain parailelism exists, and has always been emphasized,
between a detoxifying and an etminating function exerted by these two
radicals. Not only do sulfo- and glucurono-derivativesappear in the urine,
but it often has been noted that glucuronic acid intervenes when large
amounts of certain substances,such as menthol and phenol, are present
and there are insufficient sulfuric acid radicals to insure detoxification and
elimination. when mineral sulfatesare administered,the proportion of
sulfederivatives increases.
This parellelism appearsespeciallyinterestingwhen we recognizethat
sulfuric acid representsthe end result of the oxidation of sulfur introduced
into the organism in combinations in which it is a bivalent negative element. Only a smaller amount of sulfur is introduced as a hexavalentpositive element: that is, as sulfate. Sulfur is introduced mostly in bivalent
negative form, as in methionine,cystine, etc. Both sulfuric and glucuronic
acid result from oxidative processes,acting in one case upon the thiol
group and in the other upon glucose.
The relationship botweensulfuric and glucuronic acid goes still further.
It has been noted that glucuronic acid appearswhen enough sulfuric radicals necessaryfor detoxifying action are not available. However, this is
not entirely true since one processdoes not duplicate the other. Qualitative
di.fferencesintervene. (Note I9)
The significanceof glucuronic acid in the defense mechanism seems
clearer when we recognizethat, with but few exceptions such as benzoic
acid, all the substanceswith which glucuronicacid combinesare lipids or
lipoids having one or more positive polar groups. The combination with
glucuronic acid takes place through these positive polar groups. (Note 20)
In our view, glucuronic acid like sulfuric acid, has a specific role in the
defense of the organism and this seemsto be directed especially against
lipids or lipoids with a positive polar group. Bound by glucuronic acids,
the latter are eliminated as excrementalsubstances.Glucuronic acid thus
would act against many anti-fatty acid agents.We can conceiveof sulfuric
and glucuronic acids as mears by which organisms are protected against
an exaggerated activity of anti-fatty acid agents. Along the same lines,
when lipoids with positive polar groups are predominant and able to act

148 /

nEsEARcH tN pHysropATHoLocy

in an exaggeratedmanner to oppose the fatry acids physically and chemically, thc same means can be utilized to reduce this exaggeration.Thus, the
intervention of glucuronic acid as a result of an abnormal oxidation of
glucooe induced by fatty acids appears to be biologically sound. This is
also true for the sulfuric radical.
The importanceof thesesubstancesdoesnot resideonly in the fact that
the organism can easily produce them in larger quantities than fatty acids,
The fac-tthat they combine to form excrementalsubstancesis important
too, for in this way, they help in materially eliminating the anti-fatty acid
substancesfrom the organism. This would not take place if only a combination with fatty acids were possible,since the esters of fatty acids are
usually rotained in the organism and, under certain circumstances,can
again liberate their constituents.The intervention of glucuronic acid and
sulfuric acid appearsto be more effeotivethan that of the fatty acids which
have their own activity and are more toxic in exaggeratedamounts.This
appears to be especially true in the case of glucuronic acid becausethe
amount of glucose available is practically unlimited as compared with
other metabolites.Glucuronic and sulfuric acid would thus intervene in
the biological antagonism botween fatty acids and anti-fatty acid substances,inactivating and eliminating agontsfrom the last group, especialty
when in excess.Teleologically speaking,their intervention appears to be
still more interestingsince the body has, as part of its defensemechanism,
a tendencyto manufactureanti-fatty acids in excess.The interventionof
agentsother than fatty acids would prevent a vicious circle and permit an
excessof anti,fatty acidsto be removedby excretion.
FATTY ACIDS VS ANTI.FATTY

ACIDS

This study of the relationship betweenfatty acids and anti-fatty acids has
been guided by the dualistic concept. It must be recognized,however, that
the direct activity of thesesubstances
could be largely reducedto that of
one 8roup, the fatty acids. The action of anti-fatty acids is largely indirect.
They control and thus limit the activity of the fatty acids. It is within this
framework that the different anti-fatty acids selectivelyinfluencedifferent
specificfunctions of the free fatty acids.
The lipids and asscciated constituents with their multiple activities
create for each entity a balance responsiblefor many of the manifestations
of the entity. Variations in manifestationscan be attributed in large part to
qualitative and quantitative variations in the intervening lipids. A sys-

:,,;'r)

LTPTDSAND

LTPOTDS

t49

tematizationof these variations would help us understandmany of the


processesencounteredin normal and abnormal physiology.
As we have mentioned before, the balance between two antagonistic
forces, especially for normal conditions, is not static. Instead, there is
alternating predominance of the forces, which results in an oscillatory
movement. Several groups of such coupled forces, each group with its
proper rhythm, are at work. Operating simultaneously,they make for a
series of very complicated variations. Yet analysis is possible since each
of the variations follows a dualistic pattern. The variations, as they occur
at different levels and with different intensities,have been identified
through various tests. In a secondstep they have been tentativelycorrelated with changesin lipids. And next, didactically,lipid changeshave
been related to various etiologic factors, some intrinsic and some extrinsic.
^Se.r
The influence exerted by the sex of the organism upon l-ipidicbalance
was brought to our attention by a curious effect seen when cholesterolwas
administeredin an ether-oil solution to rats. only the females showed
paraplegiaand ulcerationsof the hind legs.While castrationor administration of sex hormonesdid not alter this response,it was influencedby the
administrationof two groups of lipids. The insaponifiablefraction of human placenta,for instance,was seen to induce a high sensitivityto this
preparation of cholesterol even for males, while the acid lipidic fraction
of placentapreventedparaplegiain females.(Note 2l)
Similarly,the fact that in femalesalone,adipouscells appearedquickly
in the skin of the ear after the application of sulfur mustard could be
related to the interventionof the insaponifiablefractions. (Note 22)
Starting with these observations,it could be seen that, in generar,a
higher proportion of positive lipids exists in females than in males. This
could be shown by direct analysesand by analysesof manifestationsrelated to such lipids. while many differencesare to be seen in various
manifestationsbetween females and males, only some could be related
to the direct or indirect interventionof sex hormones.In such instances.
castrationwith or without the administrationof sex hormoneswas able to
change,and even to reverse,the differencesin manifestationsseenbetween
sexes.However, in instancesin which thesemeasureswere without effect,
the differencescould be relatedto rhe interventionof lipids.

150 /

x E S E A R c Hr N P H Y s r o p A T H o L o o y

Age
The changesin lipidic balancerelated to agehave been made the object
of an extensivestudy which also sought to determine the role of lipids in
aglng processes.A general predominanceof positive lipids, more manilest
in the cellular and tissue levels than in the blood, was seen in youth. This
would be expected in view of the special motabolic influence exerted by
this group of lipids. The anoxybiotic charaoter of metabolism induced by
sterolsresultsin the intervention of dehydrogenases
which lead to an abundance of hydrogen ions. This, in turn, leads to a predomhance of the kind
of syntheseswhich favor anabolism. Growth thus could be related to the
predominanceof lipids with positive polar groups, especiallysterols.
Agng processes,on the conEary, could be related to a predominance
of lipids with negativo polar groups, especiallyfatty acids. This predominance could be found especiallyat the cellular level, as seen in cultures of
tetrahymena.(Note 23) ln complex organismsor in rats (Note 24) in which
an increasein the proportion of fatty acids at the cellular level is present,
an opposite change occurs at the systemic and even at the organic level.
There is an excessof cholesterol,this time limited to the higher levels, as
revealedthrough analysesof the blood, for instance.Changesin the blood
vesselsare related in part to this excessof sterols at the systemic level.
Many manifestationshave confirmed such an offbalance with sterol predominanceat higher levels.For example,we found the urine surfacetension
abnormally high in old age. (Note 25l Similarly, skin wheal absorption
in old people requires more than 90 minutes for completion as against
approximately 20 minutes in middle-agedaduls. A predominanceof fatty
acids at lower levels and of sterols at higher levels would thus characterizs
the changesin lipidic balancerelatedto old age.(Fig. 68)
O ther Physiological F actors
The study of the role of lipids in various physiological functions was
made indirectly for the most part, using the tests previously mentioned
which were interpreted in terms of dualistic patterns. These were related to
the general offbalances A and D and, through them, attributed ultimately
to a predominanceof sterols or fatty acids.
Sleep in itself, without relation to night or day, was found to induce
a marked change,comparableto a type A offbalancewith predominance
of sterols. Subjects with pain of an acid pattern often correlate the appearanceof pain with sleep, the pain occurring uniformly at the moment
they wake up. In these cases,the urine shows a low specific gravity with

L T P T D SA N D

LTPOTDS

l5l

a high pH and a high surface tension,correspondingto an A type offbalance.As we will see, in subjectswith an intensiveA type offbalance,
nocturnal polyuria and pollakiuria occur.
sexual intercoursein males was seento induce,in analyses,transitory
changessimilar to an offbalanceof type D, correspondingto a predominance of fatty acids.In femalesthe changecorrespondsto a transitory offbalanceof type A, manifestedby changesat the systemiclevel. Muscular
exercisewas seen to induce, in a first phase during the exerciseitself,

r00

too

60

/"

40

20

0 1 0 20 l0 10 50 60

minutes
S u b j e c t sb e t w e e n
15 and35 years

l020lo{o5060?oo

minutes
S u b j e c t sb e t w e e n
65 and87 years

F I c . 6 8 . T h e d i s a p p e a r a n ctei m e f o r t h e w h e a l i n d u c e d b y t h e i n t r a d e r m i c i n j e c t i o n
of 0.2 cc saline, varies with the age. In old age, the wheal oftcn persistsfor more
than 90 minutes.

changescomparableto an offbalancetype D. This phaseis followed by a


much longer phase of type A, indicating sterol predominance.Intensive
rnentalexerciseproducesa markedchangesimilar to offbalanceA, with all
urinary testsshowingthe patternsfound with predominanccof sterols.
The responsesattributed to influencesexerted by external lactors could
be integrated in tle same dualistic mechanism,All the data indicate the
manifest influenceexerted by the time of day. Two marked changesare
seen,one around four o'clock in the morning and the other usually around
eight or nine o'clock in the evening.The morning changecorrespondsto a
predominanceof sterols,the eveningto predominanceof fatty acids.These
changes together with the clinical manilestations related to time of day

152 /

REsEARcHrN pHysropATHoLocy

appear in a new light when interpreted not as being the direct results of
time changesbut rather of patterns of diurnal activity and nocturnal rest.
This explainswhy in rats and mice, which are nocturnal animals,most of
the analysesshow variations related to the time of day opposite to those
in humans. other variations could be recognizedmore strongly related to
time of day. variations with a 24-hour rhythm could be seen,for instance,
for urinary surfacetension in mice. But, when rats and mice were maintainedfor a length of time under artificialcondirions,with light during the
night and dark during the day, the animals changedtheir habits, becoming
active during the day and sleepingduring the night. Afrer a certain time,
most of their analytical patterns such as urinary pH, blood leucocytes,
eosinophilesetc. changed,acquiringthe type of variation seen in humans.
Urinary surface tension remained unchangedfor a long time. (Note 8
chapter IV) Even more interestingwere other changeswhich could be related to changes in externa.ltemperature.The urinary surface tension
measuredin rats in the morning for long periods of time showed variations relatedto changesin the temperatureof the environment.(Note 26)
(Fis.69)
The importanceof temperatureled to its more detailed investigation.
Variations in lipidic balancehave been found to para-llelvariations in body
temperature.The blood of normal individualsis richer in sterolsthan the
blood of those with hypothermia.Furthermore,in moments of high temperature,more sterolsare found than in momentsof low or normal temperatures.An increaseof fatty acidsoccursin conditionswith hypothermia.
These changeswere confirmed also by the correlation between blood content in lipids and temperaturein different abnormal conditions. In shock
with hypothermia the blood is rich in fatty acids, while in infections with
fever.it is rich in sterols.
The role of.temperaturewas also investigatedby studyingthe influence
upon the lipidic balance by externally applied heat or cold. Characteristic
variationscould be seenin human analysesunder the influenceof hot and
cold days, and of local applicationsof heat and cold. Manifestationscorrespondingto predominanceof lipids with positive characterwere induced
by heat, while others correspondingto predominanceof Iipids with negative characterwere inducedby cold. Variationswere seenin animalskepn
in an incubator or in a refrigerator. ( N ote 27) w e will seebelow, by studying their influence at different levels, the importance of these variations
produced by temperature.
The influence exerted by barometic change.scould be seen in changes
in total blood potassium,the two curves being paraUel.Similar changes

LTPTDSAND

LTPOtDS

153

could be observedrelated to the atmospherichumidity. Other tests as well,


such as urinary pH, calcium excretion,etc., show a similar relationship but
to a much lesserdegree.(Note 28) The influence exerted by the environment could explain the changesseenfrom one day to the other in various
analyses.(Note 28)

t2
t,
q,
L

5t

.\t ao
(-

c,
o- 32
E
o,

6'

v,

(u

.U
z

v,

(a

s5
vt

(u

(t
E
(u

6t

l!

./,
q

0aYs

Ftc. 69. The curves of the average values of surface tension of two groups of 20
malc rats cach, and two groups of female rats each, show parallel changes with thc
inverse curve of the temperatureof the environment.

ths influence exerted by changesin seasonswas studied. An increase


in fatty acids in winter and of sterols in summer could be noted. These
increa-sesalso could be largely related to the seasonalvariations in temperature. Very hot days were marked by analysesindicating intervention of
lipids with a positive character. The influence exerted by the seasonswas
sen even in the responsesof organismsto pathological conditions such as
t.:t,t:i

154 /

nESEARcHtN pHysropATHoLocy

tumors; variations in character and growth of experimental cancer could


be noted. (Note 29) The relationship of many viral infectious diseasesto
seasonalchangeswhich has been noted in many epidemiologicalstudies
coufd be rclated to the changesin lipids. (Note 30)
Efiect ol Antagonistic Lipids a Difierent Levels
A more complete study of lipids under the dualistic concept was made
by considering their activity at different levels of hierarchic organization.
This researchwas greatly facilitated by the degree of individuality which
different biological levels exhibit when they are part of the hierarchic organizationof complex organisms.It was also aided by the availability of
lower organismsin nature which correspondto various hierarchic levels.
Through this double approach,the information obtained showed the importance of the relationshipbetweenthe levels of the complex organism
and the influence exerted by lipoids. If high doses of the agents are applied, the inJluenceis exertedupon all the levels.A preferentialinfluence
is exertedupon a singlelevel if reduceddosesare used.when medium size
dosesare administered,to the preferentialeffectupon one level a reactional
responseat other levelsis added.This resultsoften in concomitantopposite
effectat theselevels.
Eflects ol Lipoids on Viruses
The antagonisticeffectsof positiveand negativelipids were evident in
the sfudy of their action upon viruses.Generally,agentswith a positive
polar group appcaredto create favorableconditionsfor the development
of viruses,while those with a negativepolar group had an oppositeeffect.
This influence,which was first seenin phagesin vitro, becamestill more
evident in viral infections.
subcutaneousadministrationof positive lipids, such as sterols or insaponifiablefractionsof organs,inducedgreaterlocal receptivityto viruses.
In experimentswith smallpox virus in rabbits,for instance,virus inoculation of the skin induced an exaggeratedresponsein those areas where
positivelipids previouslyhad been injectedsubcutaneously
comparedwith
the responsein other previouslyuntreatedareas.In less sensitivespecies
such as mice and rats, positive lipid injectionsinduced abnormally high
local receptivity to virus inoculation. Intracerebral injection of sterols
followed by subcutaneousinoculationwith smallpox virus invariably produced nervous system localizationof the virus. Intraperitonealadministration of sterolsin very high dosesin mice prior to smallpox inoculation
produced a great degree of central nervous system localization. Intra-

L T P T D SA N D

LlPOlDs

155

cerebralvirus inoculation,after subcutaneous


administrationof high doses
of anti-fatty acids, brought death earlier in test animals than in controls
given intracerebralvirus alone.
A sniking opposite effect was noted for lipids with a negativepolar
character. In rabbits, subcutaneousinjection of a polyunsaturatedfatty
acid set up a local skin area refractory to smallpox virus inoculation,although inoculation was positive in other areas of the body. Death also
occurredlater,followingintracerebral
inoculationwith a neurotropicvirus
in test animals given subcutaneousor intraperitonealinjections of fatty
acids, than in controls.This partially protectiveeffect was oppositeto the
increased receptivity seen in animals injected subcutaneouslyor intraperitoneallywith insaponifiablefractionsand intracerebrallywith the same
virus, wheredeath appearedearlier than in controls.
The antagonisticeffectsof the two groups of tipids for viral infection
appearedinterestingfrom severalpoints of view. The cffects were local,
at the cellular level, where virusesthemselvesact. Subcutaneousinjection
of lipids inducedmanifestchangcsin responsetoward the virus in the skin
at the site of injection, and little or no changeat all elsewhere.we have
utilized this fact, as we will seebelow, to obtain information regardingthc
level at rvhich various agents act. A change induced in receptivity to
viruses,limited to the skin at the site of injection, woutclindicate activity
of the agentat the cellular level. Tests basedupon the skin responseto
smallpoxvirus infection have shown that, among the lipids with a negative
polar character,a maximum of influenceis exertedby the insaponifiable
fraction of organs of exodermicorigin from speciessensitiveto the virus.
The insaponifiablefractionsof rabbit skin and rabbit brain were the most
activeof the lipids tested.Among the fatty acids,the prevcntiveeffectwas
seen to increasewith the degreeof desaturation.It was almost entirely
absent in saturatedfatty acids, notably present in polyunsaturatedfatty
acids.
The increaseand decreasein receptivityof the skin to smallpox virus
following injection of lipids also furnishedinformation about the roles of
the polar and nonpolar parts of lipids in this specificactivity. An opposite
effect was seen between two groups of substanceshaving the same nonpolar group but differing in their polar groups.While the polyunsaturated
fatty acids of saffower oil, for instance,greatly reduced receptivity, the
same polyunsaturatedmembershaving alcoholsas polar groups increased
receptivity. The polar group--negative or positive-appears to be the
factor inducingthe oppositeeffect.
The role of the nonpolar group was studied by comparing saturated

156

REsEARcH rN pHystopATHoLooy

and unsaturatedacids and alcohols. Almost no activity was seen for the
saturated.The unsaturatedmemberswere active in general,with activity in
any direction increasing with the degree of desaturation of the nonpolar
group. Thus, it appears that the nonpolar group determines whether a
substanceis active or inactive, but the nature of the activity-that is, increasingor decreasingreceptivity-is determinedby the polar group.
The influence exerted by agents with a positive charact,er upon viral
infection would explain the seasonal changes in clinical manifestations
which are especially interesting for the paralytic form of poliomyelitis.
we could show experimentally that when mice, after being inoculated
subcutaneously with smallpox vaccine virus, are kept in an incubator at
37"c, all develop cerebral involvement, while such involvement appears
in only a small proportion of other animals kept at room temperature, and
does not appear at all in those kept in a cool place. As we could also show
that one of the effects of exposure of an animal to a higher temperature
is an increase in the body of the amount of free lipids with positive character, this could explain the increasein the virus sensitivity of cells in the
central nervous sys0emwhich are especiallysensitiveto these lipids. This
relationship would also explain the increasedincidence of paralytic polio
casesduring hot weather.
The presence of greater amount of lipids with positive character in
youth helps also to explain the frequency and intensiry of viral infections
in children.(Note 31)
The study of the effects of temperature and lipids upon viruses has
shown that those effects are not limited to the host but also are exerted
upon the viruses. (Note 32) The influence of heat and cold upon virus
activity was studied in bacteriophages,where effects for virus and host
could be separated.The direct influenceupon the virus appearedrelatively
small and secondaryto the changeswhich appear in the host itself. Bacteriophage,separatedfrom microbes by filtration and kept in an incubator at
temperatures 2-3 degrees C higher or lower than controls, showed no
change in virulence. This was true as long as microbes were not present.
Microbes kept at higber temperaturewere more sensitiveto phages;when
kept at lower temperature, they were less sensitive. This influence went
so far as to change a sensitivestrain to a refractory one, and vice-versa.
The fact that microbesgrown at higher temperaturesfavor the develop
ment of bacteriophagewhile those grown at lower temperatue hinder it
could be correlatedwith the changein the richnessof lipids in the microbes
themselves.Similar results were obtained when microbes were grown for
a time in media containing fatty acids or insaponifiablefractions and were

l;r:iS$r
i

LIPTDSAND LrPolDS

157

then removed and exposed to phages.These experiments(Note 3Jl indicate the direct role played by the lipids of the hosts in the activity of
phages,and would explain the influenceexerted by temperature.Through
the change in the lipids of the hosr, the virus changestoo, becoming more
active if grown in microbes at a higher temperature and less aggressiveif
passedthrough microbes kept at lower temperature.
Efrects ol Lipids on Miuobes
The antagonisticeffectsof the two groups of lipids upon microbes were
investigated. As an example, we will mention here the characteristic
changesin Bac. anthracis treated with polyunsaturatedfatty acids and insaponifiablefraction preparations.(Fig. 70) We investigatedthe microbes
for their morphological, tinctorial, cultural and virulence characteristics.
with the fatty acids added to media, changeswhich can be consideredto
be mutationalwere induced,leadingto tiny Gram negativemicrobesgrowing on agar as transparentsmall colonies.The changes,however, were
reversible.Usually several passagesin normal media were sufficient to
produce reversal.First small and separate,then larger and more confluent
Gram positive granules were seen to appear in the microbes which, themselves, also became progressivelyplumper. Ultimately, all the characteri51is5,-rnsrphological, tinctorial and cultural---of the normal microbes
reappeared
. (Fig. 7l )
Microbes showed opposite changes when treated with insaponifiable
fractions,(Fig. 70) losing their bacillusform. Abnormally intensiveGram

6
(a)

treated.
with
sterols

i=L
\-

(b)

(c)

t?:ttio.:ilh

controI

Fro.70. lnfluence ol lipids upon microbes. Schematic drawing of the changes in.
duced in Bacillus Anthracis by the influence exerted by the two groups of lipids.
Treatcd with sterols (a) as in tbe unsaponifiable fraction of placenta, the microbes
change into cocci irregularly shaped and intensely retaining the gram stain. Treated
with fatty acids (c) from cod liver oil, the bacilli change into very tiny gram negative microbes. (b) shows untreated microbes.

..,;

t.t;.'i,*i;;',;,.

."t.

""#.;-14

158

nEsEARcH rN pHysropATHoLocy

=/--'
- -

a{a*

e&

-{.'
\

Fto' 71. Lipids and microbes. Drawing of the progressivepassagetoward normal


bacilli of the tiny gram negative microbes obtained through the treatment of Bac.
Anthracis with fatty acids. The passagetakes place usually in successivesteps. Thc
gram positive formations appear first as fine granules;they later become clumps ancl
finally give the microbes their normal aspect.

positive cocci appeared. They grew on agar as very thick creamy white
colonies. These changeswere seen to persist for a long time and seldom
were sPontaneouslyreversed.Treatment with fatty acids induced reversal
although inconsistently. we attempted to correlate the differences in
changesinduced by different lipids to the different levels of the microbe
at which they work. The changeto cocci can be regarded as corresponding
to an influenceexerted upon the membraneand the change to Gram positive to an influence upon differentiated formations present in the body.
( 3 1 3)
Effects of Lipids on Protozoa
The effects of lipids upon monocellular organisms,especiallytetrahymena pyriformis, were studied and an effort made to relate the nature
of the main changesinduced in these protozoa to changesobserved at the
cellular level of complex organisms. An initial effect was noted on the
polarity in protozoa which seemed to be oppositely influenced by long
chain polyunsaturatedfatty acids and sterols.Lipids with a positive character were seen to induce a change in the form of protozoa causing them
to become almost round, a change considered to correspond to reduced
polarity. Lipids with a negativecharacterhad an oppositeeftect;the tetrahymenabecameabnormallyelongated.
The administration of higher amounts of polyunsaturated fatty acids
was seento induce immediate changeslocalized at the anterior pole of the
organism, changeswhich ultimately lead to the breakdown of the membrane particularly at this point. This effect parallels in intensity the degree
of desaturationof the fatty acids. other changeswere seen in growth rate
and survivxfrime and, thus,in the agingprocess.(Nole 3a) (Fig.7a)
At the same time, resistanceto heat was seen to increaseas the result
of treatment with negative tipids, while it decreasedafter treatment with

LIPIDS AND

ControI

LTPOIDS

r59

with
Treated
fatty acids

Ftc. 74. In a direct action of fatty acidr on tetrahynlcna, a pas:,agcof fluicl occurs
at the surface with a break of the membrane especiallymanifest at the anterior polc
( a ) , c o n t r o l u n r r e a t e d( b ) , ( 1 2 0 0 x ) .

the positive sterols.(Note JSl The sameinfluenceupon thc aging processes,


as manifestedin a prolongation of the lifc-span, was noted for polyunsaturated fatty acids with a long chain and evcn for some mcmbcrs of the
saturated seriesbut with a shorter chain.
Effects ot' Lipids on Complex Organisnu
Morphological changes-The same level separation was uscd in thc
study of the effectsof lipids on complcx organisms.Acting at chromosomal
levels, lipids led to the appearanceof monstrosities.various lipids, especially insaponi{iablefractions of organs, rvere injectcd into larvae of flies.
While an immediate change in the cells of the larvac could be traccd to
the subnuclear level as secn in chromosornes,monstrositiesrvere seen to
be induced in the resulting flies. A similar eJTectbccame evident when
lipids were injectedinto hens' eggsbeforeor during incubation.Especially
with cholesteml but also with insaponifiablefractions of organs, a high
proportion of chickens were hatched with spasticparaplegia.
The sarneproblem is being studied, in collaboration with P. Fluss, in
Drosophila melanogaster, grown for many generations in nedia to which
an entire scriesof difierent lipids from one or the othcr group are addcd.
This study is in progressand the resultswill be published later.
We have seen that the antagonisticeffects induccd by the two groups
of lipids could be related ultimately to oppositechangesin the fundamental
biological proces$ of aging. This appeared clear for lower morphologic*l
levcls of organization and was especially evident for cells. While anti-fatty

160 /

R E S E A R c Hr N p H y s r o p A T H o L o c y

acids induce changes which can be regarded as corresponding to prolonged


youth, the polyunsaturatedfatty acids induce rapid aging with pyknosis and
karyorrhexis and death of the cells as old entities. This could be seen
clearlyin tumors,in which cellswith youthful characterlead to non-necrotic
tumoral ma.sses,
while cells that age rapidly produce necrosisin the tumors
followed by ulceration il the tumor is superficial.
The eftect of conjugated fatty acids was somewhatmore complex, indicating an abnormality in the induced processes.Their sdminisfixtion was
followed by the appe:uance of cytoplasmic and even nuclear vacuoles,
correspondingultimately to an anomaly of water metabolism.
The effect of lipids upon adipous cells appearedto be of special interest. The anti-fatty acids, especially the sterols, when injected subcutaneously in animals, induced a characteristicprocessin the adipous cells near
the injection site. These cells became very enlarged and higlrJy irregular,
with their content changed into an emulsion only slightly stained with
sudan. The fatty acids, on the contrary, imparted to adipous cells an abnormal resistanceto destruction. They remained persistently unchanged
even in the midst of very active processeswhich usually cause them to
disappear.Unchangedadipouscells were found encircled by the hvading
cancerousceus,deep within tumors in animalstreatedwith fatty acids.
On Pain-From the start of this research,the opposing effects of the
two groups of lipids upon pain has been most impressive. For the fatty
acids the degree of saturation is important. The saturated members of the
fatty acid seriesand even oleic acid are entirely without effect. Linoleic
and linolenicacidsshow a slight influence,while the polyunsaturatedmembers show a marked effect. Administration of highly unsaturatedfatty
acids and of acid lipidic fractions of certain organs,such as placenta,liver,
spleenor blood, uniformly decreasedpain of an acid pattern and increased
pain of an alkaline pattern. These oppositeeffectshave, from the beginning
of our study, contradictedthe idea that this influenceupon pain was the
result of the direct action of tlese agentsupon the nervous system.Furthermore, the opposite effects exerted upon the same pain by the other group
of lipids have confirmed the hypothesisthat the acrion takes place at the
level of the painful lesion, where the difterencesbetweentre two pains was
found to correspondto two oppositeacid-baseoffbalances.
In the study of the effectsof lipids on the pH of the secondday wound
crust,madein collaborationwith CarlosHuesca,we have demonstratedthat
lipids influence pain through changes induced on the acid-base balance
presentat the tissuelevel. The poeitivelipids constantlylowered this pH
while the negative lipids elevated it. (Note I Chapter V) Even more im-

L T P T D SA N D L T P O t D S /

151

portant than this temporary pH effect in establishingthe mechanism of


lipid action in pain was the change in the actual pattern of an existing
pain after administration of these agents. Polyunsaturatedfatty acids in
sufficient amount were found to convert an acid-pain pattern to an alkaline pattern, while sterols changed an alkaline pattern into an acid one.
We will return to this important fact later.
A pathogenic role for lipids becomesevident too, when pain can be
induced through the administration of lipids in previously painlesslesions.
Such lesions treated with large amounts of lipidic preparations became
painful. An alkaline pattern of pain was seen to appear after fatty acid
administration, while an acid pattern followed use of the insaponifiable
fraction. (Note 36)
At the tissuelevel,lipids also affectsuchacid-basesymptomsas vertigo,
itching, dyspnea,tremor, and even mental diseases.
In theseconditionsthe
same antagonismbetweenthe two groups of lipids-and the same opposite
effects upon the acid and the alkaline pattern---can be noted along with
the same possibility for changing the pattern to the opposite type if big
doses of lipids are administered.
wound Healing-The same manifest antagonism between the two
groups of lipids was also noted in their influence upon the evolution of
wounds. Changesin the sloughingand healing processwere followed by
measuring the size of wounds (Note 37) as well as by serial histological
examinations.The lipids with a negative polar group were seen to retard
the evolution of the processesby prolongrngthe first catabolic phase. positive lipids generally had an opposite effect. However, here too it could be
observedthat sterolshave relatively little effect on the healing of connective
tissue, but manifestlyfavor proliferation of the epithelia. This was especially evident in the changesin scar formation of the skin of treated animals. In rabbis treated with cholesterol,the epithelial scars were found to
have 8-10 layers insteadof the 2-3 characteristicfor the rest of the skin
and for the scars in control animals.
Regenerati6n-11 collaboration with E. F. Taskier we studied the
eftect of lipids upon the regenerationof liver in rats, after the resectionof
almost 3/q of. this organ. The rate of regenerationcould be estimated by
correlating it to the time of appearanceof fatty droplets filling up almost
all the cells. as a first step in the regenerativeprocess.In very young animals, this changein fatty liver cells was seento take place even within the
first 24 hours after resection.The change was progressivelydelayed as the
age of operated animals increased.In old animals the change in fatty liver
cells appearedonly after the fourth day.

162 /

REsEARcH rN pHyslopATHoLooy

The administration of lipids had a marked effect on appearance time


of fatty cells. Sterols induced precociousappearancein old animals. From
this point of view, sterol-treated animals appeared to react as young individuals, with fatty cells evident even on the secondday. The fatty acids
and acidJipidic fractions of organs showed an opposite effect, delaying the
time of apparance of the fatty cells. Young animals treated with polyunsaturated or conjugated fatty acids showed no fatty droplets in the liver
cells for as long as three to four days. with higher doses of the same
agents,the fatty infiltration did not occur at all.
It is interesting to note a parallelism between fatty infiltration of liver
cells and the richness of adrenals in sudanophil subsrances.An almost
complete lipid depletion of the adrenals was seen after high doses of fatty
acids and coincided with a total lack of fatty cells in liver regeneration.
(Note 38)
organic Level-Effects of the two antagonistic groups of lipids at the
organic level have been studied in terms of manifestationsclearly associated with various organs. we will review these effects briefly here, with
more details to come when the therapeuticuso of lipoids is discussed.
lnlgslings-The influence of lipids upon intestinal function is marked
by the same antagonismbetween the two groups of agents.oral administration of large amountsof fatty acids,especiallyhigher unsaturatedsuch
as obtaincd from cod liver oil, was usually fotlowed by diarrhea. Diarrhea
also occurred after parenteral administration of these substancesin large
arnounts. It was interesting to note that parenteral administration of the
acid lipidic fraction of placenta, blood or even organs had a marked influence upon the colon and rectum in particular. High doses produced
tenesmuswith a mucous or even sanguinolentsecretion.This localization
of the effectsof the lipidic fraction appearedto be especiallyinteresting
from a therapeutic point of view, as will be seen later. The oral or parenteraladministrationof the oppositegroup of lipids, sterolsand insaponifiable fractions, has an opposite effect, a constipating one, which we will
discusslater together with its therapeuticaspects.
Kidney-The manifcst opposite effects exerted by the two groups of
antagonisticlipids upon diuresis raise the question of where these effects
take place. While a systemiceffect can be recognized,a more direct intervention upon the kidney also must be considered.The addition of the acid
lipidic fraction of organs,and especiallythose obtainedfrom pork kidney,
to the perfusion fluid in a dog kidney preparation produces a manifest
decreaseof excretedurine. The administrationof insaponifiablofraction

LtPrDs AND LTPOTDS /

163

has a marked diuretic eftect which we will discuss below with its therapeutic aspect.
Nervous system-rnteresting eftects by the two groups of antagonistic
lipids upon many manifestationsof the central nervous system have been
noted.
Convulsions-Administration of sterols and insaponifiablefractions of
many organs such as placenta, liver, butter, eggs, etc., in large amounts
induces convulsionsin rats. Convulsionsalso were noted in humans when
huge dosesof these agentswere administered.But even in relatively small
amounts,theselipid agentssensitizedanimalsto the administration of other
c-onvulsantagents.In rats or mice receiving such lipids, thiamine chloride
induced convulsionsin doseswithout effect in controls. (Note 39)
An opposite effect was observed for lipids with negative character.
Saturatedfatty acids showedno influenceon thiamine-inducedconvulsions.
Such convulsions were prevented by the administration of nonsaturated
members.The effect was related to the degreeof desaturation of the fatty
acids. with increasesof the iodine number, the necessaryeffective doses
of these fatty acids becameprogressivelysmaller. While hundreds of milligrams of mono- and diethenicacids were necessaryfor each 100 gram of
body weight, the anti-convulsanteffect was obtained with only a few milliSams of clupanodonicacids,and with still lessof the nonenicacid, bixine.
The study of the pathogenesisof convulsionsalso covered the influence
exerted by these lipids of the adrenal corticoids. The administrationof
mineralocorticoids,especiallydesoxycorticosterol,
even in small doses,to
who
subjects
had receivedany one of the lipids with a positive polar group,
such as cholest,erolor insaponifiablefraction of placenta,liver or kidney,
was followed almost invariably by convulsions.we will presentmore details on this effect later in the discussionof syntheticsubstances.For the
moment we want only to note the relationship between mineralocorticoids
and lipids with positive character in the pathogenesisof convulsions.The
concomitant intervention of the two factors-an oftbalance induced by
lipids with positive character, and action of mineralcorticoids-seems to
provide new light on the pathogenic problem of epilepsy and convulsions
in general.
Coma-The role of cortical hormonesin the pathogenesisof convulsions was confirmed by the opposite effect produced by neoglucogenic
corticoids. We will see later that the administration of cortisone to subjects
receiving higher alcohols such as heptanol, octanol or octandiol in large
doses,induced a subcomatosecondition at first which progressivelychanged
into coma. (Note a0) Oppositepropertiesof the mineral and neoglucogenic

164 /

REsEARcH tN pHysropATHoLocy

corticoids,which made Seyleseparalethem accordingto their "phlogistic"


and "antiphlogistic" activity, would explain the two opposite manifestations inducing convulsionsand coma, produced in individuals previously
treated with the same anti-fatty acid agents.
On Cardiac Rhythm-The influence exerted by the two groups of lipoids upon the cardiac rhythm was studied under the same dualisl"icaspect.
The effects observed can easily be interpreted considering the role of the
differentiation of the cardiac cells for their part in the cardiac physiology.
The role of a cell in cardiac physiology is a direct function of its own
automatismwhich can ultimately be related to its degreeof differentiation.
The fact that the two groups of lipids acr antagonisticallyupon this cellular differentiation, the acid lipids exaggeratingit and the insaponifiable
fraction of sterols reducing it, has explained some of the effects induced
by these agents upon normal and abnormal cardiac rhythm. (Note 4l)
on oestral cycles-The action of the two groups of lipids at the organic level was also studied in the rat ovarian cycle. Daily, and even twice
a day, vaginal smears were made in animals treated with these agents.
when large amounts were administered,both groups suppressedthe cycle.
with smaller doses,only the lipids with positive polar groups, especiauy
sterols,produced this effect.
Systemic Level-Blood has appeared especially suitable for in vitro
and in vivo studiesof the effectsof the two groups of lipids at the systemic
level. The effects on different blood constituentswere analyzed and led to
very conclusive results. We will outline here the principal points of this
study.
Under the influence of anti-fatty acids, the erythrocytes become more
turgescent increasein volume, show a strong refringency of their crown
in dark field examination, and remain isolated from one another. The
sedimentationrate, if previously high, is reduced by treating blood in vitro
with insaponifiable fractions of organs. oxygen appears to be retained
longer in treated red cells than in controls.
The fatty acids have an opposite effect. Under their influerrce,the red
cells becomecrenelatedand develop a tendencyto form sludges.The sedimentation rate is increased.The color of the treated blood is dark and,
even after oxidation, rapidly darkens again. In vivo, lipids wittr a positive
charaoter induce leucocytosis,tlose with a negative character leucopenia.
This last effect is seen even in vitro. In Note 42, the influence exerted by
lipids upon the blood is presentedwith more details. (36)
On Temperature-The administrationof sufficient amoun{s of positive
lipids induces a frank elevation of temperature,while hypothermia follows

LrPrDs AND LtPorDs

165

thc administrationof negativelipids. The relationship betweentemperature


and lipids, however, is not so simple since changesin external lemperature
influencethe balance betweenthe antagonisticlipids. For example, animals
kept in incubators at a temperature of 35oC show an increase in lipids
with a positive character.Animals kept in a cool place, such as a refrigerator, show an increasein lipids with a negativepolar group. The organisms
are able to combat the increaseof lipids with negativecharacter by means
of the normal defensemechanism,but are less capable of dealing with an
increaseof lipids of pcitive character.Therefore, while a high proportion
of animals kept in refrigerators adapt themselvesto the new conditions,
those in incubators die in a few days.
on systemic Patterns-The influence exerted by lipids upon various
other systemic manifestationswhich are reflected in abnormal patterns in
urine analyseshas been studied. In genora.l,the fatty acids induce patterns
corresponding to the offbalance of type D, while the sterols induce patterns
of the type A offbalance.Here again we must emphasizethat any lipid, if
administered in large quantity, influencesa.ll analytical values. A certain
specificity, however, is noted since, in relatively small doses,lipids induce
changesonly in certain values. Becauseof the inherent technical problems
conceming the patterns, only a few analysescould be followed accurately
over the period of time necessaryfor a clear recognition of changes in
small laboratory animals. It is for this reason that most of our studies in
this area were made on humans where pattern changes could be easily
identffied and followed over long periods.
It is to be emphasizedthat, under these conditions, the influenceof
lipids is exerted especially upon zrlreadyexisting abnormal patterns, increasingor decreasingtheir deviationsfrom the normal, or changingthe
patterns entirely. Abnormal patternswere induced through huge amounts
of lipids, which very seldomwere administeredto patients.Tnelp X shows
schematically the analytical changesinduced by the two groups of lipids
upon various urine and blood analyses,expressedas patterns corresponding to abnormalconditions,as well as upon the manifestations
presentat
other levels.
we will discuss these effects in more detail when describins the
pharmacodynamicpropertiesof lipids and lipoids.
Mechanismol the Lipidic Biological Activity
The analysisof the changesinduced by lipids has emphasizedcertain
characlerswhich appearof capital importancefor the understandingof the
biological interventionof thesesubstances.
In one kind of activity a lipid

rirt';:l;

l.';.rr

166

RESEARCH IN

PHYSIOPATHOLOGY

acts throughits Iipoidic properties.From the data concerningits distribution


in the organismit can be seenthat, due to its solubility characters,a lipid
introducedin an organismwill be selectivelyretainedby the existinglipidic
system.When such interventionthrough its lipoidic propertiestakes place,
the nonspecificcharacterof the activity of the lipid is prevalent.A second
kind of activity results from the bond realized through the charge ol the
polar groups.The positiveor negativecharacterof thesepolar groupsdeterTrsle X
LEVEL

EFFECTS OF STEROLS

EFFECTS OF F^TTY ACIDS

Cells

Prolongsyouth character
Increases
potassiumcontent
Decreasessodium content
Reducesmembranepermeability
Reducescellularoxidation
Reduceschloridecontent

Inducesrapid aging
Decreasespotassiumcontent
Increases
sodiumcontent
I ncreasesmembranepermeability
I ncreasescellular oxidation
Increaseschloride content

Tnissaes

Lowers pH of lesions
Lowers chloride content of
lesions
Lowers water content of lesions

RaisespH of lesions
Raiseschloride content of
lesions
Raiseswatercontentof Iesions

Organs

Inducessomnolence
Inducesdiuresis
Inducesconstipation
lnducestachycardia

Inducesinsomnia
Inducesoliguria
I n d u c e sd i a r r h e a
lnducesbradvcardia

Systemic Induceshyperthermia
Induceshypertension
Blood
Increases
RC volume
DecreasesRC sed.rate
Increasespersistenceof
oxygen fixation
Determinespersistence
of
RC isolation
Determineshyperleucocytosis
Determineseosinophilia
Decreaseskalemia

Urine Induceswater excretion


Inducessulfhydrylretention
Inducescalciumexcretion
lnduceschlorideexcretion
Inducessodiumexcretion
Inducesphosphateretention
Inducesretentionof surface
active substances

Induceshypothermia
Induceshypotension
DecreasesRC volume
IncreasesRC sed. rate
Decreasespersistenceof
oxySenfixation
Determinesformation of sludge
Determinesleucopenia
Determineseosinopenia
kalemia
Increases
Induceswater retention
I nducessulfhydryl excretion
lnducescalciumretention
I nduceschloride retention
Inducessodiumretention
Inducesphosphateexcretion
Inducesexcretionof surface
activesubstances

LIPIDS AND LIPOTDS /

167

minesthus the natureof this secondkind of activity.A third kind of activity


resultsfrom the chemical constitutionof the polar group, which will induce
selectivecombinationsand consequentlywill have a more specificinfluence.
A fourth group of changesare inducedby the activity which takes place at
the nonpolar group of the lipid and more specificallyat the energeticlormations presentin it. They will have a still higher characterof specificity.
with this systematizationof the activity of the lipids, a further systematic analysisof the influenceexerted by the lipids appears possible.
Through its selectivedistribution, the administrationof a substance
having lipoidic propertieswill influencethoseentitieswhich have lipids in
an active form in their constitution.The influence exerted will thus be
proPortional to the richnessof the entity in theseactive lipids. This fact
explains why the administrationof a lipid or lipoid affectsselectivelythe
abnormalentitiesrich in free lipids and to a much lesserdegree,the normal
ones. It is this selectivedistributionwhich will further limit the activity of
the lipoid to the lipidic systemand most manifestlyto the abnormalentities.
In the frame of this limitation, this activity resultsfrom the chargeof the
polar group. Similar effectsare thus obtained for all the different lipoids
which have the same electric positiveor negativecharacterof their polar
group. This explainswhy one can usedifferentagentsfrom the samegroup
and still obtain similar results.Agentschemicallyso differentas fatty acids,
mercaptans,persulfides,aldehydesor epichlorohydrine,have similar activity becausethey all have negativepolar groups.The characteristicof the
effectsresultingfrom the electricalcharacterof the polar groups,is that they
are common for the groupshavingthe samesign and diametricallyopposite
for the agentswith a positiveor a negativepolar group.
This effect was clearly seen in fatty acids in which the negativeciuboxylic polar group was changedinto the positive primary alcohol. The
biological effects of the new substancewere opposite.
It is in the third kind of activity that the chemicalnature of the lipoids
intervene.Certain effectsresultingfrom the bond of an amino polar group
will thus be differentfrom that of the alcohols,althoughboth act as positive
energeticcentersand as such have exertedother common effects.The same
is true for the carboxyland thiol groups.
Still more specificappear the effectsresultingfrom the interventionof
the energetic factors present in the nonpolar group, such as the double
bonds,and the energeticformationsthey realizesuch as conjugated,or two
double bonds separatedby a methyleniccarbon.
The various mechanismsinvolved have explainedfurther the different
kinds of biologicaleftectswhich result.The action of the lipid by meansof

168 /

nEsEARcH rN pHysropATHoLocy

the lipoidic effect will thus influencegeneral,nonspecificmanifestations,


suchas thoseconcerningthe permeabilityof membranes.only secondarily,
will thesechangesin membraneperrneabilityinfluencethe different metabolic processes
which the membranegoverns.
In the secondgroup of changes,relatedto the interventionof the polar
groups,the antagonisticeffectsinducedwere seento concern processesresulting from membranepermeability.It is only in a third change that a
more specificaction upon the differentmetabolicprocesseshas to be considered.These are concernedwith an interventionupon metabolitesor the
agentsgoverningthem. The characterof this last lipidic interventionis its
specificinfluenceexertedupon a definitemetabolicsystem.
we tried to interpretthe influenceexerredby a lipid or lipoid according
to the above systematization.
The recent developmentof the biochemical
methodsof investigationhas put into limelight many biochemicalprocesses
by consideringthem as isolatedmetabolicentities.Most of them were seen
to result from the interventionof enzymesupon more or less specificsubstrata. One of the principal objectivesof the actual pharmacodynamic
studiesis to correlateas directly as possible,biological eftectsof diftcrent
agentsto specific metabolic processes,most of them correspondingto a
change in an enzymatic process.This approach, while very interesting,
would not take into considerationthe important role played by the nonspecificactivity of lipids and lipoids. These nonspccificinfluencesthrough
changesin the lipidic systeminducedifferentchangesin different metabolic
processes.A nonspecificchangein membranepenneability will affect many
enzymaticprocesses.It explainsthe existenceof similar influencesexerted
upon theseprocessescommon to agentswhich have nothing more in common than their lipoidic propertiesand the presenceof a positiveor a negative characterof their polar group.It is this characterwhich binds an eftect
to the nonspecificintervention.This so-systematized
analysishas thus permitted to separatethe biologicalactivity of the lipids and lipoids, the more
specificfrom the lesserinfluences,and correlateeach one to a proper or
commoncharacterof the agent.This view has amply simplifiedthe study of
the pharmacodynamic
interventionof thesesubstances.
OTHER CONSTITUENTS
In addition to the chemical elements and lipids, other constituents have
been studiedfrom the dualisticpoint of view. Although the other constituents havc receivedless emphasis.interestinginformationhas been obtained.

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LrPrDs AND LlPorDs

169

Amino Acids
Amino acids have been separatedinto groups basedupon their effects
at different levels. The first group includes the simple amino acids. In
these members the portions of the molecule which are added to [he amphoteric amino acid group, are usually electrically neutral. The amino acids
polymerizedthrough the anaphotericgroup serve as building materialsfor
the bigger protein molecules. They have appeared to be inert without
effects upon the different levels. Beyond these simple amino acids, are
two groups, energetically active, which have a second energetic center
with a negative or positive character in their molecules. while the amino
acid group servesto make thesesubstancespars of higher proteins through
the same bonds of amino acid groups as the simple members, it is the
other energeticcenter, with acid or alkaline character, which confers upon
these amino acids a positive or negativecharacter.
We studiedeffects,at difterent levels,of arginine,lysine and histidine,
which are members of the group with alkaline centers; of glutamic and
aspartic acids which have acid centers; and of methionine with a thiolic
center. Like for the lipids, the last two groups have shown similar propertias,but oppositeto thoseof the memberswith dkaline centers.The nature of their intervention appearedevident through the interesting opposite
effectsexertedupon microbes.Cultures of B. subtilis in broth containing
membersof one or the other of the antagonisticgroups show characteristic
changes.Unlike controls in which the long chains of microbes remain isolated, the microbes were seen to be kept together in media with atkaline
amino acids, forming a consistent gelatinous mass separated from the
medium. In broth with acidic or thiolic amino acids, the microbes remained separatedor formed very small aggregates.This appearedinteresting when we considered the positive character present in a.lkaline amino
acids, as related to the heterotropic,constructivetrend, while the negative,
as in the acid and thiolic members,is related to the opposite trend, We saw
further the sameantagonism betweenthe influenceexerted by histonesand
nucleic acids, the first paralleling the alkaline amino acid groups and the
second the opposite group. The more manifest effect of the ribonucleic
acids could be seen to take place at higher levels of the organization and
possibly explains the more direct action upon the genes.
We investigated the eftect of the two groups of amino acids at the
tissuelevel upon pain. Arginine,lysineand histidinedisplayedan analgesic
effect upon alkaline pain, while glutamic acid and methionine had this
effect upon acid pain. The effect could be related more to the basic tend-

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..,..b*

170

xESEARcH rN pHysropATHoLocy

ency of thesesubstancesto act through metabolicchanges,than to a direct


influence upon the acid-basesystemicbalance. The first group acts as
heterotropicagentsand the secondas homotropic, as mentionedabove.
Abnormal Amino Acids
Our researchled us to severaltentativesto defineabnormal amino acids
and the proteins they form. one concernedtheir rotatory capacity. The
naturallyoccurringamino acidsare all levorotatory.However,the organism
constantlyhas enzymesableto attackdextrorotatoryamino acid membersas
if it would have to be prepared to encounter and destroy them. Such
dextrorotatorymemberscan be conceivedto appearon a statisticalbasisas
the result of the resonanceprocessseen to occur in all the synthesisin
nature. The interventionof specificenzymesagainstthem would have the
aim to control their existenceand especiallyto prevent their interventionin
further evolution. In a work hypothesisconcerningthe cancerousprocess,
we consideredthat their persistenceand especiallytheir participation in
forming hierarchic entities would correspondto the specific abnormality
characterizingthis condition.
In another work hypothesiswhich concernsalso cancerousprocesses
and which we will discusslater, abnorma.lproteinsare thought to appearas
a resultof the bond of a carbamicradical (295) to the amino acid group.
The resultingcyclic formation having the characteristicNCNC group in
it, would correspondto abnormal amino acids which would representthe
primary characteristicformation of the cancerouscondition. (Seechapter
II,Note I)
Carbohydrates-Glucose acts as an anti-fatty acid agent, possibly becauseof the glyceryl compoundsresultingfrom its metabolism.We have
studiedit in opposition to the respectiveacids-gluconic, glucuronic and
saccharic.Glucosehas an analgesiceffect,although limited, upon pain of
an alkaline pattern, and an oppositeeffect upon pain of an acid pattern.
The acid group has an oppositeeffect upon pain. This could be correlated with the changestoward acidosisseen in the local pH of the lesions.
A manifest changetoward acidosiswas seenunder the influence of glucose
in the second day wound crust pH. We have noted previously the role
playedby glucuronic acid as an agentwith anti-positive-lipidactivity. We
believethat it is largely through this mechanismthat it favorably influences
acid pain, having an indirect action similar to that of fatty acids.

CHAPTER

DEFENSE
rTr\
REcocNtrtoN THAT multiple factors are responsiblefor abnormal
I
"t
conditions,
and that these factors can be systematizedaccording to the
concepts presented above, throws new light on a specific aspect of the
relationship between the different entities and the environment when this
tends to alter their characteristicorganization.This responseis concretized
as the defenseagainstthe noxious.The analysisof this defensehas been
facilitatedby emphasizingthe relativeindependence
of the endtiesforming
the complex hierarchic organism,the dualistic patterns of response,and
the critical role of the lipids as well as of proteins. Abnormal processesin
an organism's defense system may be better understood when they are
compared to those correspondingto normal physiologicalprocesses.For
this reason,we start with this last aspect.
The direct intervention of a noxious agent upon a biological entity can
be characterizedby its tendencyto induce heterogenization,
through an alterationof the entity'sconstituents
or the relationship
betweenthem.This, in
turn, affectsone or more of the constantsthat characterizn,the entity. The
ensuing defense response is directed ultimately at restoring the altered
constantsto their normal values.
Involved in a first stage of defenseare those very factors which normally maintain the constants,the factors which induce the oscillatory
dynamic balance. As a first response, they become exaggerated. Such
exaggeration,which takes place successively
for the opposite phases,resolves many slight noxious interventionswithout clinical manifestations.
Through a damping movement, the exaggeratedoscillationssoon return
to normal. If the normal constantsare reestablished,
the phenomenoncan
be consideredto be a physiologicalresponse.
But if the alterationsinducedby the noxiousagentpersist,an abnormal

L7r

172 /

RESEARcHIN pHystopATHoLocy

condition results. Indeed, in this casethe exaggerationof oscillatory movement can be so great that an abnormaliry may result even from this exaggeratedattempt of the entity to reestablishnormalcy. In fact, offbalances
are induced by just such changeswhich often represent,by themselvesas
will be secnlater, one of the major immediatefactors inducing the abnormalcy. As long as an abnormal condition is not resolved,the biological
entity will try to utilize new meansin order to reestablishthe normal balance. If the constantsdisturbed by the noxious intervention are fundamental, or if the changesresultingfrom the defensemechanismitself are
too great, death of the entity will result.
As expected,responseswill differ accordingto the level to which an
affectedentity belongs.However, despitethe many differencesrelated to
levels, a common and relatively simple pattern can be recognizedwhen
manifestationsoccurring at different levels as tlre result of the noxious
intervention are compared and referred to the basic pathological concepts
already noted.
Most of the information about this simple pattern was originally obtained by srudyingresponsesat the systemiclevel. Blood was parricularly
suitablebecauseof its availability,its multiple constantsand manifestcapacity to conserve them, and particularly because of the facility with
which noxious agentscould be inducedto act upon it.
The interventionof a noxious agent able to change the energeticbalance of blood sets in motion immediatelya group of successiveprocesses
which may or may not be clinically apparent,dependingupon their intensity.They have been describedas hemoclasiaby widal and hemo-shock
by many authon. Although widely investiga,ted,
the mechanismdid not
appear clcar. From our studies, we have arrived at certain conclusions
which we will briefly prasenthere.
Diphasic Phenomenon
As a noxiousfactor, we usedan intravenousinjectionof killed microbes
or of a colloidal suspensionof a metal. within a few minutes, a group of
changesoccurred.They were revealedthrough a seriesof analysesmade at
very short intervals.(Note /) The changeswere found to affect most of
the blood constituents,The most characteristicchangein our opinion is a
leucopeniawhich especiallyaffects the granulocytes.With it, there is a
lowering of serum antitryptic power; a decreaseof serum albumin; appearance of degradatedproteins,esteraseand amylase;increaseof free fatty
acids; and a lowering of coagulabilitywith reduced clot retraction.Clinically, these changesare accompaniedby hypothermia and hypotension.

DEFENSE

173

Together they representwhat we will call the "negative phase" of the immediate response.
This group of changesrepresents,in fact, only the first part of a diphasic phenomenon.The negativephase is usually followed by a second
and opposite one which we call the "positive phase" of this immcdiate
response.It results from the tendencyof the body to correct, and even
over{orrect, the changesoccurring in the fint phase. After hypothermia
and hypotension,hyperthermiaand slight hypertensionfollow. At the same
time, thc number of granulocytesincreases,as does the antitryptic power
of serum and its albumin contcnt. The serum appears richcr in frec
stcrols. Blood coagulabilityand clot retractionalso increase.After moving
rapidly to a peak, all thesevaluesreturn slowly to normal. The existence

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l'{inutes
Frc;, 75. Diphusic responsein the delense.The intravenousinjections to a normal individual of a foreign material such as of a suspensionof killed microbes or of a colloidal
metal induces a typical responsewhich correspondsto the hemoshock. A diphasic
curve seen in most of the analysescharacterizesthe occurring variations. The curve
presentedcorrespondsto the total number of the blood leucocytes.A parallel diphasic
curve is seen for other blood analysessuch as clot retraction, albumin content of the
s e r u m , a n d a n t i t r y p t i c v a l u e s o f t h e s e r u m . S i m i l a r d i p h a s i cc u r v e s , b u t o p p o s i t e i n
sens,are seen for blood coagulation time, amount of arnylase and esterasein the
\ e r u m . a m o u n t o f K , i n t h e s e r u m , a n d f o r t h e a m o u n t s o f p r o t e o s e sa n d p e p t o n e s

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174 /

xEsEARcHrN pHysropATHoLocy

of two phases can be recognized in all the changes occurring in hemos h o c k(. F i g . 7 5 )
In trying to correlatethe multiple changestaking place, it is the lysis
of leucocytes,especiallygranulocytes,which can be considcredo[ primary
importancein the developmentof hemo-shock.This is evident from the
relationship between granulocytopeniaand the intensity of the diphasic
phenomenon.The administrationof morphine or other opium derivatives
to an individual,prior to the applicationof the noxious factor, will reduce
or suppressthe granulocytopeniatogether with all the manifestations.
(Note 2/ Intensive physical exerciseconcomitantwith the application of
the noxious factor will increasethe granulocytopeniaparallel with all the
manifestationsof hemo-shock.(Note 3)
According to our hypothesis,lysis leads to liberation of proteolytic
enzymeswhich may be present as such or may be present in precursor
form in the leucocytes.And it is the interventionof theseenzymeswhich
reducesthe antitryptic power of the blood and, by digestingblood constituents,Iowers the amount of albumin presentin the serum, and induces
a parallel increaseof productsof protcin hydrolysis.The increasein anrylase as well as in esterasepresentin blood is related to the other hydrolytic enzymesliberated in this phase,and is also probably correlatedwith
leucolysis.The esterascacts hydrolytically upon the neutral fats present
and this would explain, at least in part, the liberation of free fatty acids
seenin this phase.In the changescorrespondingto the first phase,digestive
effect of theseenzymesupon the blood constituentscan be recognizedas
being one of the most important interveningfactors.
We confirmedthe correlationbetwcenthesechangesand leucolysisnot
only through thcir parallel variations, as mentioned above, but also
through in-vitro experiments.Lysis of leucocytesresultedin liberation of
hydrolytic enzymcs.An exudatc rich in granulocyteswas obtained by
injectingsterilebroth, or an aleuronsuspension,
into the pleura of rabbits.
To this exudate,removed through pleural puncture, a small amount of a
colloidalsilver-proteinpreparation(Collargol0.lvo) was added and the
preparationmaintainedat 38oC. This was seento induce the appearance
of vacuolesin the leucocytes,following the phagocytosisof silver grains.
The vacuoleswere observedto grow rapidly to huge dimensionsfollowed
(FiS. 76)
by burstingof the leucocytes.
Analysisof the pleuralfluid treatedin this mannerhas shown the same
changeas those seenin the first phaseof hemo-shock:loweringof antitryptic power with a decreasein albumin content, increasein products
formed by partial digestionof proteins,appearanceof amylaseand ester-

DEFENSE

l7S

ase, and an increaseof free fatty acids. There were also the same nuclear
"shadows" as encounteredin large amounts in the circulating blood at this
phase. The increaseof the potassiumcontent of serum seen in this phase,
and the increase found also in the supernatantpart following centrifugation of the exudate to which Collargol had been added, representsa further
confirmation of the role of leucolysisin this first phase.These data enabled
us to consider that the mechanismthrough which the blood tries to combat the intervention of a noxious agent corresponds,in the first phase,
primarily to a lysis of granulocytesfollowed by hydrolytic digestion.

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Leucocytes
traated
rtth Collarjol
FIc. 76. Drawing of the changesinducedby a colloidul su.spension
ol silver proteinate
upon leucocyles.The leucocyteswerc obtained by injecting broth intrapleurally to
rabbits. Silver proteinate was added to thc suspensionof leucocytesand thc changes
observed in a microscope heated chamber maintained at 38'C. The phagocytosisof
the silver proteinate leads first to the appearanceof this substanceas intracellular
granules, followed by the formation of vacuoles.As these grow to a huge size the
cells burst. The nucleus remains as nuclear shadow.

The second phase, which would correspond to efforts to correct the


exaggeratedeftectsof the first digestivephase,involveslargely a mobilization of reservesof those blood constituentswhich were altered during the
first digestivephase.The spleenpours a part of its storedblood into the circulation. The richnessof spleen in reticuloendothelialcells explains the
liberation of sterolswhich is seenduring this secondphase.This is recogrluzr,dby the fact that, at this time, the spleenefferent blood is richer in
sterols than the afterent blood. Other constituentscome from intercellular
and lymphatic spaces.This mobilization,characterizingthe second phase
of hemoshock, appearsto be achievedin large part mechanically,through
a direct intervention of the vegetativenervous system inducing the contraction of the smooth muscularfibers, as seen during chill, which marks
the beginningof this secondphase.Fever which follows, is in part, due to
the sterols liberated largely by the reticuloendothelialsystem.

176 /

nESEARcHrN pHysropATHoLocy

If this hemeshock, in spite of its frequently violent clinical manilestations, resolves the effects of the noxious intervention upon the blood, it
can be considered to be, to a certain degree, a physiological response.It
amounts to an exaggoration of the oscillatory mechanism through which
the characteristicconstantsof the blood are maintained. By employing the
hydrolytic enzymesstored h the leucocytes,the blood tries to resolve the
influence exercised by the noxious factor, digesting and thus breaking
down either the factor itself or the results of its direct intervention. Acting
upon blood constituents,the noxious agent often inducesthe appearanceof
micelles bigger than those normally circulating. The fatty acids liberated
by hydrolytic enzymes would insure, in the first place, a higher boundary
permeability, thus permitting the passagethrough the capillaries of substancesotherwisebarred. At the sametime the fatty acids bind the antigen
in a lipidic complex.
In the second phase, the organism tries to repair damagescaused by
the exaggerateddigestive processor by the intervention of fatty acids. If
the organism is able to resolve through a successfuldiphasic reaction the
changesinduced by the noxious agent,it returns to normal.
Prolonged Hemoshock
Inability of the organism to resolvethe noxious interventionthrough
the mechanisminvolved in the diphasic phenomenonleads to abnormal
prolongationof one phaseor another.If it is unableto destroythe noxious
factor in the first phase or to mobilize the repair process in the second
and thus correct the damage induced by the first phase, the organism remains in a prolonged first phase of hemo-shock.If the second phase is
quantitativelyor especiallyqualitativelyinadequate,the organism remains
in a prolongedsecondphase,continuingto try to resolvethe offbalanceby
a quantitativelygreater mobilization of the otherwisequalitatively inadequate weaponswhich are at its disposal.It is the predominantintervention
of the lipids which characterizestheseextendedphases.We wish to note
again that the fatty acids intervenein the prolongedfirst phasewhile antifatty acid agents,especiallysterols,are active in the second.
The adrenalsplay a particularly important role in the immediate and
prolonged defense process.In the first phase, the increasedamount of
fatty acids with four or morc double bonds found in blood and in the
organismin generalappearsto conrefrom the adrenals,which are usually
extremelyrich in thesesubstances.
In an exaggeratedlyintensiveprolonged
first phase,we found small reddish adrenalspracticallydevoid of fatty
acids.This occurrence,togetherwith the concurrentincreasein fatty acids

DEFENSE

177

in the blood, relates thesechangesin fatty acid content of the blood largely
to a Iiberation of the adrenal fatty acids into the circulation. Another important factor for the prolonged first phase appears in the intervention of
lymphocytes able to induce a lysis of compounds of very high fatty acids
such as presenteven in waxes.(Note 4) A lymphopeniacorrespondsto the
prolonged first phase. In the elevation of the amount of anti-fatty acid
agents in blood, characteristic of the prolonged second phase of the diphasic phenomenon, the adrenals seem to intervene again providing a
portion of the increasedcirculating sterols. The exaggeratedmanufacture
of sterols can be attributed to the reticuloendothelial system in general.
cranulocytosis and lymphocytosis occur in this prolonged second phase.
The interventionof sterols, which are relatively simple steroids,can explain the clinical manifestationssuch as fever, which charactcrizethe prolonged second phase, since fever can be induced by the administration of
Iarge amounts of sterols.
we can separate, from the point of view of its manifestations, the
immediatediphasichemo-shockphenomenonwith a short evolution, from
the more prolonged forms. while the former, if not too exaggerated,would
correspondto a physiologicalphenomenon,t}tc latter is always abnormal.
In the former, the principal interventionis that of hydrolytic enzymes;in
the latter, lipids play the most predominant role. pathogenically,each
phase of the diphasic phenomenon,if unable by itself to resolvethe immediateproblem, will be followed by a correspondinglipidic predominance.
The result may be either one of the two phascs,with fatty acids or sterols
predominant.We call this entire response"the antiheterogeneous
reaction"
of the defense,separatingits diphasic manifesrationsinto immediate hydrolytic and prolonged lipidic stages.
A rui heterogeneous Reaction
Although, in the prolongedlipidic stage,a certain specificiryfor particular antigens can be recognized,the antiheterogeneousresponse in
generalrepresentsrather a nonspecificeffort of the organismto resolvethe
problemscausedby the presencof any heterogeneous
factors as such.
Before going further, we want to cmphasizesomc important characteristics of this antiheterogeneousresponse related to organization. The
catabolic processespresent in the first phasc appear to result in part from
the direct hydrolytic process and in part from the biological intervention
of the products of hydrolysis, especiallyfatty acids. The hydrolytic enzymatic processis homotropic in nature by delinition, as it breaks down
difterent constituents,liberatinggroupswith anionic and cationiccharacter.

178

n EsEARcH rN pHysropATHoLocy

Enhancing the catabolic character of the first phase processesare the


anionic groups which appear to have a predominant role. The second
phase, a reparative one, is anabolic and therefore, heterotropic in character.
The study of the antiheterogeneous
responseemphasizesanother fundamentalcharacteristicof the processes.
A basic differenceexistsbetween
the direct effectsinduced by the interveningagentand thoseresultingfrom
the defenseprocessesthemselves.A direct effect of a noxious intervention
correspondsto heterogenizationof the constituents.Some changes will
appear through this heterogenizationitself, others through the defense
processes
which representthe responseof the organismto the heterogenization. While the first correspondsto a direct action, the last is catalogued
as antigenic,its manifestationsbeing grouped as defenseprocesses.The
same substancecan have a direct action and an antigenic one revealed
principally through the manifestationswhich it induces. A direct action
can be noted instantaneouslyif the changesinduced are sufficientlyintensive.The antigen effectsalways require a certain time before manifestationsappear. This time can range from a few minutes for the first
enzymaticresponseto hours or days for the prolongedresponse.
An important feature of the prolongedstageis that it persistsas long
as the noxious agent is present.This is evident in cases in which the
noxious agent can be suppressedthrough external intervention.For example, with suppressionof microbial activity by antibiotics, the corresponding clinical condition disappears.Of more interest is the effect of
antimicrobialand antitoxic immune sera. Administration of a specific
scrum, if it can neutralizethe noxious agent,producesa curative effect at
this stage.In a short time, symptomsdisappearand the organism reverts
to normal. Although nonspecific,the prolongedantiheterogeneous
reaction
shows such a straight correlation with the presenceof the antigen as to
makeus designatethis stageas primaryor toxic. In this stage,the organism
reactswith clinical manifestationsof diseaseif the antigen is capable of
inducingsufficientnoxious changes;if not, there are no clinical manifestations. Persistencein the organismof an antigenbeyond the rapid diphasic
phenomenonindicates,in general,an incapacityof the organismto achieve
its disposalsuccessfully.The need for more complex meansto combat the
antigenbecomesimperative.
with or withoutclinicalmanifestations-thatis, evenwithout a primary
toxic stageof the disease-as long as the antigcnhas not been fully neutralized, the organism will still try to resolve its intervention and return
to normal, resortingto other means.It will produce antibodieswith a cer-

DEFENSE

179

tain degreeof specificity toward the antigen. Two kinds of antibodies will
be manufactured and will differ in their fundamental characteristics,the
time of their appearance,and their role in the defenseprocesses.
CoagulantAntibodies
The first group of antibodies have a characteristicproperty. Together
with the antigen, on which they fix with a degree of specificity, they form
highly energeticcomplexes.This is manifest in a marked tendency to bind
together such complexesas well as constituentsof the blood and form huge
aggregates.When such antibodies are produced for, and act against, a
specific microbe, agglutination results. Conglutination, prccipitation and
flocculation occur when similar antibodiesact against other antigens.Due
to their tendency to establishantigen antibody complexesresulting in huge
formations, these antibodiesare generallygrouped as coagulant antibodies.
Although the coagulation characteristic is not demonstrable in vitro for
all antibodies in this group, we use the term "coagulant antibodies" for
didactic purposes.
The huge complex formation resultingfrom the binding of coagulant
antibodieswith an antigencan appear as a precipitate,agglutinatedmicrobe
or conglutinated red cells. Once established,this formation representsa
new heterogeneousentity of much larger dimensions than the antigen
alone. As srrch,it becomesby itself a new noxious agent for the organism
which consequentlyreacts against it. The organism utilizes the same processesagainst this noxious antigen-coagulantantibody complex as it uses
for any heterogeneousagent, with the same immediate diphasic or prolonged mechanism.
Teleologically,the formation of coagulantantibodiescan be interpreted
as an attempt of the organism to defend itself anew against the an,tigen.
The antigen, this time fixed through these antibodies in a new and more
noxious formation, will once again incito the nonspecific defense mechanism. First there will be the antiheterogeneousresponsewith its diphasic
phenomenonand, if once more this is not effective,a prolonged new lipidic
intervention will follow. If the quantity of heterogeneousformations is
gteat, the first phase of the diphasic phenomenoncan be so severe as to
cause death in a few minutes. If less severe,this first phase is followed by
the second,with chills and high temperature,As in all the antiheterogeneous reactions, the organism tries to combat the presenceof the noxious
factor-in this case, the flocculate produced by the antigen-antibodybond
-attempting to digest it through hydrolytic enzymes or to neutralize it
through constituents brought in during the second phase of the diphasic

180

RESEARcH rN pHysropATHoLoGy

phenomenon. If it fails, the abnormal prolonged form of this response


follows with characteristiclipidic liberation.
In terms of biological meaning, the formation of coagulant antibodies
representsa new chance for the organism to resume tho fight against antigens by using the same fundamental means, the antiheterogeneousreaction. However, since the new agent, the antigen-antibody complex, is
much more noxious than the antigen alone, the intensity of the response
will be much stronger and the chancesof disposing of the antigen will be
greater.
AIIergic Reaction
The genericterm "allergic" is reservedfor the entire group of processes
in which a coagulant antibody takes part. The reaction now against the
antigen-antibody complex is a typical antiheterogeneousresponse. A
fundamental difference exists, however, between the antiheterogeneous
reaction in the primary toxic stageand the reaction which occurs when the
heterogeneousfactor is a complex antigen-coagulantantibody usually with
more noxious character than the antigen alone. It is the nature of the antigen-antibody,the result of the bond of the coagulantand the antigen,which
gives it the allergic character. This faot explains why, although we can
possessspecificimmune sorum able to neutralizean antigen alone, this will
not influencethe allergic response.Already bound to a coagulant antibody,
the antigencannot be bound again and consequentlyneutralizrd by another
antibody. The specificneutralizing serum will have no effect upon the antigen-coagulant-antibodycomplex already formed and consequently will
have no effect upon the procssesinduced by this complex. The immune
serum does not influence the allergic manifestations which represent tbe
response to antigen-coagulant-antibodycomplexes. This would theoretically explain the favorable effects of a specific serum upon a condition
which is in the toxic primary stage,where the antigen intervenesas such,
and the lack of such favorable eftectsin the allergic stage,where the antigen is representingon.ly a part of a complex new noxious formation.
This mechanism would also explain why t}le same immune serum, although without curative effect upon the allergic stage of a condition, will
have preventivo activity. Before the onset of the allergic stage-that is,
before the coagulant antibodieshave appeared-the active immune serum
will bind and neutralizethe antigensstill free in the organism.Under these
conditions, when coagulant antibodies appear, the antigens are no longer
availableto be botrnd by them to form the noxious antigen+oagulant-antibody complexes.Without curative action, immune serum is effective as a

DEPENSE

l8l

preventive only when administeredprior to appearanceof coagulant antibodies.


An important factor for the allergic responseis the time of liberation
of coagulant antibodies. Generally, a period of 6 to 8 days is required.
Under specialcircumstances,as in casesin which the organism has manufactured the same antibodies in the past, the time necessaryfor their appe:rranceis reduced even to 4 days. In other cases,for certain antigensor
for older subjects,the time may be as long as | 4 days or even longer. For
certain antigens,or under specialcircumstances,the body appearsunable
to make coagulantantibodiesat all. In that case,no allergic manifestations
appear.
It must be emphasizedthat antibodieswill be liberatedeven if the antigen is no longer present.The presenceof antibodiesalone does not give
rise to any reaction and their appearancewill pass without any manifestations. However, they can persistunder certaincircumstancesfor months or
years and become a potential source of abnormality. At any time if the
same antigen becomes present in the body, the coagulant antibody will
form the allergic bond with it. The body will then react against the newly
formed complex with an antiheterogeneous
response.If this occurs in the
blood or central nervous system,it can appear as an immediate violent reaction which corresponds to anaphylactic shock. It is the intensive first
phaseof the diphasicphenomenonwhich kills in anaphylacticshock. such
shock can be easily producedin animalsas passiveanaphylaxyby making
coagulantantibodiesand antigensavailableconcomitantlyin the blood.
Li pi do- p rot ei c A rui bodies
Analysisof allergicantibodiesindicatesthat they have two constituents,
lipids and proteins.Electrophoreticanalysesreveal that they are displaced
mostly as beta globulins. Experiments show that such lipidoproteic antibodies lose their activity if broken into their constituents,neither the lipid
fraction nor the protein alone being able to bind the antigen.
The study of the lipido-proteic antibodies,brought us back to consider
the role of the lipids in the immediateor prolonged first phase.Moet, if not
all the natural antigenshave lipids and lipoproteinsin their structure.As
we have seenabove,someof the fatty acidsinduce defenseresponses.
The
administration of fatty acids is followed by a leucopenia-especially a
lymphopenia; administration of sterols,by a hyperleucocytosis.Some fatty
acids,suchas thoseobtainedfrom the tuberclebacilli,inducecharacteristic
lesions such as giant cells. There are both naturally present lipoproteins
and those resulting from the bond of the body's freed fatty acids to rhe

182 i

nEsEARcH lN

pHystopATHoLocy

antigen,which seemto act as specificantigens,inducing the appearanceof


coagulant,allergic antibodies.Experiments,which we will discussbelow,
have shown that, while the specificityof the antibodiesis highly related to
the protein fraction, the allergic or immune characterof the resulting responseis due to the fact that lipido-proteinsare involved in these processes.The injection of the procluctresultingfrom the action in vitro of the
acid lipidic constituentsof an organism upon proteins of another species
acts as an antigen inducing the early appearanceof coagulantantibodies.
The repeatedinjectionsof the product obtainedthrough the action in vitro
of foreign lipoacid fractionsof variousorigins upon different body proteins
also inducesallergic response.Just as we have connectedthe appearance
of the first diphasicphenomenonto hydrolytic enzymesand the prolonged
form of the antiheterogeneous
defensemechanismto the intervention of
lipids, we relate the allergicbody defenseto the interventionof lipidoproteic formations.The allergicstageof defensethus could be considered
to be a lipido-proteicdefenseresponseagainstlipido-proteic antigens.
N eutralizing A ntibodies
The unsuccessfulfight of the organismagainstan antigen through the
diphasic,lipidic or allergic responsesoften can evolve further, making use
of a more effective measurewhich correspondsto another kind of antibody, different from the coagulanttype. The characteristicof this second
type is that it forms, specificallywith the antigenagainstwhich it is manufactured,a new kind of bond, an antigen-antibody-complex,
this time entirely nonnoxious to the organism. This complex is energetically so
balancedas to corrcspondto the constantsof respectivelevelsof the body
whereit occurs.Through this new bond, the antigenis biologicallyneutralized in the sensethat the resulting antigen-antibody-complex
is entirely
harmless.
This type of neutralizingor immune antibody usually appearson or
after thc l5th day followingthe momentwhen the organismhas startedto
organizeits defenseagainstthe antigen.It can occur whether the antigen
is still prcsent or not and whether it is free or bound to coagulant antibodies.It reprcsentsthe best meansthrough which the organism opposes
the influenceexerciscdby an antigen.The appearanceof the neutralizing
antibodiescorrespondsto the last stage,the immune one, in the defense
mcchanism.If the antigenhas produceda clinical condition,the neutralizationof the antigenby the new antibodyresultsin the slow disappearance
of morbid manifestationsand a progressivereturn to normal. With or without prior clinical manifcstations,
the presenceof the neutralizingantibody

DEFENSE

183

in the organism provides a potential weapon to prevent the same antigen


from again causing trouble. This has led us to identify this part of the
defensereaction as the "immune stage" of the defensemechanism.
The action of these neutralizing antibodieshas been demonstratedbeyond doubt through passive immunity. Their administration confers protection againstthe antigen. This action is limited to the antigen so long as
it is not bound by another antibody. Clinicatly, neutralizing antibodies
have a curative value if the antigen is present, inducing the primary-toxic
response.They have, also a preventive effect upon the allergic form of the
disease if they are administered before the appearanceof the coagulant
antibodies.
lltu6alizing antibodies are globulinic in nature. They are displaced in
electrophoretic analyses as gamma globulins. Isolated zrs pure globulins,
they do not lose their activity. This would differentiate them from the
coagulant antibodies which, as previously noted, are lipideproteinic in
nature.
The defenseresourcesof organismsagainst antigensthus can be didactically separated into four fundamentally distinct groups: enzymatic
hydrolytic, lipidic, lipoproteinicand proteinic.They correspondto distinct
stagesfrom the point of view of reactionsinduced and biological meaning.
The first representsa primary, direct, immediateresponsecharacterizedby
a rapid nonspecific digestive process and followed by the exaggerated
mobilization of repair processes.If the immediate responseis inadequate,a
second stageas a prolonged lipidic defensefollows. Although it has a certain degreeof specificity,this last responseis still directed againsta heterogeneousconstitution of the agent as such. If unsuccessfulin inactivating
the agent, all these responsesare followed by another defense stage in
which action is taken against the antigen through more specific coagulant
lipido-proteinic antibodies and through the antiheterogeneousreaction to
the resulting complex. with the last stage, which is characterizedby the
intervention of proteinic neutralizingprotectiveimmune antibodies,the fight
against the antigen is usually concluded successfully.Table XI, below,
summarizes this syst,ematizationof the defenseresponse.
Defense and Hierarchic Levels
The above changes represent, in a schematic manner, what happens
when a noxious agent acts directly or indirectly upon blood. The same
basic patterns of defense,the substancesused and the processesinvolved
can be found at various hierarchic levels.It is easy to seethat, with such a

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DEFENSE

185

Jomplex mechanism occurring in different individuals and againsga great


variety of antigensr gest variations in manifestations will be evidenced.
When an antigen enters the organism and is not fully neutralized by
the intervention of immune antibodies.the mechanismof defenseis set in
motion. This can be limited to a group of entities, to one level, or can
aftect more levels and entities. According to the nature of the antigen and
the capacity of the di.fferententities to respond, the different procss will
proceed all the way to the stage of protective immunity or it can stop at
any stage.These factors, nature of the antigen, levels and entities involved
and degreeof responsefor each of them, determine the pathogeniccharacteristics of the resulting condition. The manifestationsfor a stage and a
grouP of entities can be so exclusiveas to produce a characteristicclinical
disease.The ability to respond through only a part of the defensemechanism dependson the nature of the antigen and on the conditions existing in
the different entities affected.The clinical manifqstationsfurnish important
information concerningthe defenseprocesseswhich are occurring.
C Iinical Manil estations
The first stageof the defensereaction,if highly intensive,can be manifested clinically as superacuteshock. This occurs minutes after the
intervention of the noxious factor. If the second phase of the diphasic
phenomenonis intensive,a chill with high temperaturewill appear.A state
of shock occursif fatty acids remain predominant,as in the prolongedfirst
phase. A feverish condition correspondsto a predominant anti-fatty acid
intervention in the prolongedsecondphase.while the first phase of the
condition appearsimmediately after the intervention of the antigen,the apparance and persistenceof the second phase, which corresponds to the
prolongedlipidic, dependsupon the nature and especiallythe amount of
antigen presentin the organism.Incubation time may vary from minutes to
severaldays. This relativelyshort and variable incubation time represents
an important characteristicwhich enablesus to recognizethis stage.Another characteristic of this stage is the disappearanceof symptoms after
administrationof neutralizingimmune antibodiesspecificfor the antigen.
If the antigenby itself has no toxic effectsupon the organism,its presence will not induce important manifestations.The direct response,enzymatic or prolonged lipidic, will be so limited as to have either minimal
clinical manifestationsor none at all.
we have seen that a.llergiccoagulantantibodiesusually appear after
the 6th day following penetrationof the antigeninto the organism.If the
antigen is still presnt, the appearanceof the antibodieswill induce an

186

REsEARcH rN pHysropATHoLocy

allergic condition. In caseswhere the antigen already has produced toxic


manifestations,the appearanceof the allergic stage will be marked either
by new symptoms or increasedintensity of existing ones. With a nontoxic
antigen, the presenceof which was not revealedpreviously by any clinical
manifestations,the appearanceof the allergic complex will coincide with
the appearanceof symptoms and, thus, with the appearanceof the clinical
condition. The period before the appearanceof symptoms correspondsto
the time before the appearanceof theseallergic antibodies.This incubation
time, as for all allergic manifestations,will be 6 or more days, which corresponds to the time necessaryfor the appearanceof the coagulant antibodies. An obligatory incubation time of 6 days or greater thus is an
indication that the processhas an allergic pathogenesis.
If the organism has previously manufactured similar coagulant antibodies againstthe same antigen, this incubation period could be shortened
to 5 or even 4 days. Against certain antigens,some organismsneed a longer
time to produce coagulant antibodies and the incubation time may run as
Iong as severalweeks.
Organisation and Delense
Going beyond the defensereaction in its general aspect, that is, independent of the place in the organizationwhere the characteristicprocesses
occur, we considered it in relation to hierarchic organization of entities.
conceptually, it can be acceptedthat each hierarchicentity, having a certain degreeof biological independence,
will have its own problems to resolve when it faces an antigen. Each will react for itself and. for this reason
alone, there will be differencesin the defensemechanismat different levels.
It can also be acceptedthat becauseof differencesin the means at their
dispoeal,the various entities will show individual peculiaritiesin their
resPonses.
Although some information is missing,our systematizationof the defense reaction according to the manifestations at various organizational
levels,stands.For each stage,manifestations
having common basic characteristicscan be identified at different levels such as cellular, tissular, organic, and systemic.At the cellular level, the first phase of the diphasic
phenomenoncorrespondsto a manifest increasein membrane permeability
and intracellularhydrolytic processes.
The changesseen in the first phase
of shock can be interpreted as resulting in part from such processes.we
will note here only that, for cells,the first phaseis characterizedby vacuolization of the cytoplasm and even of the nuclei similar to the vacuolization
seen in the leucocytesin the presenceof a colloidal metal. In superacute

DEFENSE

187

shock, which correspondsto the first part of the diphasicphenomenon,we


observed such vacuoles in central nervous system, liver and pulmonary
alveolarcells.(SeeShock,Chapter9) The sameprocessat the tissularlevel
causeslytic changesand, if this lysis acts upon vessels,producespetechiae.
For the systemic level, the first phase of the diphasic phenomenon is
marked by the changesoccurring in blood. The leucocytes,rich in hydrolytic enzymes,have a pronounced lytic tendency. The liberated enzymes
act upon the blood constituents and can impart to this stage the acute
dramatic aspectoften seen in clinical hemo-shock.
The prolonged lipidic phaseof the antiheterogeneous
reaction will have
different manifestationsaccording to the level affected. These differences
will correspond closely to the antagonisticinfluence exercised by the two
groups of lipids, sterols and fatty acids. we have discussedpreviously the
interventionof theselipids at differentlevels.We will mention briefly here
their role in the difterent phasesof the defensemechanrsm.
we have seen that, in general, the changesat the nuclear level correspond to prolonged youth if they are produced by the predominant intervention of sterols and to a rapid aglng with karyorrhexis and pyknosis
when produced by fatty acids. Similar manifestationscan be recognizedin
this phaseof the defensemechanismat the cellular level for the cytoplasm
and protoplasm formations, with aging signs and necrosisinduced by fatty
acids, and predominanceof youthful characteristicsinduced by sterols.For
the tissular level, intervention of fatty acids induces local alkalosis and
edema, while the sterols induce a local acidosisand fibroblasticreaction.
Lysis of vesselswith hemorrhagesoccurs in processesin which fatty acids
predominate.The predominanceof sterolsleads to a marked tendencyof
the vascularendotheliumto proliferateand this, in turn, can lead to vascular obliterationand ischemicinfarcts if the vesselsare terminal. At the organic level, the prolonged lipidic responseis more manifest than at lower
levels as the result of impaired specific function of the organ. Dualism in
clinical manifestationsis evident; oliguria or polyuria, diarrhea or constipation, insomnia or somnolenceare examplesof organic impairments seen
as clinical manifestationsof this stage of the defense mechanism. At the
systemic level, dual manifestationsare even more pronounced. Hypothermia, hypotension,cold perspiration,enophthalmia and dark-colored
blood are related to predominance of fatty acids, opposite manifestations
to predominance of sterols. Although these prolonged manifestations,part
lipidic response,can occur concomiof the nonspecificantiheterogeneous
tantly at the various levels of the organization, usually they affect one or

188 /

nESEARcHrN pHysropATHoLocy

severallevels. The manifestationscorrespondingto the one level or several


levelswill predominate.
The allergic stage shows the same clinical manifestationscommon to
the antiheterogeneousresponse with its enzymatic or prolonged lipidic
The fundamental difterencein the allergic stageis the obligatory
Processes.
incubation perid of 6 or more days. once the alleryic complex i5 lsarized,
the manifestations
are the sameas thoseproducedby highly active antigens
inducing a direct antiheterogeneousreaction.
The qualitative difterencesin the capacity of the hierarchic entities to
combat various noxious agentscan explain the diflerencesin the manifestations of allergic Processestaking place at the cellular or tissular levels as
comparedto those in the blood, which is at the systemiclevel. At any level,
the mobilization of lytic enzymesable to break down the alleryic antigenantibody complex can be so intense as to bring rapid death of the entity
or can be slow and prolonged. However, at the systemic level, as in the
circulating blood, the producs resulting from exaggeratedlysis are more
rapidly and completelydisposedof than in cytoplasmor interstitialfluids.
In the latter, they will be presentfor a long time and their noxious in-fluence
will persist.If the lytic products appear in moderate amounts in blood, the
organism may be able to disposeof them without any clinical manifestations.
For thesereasons,the presenceof antigensin the btood when coagulant
antibodies start to apPear will not induce serious manifestationsand will
even prevent them. As coagulant antibodiesappear gradually in the blood,
only small amounts of antigen-antibodycomplex will be produced at any
one time. Although highly noxious in large amounts, the complexescan be
resolved through lytic processesif formed gradua-lly,and consquently will
not provoke clinical manifestations.
At the tissular and cellular levels,a similar progressiveappearanceof
antigen-coagulant-antibodycomplex cannot be resolved in the same way.
The lytic reactions which break down this complex cannot occur with the
efficiencynoted in the blood. The complexesand tro products of lysis will
progressivelyaccumulate and the consequentmanifestationswill become
more and more intensive. It is for this reason that longJasting allergic
manifestationscolTespondto seriouslocal conditions.Even if the antigenantibody complexesare produced at a moderate rate, when antibodies appear and the antigen is present, they will induce little or no systemic
manifestations.On the contrary, seriousallergic manifestationswill arise
if the complexesare formed at the cellular,tissularor even organic levels.
Becausethe defenseProcesscsat theselevels cannot resolve them at the

DEPENSE

189

sams 1319as they appetr, as defenseprocessesin the blood can do, severe
local manifestations result. This may lead to necrosis and even rejection cif
alteredcells or tissues.These representthe very important difterenceswhich
exist between allergic processeswhich occur in the blood and those which
occur at the difterent levels following the appearanceof allergic antibodies
while the antigen is still present.
The fact that there will be no reaction when small amounts of the
allergic complex are progressivelyformed, as in casesin which antigensare
presentin the blood at the moment of appearanceof the allergic antibodi$,
is confirmed indirectly by the possibitity of prevonting severe systemic
manifestationsthrough skeptophylacticor desensitizationprocedures.The
introduction of very small amounts of antigen thus produces only small
amounts of complex at any one time, avoiding clinical manifestations.With
progressivedoses however, the antigens will fix circulating antibodies in
srrfficient proportion to prevent the formation of important amounts of the
same complexes after further administration of the antigen. The presence
of the secondphase of the diphasic phenomenon,with the exaggerationof
constituentsantagonisticto those presentin the first phase,will also act to
Prevent the occurrenceof an intensivefirst phasewhen the antigen appears
anew.
The situation changesentirely when antibodiesappear and the antigen
no longer is present. They can then accumulate in the blood in large
amounts. Thereafter, sudden appearanceof the antigen in sufficient quantity will form a large amount of the allergic complex and the subsequent
reaction can be so violent as to kill the subject.This occurs in anaphylactic
shock. When the antigenis limited to other levels,important local changes
can be induced.
The neutralizing immune antibodies, if manifestations already exist,
will prevent new ones from appearingand this will permit healing prooesses
to take place without further interference. The antibodies will prevent
manifsstationsat the respectivelevel if the antigen appearsagain.
Affinity ol Antigens
In the defense procsses,another factor intervenesto produce difierencesbetween responsesat difterent levels-the special affinity of antigens
for various cells, tissuesor organs.This affinity will determine not only the
levelbut also the individual entitieswhere manifestationswill occur. It has
to be emphasizedthat the independenceof the levelsor of groups of entities
in an organism goes so far as to allow the defenseprocessesto progressto
difierent stages.While defenseprocessesat the tissular level, for instance,

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190

REsEARcH rN pHysropATHoLocy

cannot go beyond the stage of prolonged lipidic response,those at the organic or systemic level can arrive at the allergic stage.we will see below
the importance of this unequal responseof the difterent levels.
The unequal capacity of different tissuesto manufacture allergic antibodies could be postulatedto explain the propensity for local allergic conditions. The ectodermic system appears especially inclined to allergic
responses,as seen for the skin. We tried to relate this to the natural richnessof these organs in sterols. This would explain the fact that the brain,
which is richest in sterols,seemsto show the earliest allergic manifestations,
which could be interpreted as resultingfrom early or more constant appearance of coagulantantibodies.
Besidesthese difrerencesin the responsesof various entities, an important factor intervenes in the induction of localized allergic manifestations. It corresponds to unequal affinity of the antigen itself for various
entities.This would localize the antigen in cells, tissuesor organs so that
when coagulatedantibodiesdo appear, the noxious allergic complex will
be formed locally in the sameentities.It seemsthat this localizationof the
antigen,such as upon nerves,kidney,lung, etc., is more important than the
capacity to produce antibodies in determining predilection of pathological
pr@essesfor specificcells, tissuesor organs.
one of the most interestingaspectsof the defensemechanismis the
relationshipbetween successivesteps.we could show, generally,that an
intensiveresponsein one step representsa favorable condition for appearanceof an intensiveresponsein the next step.It is a known fact that manufacture of immune antibodies is influenced by an inflammatory process.
This is the reason for the customaryinjection of tapioca, for instance,in
horsesduring their immunization for the production of therapeuticsera.
We could show that injectionsof lipids, lipid acids or insaponifiablefraction of placenta,or of organsof animalsof the same speciesfor instance,
manifestly hasten the appearanceof the next step in the defense against
the microbe.
It seemsclear that under the influenceof the lipids used,the agglutinins
appear in blood earlier and their amount increasesmore rapidly than in
the control animals.
Antigenic Factors
The interventionof different mechanismsin the defensehas led to the
suppositionthat each one would be induced by relatively specificfactors
presenteitherin the antigenitselfor appearingduring the defenseprocesses.

DEFENSE

l9l

An analysisof this aspectof the problcmof the defenschas broughtfurther


interesting
information.
The interventionof the first mechanism,that of hydrolytic enzymes
acting througha processsimilar to digestion,would have as aim to break
down the antigenitself as well as the groupsresultingfrom the bond between antigenand body constituents,
especiallyproteins.By analogywith
the processof digestion,the factorpresentin the body which would induce
this responsewould correspondto abnormallylow numberof micellcs.The
low numberof micellespresentis revealedby a cryoscopicindex near zero.
The digestivedefensemechanismwould thus intend to lower this cryoscopic index back to its normal valuesor even bclow them.
The secondmechanism,
that of the lipidic intervention,would havetwo
aims.One, to act againstfree lipidseitherprcsentin the antigenor resulting
from the hydrolytic action upon fats, and sccond,to bind hydrosoluble
constitucntsinto complexeswith a lower hydrosolubility,
and consequently
rvith lower diffusioncapacitythroughthe aqueousmedia of the organism.
This concernsthe antigenas well as the productsrcsultingfrom the lytic
intervention.The bond would take place throughthe activepolar part of
the lipid molecules.
The third mechanismis characterized
by the intervcntionof the allergic
antibodieswith thc aim of binding the antigenin higher complcxes.The
lipido-proteicantibodieswill opposea lipido-protcicfraction presentin
the antigenitselfor resultingfrom the bond bctweenlipids liberatedin the
second mechanismand proteinsof the antigenor of the body. The coagulant effcctwould resultfrom the bond throughthe polar and nonpolar
groups of the lipido-proteicantibodiesand those of the lipido-proteic
antigenicfactors.
For the fourth mechanism,characterized
by the protcctiveantibodies,
the antigenicfactor would be represented
by the proteicconstituents
of the
antigen,which leadsto an antireplication
in the specificantibodies.
It shouldbe notedthat in the complexdefensemechanismthe resultsof
the interventionof a defenseprocessrepresentantigenicfactorsfor the next
step. The presenceof productsof the enzymaticdigestionleadsto the interventionof the lipidic phase,largelyaimed to immobilizeand inactivate
them; the bond betweenlipids and antigenleadsto the appearanceof the
allergic lipido-proteicantibodies.Possibly,the occurring lipido-proteic
complexeswould intervene,facilitatingthe appearanceof the protective
antibodies.The idea that successive
antigenicfactors would induce the
of differentstepsin the defensemechanism,has led to a series
appearance
of studieswith the aim to obtain desiredreactionsthroueh the use of such

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192 /

n E s E A R c Hr N p H y s r o p A T H o L o c y

antigenicfactors. we will describehere very briefly severalsuch applications which were interestingalso becauseof the practicalresultsobtained.
Hydrolysis Products
we tried thus to utilize the productsresultingfrom the breakingdown
of body constituentsor of other materialsin order to induce through their
administration,the appearanccof the seconddefensemechanism.Applying
the dualistic concept, we separatedthus in the products of hydrolysis of
different materials,those with an acid characterfrom the group with basic
and alcoholic characters.Various materials were thus hydrolyzed using
KoH, NaoH or ammonia.The solublepart, separated,
was treatedwith an
acid and a precipitateobtained.After washingit, this precipitatewas red.issolvedby a.lkalizing
to a pH still below neutrality.This has represented
the
"acid fraction." Besidesacid lipids, this fraction containsalso acid protein
groups and even humic acids.
The part which remainedinsolubleafter treatmentwith KoH (separated from the soluble part) was treated with an acid. The part which
becamesolublewas then separated,
reprecipitated
by alkali, and partially
redissolvedby bringingthe pH, throughacidification,near 7. This representsthe "alkaline fraction." With differentdegreesof chemicalhydrolysis,
various fractions-more or less broken dsu/n-sps obtained for both the
acid and alkaline fractions.The degreeof this "digestion"has appeared
highlyimportant.The amountof the productsobtaineddecreases
for an insufficienthydrolysisas well as for a too highly pushedhydrolysis.
Accordingto the mechanisnrmentionedaboveit was expectedthat these
fractions,resultingfrom the breakingdown of body constituentsor of the
antigen and correspondingto the effect of the first enzymatic defense
mechanism,
would inducethe secondstepof the defensemechanism.This
would correspondin part to the interventionof the properdin systemand
of the lipidic defense.It has as characteristic
the fact that it would appear
only within a certaintime. The followingexperimentillustratesthis clearly.
The "acid fractions" of human blood, hydrolyzedby KoH was obtained
and then injectedintraperitoneallyto mice. At different intervalsfollowing
this injectionthe mice were inoculatedwith 3,000,000microbesof a fresh
c u l t u r eo f B a c .p r o t e u sI.n c o n t r o l st h i s i n o c u l a t i o n
w o u l d r e s u l ti n a I O O V o
lethalinfection.No protectionwas seento appearin the l6 hoursfollowing
the injectionof the "acid fraction."At thc l6th hour, J,2,,were protected.
This protectionincreasedwith time to be completeafter 22 hours, when all
the animals survived. This protection was still present after a few days.
The inoculationof thc nontreatedblood in the same proportion was

DEFENSE

le3

seenincapableof conferringthe samedegreeof defense,a fact which indicatesthe importanceof the breakingdown processin this ,.24 hours" defense.These resultsare similar to those obtainedby I. A. parfentjev with
malucidin,a product of hydrolysisof yeast.
Another applicationof the sameconcept was in the use of the lipidoproteiccomplexes.
In a group of researchstudies,we utirizedthe products resultingfrom
the bond betweenan antigenand a lipid, with the intent to obtain a lipidoproteic antigen and through it. a lipido-proteic defense response.Often
the mixture of the antigenwith the lipidic preparationsappearedsufficient.
Fatty acids, such as oleic, linoleic, arachidonicor eleostearic,acting
directly upon the killed typhoid microbeswere usually seento enhancethe
production of agglutinins and of specific immune antibodies. The same
effect was produced by lipoacids of the same spciesas the test animal.
Lipoacids of guinea pigs were especiallyactive in promoting the apparance
of agglutinins but less potent in inducing the appearanceof immune antibodies. The lipoacid fraction of bacteria such as B. subtilis, coli, diphtheria, acting in vitro upon typhoid killed microbes,led to the appearance
of antibodies against typhoid microbes but produced almoet no antibod.ies
againstthosemicrobesfrom which the fatty acidswere obtained.The lipo
acid fraction of tuberclebacitli bound to killed typhoid microbeswas seen
to induce agglutininsbut seemedto roduce and even prevent the appearance of immune antibodies.The same influencewas seen with the lipids
obtained frorn tlre seedsof Bixa orellana but was less accentuatedfor the
lipids from fish and squid. Butanol and especiallyheptanol were seen to
retard the appearanceof all antibodies,allergic and immune.
Allergic Precipitates
The injection of killed typhoid microbes agglutinated by a specific
serum was followed by rapid production of immune antisera. The serum
of rabbits injected with these mixtures prevents a lethal condition induced
in mice by intraperitonealiniectionof living microbesin much smallerdoses
than serum obtained with untreated microbes.
on the other hand, the injection of the same killed ryphoid microbes,
mixed together with a flocculate obtained, for instance, from egg protein,
and an antiegg precipitant-guinea pig serum-produces a much lcss
rapid appearance of antityphoid immune antibodies than injection of
microbes alone.
Another form of lipido-proteiccomplex, utilized as agent with the aim
to induce not a lipido-proteic responsebut a higher one in the defense

194

nEsEARcH tN

pHyslopATHoLooy

process,was that of allergic precipitates.Through a blender, we obtained


from rat and mousetumorshomogenates
in which it was no longerpossible
to seecells.After centrifugationthe supernatantfluid was separated,and
usedas antigen.Part of it was inoculatedto guineapigs,twice at 3-day intervals.The amount of appearingprecipitineswas determinedperiodically
and the animal bled when the serum had a sufficientlyhigh titre. Using the
sameantigenand the obtainedsera properlydiluted,flocculateswere obtaincd.The precipitateseparated
was injectedto animalshavingthe tumor
grafted.In a high proportion 6f g35s5-in more than Tovo in some experiments-the tumorsstartedto showchanges24 hoursfollowingthe injection
of the precipitate,to ulcerateor disappearin the subsequent
days. Similar
research,usingpooledhuman tumors,is in progress.
Intermediary Lysatesand Antigens
of interestwas a specialuseof the intermediarylysatesin order to obtain changesin the antigens,which would facilitatethe defensemechanism.
Microbes,tissuesor other products,serving as antigenswere injccted,
mixed with intermediarylysatesfrom blood or other sources.This was seen
to resultin a morc specificsecondday defenseresponse.
Mice injectedwith
such a mixture of blood intermediaryacid fraction plus killed microbes
showedresistanceto the inoculation,24 hours later, of the same living
microbesin dosesotherwiselethal.The protectionobtainedhas a marked
degreeof specificity.
In experimentsnow in course,we utilize blendedtumors mixed with
the intermediaryacid lysatefraction,to inducea defensein animalshaving
the sametumor grafted.
Thc discussionaboveconcernswhat could be called the immunological
part of the defensereaction.It has to be coupledwith many other processes
or phenomcnawhich can be systematized
as endocrine,vegetative,central
nervousor even psychologicalresponses.
Someof them could be indirectly
related to the interventionof lipids, and possibly involved through them
in the immunologicalresponses.
This concept of inrmunologicaldefense,even under its incompleteaspct has helpedus to understanda numberof importantpathogenicproblems, includingtwo which have becn of particularinterestto us: infectious
discaseand cancer.Our study of the infectiousdiseasesunder this aspect
w a s r e p o r t e di n a p r e l i m i n a r yc o m m u n i c a t i oinn 1 9 1 9 . ( 3 7 ) I n 1 9 4 2 ,t h i s
part of the researchwas presentedat the Congressof Medicine in Mexico
and publishedin the journal "Pasteur."(38)

DEFENSE

r95

INFECTIOUSDISEASES
Toxic and Allergic Conditions
In infectiousdiseasethe antigenis a micro-organismwhich may bc a virus,
microbe, protozoa,mycct, etc., or even a product elaboratedby a microorganism.Thc responscof an organism to the presenceof an infectious
antigentendsto follow the same successive
stagespreviouslyoutlincd. If
the meansat the immediatedisposalof the organismare qualitativclyand
quantitativelysufficientto neutralizethe antigen,the entire processwi.ll be
resolvedasymptomatically.
othcrwise, the first stage of the defensercaction, the primary toxic diphasicphcnomenon.will bc set into motion.
According to the qualitativeeffectiveness
o[ this response,manifestations
will vary from simplesubclinicerJ
changesto clinicalreactions.If thc second
phaseof the diphasicresponsecannottake place.a prolongedform of the
first phasewill result.It correspondsto shock,which is encounteredonly
in very severeinfections.The rapidly lethalconditionresuttingfrom transfusion of massivelyinfectcdblood is an examplc.
The secondphasebringschill and fever.If thc secondphaseresponse
is qualitativelyinsufficicnt,the prolongedform ensucs,bringing fever,the
usualmanifestationof many infcctiousdiscases.
Thc fever persistsas long
as the nonneutralizedantigcnis present.In this stageof the defcnsereaction againsta micro-organism
or its toxins,the symptoms,althoughresultingfrom thc responscof the organism,are still directly rclated to the
presenceof the antigen in sufficientquantity.The quantity necessaryto
induccthe clinicalmanifestations
can be reachcdwithin a short time after
the penetrationof the antigeninto the organism.The toxic reactionthus
can appear in a few hours. Consequently,
there is no specificobligatory
incubationtime. The manifestations
will disappcarwhen the amount of
antigenis decreased
sufficiently.For somemicrobes,antibioticshave such
action,resuitingin a decreasein the amount of the antigenprcsent,and
consequently
in the disappearance
of the clinicalmanifestations.
A similar
decreasein the amount of the free antigenprcsentcan be obtainedby its
neutralization
throughspecificimmunescra,if available.Conseqently,
such
serahave curativeeffectsin infectiousdiseases
characterized
by a primary
toxic pathogenesis.
Allcrgic antibodieswill appearafter an obligatoryincubationperiod of
6 or more days. If the antigenis still prcsent.it may be destroycdby the
new defensiveantiheterogeneous
responses
mobilizedagainstthe resulting
allergiccomplex.ln this case,the appearance
of the allcrgicantibodiesre-

196

REsEARcH rN pHysropATHoLocy

sttlts in a kind of clinical crisis which can lead to the cessationof the disease. However, if this eftect does not occur, the appearanceof allergic
antibodieswill cause an increasein symptornsor in their gravity.
In cases asymptomatic prior to the appearanceof the allergic antibodiesbecauseof low direct toxicity or insufficient quantity of the antigen,
the diseasewill become cl.inically apparent only with the appearanceof the
allergic manifestations.The clinical condition thus will have an obligatory
incubation of 6 or more days, since this representsthe time necessaryfor
the coagulant antibodies to be produced. Since the manifestationsin such
casesare due to the allergic complex and not to the direct action of antigen,
they will be nonexistentor minimal during the incubation time. Due to the
allergic complex, the condition will not respond to specific immune sera
able to neutralize the antigen but ineffective against the allergic complex.
Specific immune sera are not curative for these infectious conditions of
alleryic pathogenesis.As already noted, only when administered before
allergic antibodies have appeared,during their incubation period, do these
sera have a marked preventive effect.
Thus the pathogenesisof an infectious diseasecan be toxic or allergic
in nature. The two pathogenicmechanismscan be identified easily tfuough
incubation time of major clinical manifestations. An infectious disease
which appears shortly after the entrance of the antigen without an obligatory incubation has to be corcidered, according to our concept, to be ol
toxic pathogenesiswhile one which appears alter an incubation time obligatory greater than 5 or 6 days has to be considered allergic.
Applying this concept, we have separatedthe clinical infectious diseases
into two groups, toxic and allergic, using incubation time as the criterion.
We wish to note here the great similarity in the incubation time for the
diseasesin each group. Most of the allergic group have an obligatory incubation time ranging from 6 to 14 days, which coincides with the usual
time neededfor the appearanceof the allergic antibodies.The incubation
time is independent of the fundamental nature of the etiologioal agentvirus, microbe, protozoa, etc.---or of the nature of their products+xo.
toxins, endotoxins, etc. This indicates that the principal factor in the
incubation time is the allergic pathogenic mechanism itself.
Based upon the criterion of obligatory incubation time, the following
diseaseswith brief incubation time have been consideredas having a toxic
pathogenic mechanism: diphtheria, botulism, anthrax (Bac. anthracis),
meningococcalinfections, cholera, some streptococcalinfections, dysentery
(especiallyShiga Kruse bac.), plague, scarletfever, pneumonia,etc. In the
allergic group, with an obligatory incubation time above 6 days, we find:

DTFENSE

l9'7

typhoid, typhus, tetanus,prertussis.


rabies,mcaslcs,poliomyelitis,glanders,
etc. (TnnlE XII) In both groups.thereare variedetiologicalagents.Thus,
in the allergicgroup, for example,the antigensinclude a microbe with an
exotoxin (tetanus) with an endotoxin (typhoid), a rickcttsia (typhus),
and a virus (rabies).
'f,rsLe

Xll

lNrrctrous Dtselsus
Incubation
Low
Dyphtheria
Anthrax
Botulism
GaseousCangrene
Plague
Erysipelas
Dysentery
MeningococcicInf.
Cholera
Pneumococcus
Inf.

Ohligatoryubove 6 duys
Typhoid
-f
etanus
Pcrtussis
Glander
Tularenria
Leprosis
Typhus
Rabies
i\'teasles
Munrps
Poliomyelitis
Smallpox
Chickenpox
Recurrentfever

The concept of toxic and allcrgic pathogenesisfor these diseasesis


impressivelyconfirmed when we consider the cfTectsof specific immune
seraupon their evolution.The specificserademonstratecurative properties
for all diseasesin the first group with brief incubationtime, consideredin
our conceptbecauseof this incubatingtime as toxic. Not one of the conditions of the secondgroup, consideredas allergicon the basisof their
incubationtime alone,can bc curedby immunescra.Still more impressive
is the fact that, in spiteof the lack of curativeeffect,the sameserahave a
marked preventiveeffect upon the sameallcrgic conditionsif administered
beforethe onsetof the symptoms,that is, duringthe incubationperiod.This
confiffnsour explanationthat the therapeuticinefficiencyof the sera in the
secondgroup is due to the allergicpathogenesis
of the diseaseand not to a
lack of active antibodies.Moreovcr, the sarnesera have a curative action
upon infectionswith brief incubationperiods induced experimentallyin
animalswith the same agent.Thc concepthas been confirmed in most of
the infectiousdiseasesand we will discusssomeof thesediseasesbriefly.
Before discussingthis aspectof infectiousdiseasesin more detail, we
want to mentionanotheroccurrencewhich can bc interpretedalso through

198

p , E s E A R c Hr N p H y s r o p A T H o L o c y

the concept of allergic conditions. It concerns a kind of recurrence of


symptomsseenoften around the 7th day after the beginningof the clinical
condition in infectiousdiseaseswhich, by themselves,have allergic pathogenesissuch as typhoid, mumps,measles,pertussis,etc.
While in thesecasesthe condition itself can be consideredan allergic
manifestationagainstthe infectiousagent as antigen,a 7th day exacerbation in the course of the clinical condition can be interpretedas a second
allergicreaction.This time a new antigenhas to be considered.This appears
to occur with the first allergic manifestation,the new reaction appearing
7 days later. The complex antigen-coagulant-antibodies
responsiblefor the
clinical manifestationsof the allergic condition could representthis secondary antigen. Besides the antiheterogeneous
reaction-+nrymatic and
lipidic-which determine the symptoms of the condition, this complex
inducesthe appearanceof a new group of coagulantantibodiesagainstit.
Around the 7th day after the beginning of the clinical condition when
thesenew coagulantantibodiesagainstthis secondaryantigen appear,they
induce the exacerbationseen.
The existenceof these secondarya.llergicreactionstoward secondary
antigens,often themselvesof allergicnature, explainsmany of the tardive
manifestations
seenin the courseof infectiousconditions.
Pneumococcic P neumonia
This disease,which appearsafter a very short incubation pcriod, has
the characteristicsof a primary or toxic condition, with chill marking the
beginningof the clinical manifestations.Antipneumococcicimmune sera,
correspondingto thc type or even to the subgroupof the etiologic microbe
are curativewhen administered
in time and in adequatedoses.In the natural evolution of the disease,a crisis usually appearson the Sth day. This
correspondsto a marked aggravationof symptomswhich can be so intense
as to lead to death.The term "crisis" indicatesthis characteristicexaggeration of the manifestations.The coincidencebetween appearanceof the
crisis around the 8th day and the moment when allcrgic manifestations
gcnerallyoccur has suggestedan allergicnature for this crisis.
usually, the courseof the diseasechangessuddenlyafter the crisis.
Most of the marked manifestationsdisappearin a short time. This allergic
crisis, with an initial increasein the severityof the condition, is not seen
in patientswho have receivedspecificserumwhich has actedto preventthe
crisis as allcrgic reaction.In pneumoniathe crisis has its beneficialeffect,
a fact which accordswith the conceptthat allergic interventionprovides a
new opportunity to resolvc the interventionof the noxious agent. As we

r::l::.
'';."";..
,

DEFENSE,

,/

199

have seen,the allergicstagerepresentsa second,more complex method of


combattingantigens.In the caseof pneumococcic
pneumonia,this allergic
defenseeftort is often successful,The evolution of the diseascis stopped.
Diphtheria
We considereddiphtheria,becauseof is characteristicshort incubation
time, to be a typical primary-toxic discase.This is also confirmcd by the
curative effectof its immune serum.However,the diseasehas a manifestation in which we recognizean allergic nature: diphtheria paralysis.with
a usual obligatoryincubationperiod of about g days, and always of morc
than 6 days, diphtheriaparalysisis a typical allcrgic condition. once present, it resistsdiphtheria immune serum,yet it can be efficientlyprevented
by the same senrm. In animals such as guinea pigs and hamsten, such
paralysiscan be induced and its allergiccharacterclearty recognized.The
heatingof toxin at 56"C reducesits direct toxicity without impairing its
antigenicproperties.Administrationof even huge amountsof heatedtoxin
does not produce any immediatetoxic effect. yet the heated toxin, evcn
though it has lost its toxic effect,inducesparalysis,
classically, this para.lysishas been related to a hypothcrical thermostabilefractionof the toxin, with a high incubationtimc. This view does
not agree with thc resultsof our cxperimcnts.When different amounts of
the same heatedtoxin are injectedin guineapigs of the same sex, age and
weight, the incubation time for paralysis,although always above 6 days,
changes,becomingparadoxicallylonger when higher dosesare used. with
great amountsof the heatedtoxin, correspondingto 20,000 lethal dosesof
the nonheated toxin, incubation time becomesas long as 14-17 days in
contrastto 8-9 days for relativelysmall amounts.TesLr XIII showsthis
relationship.
T,rsle XIII
Changes
in theincubation
timeof paralysis
incluced
by diftcrentamounts
of heateddiphtheria
toxin
Arnoutrt used

.5 LD
I L D
5 L D
20 LD
IOO LD
1000 LD
5000 LD
20000 LD
'Onein [our animals
didnotshowparalysis.

Incubation ti,tleurcrage ol 4 anintals


8.33 daysr

8.25
8,00
8.25
9 . 75
I 1.00
13.75
t5.75

200

nEsEARcH rN PHysropATHoLocy

If the paralysis were induced by direct action of a thermoatabilefraction of the toxin, then higher doses of the fraction should reduce, or at
least, not increase the incubation time. This paradoxical fact can be explained simply through the mechanismof allergic pathogenesis.It is a fact
common to immunological reactionsthat an organism has gteater difficulty
in manufac{uring any antibody when very large amounts of antigen are
Presentthan when smaller amountsare involved. This difficulty is translated
into a longer time necessaryfor the appearanceof the antibodies.As seen
in our experiments,in the caseof an a.llergicreaction, this difficulty in the
manufacture of coagulant antibodies would result in a longer incubation
time.
The localization of the auergic manifestation as paralysis can be explained in part through the affnity of toxin as antigen for nerves and in
part through the participation of the nerves in the allergic reaction. The
levels at which the diphtheria toxin acts seem to be tissular, organic and
systemic,with preferencefor the adrenals,inducing characteristicsuprarenalitis. When coagulant antibodies appear, no manifest systemic allergic
reaction will occur with the antigen still present in the blood. The allergy
will be manifest,however,at the lower tissuelevel and especiallyin the
nearby nerves. Antigen must be presentin the nerve at the moment of
appearanceof the coagulant antibodies if paralysis is to occur. This can
be demonstratedby using sensitizingand triggeringinjectionsof toxin in
animals.
we sensitizedguinea pigs to heated and unheatedtoxin by injecting
relatively small amounts intravenously.On the sixth or seventhday, another small quantity of the same toxin was injected, this time near the
sciatic nerve. The total amount of toxin was far below the lethal dose. Two
or three days later, paralysisdevelopedin the injeaed timb in a high proportion of animals while no such paralysiscould be observedin animals
injected only intravenouslyor with the same total amount of toxin at once
in the limb. In other experiments,the daily injection of small amounts of
toxin, whetherheatedor nonheated,near the sciaticnerve, induced paralysis although the total quantity of toxin was much lower than that which
ordinarily would induce para.lysis
in any similar animal. paralysisappeared
in thesecasesafter an incubation period of about 12 days. The animals
sensitizedby one or more injectionsof heatedtoxin respondedto the nonheated as triggering injections and vice versa, indicating that antigenic
propertieswere responsiblcfor rhe paralytic allergic manifestation.
In humans,anti-diphtheriaserum,eftectiveagainsttoxic manifestations.
had no effect upon paralysisonce it had appearedbut is very effective in

i tr',riilliiiiLiliill

DEFENSE

201

preventingit. The s,unewas true in animal experiments.Injected 24 hours


before the appearanceof paralysis.the serum had a consistent preventive
action. This fact confirms again the allergic pathogenesis
of the paralysis.
In anotherexperiment,we showedthat administrationof cortisone,with
its anti-allergic action, also reducesthe incidenceof paralysis without having the same effect upon the direct toxic action. It can be noted, too, that
among small laboratory animals,diphtheria paralysiscan be induced readily
in guineapigs,lessreadily in hamsters,and not at all in adult rats and mice.
In addition to the sensitivityof guineapigs to diphtheria toxin, this can be
related to the great capacity of theseanimals in general to produce allergic
antibodiesand thus to be subjectto anaphylacticreaction.Basedon these
considerations,we can classify diphtheria paralysisas a typical localized
allergicreaction.
Typhoid
Typhoid, as seen in humans, is an allergic condition with an incubation period obligatory longer than 6 days. However, in experimentalanimals the samemicrobe inducesa condition with a short incubationperid,
a fact which indicatesa primary toxic pathogenesis.
This differencecan
explain the striking difterence in results with immune sera. The literature
emphasizesgreat efficacy in experimentalanimals for various immune sera
prepared againstthis microbe and its endotoxin (Chantemesse,Besredka,
Kitasato, wassermann,etc.), but no efficacyin the human disease.
In our experiments,
whena suflicientamountof microbes,aliveor dead,
was injectedat once, a primary-toxiccondition was induced in guineapigs.
The incubation period was brief. with the same microbe, alive or dead, we
were able to obtain the allergic form in guinea pigs with repeated,daily
injections of small amounts.The ailergic form similar to that seen in humrns was induced. After about 12 days, temperaturestafled to rise and
usually remained high for more than two weeks, even with the dead
microbe if the injectionswere continued.lf living microbeswere used,the
condition continuedeven without new injections.We even obtainedpositive
hemocultureat this time. Under similar conditions,the same allergic form
of typhoid also was induced in rabbits although much less consistently
than in guinea pigs. An antityphoid serum obtained from rabbits showed
activity against the toxic form of infection induced in guinea pigs. It was
entirely ineffective againstthe allergic form when that was already present,
although the total amount of microbes injected over a period of many days
was smaller than was used to induce the toxic form. Injected before the

202 /

R E S E A R c Hr N p H y s l o p A T H o L o c y

8th day, the same serum prevented the appearanceof the allergic form of
the experimental disease.
Tetanus
In the light of our concept, we studied tetanus pathogenesisin an effort
to explain the classicallyemphasizedseparationbetweenthe so<alled small
and big animal disease.(39) In mice, tetanushas a short incubation period
and is manifestedby localized contractions,while the diseaseof so-called
"big animals" starts with trismus after an incubation period of more than
6 days. Based upon incubation times, we consideredtetanus to be the toxic
form in small animals, the allergic form in large animals.
we could, in fact, induce a condition in mice manifested by trismus
and epistotonus,and having an incubation period longer than 8 days, by
daily repeatedintravenousinjectionsof small amountsof toxin. The special
affinity of this toxin for the nervous system,and the strong bond between
nervous tissue and toxin, limited the response to the antitetanic scrum
even of the primary form. If injected in time, however, the antitetanic
serum controlled this primary form. The same serum appearsto be highly
effective in the prevention of the condition in animals which have been
prepared for the allergic form, provided it is administered before the allergic manifestations have appeared. The same senrm is totally inaaive
once the allergic condition is present.
Rabies
Under all circumstances,rabies needsan incubation time of more than
5 days. From our point of view, therefore, it has to be considered an allergic condition. when the rabies virus was passedrepeatedly through the
brains of rabbits, incubation time became continuously shorter and ultimately was fixed at 6 or even 5 days. Classically,this progressivelyshortened incubationtime is interpretedas being due to progressivelyincreased
virulence of the virus after these passages.In the tight of our concept of
the pathogenesis
of infectiousdisease,a reductionof incubationtime is the
result of increasedvirulence onJy in casesof primary direct toxic pathogenesis.In the allergiccondition,which has an entirely differentpathogenic
mechanismwith the incubation period related to the time necessaryfor
the body to producecoagulantantibodies,a shorterincubationwould correspond to a different change.It resultsfrom a greaterfacility of the organism
for manufacturingantibodiesagainstthe infectious agent. In the case of
rabies, a short incubation period of 5 days for a "fixed virus" would mean
that the organism is able to manufacture allergic antibodies more easily,

DEFENSE

703

and consequentlyearlier. Apparently,antibodyproduction is more difficult


for the "streetvirus" which has a longer incubationtime. The fixed virus
consequentlyappears to be not only a brain-adaptedvirus, but also a
weaker antigen,againstwhich the body and especiallythe nervoussystem
is more easilyable to manufactureallergicantibodies.
The possibility of using the fixed virus as a vaccine in an individual
already infected with street virus can be explained by differencesin the
relationshipbetweenthe organismand the two viruses.Usually the street
virus has a much longer incubation time, indicating that the body needs
much more time to manufacture the allergic antibodies.The same as the
animal is able to make allergic antibodiesin a short time against the fixed
virus, the vaccinatedindividualwill be able to manufacturemore rapidly
also the protectivcneutralizingantibodicsagainstthe same changeclfixed
virus. The neutralizingantibodieswill thus appearearlier and act against
the "street virus" before the organismin generaland the nervoussystemin
particular has made allergic antibodiesagainstit.
It is possible,however,that an additionalfactor may intervenein this
case. In vaccine, we use an allergic complex as it is present in the nervous
system. This corresponds to a further step in the general process of immunity and its presencecould shortenstill more the time necessaryfor
the appearanceof protectiveneutralizingantibodies.
The conceptof rabies,clinical and experimental,as an allergic condition has recent-lyreceivedconfirmationthrough the results obtained with
a specific antirabiesserum (Koprowski). With no curative capacity, this
serum is able to prevent the diseaseif it is injectedbefore the appearance
of the clinical condition,even if onJyshortly before.It helps in caseswhere
no more time is left for active immunity to be establishedby the body itself
as a responseto the vaccine.This passiveimmunity is consequentlyindicated for the case in which vaccinationstarts late. The serum, with no
curative effect once the clinical condition has started, has a preventive
eflect, a fact which accordswith the conceptof rabiesas a condition with
allergic pathogenesis.
Syphilis and Tuberculosis
Syphilitic chancre has the characteristics
of an allergic condition. The
first lesion,often a small blisteron a noninduratedbase,showsa minimal
reaction in spite of its richnessin treponemas.It is only after an incubation
of about 9 days that the intensivereaction appears,with the characteristic induration. Becauseof this, tle chancre can be consideredto be a
specificallergic manifestation.The positive lutein reaction also corresponds

204

nEsEARcH rN pHysropATHoLocy

to an allergic response.The appearanceof secondarymanifestations also


can be indicative of allergic pathogenesis,but with another antigen than
the treponema involved.
Severalpossible antigens have to be considered.One would consist of
constituentsof the microbe itself againstwhich the body needsalmost one
month to manufacturespecificcoagulantantibodies.Another antigen would
correspondto constituentsof the body itself becoming heterogeneousunder
the influenceof the treponema.A lipido-proteinicantigenseemsplausible.
Complement fixation, flocculation, and other diagnostic tests for syphilis
use antigenswhich are not directly obtained from the microbes but usually
correspondto lipido-proteic fractions of organs,such as heart. In the origlnal reaction of Wassermann,the antigen was an extract from organs rich
in treponema,such as the liver of stillborn infants with heredosyphilis.
This would favor the hypothesisthat a secondaryantigen is involved and
that it has its origin in the body constituents heterogenizedthrough the
influenceof the spirochetae.A similar antigenwould accountfor the pathc.
genesisof the secondarymanifestations.By extending this concept, the
tertiary lesionsand parasyphiliticmanifestationscan also be seento be of
similar allergic pathogenesis,with other newly formed antigensinvolved.
Secondaryantigens would conceivably develop in tuberculosisas well.
White the primary tuberculouschancrecan be seen as an allergic manifestationhaving the lipido-protein of the microbe as antigen, the cavern
formation can be attributedto a secondaryantigen.
It appears highly probable that it is these "secondary" antigens,together with the inability of the organismto manufactureefficient immune
antibodies,that keep the defensein the allergic stageand impart to both
tuberculosisand syphilis not only their chronic character but also their
clinical gravity. The allergic pathogenesis
explainsalso the inefficiencyof
all the tentativesto obtain sera againsttheseconditions.
Streptococcal I nl ections
Erysipelasand many other streptococcicinfectionsappear as primary
toxic diseaseswith a short incubation.Active immune sera in sufficient
amountsinjectedin time give good results.Often a marked changein the
symptomsis seen toward the Sth day, a fact which could be considered
to indicate passageinto the allergic phase.Many other manifestationsof
streptococcal infections, such as those seen in rheumatic fever, can be
consideredallergic.
The glomerulonephritiswhich appearsas a complicationof scarletfever
or of pharyngealstreptococcicinfection is especiallyinteresting.While a

,;
:.

";*:
, " .si-"":^:,,, ;- .1: ;.
'
'-\;ij
uigfff;
-:i. * s

: . ,, :is ,'

DEFENsE

205

change in gencral symptomsin theseinfectionsis seen toward the ltth


'l-he
day, this complicationusuallyappearstoward the 24th day.
intervention of a secondaryantigenresultingfrom the bond betweenlipids and thc
renal tissuescan be hypothecatcdin the light of studics concerning immunologica.ldefense processesagainst tissues,which we present in the
following pages.
We do not want to leave the problem of infectiousdiseaseswithout a
few more words about the use of lipids in the defensemechanismagainst
microbes.The fact that lipids liberated in the first defenseresponsesare
bound to microbes and intervene in this complex form to promote the
appearance
of higherdefenseprocesses
has led us to use similar bonds in
order to stimu.latethis defense.We have seenabove how lipids other than
thoseofferedby the infectedorganismcan be used.The injectionof killed
microbes treated with lipoacid from hetcrogeneous
sources,such as from
speciesnaturallyrefractoryto the microbe.hasenhancedthe defensemechanism. Microbestreatedwith lipoacidsof the tuberclebacilli or of Bixa
orellana were seento induce a strongspccifica.llergicresponse.
Interestingresultswcre obtaincdthroughthe usc of insaponifiable
fractions bound to the microbes.The fractionsobtainedfrom refractoryspecies
appearedto be most effectivein enhancingthe defensemechanismin general. The insaponifiablefractionsobtainedfrom the entire body of rats,
animals refractory to most infections,gave the best resultsfor most of the
infectionsstudied.
in addition to using killed microbestreated
In these investigations,
with lipids in vitro, we employedanothermethod to treat the microbes.
Lipoids were added to the media in which the organismswere grown.
Somc of the lipoids were seento increase,and othersto decre:u;e,microbial
virulence.Killed and usedas vaccinesin casesof resistantinfections,these
lipoid-treatedmicrobeswere seen to induce more eflectiveimmunization.
Experimentsin progressindicatethe possibilityof using such microbes
-and even virusesso treated-to obtain long-lastingimmunity. Microbes
with very reduced virulence are used as live vaccines.Their capacity to
induce effectivedefenseresponsesin a short time also his led to their use
as "late" vaccines,i.e,, vaccineswhich can be administeredduring the
incubationtime of an infection.As thesestudiesare still in progressan
evaluationof the resultsis not yet possible.
An interestingaspect of the influenccexerted by lipids upon microorganismsis their use in producingqualitativechangesin antibiotics.Prcliminary researchshowsthat the addition of lipids of the microbesagainst

I iil

.,.iii

206 /

n E S E A R c Hr N p H y s r o p A T H o L o o y

which more active antibioticsare sought seemsto alter the antibiotics


that they have a higher degreeof specificityagainstthesemicrobes.
IMMUNOLOGICAL DEFENSE AGAINST
CELLS AND TISSUES
Heterogenizationof the Transplants
It is known that the introductionin a normal subjectof cells or tissuesfrom
an animal of another speciesor even a transplantfrom thc same spccies
will induce the appearanceof defenseprocesses.These differ with the degree of heterogeneityof the transplant.Experimentally,we can vary this
degreeof heterogeneityof transplantedcells and study the different responsesin the frame of the normal and abnormaldefensemechanism.
For the highly heterogeneous
transplant,such as cells or tissue of a
strangespecies,a primary responseoccurs, with liberation of hydrolytic
enzymes and lipids. If sufficiently strong, this response will destroy and
eliminate the transplant. If the transplant is moderately heterogeneous.
such as one from an individual of the same species,the primary reaction
is milder so that the transplantedtissuesurvivesthis attack. It will. however, be killed and rejected with the appearanceof the second defense
stage,i.e., that of the allergic reaction.The damageto the transplantcan
be attributedto the antiheterogeneous
reaction,which this time appearsto
be directedtoward the product resultingfrom the bond betweenthe transplant and tissueallergic antibodies.
For a still less hetcrogeneous
transplant,such as one from young animalsof the samespecies,
the two defenseresponses
arc nrild. However,the
transplantis often destroyedthrough a later interventionof immune antibodies. This is seen to occur after some months for organs or for cell
transplantssuch as bone marrow cells for the treatmentof severeradiation
damage.In these cases,the defensemechanismwhich intervenesmonths
after grafting can be correlatedto the immune stage.An autograft,which
is a perfectly homologoustransplant,usually will survive. The fate of a
transplantthus appearsto be determinedby its heterogeneity.
This heterogeneity, howcver, does not result only from the differenceswhich exist
betweendonor and receiver.Even an autotransplantcan be heterogenized
by surgicalmanipulation,heat or other treatment,or by changingits organizationalrelationshipto other entities.to such a degreeas to be destroyed
by an immune, allergicor even a primary defenseresponse.
The heterogeneityof the transplant-intrinsic or induced by the appli-

DEFENSE

2O7

cation of external agents-represents only one factor which determines


the nature of the defense processes.Another factor corresponds to the
changesin the antigen or constituentsinduced by the intervention of prifl&rJ, allergic or even protective irnmunological reactions. The study of
the defense reaction against the organism's own tissues or cells heterogenized by previous immunological responsesis of special interest, in view
of the role of such heterogenizedentities in a more complex defensemechanism. The organism often heterogenizesits own entities through the agents
used in the defense against foreign entities. primary, allergic and even
immune reactions induce various degreesof heterogenizationof the organism's own constituentsat various levels of the organization.
Through the intervention of hydrolytic enzymes,lipids, allergic antibodiesor even neutralizingantibodies,differentchangesin an organism's
orvn entitiescan be induced.From these,the heterogenization
of body entities by lipids was studiedin particular.The heterogeneous
effectof lipoacids
could be shown in many experiments,
as in the following: Suspensions
of
cells of differentorgansof guineapigs,in a concentrationof I gram of cells
to l0 cc.of saline,were prepared.At the sametime lipoacidsuspensions
in
saline were obtained starting from ZVo solutionsof different lipoacids in
alcohol. Four weekly administrations
to guinea pigs of the separatecell
suspensions
or of the lipoacid suspensions
were not followed in most of the
animalsby any seriousmanifestations.
A heterogenization
of the cells was
obtainedthroughthe actionof the lipoacidsuspension
upon the cetls.While
one singJeinjection of the so-treatedcells showed no noxious manifestations, consecutiveinjectionsat weekly intervalswere seento induce,in less
than a month, importantchangesgenerallyconcerningthe respectiveorgans
from which the cellsderived.
The lipoacid-cellcomplexactsas an antigen,with the type of cell determining the organ where the abnormal changeswill occur, and the lipid
determiningthe characterof the occurring reaction.Depending upon the
lipoacid, the effect will vary from minimal tissular lesions all the way to
changesleadingto death.
massivedegenerative
The degreeof heterogeneityof the lipoacid appearsto be one factor
which determinesthe stageof defenseinduced.Oleic and linoleic acids,and
the lipoacidsfrom human placenta,cow liver or total body of guinea pigs
had a slightereffect in inducing organ lesionsthan the lipoacids obtained
from Bixa orellanaand especiallyfrom the tuberclebacilli which led to
seriousdamagein the respectiveorgans.Tuberculinacting upon the cells
had the sameeffect as lipids obtainedfrom tuberclebacilli.
Through variations in the nature of the autogenousfactors-hydrolytic

208

R E s E A R c Hr N p H y s r o p A r H o L o c y

enzymes,lipids, allergic antibodiesor even immune antibodies-a graduated seriesof changesin an organism'sown entities can be induced. of
the factors which intervene in the heterogenizationof such entities, we
have studiedthe lipids in particular. An antigenicrole for lipoacidscould
be shown in many experiments.For example,suspensionsof red cells or
cellsof different tissuesof guineapigs in a concentrationof I gram of cells
to l0 cc. of saline were prepared,At the same time lipoacid preparations
wereobtainedin the followingmanner.5 cc. of a Z% solutionin alcoholof
difterentlipoacidsor mixturesof lipoacidswere added to ll0 cc. of water
and the preparationboiled under low pressureuntil reducedto 100 cc.
The cell suspensions
were administeredto guineapigs in four injections
at weekly intervalswith no seriousmanifestations.
The same was done for
the lipoacidsabove. A preparationwas obtainedthrough the action of the
lipoacidsupon the cell suspensionin the following manner. 5 cc. of the
colloidallipoacid aqueoussuspension
were addedto 5 cc. of the suspension
of cells of different tissues.To 5 cc. of red cells, only I cc. o[ the lipidic
suspensionwas added. The mixture in each case was incubated for two
hours at 37"C and centrifuged.The cellular residues,separatedfrom the
supernatantfluid, were resuspendedin saline zr.ndkept frozen. While one
injection of the so-treatedcells showed no noxious manifestations.consecutiveinjectionsat weekly intervalswere seen to induce, in less than a
month, manifest changesin the respectiveorgans. with the red cells, a
marked anemia was induced. oleic and linoleic acids, and the lipoacids
from human placenta,cow liver or total body of guinea pigs had only a
slight effect in inducing organ lesions.The lipoacids obtained from Bixa
orellana and especiallyfrom the tuberclebacilli led to seriousdamage in
the respectiveorgans and resultedin death usually in less than 3 weeks.
Tuberculin in thesecaseshad the same effect as lipids obtained from tuberclebacilli.
The degreeof heterogeneityof the lipoacid appearsto be the factor
which determinesthe stageof defenseinduced,The lipoacid-cellcomplex
acts as an antigen.with the type of cell determiningthe organ where the
abnormal changesoccurs, and the lipid determiningthe character of the
occurringreaction.Dependingupon the lipoacid. the effect will vary from
minimal tissularlesionsall the way to massivedegenerative
changesleading
to death.
The intervention of a bond betweencells and lipids apparsevident
when acid lipid preparationsare injected repeatedlyat week.lyintervalsin
the sameorgan. Lesionsare obtainedwhich are similar to but lessintensive
than thoseproducedby the cell-lipoacidcomplexes.

DEFENSE

20c)

It was highly interestingto note the differences


in lcsionsdependingon
the origin of the cells injected.Zones of necroeis.often with subacute
cellular degenerationand even with inflammatoryprocesses.were induced
by not too heterogeneous
fatty acids.Acute glomerulonephritis.
Iiver degeneration,pneumonia,enteritis or encephalitisresulted from repeated
injectionsof cel.lsfrom kidney, Iiver. lungs, intestinesand brain treated in
vitro with bixin, lipoacidsof tuberclebacilli,lipoacidsof fish or even fatty
acid mixtures of cod liver oil.
Against tissuetransplants,injectionsof the host with lipoids with negative character-such as fatty acids.mixture of lipoacidsof differentorigins,
and lipids with SH or SeH as polar groups-have exaggeratedall phasesof
defenseprocesses.
skin transplantsbetweensiblings,which usuallygive a
high percentageof acceptedgrafts,were rejectedcompletelyafter treatment
with some of theseagents.The degreeof heterogeneityof the agentsap
peared particularly interesting.with lipoacids of the same species,very
higl doseswere requiredto induceonly minimal changcs.on the other
hand, preparationsof lipoacidsof fuh, mollusk, molds and microbesproduced marked effects.Transplants treated with these preparationswere
rejected or absorbedafter eight or more days. Seldom was an immediate
rejection seen.
Even more interestingwere the resultsobtainedby direct action of the
lipids upon transplants,achievinga bond betweenthem. For theseexperiments, the agentswere usedin oil solutionsas weUas in salinesuspensions.
Transplants were dipped into diflerent preparations.Even autografts if
treated with lipoacidsof the same speciesoften werc rejected.This took
place even after more than three weeks.when more heterogeneous
lipoacids were used, such as those obtained from other species,autografts
were rejectedas completelyas transplantsof the same species,i.e., around
the eighth day. This also occurred with relatively heterogeneousagents,
such as the lipoids of microbes,especiallythose of the tubercle bacilli.
with still more heterogeneousagents,such as lipoids with SH or seH
polar groups, the treated transplantswere rejectedthrough an immediate
direct inflammatoryreaction.
The influenceexerted by injectionsof the opposite group of ripoids
with positive character was in the opposite direction. The percentageof
acceptedtransplantswas increased.
By dipping skin transplantsof animalsof the samespeciesin preparations of the insaponifiablefractionsof the species,the percentageof persistentgraftswas highly increased.ln someexperimentsall the transplants
between siblingswere positive.Even betweendifferent strains of mice,

210 /

R E s E A R c Hr N P H Y s T o P A T H o L o c Y

such positive results were obtained. The treatment of transplants with


butanol alone was not effective.Adding butanol to the preparationof insaponifiablcfractions,however,enhancedthe effectof the latter.
The most interesting results were obtained by cross-treatment-in
which the transplant was treated with the insaponifiablefraction of the
strain of the host and the host with the insaponifiablefraction of the donor.
An unusual number of positive grafts were obtained between strains of
mice and, in exceptionalcases,even betweenspecieswhen a mixture of
the two preparationsof insaponifiablefractionsof the donor and host was
usedfor the treatmentof both transplantand host.
Even more interestingresultswere obtainedwhen, in addition to these
treatmentsof transplantand host, anothertreatment-that of the "bed" of
the transplsnf-"rin5added.The wound receivingthe transplantwas soaked
with the mixture of insaponifiablefractions. Often aJter the graft, treatment with the fractions was continued through small injections into the
bed of the transplant.Injections into the transplant itself, if possible,increasedthe number of positiveresults.
Before pursuing further the study of these interestingproblems, an
analysisof another aspectof the responseto hcterogenizedmatcrial has
appearednecessary.It concernsthe interventionof different levels of the
organizationin the defcnsemechanism.This was seen to vary according
to the degreeof heterogeneity
of the transplant.The defenseprocessesthus
can be limited to the heterogenized
entity or to entitiesof the samelevel.
or they can extcnd far into the hierarchic organization.With a highly
heterogenizedmaterial, a broad hierarchic reaction occurs with several
superiorlevels intervening.In thesecasesprimary enzymaticor prolonged
processes
involve the tissular,organicand systemiclevels.With lessheterogcnization,the ensuingprimary reaction is not strong enough to destroy
and eliminatethe heterogenized
entitiesand an allergicreactiontakesplace.
This involvcsother levelssuch as tissularand even organic.With still less
intensiveheterogenization,the defenseremains localized at the affected
level itself and is weaker.With the defenseinefiicientin its primary or allergic stages,a protectivestagebecomesnecessaryin order to take care of
the heterogenized
entities.
The analysisof many conditionsindicatesthc importanceof the different factorsfor the developmentof the clinical manifestations.
Seventh Day Manilestations in Trauma
we have studied these defensereactions for trauma, the degree of
intensityof the trauma indicatingthe extentof the exogenousheterogeniza-

DEFENSE

2lI

tion. Very intensivetrauma can produce a lethal superacuteshock which


corresponds,as we shall see below, to a generalizedprimary response.A
less intensive trauma may induce a tissular necrosis with consecutive
sloughingas a localizedprimary defense.A still less intensivetrauma may
induce an allergic tissular response.The importanceof these changesfor
clinical manifestationsis such that it appearsnecessaryto emphasizethem.
After surgery,for instance,a slight temperatureelevationis often observed
betrveenthe 7th and 9th day, This has to be interpreted as an allergic
reaction. When intensiveenough, this allergic responsewith the ensuing
l}tic action passesfrom thc tissular level to the higher level of the blood
vesscls.Along with inflammation and pain, local hemorrhagesoften appear. Severehemorrhagesoccur at this time after various traumatic incidents. The most disturbing complicationsfor plastic surgcry of the nose,
for instancc,are the sevcre "7th day hcmorrhages."The fact that they
start at this critical moment indicatesthcir allergicpathogenesis.
The study
of theseallergicchangeshas shown that they occur in the evolution of all
traumatic lesions. They can occur and remain clinically inapparent and
uneventful,as seenin the followingexperiment.
In groupsof rats of the samesexand agekept under similarconditions,
parallel skin incisions 3 cm. long at t/z cm. intervals were made. The
lesions were excised at differcnt times and chloride content determined.
Fig. 78 shows the curvc of averagevalucsof the total chloride content
of theseskin woundsin goups of six animalsfor each day. It can be seen
that intcnsivelocal chloride retentionoccurswith the first <lefense
reaction,
with valuesas much as four times greaterthan thoseof normal tissues.On
the third day, chloride content falls. It goes below normal tissue values
after thc fifth day during the hcarlingprocess.However, in an othcrwise
regular curve, thcre is a distinct temporaryincreasein chloride content on
the 8th day. Its occurrenceat this time, when coagulantantibodiesappear,
indicates its allergic nature.
The same allergic pathogenesis
explainsthe exacerbationof symptoms
seen about the 7th day in many conditions.In patientswho have suffered
a myocardialinfarct, for example,recurrenceof pain is often scenthe 7th8th day aftcr the infarct.
Part of the effectsof chemical,physicaland hormonal agentscould be
interprcted in terms of influenceexerted upon thc different factors which
intervenein the defenscmechanism.Someagentssuch as opium derivatives
were seen to affcct the liberation of hydrolytic enzymeswhile others interfere with the manufactureof allergicor immune antibodies.The influence
exerted by radiation upon the defensemechanismcan be related to its

2r2 /

RESEARCH IN

PHYSIOPATHOLOGY

o
f
!
j

g
o

E
:

|J

Flc 7E. Wound chloride ctrrve. The curve of the amount of the chlorides present
in skin wounds in rats correspondsto the average value obtained in 6 rats for each
figure. A first phase, with high values correspondsto tbc offbalance D. This is followed by a second phase characterizedby an offbalanceA. A variation in the curve
correspondingto the 8th day is constantly seen as corresponding to an allergic reaction. The values representmgs. of chlorine per 100 gr. of weight of the wet malerial.

effect upon granulocytesand lymphocyteswhile neoglucogeniccofticoids


affect the connective tissueand lymphocytes.
Researchin all these directions is still in progressand the results will
be communicatedin further publications.
For the time being,they havebrought more information and suggestions
of researchin the specialcaseof the immunologicalproblem of cancer.
IMMUNOLOGICAL

PROBLEMS IN CANCER

We usedthe data obtainedfrom the analysisof the defenseprocessagainst


cells and tissuesin the study of the immunologicalproblem of cancer.The
different canceroushierarchicentities,as defined previously may be con-

l}.;gii:r,+.#t
;,*4dliiljiliaq$;$,f,j##i;.;issi

DEFENSE

213

sideredto correspond,up to a certain point, to heterogeneous


entities-the
grafted tumors to transplantsand the spontaneoustumors to heterogenized
entities of the individual.
We tried, in a first seriesof experiments,to follow the interventionof
the differentmechanismsof defenseon graftedtumors in animals,employing transplantsof various degreesof heterogcneity.Different types of tumors were used. Highly heterogeneous
tumors obtainedfrom speciesother
than the host would not grow when transplanted.The death of the transplant. even its rejection if mechanicallypossible.occurs in a short time.
The necrosisof the transplantand the relativelywide inflammatoryprocess
that developsaround it immediatelyafter the graft indicatethe intervention
of the first stageof the defensereaction from the cellular to the organic
level.
with a secondgroup of tumors, usuallyfrom the same speciesor even
moderatelyheterogeneous,
the grafts take and the tumors grow for a time.
often, around the 8th to the l5th day. the transplantstarts to show pro
found changes.The changesaffectthe entire tumor which involutesrapidly
and is often expclled.That this is due to allergicreactioncould be shownby
the following experiment.Fragmentsof the samekind of tumor which had
been obtained from different animals were transplantedat 2 to 3 day intervals in the same host. In spite of the different agesof the transplants,
the death and rejection of all occurred at short intervalsand in the same
manner, indicatingthe interventionof a mechanismtaking place in the
host and relatively independentof the evolution of the transplant itself.
Such a mechanismwould be the interventionof allergic antibodies.
In a third group of grafts of low heterogeneity,
the tumors continue to
evolve for an even longer period of time, and it is only in a few animals
that these tumors are entirely rejccted.This change,which consistsof
cytolysisof the tumor, takes a certain time to be completed,which indicatesthat it probably resultsfrom the interventionof protectiveantibodies.
The first and the seconddefensemechanismsappearedto be inadequateto
conquerthe tumor and it was the third stage,with formationof protective
antibodies,which apparentlywas able to accomplishit. This mechanismis
confirmedby the fact that later grafts are negativefrom the time they are
transplanted,through cytolytic changesin the tumor and not through an
intensiveinflammatory processas seen in the primary reaction. The immunologicalnature of the defensein thesecasescould be seenalso through
the passiveimmunity which could be inducedin other animals with the
serumof the host.

214 i

xESEARcHtN PHYsropATHoLooy

Grafts in Humans
The grafting of cancerouscells in normal humans usually leads to the
appearanceof a growing tumor which (315) after a period of days, almost always sufters the same fate as a moderately heterogeneoustransplant: death followed by resorption or expulsion. The time when this processo@urs indicatesthe allergic natureof the defensemechanism.It is highly
probable that, without the intervention of this efficient allergic defense,the
cancerousprocess would have continued to evolve.
Such continued growth occurs if grafts are made in subjects already
having their own cancerousprocess.This would indicate that the allergic
defensemechanism against the graft is no longer operating in these cases.
The fact that a canceroussubject acceptsa new tumor graft while the
normal one rejects it indicates that defenseprocessesare different for the
normal and this canceroussubject.The inability of thesesubjectsto reject
a grafted tumor through an alleryic responseappears to be the major immunological difference between the normal and the cancerous subject. Still
more important is the fact that in cancer patients,the anomaly would correspond to a lossof the capacity to reject the grafted tumor which the normal
subjectseemsto have.
In trying to determine the nature of the immunological anomaly in the
evolution of a spontaneouscancr in patients, we have to relate it to this
loss of the defenseprocessesas seen above.
In studying this occurrencein general,a loss of defenseagainst:rn antigen can be conceived to occur for any of the three different mechanisms
involved in defense:primary, allergic or protective.In caseswhen this takes
place, the loss of the protective stagewill take place first. The allergic defense will be affected next and finally, the primary response.This explains
why the inability of an individual to achieveone stageof the defenseleaves
the defenseresting in the immediately previous stage.The inability to manufacture protective globulinic antibodies,for instance, will leave an individual in the allergic stagewhich, in the developmentof defense,preccdes
manufactureof the immune antibodies.This results in a potential allergic
condition if the antigen is absent or an actual allergic condition if the antigen is present.We have seenthat this occurs in most of the chronic infectious conditions.Similarly,with the inability of an organismto manufacture
coagulantantibodies,the defenseremains in the previous defensestage,the
primary lipidic one.
Before going further, we have to discussa factor believed by many
authon to be involved also in the defensemechanismagainstcancer. A few

..,:::: :,,::.

.)

DEFENSE

215

yearsago, the defensemechanismin generalhad beenrelatedto the properdin system.Howcver,whenconsidered


in termsof the systematization
of thc
defenseprocesses,properdin has to be regardedas a nonspecificdirect antinoxiousreaction.It appearsto be involvedin the antiheterogeneous
defense
response,appearingafter the enzymatichydrolytic attack and at the bcginning of the prolongedlipidic intervention.
The decrease
in the properdincontentof thc blood of subjectswith cancer has causedvarious authors to try to explain through it the differences
in the reaction of cancerousand normal subjectstoward a new transplant.
The analysisof the conditionsunder which this occurs,however,has shown
us that the anomaly does not residein the antiheterogeneous
processesof
defense,which are the same againstantigen and allergic complex, but in
the allergic reaction itself. From the immunologicalpoint of view, the differencebetweena normal subjectand one with invasivecancer residesin
the loss of capacity to induce the secondtype of defense,the allergic,toward the canceroustissue. Correspondingto an inability to manufacture
coagulantantibodies,this deficiencywould explain the lack of respective
antiheterogeneous
reaction toward the antigen-coagulant-antibodies
complex and consequentlythe low blood content of properdin seen in these
cases.
Failure of the allergic defensemechanismspecificallyagainst cancer
entitiesneed not mean generalfailure of allergic defense.The failure may
be limited to inability to manufactureallergic antibodiesagainsta specific
antigen.We have seen,especiallyfor the infcctiousdiseases,that primary
and allergicprocessescan occur with great intensityand still not be qualitativelyefficient.The agent,the microbe,for instance,can still rcmain present despiteeven violent ailergic reactions.The mere presenccof defense
processes
does not implicitly mean successful
defense;they may be qualitatively insufficient.
In cancer,if the allergicdefcnseis insufficicnt,
two eventualities
havc to
be considered:either the organismin generalcannot passinto the allergic
stageof defenseand thereforeis unableto manufactureallergic antibodies,
or this responseis only qualitativelyinsufficient.In thc latter casc,the general and even local reactionscould be quite intensivebut still be ineffective.
This seemsto occur only in certain forms of cancer such as those with a
high inflammatoryprocess;for instance,in the inflammatoryform of breast
carcinoma.As this cancer starts and evolvesas an acute mastitis,very intensive defensiveprocesses,apparentlyonly of the primary stage,occur.
But they are unable to check the diseasewhich usually cvolveseven more
rapidly in these cases.This is also true for other cancerswhere fever is

216

nESEARcHrN pHyslopATHoLocy

present,indicatinga prolongedprimary, toxic stage.The lack of local reaction seenat the site of the growing transplantin the canceroussubject at
the time when the normal individual kills or rejects the transplant points
to the fact that the anomaly residesin qualitative inability to manufacture
allergicantibodies.
The next problem was to investigate the reason for the failure of
allergic defense against the tumors. we could show that the cancerous subject has not lost the capacity in general to manufacture coagulant antibodies.Even subjectswith very widely spread cancer were able
to respondwith a local skin allergicreactionto a secondinjection of an antigen (proteins from mollusks) made more than ten days after a first preparatory one. (Note 7) Their inability to fight transplantedcancer cells
through a similar allergic reaction indicatesthat the loss of this capacity
is not generalbut relatively specifictoward the cancerouscells. The lack
of an intensive inflammatory process, as well as the existence of high
amountsof lipids in the canceroustissue,also would indicateindirectly an
inability of the canceroussubjectto resolvethe existing immunological
problem of fighting cancer through an allergic reaction. The presenceof
large amountsof lipids indicatesthat thc defensemechanismhas been
arrested in the stage of pronounced lipidic predominance. Abnormal
amountsof lipids thus could representan indirect meansof recognizingthe
failure of an allergic responseto cancerousentities.
The next problem was to try to determinewhere in the organization
the failure occurs.The different levelsof the organizationare independent
to a certaindegreeand passageof an abnormalityfrom one level to another
induceshierarchicprogressionof the condition. This has posed the problem of the progressiveloss at the differentlevelsof the natural capacityto
defend againstcancer. Recently many investigatorshave shown that cancer cells passinto the lymphatic systemand into the generalcirculation in
a much higher proportion than had been suspectedbefore. Malignant cells
in the circulation are destroyed,however,by the defcnsemeanswhich are
not lost at this level.The samepatientthus may still have an activelygrowing cancerat the tissularlevel,indicatingthat this defenseprocess,although
successfulfor the higher levelsof the organization,does not interveneat
this lower level.
The hierarchicprogressionof cancercan be seenas a proefessiveloss
of the immunologicaldefensecapacity.while the organism conservesthe
capacityto fight at a higher level, a lower hierarchicentity no longer opposesthe cancerouscondition. It is not the absenceof cancerouscells in
blood or organs which explainsthe lack of an explosivespreadof the dis-

DEFENSE

217

ease,but the presenceof efficientdefensemeansat theselevelswhich keeps


a cancer still localized.
Metastases
The relativeindependence
in the lossof the defensecapacityof different
entities would explain one of the most baffing problems of cancer-why
certain cancerstend to metastasize
to certain organsor tissues.Somecancers show bone metastases,
othersspreadto many organs,while still others
spreadonly to certain specificorgans.This can be explainedby a loss of
the defensecapacityat the organ level. While some organslose,others still
maintain their allergicdefensecapacity.The circulatingcancer cells will
inducemultiplc metastases
in the first but will not be able to take hold in
the latter.
A similarmechanismcan alsoexplainthe persistence
of inactivecancer
cells for years after an operation.The defensemechanism,while it is not
able to aftect the cells and destroy them, is still sufficientlyactive at the
tissular level to prevent the condition from progressingat this level. The
cancr cells will start to invade this level only when the tissular level is
unable to defend itself further through an allergic responseagainstthe invading cells. By losing its allergic responsecapacity,the tissular level will
even exaggeratethe correspondinglower primary stageof defense,that is,
the prolongedlipidic phasewith the consequentchangeswhich this brings
on. Among them would be the appearanceof pain.
Under thesecircumstances,
the generalimmunologicalcondition favorable to the hierarchically progressivedevelopmcntof cancer has to be
regardedas the loss of the capacityof the different levels for an effective
allergicresponsetoward cancerousentities.The immunologicalproblemof
cancer consequentlyappearsin a speciallight, different from all the other
known conditionswherean unsuccessful
immunologicalresponseis present.
In the other conditions,the problem of the inability to conquer an antigen
is one involving the incapacityto mobilizeor developan eftectiveimmunological response.In cancer,the body appearsto have lost a previouslyexisting capacitythat was presentbeforethe diseaseappeared,In other diseases,
the immunologica.lproblem is to create a new and favorable condition in
the fight againstan antigen,by developingmeanswhich do not exist in the
normal individual. In cancer. the immunologicalproblem would be to
prevent the loss of a property possessed
by normal subjectsor. if already
Iost.to find somemeansto regainit.
Theseconsiderations
and the study of the differentfactorsinvolvedin
the developmentof the progressive
defensestagehas led us again to the

218

RESEARcH rN pHysropATHoLocy

role of the lipids in these processes.The appearanceof a stage in the


defense mechanism seems to be strongly related to the frrlfillrnent of
the qualitative requirementfor the previous stage.Deficiency of essential
factors in one stage representsan impediment for the next stage. In the
case of cancer, failure of the specffic allergic phase thus could be traced
to a qualitativelyinadequateprecedinglipidic phase.A level is unable to
surmountan allergicdefensebecausethe lipids which can mobilize arequalitatively inadequate.Even abnormal richnessin lipids thus could be interpreted as resulting from their qualitative inadequacy.This very excess
indicatestheir importance.
Imntunological Therapeutic Approach
The fact that effectivedefenseresourcesare present at a highcr level
does not mean that they inevitablywill act at a lower level.
This view of the abnormal immunological processesin cancer has
pointed the way for some new therapeuticapproaches.From the therapcutic point of view, the problem becomesone of how to induce the body
to regain, at the necessarylevel, the lost immunologicalcapacity,and
througha specificallcrgicdefense,to combatthe cancerousentities.Furthermore, sincethis specificallergicdefensecapacityis lost independentlyby the
various levels of organization,the immediate problem would be how it
could be recoveredfor the particular level where the loss occurs. The existenceof adequatedefensecapacityat a higher level, such as the systemic,
docs not provide a solution for cancer presentat lower levels such as the
tissular,becauseof the independence
which exists betweenlevels.only
the manufactureof coagulantor immune antibodiesagainstcancer at the
proper levcl would put the individualin a sufficientlyactivedefensephase
to enablehim to resolvethe condition at that level.
The problem of immunity at the proper level thus appearsto be critical
for any immunologicalattack againstcancer.It is evident in other conditions as well and has inspired the use of local vaccination in localized
infcctiousconditions.( 307, 308)
The study of immunity againstviruseshas permitted us to recognize
the importanceof immunity at differentlevels.Virus infection is a typical
cellularcondition,the virus multiplyingonly within a cell. Theoretically,an
immunity at all levels can be induced for viruses.According to the view
discussedabove,however,a systemicimmunity with circulating antibodies
will not insure a cellular defense.It would interveneonly when the virus
is passingthrough the systemiclevel and its activity would last only during
the time when circulating antibodiesare present. During this time, the

DEFENSE

219

virus will be prevented from reaching the cells. Once tbe circulating antibodies iue no longer present, the cells cease to be protected. For an efficient defenseagainst viruses an immunity within the cell appears thus to
be indispensable.The use of dead virus vaccine will induce only systemic
immunity, which can be recognizedthrough the circulating antibodies. It
is unlikely that the killed virus enters the cells. It does not aftect them,
and consequentlydoes not induce cellular immunity. Even a mild cellular
infection with living virus will give the necessarylongJasting cellular imrnunity. This would explain the need for living and not killed vaccinesfor
viral infections, as first postulatedby Pasteur.
A similar level immunity can explain the differencesseen between the
immunity resulting from the use of microbial vaccines and that produced
by natural disease.Typhoid infection gives lifelong immunity; the vaccination, only relative and temporary immunity. An explanation can be found
in the fact that, in the disease,along with the septicemia,manifestchanges
occur in organs and tissues.Spleenand lymphatic tissuesare highly affected
in typhoid and it is possible that the developmentof the defense at their
level would explain the lifelong immunity that follows the natural infection.
rn cancer, the problem would be to induce not a systemic defense,
which is still present for invasive cancer, but an effective tissue or even
cellular defense.Immunologicaltreatmentof cancer would have to make
tissular and possibly cellular levels regain their capacity to defend themselves through efficient allergic responses.The immunological prevention
of cancer would lie not in the creation of this defenseor in increasingit
quantitativelybut enhancingit qualitatively.A successfulallergic defensc
at this level apparently would have a preventive and even curative effect.
The use of lipids in the induction of the defensemechanismagainsttissues
has an interestingapplicationin cancer.A systemictreatment with lipids
or lipoids can change the defenseresponseso that it can be effective at
a specific level where it is otherwise inadequate.For invasive cancer, the
lipid activity must be induced at the cell level. The active lipoids for this
purpose are those with a high affinity for the cancerouscell.
As abnormal cells in general show similar capacity to bind the lipoids
administered,this general affinity becomesa handicap if abnormal entities
other than cancerouscells are present. These considerationshave led us
to attempt to use methodswhich will insure the activity of lipids at the ccll
level.
In one of these methods,the chosenlipoids are brought directly into
contact with cancerouscells through local injections into the tumors. SingJe
injections produce only limited changes in tumors. Local injections re-

22O /

REsEARcH rN pHysropATHoLocy

peatedso as to insure the presenceof the lipoids once, and then again 15
days later, are required to induce an eftective response.The lipoids or
lipids are so chosen that, when bound to body constituentsthey will induce allergic or immune defenseresponses.The acid lipids of tubercle
bacilli, bixine or guinea pigs are especiallyprone to induce allergic reactions,while the lipids of microbes-such as coli, typhoid or diphtheria
-produce immune responses.
In another method, lipids chosenwere bound to cancerousentities in
vitro. Cancerouscells were obtainedand treatedin vitro with lipids under
whoseinfluencethe body is able to manufactureallergic or immune antibodies. colloidal suspensionsof the lipids or lipoids were prepared as
mentionedabove,mixed with suspensions
of cancerouscells, kept at 37.C
for a few hours, then separatedfrom the non-fixedlipids and injectedinto
patients. In order to obtain good results it was necessaryto inject this
material at least twice, at an interval longer than two weeks, in order to
insurean allergicreactionagainstthc cell-lipidpreparation.While a single
injection produced good resultsonly in a very small number of cases,repeated injections were manifestly more effective.When canceroustumor
cells could be obtained through biopsy from the patient, we used thenr
for the in vitro treatmentwith lipoacids.when biopsy material was not
available,we used cancerouscells of similar origin as the tumor of the
subject,preferablypooled.
The condition for successof these methods has appearedto be the
presenceof the cell-lipidcomplex at the moment of appearanceof antibodies. This is assuredonly by the repetition of the injection. Another
interestingaspectof the immunologicalproblem in cancer, related to loss
of the natural defensemechanism,is the loss by cancerentitiesof their
capacity to utilize certain elements known to intervene in the defense
mechanism.The role of magnesiumin the properdin system, copper in
cytochromeoxidase,of calcium in generaldefense,suggestsa correlation
betweentheir deficientutilizationin cancerand the loss of the defense.We
will discussthis problembelow,after reviewingthe pharmacological
aspect
of theseelements.

CHAPTER

T H E C O R R E L A T I O Nt s E T W E E N
T H E B A S I CC O N C E P T S
rTrl
rs A cLEARinterrelationship
l[
betweenthe four basicconceptspre"r^u
viouslydiscussed
which permitsus to considerthem togetherand to establish a unified viewpoint.For all four can be scento representdifferent parts
of the same fundamentalproblem in biology: the manner in which an
entity resolvesenergeticdifferencesbetweenitself and the environment.
we have seen that, in the framework of fundamentallaws governing
nature, matter can be consideredto correspondto isletsof heterotropyopposingthe homotropictrend of evolution.Conservationof an existingentity
appe:us to be the principal meansby which heterotropycan be achieved.
And heterotropyis fulfilled, specifically,through maintenanceof the constantsof entitiesas valuesdifferentfrom thoseof the environment.
H i erarchic Organization
The continuoustendencyof natureto progresstoward maximum homotropy has made the conservationof existing entities a persistentlyacute
problem. The problem posed by the progressivelychanging environment
cannot be solvedthroughchangeswithin entitiesthemselves.
Any "adaptation" of the entity itselfwould affectits constantsand, consequently,
would
be contrary to the fundamentalpurposeof heterotropy.Nature has resolved
the problemin an entirelydifferentway. Sincethe entity itself must remain
unchanged,and yet the influenceof the environmentmust be conteracted,
nature has made use of hierarchic organization.Secondaryparts, reproducing the immediateenvironment,are joined to existingentities.often
surroundingthem and acting as buffersagainstenvironmentalinfluences.
Through theseadded secondaryparts,hierarchicentitiesare organizedso

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; *- 1;

222

xESEARcH rN pHysropATHoLocy

that they reproducethe characteristicsof the environmentpresent at the


time of their formation. Through this means successivelyrepeatedmany
times, an entity can be kept unchanged,in a medium similar to the original
one, despitecontinuingchangesin the environment.
Hierarchic organizationthus representsthe main mechanismthrough
which the heterotropic achievements,representedby entities, counteract
the influence exerted by the homotropic force. Conceptually,hierarchic
organizationcan be seento representa form of defensedevelopedin time
by entitiesagainsta specificfactor, progressivehomotropic changesin the
environment.The successivesteps of the hierarchic organization respectively the hierarchicentities,reproducein short, the evolution of the relationship which has been developedbetweenthe entity and the changing
world. Hierarchic organizationcondensesthe phylogeneticevolution of
this specificpart of the dcfenscbetweenthe entity and the changingenvironment.Through this view, we can integrateorganizationin the general
dcfense,the hierarchicorganizationbeing part of the mechanismused
againstprogressinghomotropy.
Constituents
In the same manner, we can further integrate into the same defensc
mechanismthc various constituentswhich form the secondaryparts of the
hierarchicentities.We have thus tried to correlatethesc constituentsmore
directly to the successive
environmentsin which entitiesevolved.We have
seenabove, how this appliesto elementswhich are common to, and predominantin, both the entitiesand the environmentswhich correspondto
the media in which theseentitiesevolved.Through the correlationbetween
elementswhich enter into hierarchic organizationat various levels, and
their positionsin the periodic chart, the successivephylogeneticpassage
from one environmentto anotherhas a specificmeaning.In media formed
by elementswith lower atomic weight,the influenceexertedby progressive
homotropy is less manifest.The changesin the elementsas body constituents can bc thus also integratedin the samedefensemechanism.
Besidcsthe elements,other constituentscan be similarly integratedinto
the defenseagainstthe changesof the environment.In the immediatedefense processagainstnoxious agents,we have seen the successiveintervention of different constituents---{nzymes,lipids, lipido-proteins and
proteins,in that order. The high degreeof individuality and independence
of the entities in the hierarchicorganizationhas permitted us to conceive
of theseconstituentsas participatingwith a certain independencefor each
cntity. The presenceof all these constituentsin each higher biological

T H E C O R R P L A T I O NB E T W E E N T H E B A S I C C O N C E P T S

223

cntity, which is part of the complex organism,suggeststhat theseconstituents entered into the formation of these entities as the result of thcir
interventionin the defensemechanism.Thus, it can be conceivedthat. in
its phylogeneticdevelopment,each entity has passedthrough a succession
of defense phases in which specific groups of constituents-nzymatic,
lipidic, lipido-proteicand proteic-have been predominant.In actual organization,while all higher entitiescontain fundamentallythe sameconstituents, different substancesare predominant at different levels. This can
be explainedby the predominanceof a particular defensemechanismat a
particularlevel.According to this view, this defenseis principally in the first
stage,that is, of enzymaticnature, for most endodermicformations. It is
in the prolongedlipidic stagefor ectodermicformations,lipido-proteicfor
the reticuloendothelialsystem,and proteinic for cells. Through thesecorrelations,constituentscan be more completelyintegratedin defcnse.
The kind of special defensedevelopedfor the different levels of organization, through predominant specific constituents, has not been
followed by a total discard of the other constituents,which do not have
such roles. Instead,the latter have been retainedin the entities in smaller
amounts and in inactive forms. This confers upon the entity the capacity
to mobilize these constituentsand use them when the need to respond
to an acute emergencyarises.Pre-fermentsand cven ferments in mitochondria; fatty acids and anti-fatty acids bound as esters;lipido-proteins
and proteins in various combinations-all these are inactive constituents
which can be changed easily into active agents. when fighting a new
noxious intervention,an entity will resort to liberating or activating these
constituentskept in reserve.Each entity and level of organizationdoes
this indepcndentlyof other entitiesand levels,yet constituentsactivatedat
one level can act at other levels, too. The successor failure of defense
especiallyin its first stages,dependsnot only on the intrinsic value of thc
constituentsavailable, but also on the capacity of the affiicted entity to
utilize these means by activating them. Although activation processesbecome strikingly evident in abnormal conditions similar processessecm to
be important even in the maintenanceof existingentities.
Dualism, as we have seen, characterizesboth normal and abnormal
physiology.That which is considered"normal" is the result of an altcrnating intervention of two groups of opposite constituents,producing an
oscillatory movement and a dynamic balance. The dualism seen in abnormalities,when one or the other opposedfactor is persistentlypredominant, is related to hierarchicorganizationand the defensemechanism.
Dualism results from the intervention of two fundamental forces in

224

nESEARcHrN pHystopArHoLoGy

nature-homotropy and heterotropy. Even the simplest analysesmake


evidenteither the homo- or the heterotropiccharacterfor many manifestations and processes.
For instance.an ulcerationor an enzymatichydrolysis
of a protein has to be interpretedas an homotropiceffect while a growing
tumoral mass or the synthesisof a protein can be seen as an heterotropic
one. For other manifestations,this characterappearsless immediatelyevident and it is through further analysisthat it can be recognized.Dualism,
like the other concepts,thus can be integratedin the defenseof entities
against an environment progressivelychanging toward maximum homorropy.
We have used this conceptual fundamental view in studying many
problemsin biology. It has aided us to formulate helpful working hypotheses.Despite its shortcomings,when applied to particular situations,
this basic concept has served as a guide in correlating specific problems
with the fundamentallaws governingnature. [t has also engenderedhelpful new interpretationsof availabledata. Through the relationshipof the
four conceptsdiscussedabove and the fundamentaldefensemechanism,
we have been able to analyze many problems without reverting to empiricism. Certain of these problems. to whose better understandingthis
approach appears to have contributed, are discussedin the pages that
follow.

CHAPTER

S H O ( ]K
r
ll* ro,r, oF THE pRocRESs
rcalizedin the lust decade,shock remains
one of the most challengingproblemsin medicine.That lipids have a
critical role in shock pathogenesis
seemsclear from a long-term study
which beganwith an investigation
of the activity of fatty acids in the induction of the abnormallydark color of blood secn in shock.The results
of this study will be discussedhcre not only becauseof the intrinsicinterest of the problem of shock itself but a.lsobecauseshock often represents
the terminal phaseof cancer as it does of many other diseases.In this
presentation,we will try to remain as much as possiblewithin the framework of our direct contributionto an understanding
of shock. A portion
o f t h e s er e s e a r c h ewsa s p u b l i s h e di n 1 9 4 3 .( 4 0 )
In studyingthe very complex phenomenonof shock, one has to consider a seriesof well-definedproblems.Shock has been related not only
to a large number of causesbut also to a seriesof vcry varied clinical
manifestations.
An initial problem was to determinewhetherthere is any
common relationshipbetweenthe different types-between the shock, for
instance,which kills a subject within a few minutes after a severesudden
trauma, and the shock that kills in days through profound systemicmetabolic impairments.What is common to, and what is different between
them, from the point of view of pathogenesis?
What constituentsintervene
and how, in shock?Theseand many other problemshave beenapproached
systematically.
T-vpesol Shock-As a starting point, we attenrptedto classify the
types of shockand found an interestingrelationshipaccordingto the time
that is, the intervalbetweenapplicationof thc noxious
of their appearance,
Three typescould be identifiedwith
stimulusand onsetof manifestations.
this criterion.
225

226 /

R E s E A R c Hr N p H y s r o p A T H o L o G y

There is an immediate type of shock which appears within a ferv


minutes after the application of the noxious agent. It is induced experimentally in animals by intravenousinjection of a noxious substance,by
scaldingthe animal in hot water, or by strong mechanicaltrauma. It has
predominantcentral nervous system manifestations,including exophthalmia and paralysis of the posterior limbs, followed by clonic convulsive
movements,and usuaUy is terminated by death. A similar superacute
type of shock is occasionallyseen in humans following transfusionsof
blood with an incompatible group. It also may be seen following very
severetrauma. In the case of bullet wounds, for example, large calibre
bullets may bring rapid death. Neither immediate hemorrhage nor any
organ impairment is sufficient, in itself, to account for the speed of death
in many of these cases.However, it can be explained by the rapid and
intensive participation of the central nervous system in this superacutc
type of shock. Sometimessuch shock is not lethal in animals or humans
and is followed by a period of prostrationand ultimate but slow recovery.
We called this type of shock the "superacute."
In a second type of shock, more frequently encounteredin humans,
the manifestationsappear after a certain period of time. Such shock often
is seen after direct transfusions,when the rate of injection has been too
rapid or when the syringeand tubeshave not been well coated with oil or
paraffin,or when thcre has been a subgroupincompatibilitybetweendonor
and receiver. The patient usually experiencesa severe chill within 30
minutes, The chill is succeededby a rise in temperature which usually
Iasts 15 to 60 minutes or more. The patient next experiencesdiaphoresis,
after which the episodeusually is concluded.In some casesthe symptomatology is different. At about the same time-30 minuts5-after transfusion, for instance, hypotension with hypothermia, cold and clammy
perspiration,and intensivedyspneaare noted. In thcse casesdeath can
follow in a short time. The same reaction is sometimesseen to occur,
usuallyalso in about 30 minutes,after the releaseof a tourniquet.We have
employedthe term "acute shock" to describethis secondtype characterized by its appcaranceat approximately30 minutes after the noxious intervention.
A third form, the "state ol shock," is considerablyslower in onset and
persistsmuch longer.Characterized
by hypotension,impairmentof circulation, cold and clammy perspirationand markedenophthalmia,it may lead
to death after scveral days during which the condition progressivelyincreasesin severity.It can, however,also end in recovery,This is the form

sHocK

227

most often encounteredin clinical medicine, in cancer and many tenninal

conditions.
The next problem was: could a common pathogenicmechanismbe
recognizeddespite the greatly varied manifestationsof these three forms
of shock?
Shock Mechanism
We saw one primary correlation between the three clinical types of
shock in the fact that sometimesone type is followed by another. Superacute shock, if not lethal, may be followed by acute shock which, in turn,
can change into a state of shock.
But it was the chemical analysisof blood, organs and entire bodies of
animals killed by any of the three types of shock which indicated the possibility of a mechanism common to all three. A low antitryptic power of
the blood, and the presenceof substancesresulting from protein hydrolysis
were found to characterlznall 3 types of shock. Additionally, ut increase
in the amount of free fatty acids, and the presenceof abnormal members,
occurred h all three types.
Fatty acids were studied from the point of view of the reciprocal position of their double bonds, through the oxidative fission method mentioned
previously. The appearanceof oxalic acid following oxidative fission indicates the presence of conjugated double bonds. The oxalic acid index
obtained indicates the proportion of these conjugated double bonds. In
normal rats, this oxalic acid index usually is zero in the total amount of
fatty acids; in normal mice, values below I are seen. In all animals in
shock, even in cases of superacuteshock followed by sudden death, the
oxalic acid index is invariably much higher. Furthermore, the death of an
animal in acute shock or state of shock appearsto be related to the presence of a critical oxalic acid index, indicating a concentration of abnormal
fatty acids incompatible with life. Whether it appears in a relatively short
time as in acute shock, or many days after the noxious intervention as in
the state of shock,the oxalic acid index found in dying animals is between
14 and 17. Such high values are not found in superacuteshock but the
oxalic acid still is markedly increased.Thus, the presence of hydrolytic
processestogether with abnormal fatty acids appears to be a common
pathogenic factor for the difterent forms of shock.
Pathological Clanges
The three types of shock-because of the presencein all of hydrolytic
processesand abnormal fatty acids-<ould be related to the first phase of

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228

R E S E A R c Ht N p H y s t o p A T r { o L o c y

the immcdiatediphzsicdefensephenomenonor its prolongedform. The


next problemwas to determinewhat othcr factorsmight influencethe developmentof diflering manifestationsso as to make shock appear in three
forms.
The study of pathologicalchangescharacterizingeach of these forms
was undertaken.We found cellular vacuolationa characteristiclesion in
animals in superacuteshock. Vacuoles are present in the parenchymal
cellsof the liver, to a lesserextentin the alveolarcellsof the lung, and to
a still lesserextent in kidney ceUs.Of special interest was the fact that
thesevacuolesare often seen in the cytoplasmand even in the nuclei of
brain cells. These findings explain the predominanccof the neurological
symptomsin this form. [n a publicationin 1943,we describedthis vacuolation as a characteristicof the superacuteshock. The fact that the characteristicpathologicalchangeencounteredin superacuteshock is the presence
of vacuolesin different cells suggeststhat this form of shock occurs principally at thc cellularlevel.
ln the acute type of shock, which usually appearshalf an hour after
noxiousintervention,there may be someevidenceof cellularvacuolization,
but the principal changesare at the tissularlevel. The changesare largely
localizedin the immediate areasdamagedby the noxious agent and are
manifestedby vascularand interstitialpathologysuch as marked edemaor
capillary hemorrhage.Splanchnicvasodilatationand pctechiaeat the surlace of pleura or peritoneumappear when the noxious agent acts indirectly in the blood or is applieddirectly to it through intravenousinjection.
The degreeof generalizedvasculardamagecorrespondsto the degree of
direct participation of the blood. We have discussedpreviously, in the
chapteron defense,the changesoccurringin the blood which characterize
hemoshock.Thc characteristicleucolysis,which is followed by hydrolytic
digestion,explainsthe high degreeof breakdownof blood constituentsand
vessels
observedin this kind of shock.While the participationof the cellular level-and especially of the central nervous system----characterizes
supracuteshock, particiPationof the tissularlevel leadsto the acute form.
We considerpathologicallycharacteristicof the stateof shock-in addition to the changesseenin blood, such as hemoconcentration,
dark color,
tendencyto form sludges,etc.-two other specificmanifestations;
milliar
lesionsin the gastric mucosaleadingto hemorrhageand ulceration,and a
manifestfluid accumulationin the first portion of the small intestine.Since
the variouschangesin the stateof shock affect the blood and two organs,
the stomachand duodenum,they can be consideredto involve the organic
and systemiclevels.

This analysishas permitted us to continue to u.";;;rt*r;,r:t:


that all three forms of shock stem from the samefundamentalmechanism
-the appearancof abnormal fatty acids as part of the first phaseof the
diphasic defense reaction. The differencesin manifestations between the
formsof shock are due to the level at which the mechanismoperates,cellular for superacuteshock, tissular for acute, and organic and systemic for
the state of shock.
The study of a special condition, hemoglobinuria "a frigore," or
paroxystic hemoglobinuria, has helped us to understand the time factor
in shock. In this condition, immersion of the hand in ice water, for instance,induceshemoglobinuriaand violent chill about half an hour later.
We have been able to demonstratethat in the developmentof such a
manifestation,two or often even three hemoshocksoccur, each one characterizedby a diphasic phenomenon.The first shock appears within ten
minutesafter immersion of the hand in icy watcr. Usually the first sensation and chill are very slight and while a reduced hemoglobinemiais present, hemoglobinuria is almost nil. It is the second hemoshock, appearing
approximately 30 minutes later, which is usually very intensive with manifesthemoglobinuria.The third shock,which appearsabout two hours after
immersion in ice water, is usually clinically inapparent and is revealed
only by blood analysis.
The study of this condition has indicatedthat in the appearanceof the
three episodesof hemoglobinuria,besidesthe changesin the red cells under
the influenceof cold. which are characteristicfor the condition as seen in
test, the importantfactor is the leucolysisoccurthe Donath-Landsteiner
ring as part of the hemoshock.The subsequenthemolysisleads to free
hemoglobin in the blood which, if in sufficient amount, passesinto the
urine. The changesinduced in leucolysiswill determinethe degreeof consequent hemolysis. The suppressionof leucolysisby administration of
morphine or other opium derivativesprevents any manifestation,while
physicalexerciseundertakenfollowing the immersion of the hand in icy
water induces,in addition to a very intensiveleucolysis,exceptionallyintensive clinical manifestations, The time when the three hemoshocks
appearalso marks the time when the threc forms of pathogenicshocksuperacute,acute and state of shock-are seen.The interventionof three
different noxious heterogenizedconstituentsappearsplausible.(Note l)
Fatty Acids and Sodium Chloride in Shock
We noted that in all three types of shock, abnormal fatty acids can be
found. A studv of the role of thesefatty acids permitted us to further un-

230

RESEARcH rN pHysropATHoLocy

derstandthe mechanisminvolved in thesethree types of shock. Since these


samefatty acids have been seento figure in abnormal metabolismof sodium
chloride, the next logical step was to investigate the correlation between
the latter and shock. Following this line, efforts were made to see if the
difterencesbetween NaCl metabolismat different levels of organization
would help explain the peculiaritiesof the different types of shock.
As we have noted, when abnormal fatty acids impair sodium chloride
metabolism,two processesoccur. First, there is abnormal fixation of chloride ions by abnormal fatty acids; then, sodium ions, freed following this
chloride fixation, become bound to carbonateions, resulting in alkaline
substances.The pathological nature of chloride fixation results principally
from the fact that the binding taking place at the conjugateddouble bonds
is abnormally strong. Occurring in two steps,with a displacementof the
double bond in the first, the bond betweenthe conjugatedfatty acids and
chloride ions appear nonreversible.(Note 8, Chapter 6)
The great inequality in the ability of chloridesand sodium ions to pass
through membranescan serveto separate,anatomically,the fixed chlorides
from the free remainingcations.When this occurs, two distinct processes
can be recognized,one involving the binding of chloride ions by abnormal
fatty acids,the other involvingthe binding of carbonateions by sodium ions
and the resultingappearanceof alkaline compounds.In the cells, the two
processestake place separately,the sodium alkaline compound inducing
the appearanceof vacuoles.In tissues,the chloride fixation takes place
predominantlyin the cell, while the binding of carbonate occurs in the
interstitialspaces.This leadsto a localizedintercellularalkalosiswith consequentedema.
The same mechanism is involved in the changes associatedwith the
state of shock, except that theseprocessesoccur at the systemiclevel. It
is the part played by the sodium chloride of the blood in normal physiology,
especiallyin the processof digestion,which explainsthe abnormal changes
seen as characteristicof the pathologicalmanifestationsin the state of
shock.
Normally, chloride ions are excretedinto the stomach,where they are
bound to hydrogento form hydrochloricacid. An almost equal amount of
sodium ions, bound to carbonateions, is eliminatedin a secondstep into
the intestinesvia the pancreaticand intestinalsecretions.The chloride and
sodium ions are later liberated to form sodium chloride which is entirely
reabsorbedin the distal portion of the intestinaltract, the colon. The sodium and chloride ions are not simultaneouslysecretedin the digestive
tract. The interval betweenthe excretionof chloride ions into the stomach

sHocK

231

andof sodium ions into the intestinesa@ountsfor the physiological"alkalinetide" associatedwith digestion.
when chloride ions are pathologicallyfixed to abnormal fatty acids in
theblood, they can no longer be dissociatedand secretedby the stomach
in the form of hydrochloric acid. Instead,they remain bound to the fatty
acidsand accumulatein this form within the gastricmucosa.The multiple
rnilliar gastric mucosal ulcerations in the state of shock results from the
interventionof these abnormal fatty acids brought into the mucous membraneby the chloride ions to which they are bound. The ulcerationsare
causedby the catabolicaction of fatty acids.Thus, the first phaseof abnormal sodium chloride metabolismleadsto the characteristicmultiple gastric
ulcerations.
The secondphaseis relatedto the metabolismof sodium. The sodium
ions are secretedas carbonatesby the pancreasand intestinal mucosa in
the fint part of the small intestine.In the state of shock, becausethey do
notencounterthe chloridesnormally coming from the stomach,they remain
as carbonates.As sodium carbonateis accumulatedin the first portion of
the small intestine,a local alkalosisoccurs, leading in turn to an important
loca.lretentionof water. It shouldbe noted that this is a very differentsituationfrom achlorhydriaor hypochlorhydria
in which,while the chlorideions
arenot secretedinto the stomach,no excesses
of sodium ions appearin the
blood or in the intestines,and consequentlyno local alkalosisor fluid
accumulationoccurs.
The differencebetweenthe systemicand tissueprocessesin shock lies
in the localizationof the abnormal sodium chloride metabolism.In tissue
anomaly,the separationof sodium chloride takes place between the cells
andthe pericellularstructures.At the systcmiclevcl, it occurs betweenthe
stomachand intestines,
with the blood servingas intermediary.This mechanismexplainsthe larger amountsof water which distendthe upper parts
of the intestine,as observedin autopsiesof animalswhich have died in this
form of shock.
The close similarity betweenthe abnormal processesthat take place in
sodiumchloride metabolismat the tissueand systemiclevelsprovidesthe
basisfor another working hypothesisconcerningthe mechanismin superacuteshock. We have seenthat the productionof vacuolesin cells characterizesthis latter form of shock. The unequal cellular permeability for
chloridesand sodium in their dissociatedform is known. Chloride ions can
circulatemuch more easily betweencells and the pericellularspacesthan
can sodium ions. An initial effect of the intervention of abnormal fatty
acidsin cellularpathologyis the fixationof chlorides.At the sametime, an

232

RESEARcHrN pHysropATHoLocy

increasedpermeability in membranesoccurs. This would permit more


sodium ions to passthrough cell membranesand to accumulateintracellularly, inducinga liberationof potassium,the cellular cation. As the chloride
ions are bound to fatty acids in the cells, the sodium ions in the cetls liberate potassium and join it to form alkaline compounds. Isolated in vacuoles, thesecompoundsalso accumulatewater.
Thus, we have a concept of single pathogenesisfor all three forms of
shock basedupon abnormal sodium chloride and water metabolism,with
the abnormalitytaking place at differentlevelsof the organization,cellular
for superacuteshock,tissularfor the acute form, and systemicfor the state
of shock. The displacementof potassiumby sodium in cellular physiology
contributesto the increasein serumpotassiumfound in all forms of shock.
Water Metabolism
The localized retention of water, prompted by the alkaline sodium
compoundswhich result from abnormal sodium chloride metabolism,occurs in the cells. tissuesor intestinesin the different types of shock. Many
of the differencesin manifestationsbetweenthe three forms of shock can
be explainedin terms of localizationof this abnormal water metabolism.
The sensitivityof the cells of the nervoussystemto intracellularchanges
explainsthe predominanceand severityof the nervous system manifestations in superacuteshock, Abnormal tissuewater metabolismexplains not
only the predominantlylocal characterof the manifestationsseen in acute
shock,but also the hemoconcentration
valuesin thesecases.As often seen
in burns, important amountsof water are driven out of the blood into the
damagedtissues.
The abnormal water metabolismhowever,appearsto be the principal
manifestationin the state of shock. Upper intestinal water accumulation,
rather than a general unlocalizedfluid loss, can be demonstratedin the
pathogenesisof this form of shock. In opposition to the local lesion with a
high retentionof water, the generalsubcutaneoustissuessustaina loss of
water rather than an accumulationduring shock. This would not occur if
therewere a generalincreasedpermeabilityof all capillaries,allowing water
to pilss freely. The role of water accumulationin the first portions of the
intestinedue to the abnormal loss of systemicwater was demonstratedin
animal experiments.When the small intestinal tract had previously been
removed, and a state of shock was later induced by trauma, no hemoconcentrationoccurred.
It is the participationof one or anotherof the three principal levels of
tissular or systemic-which explains why the
the organization----cellular,

sHocK

233

same pathogenicprocess,abnormal sodium chloride and consequentabnormal water metabolism, produces such different manifestations in the
various types of shock. It must not bc forgotten howcver, that in the last
analysis,the abnormalitiesin sodium chloride and water metabolismresult
from the interventionof abnormal fatty acids.Fatty acid intervention,together with the abnormal sodium chloride and water metabolism confirm
the unitary pathogenesisof the three forms of shock.
Other Changes
Other changesassociatedwith shock also can be related to the influence
exercisedby abnormal fatty acids. The appearanceof rouleaux of red cells
may be easily explained by fatty acid intervention. It is the replacementof
the nonpolarity normally present at the surface of the red cells by a dipolarity which results in the formation of the rouleaux. This can be induced
by fatty acids in vitro. Sludge formation would represent a still more advanced step in this same processand would apparto result from a polypolarity at the surfaceof the red cells.Sludgeformationshave been induced
in vitro by fatty acids added in larger amounts to plasma. (Note 2) They
contribute to the circulatory impairment considered to be an important
factor in the tissular respiratory troubles seen in shock.
We have noted that the richnessin free fatty acids interferes with the
ability of the red cells to keep oxygen fixed, a fact which would impair its
transport. This, together with hemoconcentrationand circulatory impairment, has been found to account for the black color of the blood in shock.
(Note 3/ The clinical manifestationsare characteristicof offbalanceD.
Experimentally Induced Shock
The hypothesisthat the three types of shock are caused by the intervention of the same factol-3lnospal fatty acids-has been further confirmed experimentally. The cellular changes that characterlze superacute
shock can be induced by the rapid introduction into the blood stream of
c-ven minislsl amounts of fatty acids in preparationsin which they are
bound to plasmaconstituents.
Pooled heparinizedplasma of mice was treated by stirring it in a nitrogen atmospherefor one hour with a preparation of conjugated trienic fatty
acids. The nonbound fatty acids were separatedthrough short centrifugation. The plasma was injected intravenouslyin mice. For control, plasma
treated under the sane conditions with stearicacid was used. While control animals did not show any apparent discomfort, the mice injected with
the plasma treated with conjugated fatty acids died immediately, in most

234

xEsEARcH rN PHYsToPATHoLooY

caseseven during the injection itself. With such preparations,superacute


shock was induced in what we consider a direct way, the sudden death
contrastingwith the casesof hemoshockwhere death occurs usually after
an interval of a few minutes.This characteristicof direct immediatedeath
is consistentwith the pathogenicrole of fatty acids in superacuteshock.
The tissuechangesthat character'tzs,
the acute type of shock also may
be induced by local administrationof abnormal fatty acids with the condition that sufficientamounts are used. (lt'lote4) The systemicchangesthat
typify the state of shock can also be produced by prolonged absorption
of fatty acids, as when they are repeatedlyintroduced intraperitoneally.
(Note 5 )
The relationshipbetweenshock and lipids can be further seen in the
antagonisticeffect exercisedupon shock induced with standardizedtrauma
by two groups of lipids with positive and negative characters.We have
utilized the Noble-Collipp drum on a large scale to induce shock in rats.
In some groups of animals shock induction was constant;in other goups
under the sameconditions,shockcould be inducedonly in some animals.
Nevertheless,
it was still possibleto recognizeopposite effectsinduced by
the administrationof the two groupsof lipids. In some animalseven ap
parently little influencedby the trauma, the injection of a mixture of conjugated fatty acids immediatelyafter trauma brought death within a short
time. In no other animals, traumatizedunder the same conditions, have
we seendeath occurring within the sameshort interval of time. This also
appliesto animalsinjcctedbeforetrauma.In thesecases,the animalsdied
evenduring the trauma,that is, in the drum. (Note 6)
Conversely,the administrationof sterols,especiallypreparationsof the
insaponffiablefraction of human placenta,before induction of trauma prevcnted lethal shock alnrost without exccption,whereasunder the same
conditions the same trauma produced death in a high propoftion of the
controls.Evcn when injectedimmediatelyafter trauma,this sterolpreparation preventedthe developmentof lethal shock in a high proportion of
cases.(Notc 7)
The different forms of shock, althoughresultingfrom the same fundamental abnormal process, appear to respond differently to therapeutic
agents-again becauseof the localizationof the abnormal processesat
differentlevels.Adrenalin and relatcdcompounds,when administeredin
time, are able to control superacuteshock, but they are almost entirely
without influcnce upon the other forms. While acute shock can be influencedby the administrationof a largeamountof sterolsand butanol, superacute shock is unaffected,possiblyalso becauseof the slow absorption of

sHocK

235

the sterols.None of theseagentsis of significancein the treatmentof the


stateof shockwhich is only mildly influencedby butanoland ccrtaincortical
hormones such as hydrocortisone,especiallywhen introduced directly in
the circulation.
In order to act upon the fatty acids and sodium which produce the
abnormal water metabolism,we have utilized glycerophosphoricacid administeredin large amountsparenterally.Diluted with saline,it was usually
injectedintravenously.The good resultsobtainedare discussedlater.
The use of heptanoland of polyunsaturated
alcoholshas also led to
interestingresults.It was howeverwith preparations
havingsevcralof these
agents,working at differentIevelsof the organization,
that the bcst results
were obtained.
The measurement
of the chlorideindex and of the surfacetensionof
the urine haverepresented
valuablemeansto judgethc changesoccurringin
shock,in their clinical evolutionand especiallyin the action of the agcnts
in relationshipto the occurring recovery.
The study of shock has contributedto the knowledgeof the therapeutic
problemsof cancer and other conditions.The causeof death, when a predominance of fatty acids occurs as a systemicmanifestation,corresponds
to the state of shock.The possibilityof successfully
influcncingthis form
of shock would furnish a valuabletool for the treatmentof all severemanifestationsrelated to predominanceof fatty acids.

O
C H A P T EI R
RADIATION

"ln
lL Hp sruoy

oF rHE efiects produced by irradiation upon all biological


entities has resulted in the accumulationof a large amount of data. Being
unsystematized,
this information has helped but little to resolvethe many
physiological and therapeutic problems connected with radiation. New
light on theseproblemscan be providedby relatingthem to the basic concepts we have been discussing.
Other factors also have led us to study the problem of radiation. Its
widespreaduse for the treatment of cancerouslesions with indisputable
successin many cases,and the fact that some of radiation'seffectsappear
to be quite similar to those induced by the administration of different lipids,
led us to investigatethe mechanism through which radiation works and,
especially,the possiblerelationshipbetweenradiation and lipids. We will
discusshere briefly, someof the resultsof this investigation.
Irradiation ol Lipids
We began by trying to determine the eftects of radiation upon normal
lipids in vitro. As always, we tried to guide the researchby theorctical considerations.
Investigationof in vivo and in vitro effectsof radiationupon
proteins in general showed that histones,protaminesand alkaline amino
acids are most sensitive.These constituentsof complex protein molecules
have positive electricalcharacter.This relationshipbetween sensitivityto
radiationand positiveelectricalcharacterprovideda clue as to whereto look
in fatty acid moleculesfor changcsinducedby radiation.Severalpositive
centersare presentin the energeticstructureof fatty acid molecules.One
is representedby the carbon of the carboxyl. Its positive characteris due
to its bond to two oxygen atoms. This would explain the exaggeratedion-

izationwhichtakesplaceat the levelof this carboxylgroup.

RADr^rtoN

237

other positive centers also can be recognized.we have mentioned


previously that the positive character of carbon propagates through the
chain in an induction effect that causesalternate odd carbons to be positively charged, although the strength of the positive characrcr decreases
rapidly with distance from the carboxyl. Since a double bond geatly enhances the energetic character of the carbons linked by it, induction will
result in a center in which a more intensivepositive carbon is present.
Study of the reactions that take place at the double bond in a fatty acid
molecule confirms this view, since an electrophiliccharacterpredominates

g
o

v,
0

I
6
t
.6
L

fa

I rradiated
fbnirradiated

i l aY e

Length

(np)

Fto. 79. Irradialion ond conjugation in vilro. Spectral analysesin ultraviolet of samp l e s o f c o m m e r c i a l l y a v a i l a b l el i n o l e i c a c i d ( w i t h s m a l l a m o u n t s o f l i n o l e n i c a c i d
present) irradiated with gamma rays from 80 mgr of platinum filtered radium/10 cc,
f o r 6 d a y s a t r o o m t e m p e r a t u r eF. o r t h e a n a l y s e sd, i l u t i o n so f 0 . 0 0 2 9 bi n a l c o h o l ,w i t h
alcohol as reference, were used. The absorption spectra of the irradiated (-)
linoleic acid compared with the nonirradiated ( . . . . ) shows the appearanceof conjugated trienes recognizedthrough the characteristicpeaks.

238 /

n E s E A R c Hr N p H y s l o p A T H o L o c y

at this point. when treated with sodamine,carbons forming double bonds


combine selectivelywith it, indicating the positive electrostaticcharacter
of this formation. Consequently,we thought that the effect of irradiation
would be most likely to occur here. This has been confirmed experimentally. We could demonstratethat radiations causechangesespeciallyin the
reciprocal position of the double bonds in the molecule.
The results of this researchwere originally presentedbefore the Fifth
InternationalCongressof Radiologyin London in July 1951. We will timit
ourselveshere to a short resumeof the proceduresand findings:
Irradiation in Vitro
a) Radiation of polyunsaturatedfatty acidsin vitro inducesa conjugation of their double bonds, which increasesquantitativelywith the intensity
of the radiation.This has beenshownby spectralanalysisand by the oxidative fission method. (Note // Samplesof commercially available linoleic
acid which, through analysis,have beenfound to contain variable amounts
of linolenic acid, or cottonseedoil were treatedwith radiationsof different
sources,such as radium in platinum needlesfor gamma radiation,in monel
metal for beta radiation,thorium X for alpha radiation and X rays. Table
XIV showsthe resultsof analysisof the oxalic index. Figs. 79 and 80 show
the direct spectralanalysisof the samplesbefore and after irradiation as
well as the result of their chenricalconjugation.
Comparison of direct spectral analysesshows the appearanceof an
important amount of conjugatedtrienesin the irradiatedsamples.Analysis
after chemical conjugation of irradiated and control samples shows a
greateramount of trienesin the irradiatedsampleindicating that a process
of desaturationa.lsohas taken place through irradiation.
A dirc.ctrelationshipwas observedbetweenconjugationand amount of
radiation. (Tnalr XV) The quantity of conjugatedisomers,determined
by spectralanalysesand measuredby the oxalic index was seento increase
as radiationwas increasedeither by prolongingexposuretime or increasing
the amountof radium used.
b) Irradiation of fatty acids appearsto induce the appearanceof conjugated trienes. when a mixture of polyunsaturatedfatty acids, such as
thosefound in cod liver oil, was exposedto a radiation sourceconsistingof
platinum filtered radium, the changeswere limited to the appearanceof
conjugatedtrienes.Conjugateddieneswere seenin only some experiments
and then only in small amounts.The presenceof conjugated members was
recognizedthrough their characteristicabsorptionpeaksin ultra-violetanal-

-l
,tl

RADIATION

I
,t-1

30

E
an

c
o
F

"-*

"l
-l

uo']

t
I
I

'ol
'ol

too

rl

I
I
I
I
I
I
I
I
I
I
I
I

oo1

tn

t-'\

,
t
,
t
,
I
I

20
20)

.9

239

-aa

c
.9

t
a

t
t
t

320

300 ?30

240

220

200

Wove Length (my)


Frc. 80. Irradiation and desaturalion. Spectral analyses in ultraviolet (0.002% in
cthyl alcohol with alcohol as reference) of untreated sample (-)
and of the
radiated sample (. . . . ) both after alkaline isomerization.They show that the irradiation has induced also an increase in the amount of trienes present, which indicates
that a desaturation also occurred.

ysis. When the same mixture of fatty acids was treated by the usual
chemical methods ernployedto produce conjugation,i.e,, with potassium
hydroxide in ethylene glycol or glycerol ( 4l ), the spectral analysis
showed that the preparation contained fatty acids having between 2 and 6

240

RESEARCH TN PHYSIOPATHOLOGY

T,rsle XIV

Fatty Acid

Type of
Radiation

Sourceof Radiation

Exposure
Time
(Days)

Oxalic
Acid
mg/E
Fatty
Acid

6
8
l5
4

0
8.3
9.9
13.3
9.85

7
27

4.3
6.8

53

12.4

5
8

0
5.2
8.0

Linoleicacid
tt

tt

tt
t

tt
r

tt

gamma
gamma
gamma
beta
alpha
x-ray

tt

x-ray

50 mg Radium
120 mg Radium
120 mg Radium
25 mg Radium
in Monel metal
150 uc Thorium X
5000r daily--deep
therapymachine
5000r daily--{eep
therapymachine

Cottonseedoil
t
tl

t
tt

gamma
gamma

80 mg Radium
80 mg Radium

doublebonds.Figure 8l showsthe curvesof spectralanalysisfor such an


experimentin which 3 cc. of a cod liver oil fatty acid preparation were
treated for six days with 100 milligrams of radium filtered through platinum. In curyo "a," of the untreatedsample,it can be seenthat there is no
absorption due to the presenceof conjugatedmembers.Curve "b," for the
irradiated fraction, shows the typical conjugated rrienes, while Fig. 82
showsthe result of the chemicalconjugationof the nonirradiatedpreparation with membershaving from 2 to 6 double bonds.
Trst-e XV
Errecrs or Inn,rnrrrloxUpoNrHe Qurxrlry oF Oxrlrc Acro
PneseNrArten Oxto,rtrveFrssroN

Fatty Acid

Linoleic Acid
l0 cc.

l0 cc.

Exposure
time:
Days

Oxalic
Acid
mg g
Fatty
Acids

0
4
6
8

0
4.5
6.2
9.9

tt

15
20

r3.3
t4.4

1 5 0m g R a
,,

6
15

8.2
16.3

Sourceof
Radiation
0
1 2 0m g R a

RADIATION

241

IO

20

30
c
o

10

(a)

tn
.n
E

50

v,
E
.tt
t-

60
ia

70

I rradiated
Noninradlated

80

s
28o
260
i t a v e L e n g t h

2,to
(D!)

220

Frc. tl. Spectral analysis (0.002Vo in alcohol) of a mixture of. latty acids lrom cod
with 100 mg platinum
l i v e r o i l , u n t r e a t e d( . . . . ) ( a ) a n d i r r a d i a t e d( b ) ( - )
filtered radium/3 cc, at room temperature for 6 days. The analysis shows that the
conjugation which takcs place leads to the appearanceonly of conjugated trienes,
in spite of tbe presence of di-, tri-, tetra-, penta- and hexaenic unsaturated fatty
acids as shown by the absorption spectrum of the same mixture after chemical conj u g a t i o n w i t h p o t a s s i u mh y d r o x i d e i n e t h y l e n eg l y c o l . ( c ) a s s e e n i n F i g . 8 2 .

c) The changesinduced in fatty acids are essentiallythe same regardless of radioactive source. Thus the effect upon a linoleic acid preparation
containingsomelinolenicacid was the samewith alpha particlesof Thorium
X, beta rays from radium in monel metal, gamma radiation from platinum

242

xESEARcH IN

PHYSIoPATHoLocY

by a 400 kw. therapyunit. Figures


fi.ltered
radium,and X-rays generated
83 and 84 and TableXIV showtheseresults.
Irradiotion in vivo
d) Irradiation of the body of normal animals killed by decapitation,
producessmall amountsof conjugatedfatty acids.
under ether anaesthesia,
In early experiments,different organs obtained from slaughterhouseswere
irradiated and fatty acids examined. In general,even after intensive radia-

30

40

50
C

.9 -^

.3bu

c
o

Fzo
)
80
90

400

380

36U.

340

320
300
28c
WoveLength(mp)

260

240

220

F r c . 8 2 . C u r v e o f s p e c t r a l a n a l y s i sof the cod liver oil fatty acids after chemical


conjugation, shows the presenceof d i - , t r i - , t e t r a - , p e n t a - , a n d h e x a e n i c m e m b e r s .

tion, correspondingto 4,000 r. in one treatment,the oxalic index of fatty


acidswas neverfound to be above l, correspondingto I mg. of oxalic aciJ
obtainedfrom one gram of fatty acids.
e) On the other hand, the amount of conjugated fatty acids in the
bodies of living animals receiving radiation increases significantly. The
following experimentis illustrative.Eighty rats of the same sex, age and
weight(about 180 gms.), separated
into severalgroups,weregiven 1500r..
delivered by a therapy unit with no fi.lter.Four control animals were killed

243

RADIATION

l0

20

30

/r'\'.

f .,

I '

c
o

.a

40

att

/,'

l t

\'.\

\'..i
t v

t
t
,

ah
t

E
U'

)
,
l

bU
I

g
I

C'

I
t
I
I

i-

r-

60
t

}Q

m
I

l i
I r

80

l i
I t

t^'

Ir r a d i a t e d
llonirradiated

i
90

---.-'2
oo

2qo

U a v e l e n g t h ( m p )
Fro. 83. Absorption spectra (O.OO2Voin ethyl alcohol/ethyl alcohol) in ultraviolet
o f c o m m e r c i a l l ya v a i l a b l el i n o l e i c a c i d ( w i t h s m a l l a m o u n t o f l i n o l e n i c a c i d ) n o n with beta particles from 25 mg monel metal
irradiated ( . . . . ) and irradiated (-;
filtered radium/10 cc at 37'C for four bours. Some conjugation occurs in thc control
when kept in the incubator,

raiff$.r,4,{a{tfr
{iffi#(ffi'}*$itl's i#t1'

244

nESEARcH rN pHysropATHoLocy

beforeexposure.Groups of four treatedanimalswere sacrificedperiodically, startingimmediatelyafter irradiation,at 2, 6 and 24 hours aftcr irradiation,and eachday thereafteruntil all animalsdied or had beenkilled.
During this time, nontreatedcontrol animals,kept under the same
conditions,were also sacrificed.The quantity of conjugatedfatty acids

c
o

e
c
C'
L

>e

f a v e l e n g t h ( z r A )
F r c . E 4 . A b s o r p t i o n s p e c t r u m o f t h e s a m e m a t e r i a l a s i n F i g . E3, non-irradiated
( . . . . ) a n d i r r a d i a t c dw i t h o l p h a p a r t i c l e sf r o m 1 5 0 m c T h o r i u m X / 1 0 c c a t r o o m
t e m p e r a t u r ef o r 7 d a Y s . ( - )

RADr^rtoN

245

found in the entire body of each individualanimal at rhe time of death


was determinedby meansof the oxalic acid index method.
The oxalic acid index for fatty acids in the bodiesof untreatedcontrol
animalswas usually zero.occasionallythere was a variation from 0 but it
was always below 0.6. Irradiated animals killed within the first two days
showedan irregular increasein conjugatedfatty acids, with oxalic acid
indexvaluesof between0.6 and 5.1. Threedaysafter irradiation,the oxalic
index was above 3 in all the dead or sacrificedanimals. Thc index rosc

*
r

xt
X

r r r
l

,
x

8
r.ft
:

x
c

;
.!
x
o

I
E

(t
L

Ftc. E5. Futty acid conjugution indttted hy-irradiation in vir'<t.Changes in the oxalic index of total fatty acids of rats irradiated with a letlrcl dose (1500r). Sacrificed
at different intervals, the oxalic index of their fatty acid shows progressivelyincreasi n g v a l u e s ,T h e a n i m a l sd i e w h e n t h e i n d e x h a s a r r i v e d a t a c r i t i c a l v a l u e b e t w c e n l 4
and 17.

246

npsEARcH tN pHysropATHoLocy

even in animalswhich showedno visiblei.[l eftectsat the time they were


killed.
The index increasedcontinuouslywith the passageof time until the
animalsdied. By the fifth day, it was above6 for all animalsand, by the
seventhday, with few exceptions,it was around 10. After the twelfth day,
it had risen above 12 rn most of the animals.In all animalswhich were

oigl&
I ndex

7 days 12,2

!o
I

1.4
4.L
3.t
c
o { o
6

o
c
6

Eao
la

Ncrnal

3 hrs.

3,2

7o

Eo

Joo

"o ,.r. Llf,itr.,(rA'"

22o

F t c . E 6 . S p e c t r a l a n a l y s i s ( . 0 1 i n e t h y l a l c o h o )l o f t h e t o t a l f a t t y a c i d s o b t a i n e d
from tbe body of rats irradiated with 1500r shows changes more manifcst around
270 mp. The oxalic index of the preparation is indicated and shows a parallel increase
with the changesin the curve.

RADtATTON

247

sacrificedafter the l3th day or which had died at any time, the index
showedvaluesbetweenl4 and 17. (Note 2/ Figure 85 showsthescresults
in the group of rats describedin this experiment.These changeswere observedwhen the same procedurewas repeatedin other goups of animals.
These experimentsclearly indicated that the quantity of conjugatedfatty
acids progressivelyincreasesin the days following the exposurein animals
lo

[\.*'

,"-

'iiradtated

o
an
o
e
-q

,O

E
(!
L

'a

Eo

Jto

tto

.t?o

frl"ue
tenqffir1l

,r<

Frc. 87. Spectral analysis (.01 in ethyl alcohol) of the fatty acids of the lotal body
of a mouse irradiated with l50Or, shows an increasein fatty acids with the absorption
correspondingto 270 mp., as compared with the control.

treated with one lethal dose of X-ray. Death occurred when the amount of
conjugatedfatty acid reacheda critical level equivalentto an oxalic acid
index value betweenl4 and 17. The spectralanalysisof fatty acidsof animals treatedwith radiation showedchangescorrespondingto the presence
of conjugatedisomers.These appearin the samplesof fatty acids obtained
from the entire body of theseanimals.(Figs. 86 and 87l Still more evident
werethe conjugatedtrienesin the fatty acidsof organs.Figures 88, 89 and
90 show the differencein such analysesas comparedto correspondingun.

248

xESEARcH rN pHysropATHoLocy

treatedcontrols.The presence
of conjugated
trienesappearsclearlyin the
characteristic
peaks.
The concept of a critical value for the oxalic index is supportedby
other studiesin which the same value is found in animalsdying after
adrenalectomyor after thermal, chemicalor traumatic statesof shock.
Even in animalsdying in superacuteshock,within 3 to 5 minutesafter
beingseverelyscaldedin hot water,the level of conjugatedfatty acidsis
higherthan in controls.

2c)-+-

0 . 0 1 2t n e t h y l a l c o h o l / e i h yal l c o h o l
Imariiated

,o

(o

c
o
q
vl
c

E6o
|9
t

Da

70

9o

,.o

3@

2to

waritnothr#Ft

22o

F r c . 8 8 . S p e c t r a la n a l y s e s( . 0 1 i n e t h y l a l c o h o l ) o f t h e f a t t y a c i d s o f t h e k i d n e y o f a
normal rat and of a rat irradiated 6 days previously with 1500r. The peaks characteristic for conjugated trienes are seen.

R^DrArtoN

249

when the irradiated


dosewasnot a lethalone,that is, below600 r. in
our experiments,
the oxalic index increasedat fint but decreased
after
about two to three weeks.It neverreachedthe critical value of. 14-17.
( F i g.9 l ,N o te3 )
Local Efiecls
f) We completedthe studiesof the eftectsof radiationupon fatty acids
in vivo by considering
them at the localtissuelevel.The first requirement
20

0 . 0 1 2i n e t h y l a l c o h o l / e t h yall c o h o l

- lmadiated

Jo

t
a
I
t

to

c
o
t^
vt
E
tn
c

t>e
76

8o

9o

5oo

"?.r. 1.61fr
@r)t""

220

Frc. 89. The spcctral analyses (.01 in ethyl alcohol) of the fatty acids of the liver
of a normal rat and of a rat irradiated 6 days previously with 1500r shows the appearaoceof the cbaracteristicpeaks of conjugated trienes,

xEsEARcH rN pHysropATHoLocy

250 /

was to establisha radiation procedurewhich would producea standardized lesion.When radiationswereapplieddirectlyto the skin of animals,
the individualdifferencesin responsewere quite marked.Thesecould be
explainedin part on the basisof ageand particularlyof sex,the difference

,o

0.01%
i n e i I alcohol/ethyl
alcohol
,l r r adiated

(o
,
,
I
I

,
,
I

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o 6o
an
v,
E
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c
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r

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/""

ba

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ve Lenoth
(np;
F t c . 9 0 . S h o w s t h e s p e c t r a la n a l y s e s( 0 . 1 i n e t h y l a l c o h o l ) o f t h e f a t t y a c i d s o f t h e
lung of a normal rat and of a rat irradiated with 150016 days previously. Pcaks correspondingto coDjugaledtrienes are present.

betweenthe skin of male and female rats being manifest.However, there


were also pronouncedindividualdifferencesin animalsof the samesex, age
and weight living under the sameconditions,so that even when the experimental procedurewas carefully controlled,the same arnount of radiation
causedreactionsthat varied widely from slight erythemato ulceration.

RADIATION

,/

251

The problem of variability was satisfactorilyresolvedby radiating abnormal tissues,such as thoseof a wound, insteadof normal tissues.Standardizedlesionswere first producedand then irradiated.we usedthe following technique:an area of the skin on the back of male ratsweighingaround
200 grams was epilatedand, under ether anaesthesia,
a 2 cm.long incision

a
c)
E
q,

(o

P
L
r

'

x
T

Ccntrol

'H.r.

,
2
Oays

Ftc. 91. Fatry acid coniugation and irradiotion in yiv'o. The changes in the oxalic
i n d e x o f t o t a l f a t t y a c i d s o f r a t s s u b m i t t e dt o s u b l e t h a li r r a d i a t i o n ( 6 0 0 r . ) . O n l y a
temporary increasein the oxalic index of the fatty acids of the animats is seen, the
a m o u n l s n o t r e a c h i n gt h e c r i t i c a l v a l u e s .

was made, penetratingthe skin and subcu'raneous


tissuesdown to the
dorsal aponeurosis.A needlecontainingradium was then placed between
the lips of the cutaneouswound. A thread passedthrough the hole at one
end of the needlewas used to fix it to the skin. Two retainingsutureswere
also used to maintain the radium needlebetweenthe lips of the wound,
The needlewas left in place for the desiredlength of time and then easily

252

R E s E A R C Hr N p H y s r o p A T H o L o c y

removedwith the help of the thread passedthrough the hole of the needle.
The retaining sutureswere also removed and the wound left open and undressed.
The length of time that the needle was left in place varied with the
amount of radium, the nature of the filtering metal, and the radiation burn
desired.we found that l0 mg. of platinum filteredradium had to be left
in place for about 90 houn in order to produce a standardizedulceration
that would last about four weeksbefore healing.The same effect was obtained when 25 mg. of monel metal filtered radium was kept betweenthe
lips of the wound for only two hours. when monel metal needleswere
used for only one hour, too great difterencesappearedbetween the ulcerations obtainedand the time necessaryfor their healing.A two-hour exposure causedan ulceration which usually required 4 to 5 weeks to heal in
control animals.lf the needlewas left in place for 3 hours, the ulcerations
were quite uniform but they required over two months to heal and more
than half of the wounds never healed.Failure to heal and more extensive
necrosisresultedfor periodsof exposurebeyond 3 hours.
Therefore,we utilized l0 mg. of radium in platinum for 90 hours in
one group of experiments,and 25 mg. of radium in monel metal for 2
hours in another group, in order to produce standardizcdulcerationsthat
would generally heal spontaneouslyafter 4 to 5 weeks.This technique has
been used in several hundred animals for various experiments. The fatty
acids of thesestandardizedradiation lesionswere studied.
Days after irradiation, the ulceratedlesionswere removed along with
about one cm. of surroundingtissueand submittedto analyses.It was always necessaryto use as many as 5 or 6 lesionsto obtain the quantity of
fatty acids needed for an oxalic index determination.The lesions werc
found abnormally rich in conjugatedfatty acids. Commonly, indices as
high as 4o-and in exceptionalcasesas high as 65-were found (Trnle
XVI) in comparisonwith 0 or 0.3 for normal skin with its subcutaneous
tissues.
Lipids and Radiation Burns
S) T'1t" apparanceof conjugatedfatty acids as an effect of radiation
has posed the problem of the role of theseabnormal fatty acids as intermediary agentsin the biological changesinduced by radiation. In trying
to solve it, we compared the eftectsobtained by administrationof conjugated fatty acids with those of radiation at different levelsof organization.
This study was facilitatedby consideringthe changeswhich take place in

R A D I A T I ( ) N

2s3

Trnle XVI
Ox,rlrc lNoex on Frrry Actos or Reorrrrox Bunxs
ElapsedTime

NormalSkin
Non-treated
wound

Woundwith 25 mg. radiumin


monelmetalfor 2 hours

24 hours
48 hours
7 2 hours
6 days
2 hours
24 hours
4 8 hours
4 days
I week
')

weeks

3 weeks

Average
0.1
2.2
3.9
2.3
1.8
t.7
6.1
l3.9
l9.1
31.0
46.0
49.4

the cellular cytoplasm and nuclei as induced by various substancesdesignated as radiomimeticagents.


It could be seenthat apparentlyall agentswhich induce radiomimetic
effectsare lipoids with negativepolar groups.The effectsof higher polyunsaturatedfatty acids, and especiallythe conjugatedisomers,appar to be
the same as thoseof known radiomimeticagents.The similarity between
the effectsof these fatty acids and those of radiation makes it logicd to
consider that at least some of the radiation-inducedchangesresult from
the interventionof theseabnorma.lfatty acids,
We have seenthat the changesinducedby fatty acids upon cell metabolism are in large part due to an increasein cell membranepermeability.
A similar change of cell membrane permeabi.litycould be recognized
among the eftectsof radiation. Following radiation, it could be seenthat
sodium of the interstitialfluids penetratesinto the cells more readily. This
was observedwhen radioactivesodium was used. (42) The cellular vacuolization seen to follow radiation, especiallyhigher doses, representsa
corollary of the abnormal penetrationof sodium into the cells which partty
resultsfrom the increasein membranepermeability,
h) At the tissularlcvel,the influenceexercisedby radiationupon pain
was seento $eatly resemblethat inducedby administrationof fatty acids.
Radiationefficientlyrelievespain that has an acid patternbut it may increasepain of an alkaline pattern.Furthermore,pain which appearsfollowing radiationhas an alkalinepatternand consequently
is increasedby
further radiation, or administrationof unsaturatedfatty acids. (Note 4)
i) At the tissularlevel,it could be seenthat the area of ulcerationof
the standard lesions obtained throueh irradiation of skin wounds was

254

REsEARcH rN pHyslopATHoLocy

increasedby the administrationof polyunssluratedfatty acids in general.


In somecasesthe ulcerationdoubled in size as comparedto controls. The
administrationof fatty acids also markedly delayedwound healing.when
the quantity of fatty acids administeredwas great enough,the wounds did
not heal at all. Six daily subcutaneous
injections,each of I cc. of a l\vo
oily solutionof cod liver oil fatty acids,preventedhealing.(FiS.92) The
area of ulceration was even greaterwhen only rA c*. of. a ljVo solution
of the conjugatedfatty acid isomers,obtained through an in-vitro conjugation of the preparation of cod liver oil fatty acids, was administered
under the sameconditions.This showedthat conjugatedfatty acid isomers
had a more manifest effect upon radiation wounds than the unconjugated
acidsobtained from the samesource.
j) we followed effectsof intensiveradio and radium therapy in humans
at organic levels. In caseswith radiation-inducedproctitis, mucositis, or
epidermitis,the changesobservedwere seen to correspondto the pattern
encounteredwith fatty acid predominance,
and especiallyto the pattern
induced by abnormal fatty acids.The appearanceof oxidizing substances
in the urine is frequently observedin patients with radiation burns after
extensiveX-ray therapy.They were almostconsistentlyseenin those cases
in which radiationlesionswere produced.The administrationof fatty acids.
and particularly of conjugatedfatty acids, to these patients increasedthe
intensityof the lesions.
k) Systemic changes induced by intensive radiotherapy were also
analyzed.Here again, the changesfollowed the pattern observed when
there is a predominanceof fatty acids,particularlyof abnormalfatty acids.
The appearanceof oxidizing substancesin the urine was frequently noted
after intensiveX-ray therapyand, as mentionedpreviously,was consistently
observedin those casesin which a radiation lesion was produced. Other
systemiceffectsof intensiveradiotherapywere seento include an increase
in urinary cxcretion of surfaceactive substances,
an increaseof potassium
in serum,a retentionof chloridesand water, and particularly,an increase
in the sulfhydryl index indicating an exaggeratedexcretion of this group.
Thesechangcsfollowing intensiveradiation are, as previouslynoted, similar to thoseseenwhen a predominantinterventionof fatty acids occurs.
Certain of these changesappear to have prognostic importance for
radiationtherapy.For example,in severalcaseswith very low urinary surface tension,high retentionof chloridesand absenceof urinary peroxides.
The continuationof irradiationled to death. (Note 4/ This is consistent
with the findingsin animalsthat lethal effectsof inadiation are directly re-

BADIATION

255

,,.,
,;,]]:,,i.

u
C

Ftc. 92. Lipids anct radiatian *'ounds. Radiation rvounds 5 weeks aftcr exposure to
l0 rngr. radium in platinum-for 96 hours. (a) Untrcated conrols: (b) treatcd tlnily
with I cc of a cod liver oil fatty acids l0{?. solution; {c) treated daily with 0.5 cc of
a 109* rolution of unsaponifiable lipid fraction cxtracted from human placcnta. The
lrcatmenl with fatty acids results in larger lesionsthan in controls, with no tendency
to hesl. Thc treatmcnt with the unsaponifiablc fraction lcads to a hcaling of the lesion
in rround threc wocks.

256

*ESEARCHrN pHysropATHoLocy

lated to the appearanceof large amountsof surfaceactivesubstancesin the


urine.
ROLE OF ANTI.FATTY.ACIDS
The study of the biological effectsof radiation also has revealedan
important role for anti-fatty-acid agents. The intervention of these substancesin the physiopathologicalprocessesthat occur in the organism
under the influenceof radiation can be consideredto be reactional.They
correspondto a responseof the organismto changesinduced by radiation
upon the constituentssuch as fatty acids.
This antagonismis clearly shown in experimentswith animals. The
administrationof sterol preparationsnot only reduccsthe size of ulceration
in standardskin radiation lesionsbut also significantlyimproves the rate
of healing. Insaponifiablefraction preparationsfrom human placenta, beef
liver, spleen or blood, as well as from butter, produce such favorable
effects.(Fie. 92)
If sterols are administered24 hours after the radium is inserted (or
later), the influence upon the dimensionsof the ulcer that develops is
reducedand is further reducedwith increaseddclay. In some of the animals treated with I cc. of a 5% oily solution of the insaponifiablefraction
of human placenta in sesameoil for seven days a week, healing with a
normal scar was complete within two weeks. Controls, treated with the
oil vehicle only, required an averageof more than four weeks to heal.
similar eftects were obtained with the administrationof I cc. of. a 7vo
solutionof butanol in salinetwice a day, beginningwith the day of radium
application.
The useof small amountsof radiationhas,in general,a differenteffect
to that of intensiveradiationfrom this point of view.This can be attributed
to the reactiveinterventionof anti-fatty acids.An exaggeratedscar-forming
effect,prolongedfibroblasticreaction,exaggerated
connectivetissueformation, and vascularsclerosisand thrombosisresultingfrom endothelialproliferation are all part of this long-term responseto moderate amounts of
radiation.The same effectsare producedby anti-fatty acid preparations.
All the manifestationsare opposite to those obtained with high doses of
radiationor fatty acids.
From a clinical point of view. the administrationof the insaponifiable
fractionpreparationhad a beneficialradiationeffect.Even in lesionsthat
had persisted
for years,the pain was observedto disappearafter a few days
with t.i.d. dosesas low as I cc. of a 59t solution of the insaponifiable

R^DrArroN /

257

fraction of placenta in oil. In severalcases,chronic lesions three to five


years old healedin only a few months of treatment.
l) The oppositeclinical responseto high or low doseswas frequently
observedin the systemicchangesin patientsreceivingX-ray therapy. While
high dosesled to a manifest lowering of the surfacetension of urine and
an increaseof the sulfhydryl index, togetherwith the other changes@rresponding to offbalanceD for small doses of radiation, certain opposite
effectsrelated to a predominanceof sterolswere noted. Of particular interestwas the absenceof oxidizing substances
in the urine, and the changes
in urinary surfacetension. In all casestreated,a first reactionto radiation
was a higher sulfhydryl index and low surfacetension correspondingto a
fatty acid predominance.When small or moderate amounts of radiation
were used, this reaction was very slight and rapidly disappearedin favor
of a secondchangecorrespondingto a predominanceof sterols with high
urinary surface tension, for instance. It is interestingto note at this point
that this secondaryresponsehas been observedespeciallyin those patients
for whom radiation also has had a limited therapeuticeffect. As we will
see, the dualistic interpretationof data furnished by urinary analysis in
patientsundergoingradiationtherapycan be usedto guide this therapy.
Role ol Adrenols
m) The study of the systemicsecondaryanti-fatty acid responseto
radiation has led to an evaluationof the interventionof the different antifatty acid agentsand to the role of the adrenals.It is known that adrenal
hormoneshave a peculiar effect upon the lymphatic system.They induce
a shrinkageof the thymus, spleen,and lymph nodes, along with blood
lymphopenia.Sincesimilar effectsare producedby irradiation,the problem
of the part played by an interventionof these adrenal hormones in the
radiation responseis of interest.
When the adrenalswere removed,shrinkageof the thymus and spleen
and lymphopeniastill occurred after radiation,but was markedly reduced
as compa.redto nonadrenalectomized
irradiated controls. Since shielding
of the adrenalsduring irradiationdoesnot alter the effectupon the lymphocytes and lymphatic organs,the role of the adrenalsappearsto be an indirect one. Adrenal hormonal secretionappearsthus to be a responseto
the systemicchangesinduced by inadiation. Adrcnalectomywould eliminate this secretionand thereby diminish the degreeof lymphopenia and
the involution of lymphatic organs.However, the secretiondoes not result
directly as an effect of irradiation of the adrenalssince shieldingdoes not
influenceit. Another factor seemsto intcrvenc to stimulate adrenal hor-

258

RBsEARcH rN pHysropATHoLocy

monal secretion. The difterences between the effects seen in adrenalectomized and nonadrenalectomizedinadiated animals correspondsthus to
the adrenal responseto the systemicchanges.
In experimentson rats, we have shown that polyunsaturated,and especially conjugated,fatty acids inducechangesin the number of lymphocytes
a short time after their administration,and that this is followed by involution of the thymus, spleenand lymph nodes.This seemsto occur through
the intervention of the adrenals since it takes place to a greatly reduced
degreewhen the sameamountsof fatty acidsare administeredto adrenalectomized animals.The abnormal fatty acids seem to influencethe adrenals
and their responseelicits lymphopeniaand involution of lymphatic organs.
However, this indirect action through the adrenal glands is only part of
the story. Large dosesof the samefatty acidswill directly induce a certain
amount of lymphopenia and involution of the lymphopoeticorgans since
these changesalso occur when these fatty acids are administered in large
dosesto adrenalectomized
animals.
n) All of this researchindicatesthat two of the mechanismsthrough
which radiation acts upon the organisminvolve changesin lipids. In one,
the action is directly through fatty acids; in the other, as a responseto
thesefatty acids, anti-fatty-acidagentsintervene.The role of the adrenals
appearsto be still more interestingconsideringthe nature of the fatty acids
produced by radiation. As seen above, the conjugatedtrienes appear almost specificallyas a result of irradiation of mixturesof fatty acids.It was
also seenthat the corticoidsintervenespecificallyagainsttheseconjugated
fatty acids.This correlationseemsto representthe link betweenradiation,
conjugatedfatty acids and the adrenalresponse.(Ch. 6, Note l7)
Direct Action ol Radiation
o) In spite of the importanceof fatty acids and anti-fatty acids, they
representonly one part of the mechanismthrough which radiation acts.
The direct and indirect action of radiationon other constituentsalso must
be considered.Thc influence exercisedupon these constituentscan be
largely relatedto variouschanges.There is a quantitativerelationship,for
instance,betweeninductionof mutationsand the direct impactof radiation
on proteins.Changesin fatty acids also are the result of such a direct impact. It appearedinterestingto ascertainhow much and which of the pathological changesthat follow irradiation are due to the direct impact upon
lipids and how much to the impacton the other constituents.
The three kinds of biologicalactivity of radiation-through other constituents,through changed fatty acids. and reactional through anti-fatt)'

R^DrArroN /

259

acids---couldbe studied at different levels of the organization. We note


here a few of the resultsof thesestudies.
Below the cellular level, the influenceof lipids seen$to decrease,causing the direct eftect of the radiation on other constituentsto appear predominant.For nucleo-proteinsand below them, only this last eftect seems
to occur, the changes induced apparently affecting histoncs and alkaline
amino acids. The close mathematicalrelationshipbetweenthe amount of
radiation and mutation would seemto indicatethat, even at the genelevel,
only the effect upon constituentshas to be considered.
The introduction of anti-fatty acids into the medium in which tetrahymenaor suspendedcells (as from ascitestumors) were irradiated,served
to distinguishthe direct effectsfrom those induced through fatty acids. In
the presenceof anti-fattyacids,vacuolizationand even changesin the nuclei
seenin the irradiatedcontrols are prevented.The fact that these changes,
which characterizethe radiomimetic effects,were reduccd by anti-fatty
acids, indicatesthe role of fatty acid changesin the pathogenesis
of these
effects.Among other agentstested,the insaponifiablefraction of organs,
and especiallyof placenta, appearedto be most effective in preventing
radiomimeticeffects.The high alcoholsor glycerolalso showedsuch influence,but to a lesserextent, In complex organisms,the diflerenceof
the effect of radiation on fatty acids and on other constituentsis increased
at the higher levels. At the systemiclevel, this effect is almost limited to
the fatty acids.
The introductionof polyunsaturated
fatty acids to the medium greatly
increasethe toxic effectsof radiation,as comparedto controls exposed
to radiation or fatty acids a.lone.The proportion of mutations was not
changed,however.
The ultimate effect of radiation at difterent levels dependsupon the
relationship between three factors: changed fatty acids, other changed
constituents,and the intcrventionof adrena.ls.
The effect of the adrenalsis
progressively
more manifestat the higher levelsof the organization.At
lower levels,the direct interventionof the abnormallipids becomesmore
important than the adrenalresponse,the latter being less able to act at
theselevels.At the cellularlevel,the influenceof lipids is still predominant.
At the tissularlevel, the direct lipid effect is still striking, while the influenceof the adrenalresponseis limited to the connectivetissue.Although
(as part of the adrenalresponse)
the effectupon the lymphaticconstituents
is important at the organic level, the steroid responsebecomesmore important at the systemiclevel.
p) On the other hand, it appearspossibleto vary the amount of the

260

xESEARcH rN pHysropATHoLoGy

lipidic effect by changing the nature of the radiation. The use of more
penetrating rays or of different corpuscleshas to be investigatedin terms
of the relationship betweeninfluenceupon fatty acids and the effect on
other constituents.It could be seenthat, in correspclnding
dosages,the less
penetratingradiations had a greater influence upon fatty acids than the
more penetrating.The fact explainsthe reductionof radiationburns directly
relatedto the interventionof fatty acids.Similarly,in a systemicprocedure,
such as teleradiotherapy,
the effecton other constituentsis reduc'edas compared with the direct influenceexertedupon the fatty acids.
It is possiblethat radiationsusing neutronswould induce an increascd
direct impact on other constituentswithout a correspondinglyincreased
effectupon the systemicfatty acids,The skin effect,which is minimal with
theseradiations,would indicatelittle interventionof fatty acids.
The unequalpart playedby lipids at the different levelscan be utilized
to obtain variationsin radiation eftects.If effectsupon the lowest levels of
the hierarchicorganization,such as upon histonesand basic amino acids,
are desired,radiation could very well be the tool to be chosen,becauseof
the small amountsof lipids prcsent at these levels. If the influencecould
be limited to such action, radiation could be consideredideal for such
therapeuticeftects. Unfortunately, this is not possible even when very
penetratingradiation is used, and the eftect of radiation upon lipids still
constitutesone of the principal factors which must be consideredwhen
radiationis usedas a therapeuticweapon.
Thus radiation is not the ideal meansfor affectingthe subchromosomal
level, in spite of the fact that it may, througlr its effect upon proteins,have
a profound influencebelow this level. Its ability to causea conjugationof
fatty acidsrepresentsthe seriousobstaclcto its use. In view of this. the
effectof radiation upon lipids actuallycan be consideredas an undesirable
epiphenomenonwheneverthe purposeof the therapy is to achievea local
effect at the lowcr levels. Frequently, the changeswhich require discontinuationof radiationtherapycan be recognizedto correspondto abnormal
local or systemicmetabolismproducedby the abnormalfatty acids.
It must, however,be recognizedthat the appearanceof abnormal fatty
acids has some advantageseven upon protein effects,since indirectly they
can make local tissuesmore sensitiveto radiation.We have previously
noted that abnormalfatty acids causcchangcsin the tissueand cellular
metabolismwhich lead to local alkalosis.This local pH changemay have
favorableresultsby acting upon the amphotericproteinsand by increasing
the positivelychargedmemberswhich apparentlyare the only ones sensi-

R^Dt^rtoN /

261

tive to radiation. Indirectly, the interventionof the abnormal fatty acids


will thus increasethe sensitivityof tissuesto radiation.
Before going further, we wanted to emphasizean aspect of the offbalanceD for which the studyof shockand radiationbroughtan important
contribution.A separationcan be made betweentwo phasesof offbalance
D. one in which oxygenis principallyfixed and anotherin which chlorine
is fixed. The first phase,"oxygen,"has as characteristic
the appearance
of
peroxidesin the urine, and clinicallyhas little noxiousmanifestation.
The
othcr phase,"chlorides,"with fixationof this ion leadsthe seriousmanifestationsas seen,for instance,in shock.For this reason,in radiationthe
disappearance
of urinary peroxideswith persistingoffba.lance
D, as seenin
the other patterns.will indicatea passage
"oxygen"
phase
from
into phase
"chlorides,"which correspondsto the appearanccof a seriouscondition.
(See also Note 4. Chapter l0)
Radio-Therapy
The above considerationsappear inrportant in the radiotherapy of
tumors. The tissular and systemicchangesrelated to the intervention of
fatty acids,especiallywhen thesechangesare sufficientlyintense,in themselvescan act upon tumors. However, when abnormallyintense,they can
constitutea seriouslimitationfor continuationof radiation.The manifestations that result from the pathogeniceflect of abnormal fatty acids, if intense, can prevent the use of large doses of radiation which would
otherwise be necessaryto influence a tumor through a direct effect upon
the lower levelsof the biologicalorganization,histones,nucleo-proteinsand
even genes.Consequently,the appearanceof abnormal fatty acids, which
represent an important factor in the biological effect of radiation, can be
consideredas a favorable effect when we seek to bring about systemic
changesand influencepain and metabolism,particularly at higher levels.
At the sametime, they can also representa principal obstacleto the more
effectiveuse of this same therapeuticagent when one wants to obtain an
effect at lower levels.
As for the effectsobtainedthrough the influenceexertedby fatty acids,
they can be decreasedby changingthe antagonisticrelationshipbetween
the abnormallipids and the defensemechanismof the adrenals.With small
amounts of radiation separatedby long intervals,the interventionof the
adrenals,as long as they function normally,can overcomethe effect of the
fatty acids. With higher dosesapplied more often, the fate of the irradiatcd individual dependsupon whatever antagonisticfactor predominates.
With high dosesor with a relativeadrenalinsufficiency,the direct effect of

262

xESEARcH rN pHysropATHoLocy

the abnormal fatty acids can becomepredominant.In that case,the type


D offbalancewill be more pronounced.It is in such offbalancethat subjects
die from too intensiveradiation.Thesefactors can be of major significance
in the intervention against accidental radiation as well as in guiding the
therapeuticuse of radiation.
Becauseof the interventionof abnormal fatty acids,systemicradiation
does not seemto be the best procedureunlessa very intensesystemiceffect
is sought. If this effect is desired,it can be obtained through a method
other than radiation.Furthermore,as we have noted above,the conjugating
effect of radiation upon fatty acids is almost entirely limited to the production of trienes and dienes.The biologicaleffectsof such conjugatedfatty
acidsare more apparentat the tissuelevel and aboveit. The energeticvalue
of conjugatedtrienesand dienesseemsto be too meagerto permit them to
act intensivelyat levelslower than the cellsor nuclei.
In order to have a manifestfatty acid effect, it appearsnecessaryto
have an adequateapplicationof radiation. since an exaggeratedsystemic
action of the abnormal fatty acidsmay even induce lethal eftects,radiation
does not appear to be the therapeuticmethod of choice for an influence
exercisedthrough fatty acids.Radiation,however,is more compatibleeven
with a desiredlocalizedeffect through the limitation of the field in which
the changesin fatty acids are induced.In this case,fatty acids may intervene with a lower systemicinfluence.This accountsfor the analgesicaction
of radiation which probably is relatedto an effect exercisedby local fatty
acids. Even here, however.the appearanceof an alkaline pattern of pain
can lead to undesiredchanges.In this case,radiationwill increasethe intensity of pain. TNs fact reducesthe indicationsfor use of radiation even
at the tissuelevel.
Biologically-Guided Radio-Therapy
The knowledgeof the important roles played by abnormal fatty acids
and anti-fatty acids in the biologicaleftectsof radiation has suggesteda
biologicalguide for radiotherapy.Urinalysis,by reflectingvarious systemic
changes,can serveas a valuableindicationof manifestationsand processes
presentin subjectsundergoingradiation. The persistenceof a pattern related to predominanceof fatty acids indicatesthat the patient has passed
into an imbalancethat can only be increasedby further irradiation and, if
it becomessufficientlyintense.may even prove to be lethal, causing the
patient to die with symptomsof severeshock. [n contrast,a pattern correspondingto the predominanceof sterolscould be consideredas being consistentwith a preponderantreactive response,which would indicate that

R^DIATION

263

higher amountsof radiationcould be usedwithout dangcr.From a practical


point of view, the information given by the urinary surface tension has
appearedvery valuable.The moment when, and the amount of, irradiation
to be given can be determinedby these analyses.A low surface tension
would contraindicateadministrationof radiation while high values would
indicatethat radiation should be increased.
The administrationof lipids or lipid-like substanceswould representa
method of controlling undesirableprocessesand a-llowingmore effective
useof radiation. If the reactiveinterventionof adrenalsappearstoo strong,
a lipoid with negativecharacter could be added to counteract this and.
consequently,could increasethe desirableeffect of the radiation. Subjects
receivingfatty acidsor sulfur preparationsalong with radiation have shown
intensivelocal effectswith very small dosesof radiation. Epidermitis and
mucositiswere seenin such patientseven with dosesas low as 600 r. The
sameintensiveeffectcould be seenin the tumors.The useof lipoids appears
indicatedwhen an intensiveeftect through fatty acids is sought,as in lymphatic tumors. On the other hand, if the effectof fatry acids is higher than
can be accepted,and representsa handicapfor the desiredeffect on proteins, then adrenal hormonesor other anti-fatty acids must be added. By
reducing the effect of abnormal fatty acids, it becomespossibleto obtain
a more intense impact on proteins and, at the same time, to avoid the
otherwiseinherent undesiredside effects.The choice of the anti-fatty-acid
agent must be guided by the level at which the effectsof the abnormal
fatty acids would make themselvesfelt. While corticoids act especially
upon systemic and organic levels, sterols and other positive lipoids act
upon the lower levels.Butanol and similar agentsare effectiveupon local
changes,such as pain.
The guidanceof radiationtherapy,as an exampleof how this new view
may be used to improve therapeuticapproaches,will be discussedlater.

C H A P T EI RI
P R O B L E M SI N C A N C E R

NT

I \ ew lNsrcHrs rNTo many pathologicalproblems-and those of cancer


in particular'-are oftered by the conceptswe have been discussing.Let us
take, for instance,the problem of just what cancer is. Classically,one is
entitled to speak of a condition as cancerous when cells with cancerous
character are present in the body. whether, on the one hand, only cancerin-situ cells arc identified or, on the other hand, the patient is dying and
has almost no organ or function left untouched-the condition is considered cancerous. Yet, so long as the concept of cancer is associatedimplicitly with the concept of malignancy, to consider clinically healthy
individuals to be cancerous only because of the presence of cells with
cancerousnuclear characters,when most of them will never show further
developmentof the disease,is entirely confusing.It is essentialto separate
the two concepts,the presenceof cancerousentities and actual malignancy.
The fact that the hierarchic levcls of the organism participate in the
various manifestationsof cancer puts the problem in its true light. A cancerouscondition does not implicitly mean malignancywhen it involves only
the prcsenceof an entity with cancerouscharacter. Other attributes must
bo considered.In the hierarchic progressionof cancer, malignancy begins
to be manifestedwhen the cellular level participatesand inducesinvasive
canoer. With malignancy an attribute of only some of its phases,cancer
can be seen to embrace many changes,beginning with those of the lowest
hierarchicentitiesand terminatingwith the systemiclethal condition.
The plurality of phasesof cancer,with the broad variationsin time and
other factors which de0erminethe passagefrom one phase to the other,
logically raises several immediate questions.
In view of the multiple phases,one cannot speak of pathogenesisof
of the different phases.Concancerin general,but rather of pathogenesis
264

PROBLEMS tN

CANCER

265

sequently,even postulatingthe existenceof somespecificoriginal cancerous


change,such a change would not, alone, induce the entire diseaseand
determine the passagethrough successivephases. Different pathogenic
factors must be consideredto intervenein order to have cancer passfrom
one phase into another. The evolution of the cancerouscondition has to
be relatedto thesedifferentfactors,someof them possiblymore important
than the original specificchange.The passageof a cancr from the noninvasiveto invasivephase,or from tissueto systemic,is surely more important than the appearanceof a low level cancerousentity. An original
change at a lower level appears,in fact, to be of very little importance, not
only becauseof its ubiquity but also becauseit is not implicitly related to
malignancy.From this point of view, then, cancerno longer can be defined
as some singlespecificchangein a cell, nucleus,chromosome,geneor other
biological entity.
has to be conceivedof in an entirely new way, in terms
Carcinogenesis
of plural factors and their relative values.Acceptingthe phasesabove invasive cancer as the only ones which correspondto clinical malignancy,
they have to be regardedas the end result of a seriesof cancerouschanges
developedat progressivelevels,with the interventionof many factors, not
just one.
Diagnostic Tests
This view puts the problem of diagnosisof cancer in a new light. The
recognitionof a cancerouscondition by itself, althoughimportant, has little
clinical meaning.The presenceof "cells with cancerouscharacteristics"in
the prostate of almost all men 40 years of age and older, and in the
thyroid, lung and stomach in a high proportion of the population, has
fai.ledto produce a general feeling of despaironly becausesuch findings
are commonplace.While they still mean cancer,they do not implicitly indicate malignantdisease.It appearsvery clear that a diagnosisof cancer
is incomplete without immediate qualificationas to its phase. We can no
longer speakof cancerwith any degreeof practicalmeaningunlesswe add
a descriptive adjective-noninvasive, invasive, tissular, organic or systemic.
And the searchfor a test to detectcancerwill have no meaningas long
as we have not definedin advancethe informationwe want. A test, biochemicalor immunological,which indicatesthe existenceeven of a specific
anomaly in the noninvasivephase or before, while interesting,wiU have
little significancesincethis anomalyexistsin so many subjectsand for most
doesnot go beyond the noninvasivephase.The test will not indicatemalig-

266

xesEARcH rN PHYsToPATHoLocY

nant cancer in the clinically frightening sense.On the other hand, the
processeswhich are addedto a noninvasivephaseform of cancr and turn
it into the invasive,tissue,organicor systemicphase,have no characterof
specificity. Similar growth changes,or the appearanceof lipidic predominance, which representadded factors are seen in many other conditions.
By using them for diagnostic purposeswe will not recognizn,the cancerous
condition but only nonspecificinterveningfactors. While these factors are
responsiblefor the changes,malignancydevelopsonly when, and because,
thesefactors operateon already abnormalentities,i.e., cancerousentities.
This explainsthe nonspecificityof many proposedtestsand the misleading
positive results obtained in conditions such as pregnancy where one of
theseadded factors, (active growth processes)is always present.
A test for cancer, to have clinical value, would have to indicate two
things: one, the specificearly changewhich is widely distributedbut representsthe cssentialcondition for the potential developmentof malignancy;
and, two, the concomitantprcsenceand concomitantoperationof the nonspecific factors which can cause the actual developmentof malignancy.
This kind of diagnostictest undoubtedlywill come from further systematized study of biochemicalchangesinducedby the simultaneousaction of
the two groups of factors.
Immunological studies reprcsentan approach of value for diagnosis.
The different phasesof cancer can be interpreted,in the final analysis,to
correspondlargely to the interventionof the defensemechanismat different stagesat the different levels.As mentionedabove, a changein a phase
resultsalso from a changein the defensestageat the respectivelevel. We
have seenthat the immunologicalaspectof cancer cannot be understood
without acceptinga relative independenceof the levels in their different
stagesof defense.This view explains somc seeminglyparadoxicaloccurrences.
Cancerouscells are frequently found circulating in the blood yet this
does not indicate generalizedcancer. While the organism defends itself
successfullyat thc systemiclevel againstcancerouscells, the cancer can
still progressat the lower level of the tissues.The loss at this Iow level of
an effectivedefense,principally primary or allergic,which is still persistent
at the systemiclevel, explainswhy the cancerouscells invade the tissues.
A test indicating the presenceor absenceof any immunologicalreaction
would consequentlyhave value only when related to hierarchic levels. It
must furnish indicationsonly of what is happeningat a specificlevel. The
nature of the immunologicalreaction in cancer is also different from the
reactionin other conditions.Defensecapacity-natural defensecapacity-

PROBLEMstN cANCER

267

at differentlevels is lost as the respectivelcvel participatesin the disease.


This is in distinction to the immunologicalpr(xessesin other conditions
in which the normal individual lacks specificimmune bodies.An immunologicaltest for cancerwould have to revealthe lossof a previouslyexisting
defensemechanismrather than the appearanceof an immunological response.This loss can be revealedin different ways. In one, the response
to a cancerousantigen is investigated,and its lack would indicate the
existenceof a cancerouscondition at this level.
In a study now in progress,we utilize pooled human tumoral tissuesas
antigen,and try to see if an allergicreactioncan be induced with it, in two
administrations,
sensitizingand trigger. Two intradermic injections at the
samesite are made 12 days apart. They induce an allergic reaction in
normal individuals, but are without eftect in patients with active malignancy.If the eftect is negative,a third injection is given l5 days later. A
similartest is made for the conjunctiva,with sensitizingand trigger instilIationsof a similar antigen.No allergic reactionindicatesa positive result,
whilea reaction is consideredto be normal.
Another test which we are studyingis basedon the same lack of efficientdefensemechanismat the tissuelevel. Such a lack of defensewould
permit a cancerousantigen to be present without the body offering a
sufrcientlyeffectivedefenseagainstit. The presenceof such an antigen in
the tissuesis revealedby inducing an allergic reaction, through the administrationof specificcoagulantantibodies.Scra of guinea pigs injected
with pooled human tumors and having a high precipitin content are used
in intradermicinjectionsor in eye instillation.An immediatereaction indicatesa positive result. As control, we use normal guinea pig sera. The
studiesare now in progressand the diagnosticvalue of thesetests will be
reportedin a later publication.
Circulating Cancer Cells and Surgery
These immunologicalconsiderationshave appearedimportant in consideringa problem related to the use of surgeryin cancer. The existence
of a veritable flow of cancerouscells in the general circulation, largely
inducedby the manipulationsinherent in operative procedures,has producedgrave doubts as to the value of the measurestaken by surgeonsto
preventlocal spread of cancerouscells through the surgical act itself.
Paradoxically,however, these precautionshave been followed by good
clinicalresults.Analysis from the point of view of the defensemechanism
involvedcan clearly explain this situation.In the invasivephase,the systemicdefensemechanismis stiU adequateto insure destructionof cancer-

268

RESE^RcH tN pHystop^THoLocy

ous cells which get into the blood. This is not true at the level of the
interstitial formations, that is, at the tissular level, where such defense
means are failing. The real danger during surgeryconsequentlyis not so
much the presenceof cancer cells in the blood, since the blood can still
take care of them, but the spreadof thesecells at the tissular level where
the defensecapacityhas beenlost. and where a cancerouscell consequently
has every chancenot only to remain alive but also to grow.
The independenceof the defensemechanismat different levels also
must be takeninto accountin explainingthe differences
in the eventswhich
follow the appearanceof a spontaneous
tumor and those which occur after
cxperimentaltumor transplantation.

EXPERIMENTAL

CARCINOGENESIS

The problem of carcinogenesis


apparsin a new light when cancer is consideredunder the conceptspresentedabove.Classically,the experimental
induction of cancer is judged successfulonly if the result is a tumor in
the invasivephase,that is, with abnormal cells invading normal surrounding tissues.This is consideredto correspondto a fundamental specific
changewhich transformsthe normal cells into cancerousones. The entire
diseaseis held to stem from the relationshipbetweenthese abnormal cells
and the organism. (29O, 291, 303) To these simple views of the abnormality, we have proposedanotherone.
In our view cancerrcpresents
a hierarchicallyorganizedcondition.Its
invasiveform is only one phasein a long sericsof changeswhich transforms
successive
hierarchicentiticsinto cancerousentities.Carcinogenesis,
thus.
is not simply a changeof a normal cell into a cancerousone but a step by
step progressivehierarchicdevelopment.A cell is cancerousonly if it has
a cancerousnucleusjust as a nucleusis cancerousonly if it is formed by
cancerouschromosomeswhich, in turn, are cancerousif they have cancerous genes.With the same reasoning,it is possibleto go far down in the
organization,below gcneseven to nucleo-proteinsor still lower to histones
or even alkalineamino acids,to find that the first changes,which can be
consideredto be specificfor cancer,take place at the bottom of the organization of the biological realm. In other words, a cell becomescancerous
after specificcancerouschangeshave occurred in all the hierarchicallyinferior entitiesthat composeit. Thus. a successfulexperimentallyinduced
cancer.i.e., one that is alreadyin the invasivephase,meansthat changes
would have affectedthe entire seriesof hierarchicentities,including the
cells,whoseparticipationresultsin the invasivecharacter.Seenunder this

i,ffi#l:*

PRoBLEMS rN c^NcER

269

aspect,carcinogenesis
no longer can be acccptedas a simple processoccurring in the cells, but must be regardedas a successionof organizcd
processes.
This becomesstill more interestingwhen it is realizedthat changesin
the constituentsat the lowest levels of the organizationcan occur on a
statisticalbasis,that is, independentlyof the direct interventionof external
agents.As thesechangeshave to be developedfor many successivehierarchic entities, it takes a certain time for them to be realized. This would
explain why most cancers appear after a certain age. Cells with cancerous
nuclei, i.e., ln the noninvasivephase,frequentlyare present,in older people, in many organs without producingclinical manifestations.Conceptually, in order for an agent to be considered a successfulcarcinogen, it
must act upon these noninvasive entities to such an extent as to change
them into invasiveones. It can thus act upon entities which have already
progressed,by themselves,far enough in the hierarchicdevelopmentof a
cancerous processand have arrived at the noninvasive phase without any
manifestation.The excessivelength of time necessary,even for the most
active agents,to induce invasive cancer would suggest,however, that more
than a simple passagefrom an already existing noninvasive cancer into
an invasiveone is involved. A plurality of changesmust be induced,some
or all at levels below the cell.
We are inclined to favor this last hypothesiswhich obliges us to consider that a carcinogeninduceschangesat differentlevelsof the organization. It is supportedby a seriesof facts. In addition to having the capacity
to induce invasive tumors, carcinogenic agents also induce precancerous
lesions which correspondto cancerousentities below the invasive phase.
Cells with abnormal nuclei or with only abnormal chromosomesare almost
constantly seen in induced carcinogenesis.
Even agents which produce a
high proportion of invasive cancer consistentlyinduce such changes at
Iower levels as well. For carcinogenswhich induce a low proportion of
invasive cancers,the effects often appear to stop at Iower levels. Such
activity at subnuclearlevels of the organizationis seen in the capacity of
most of the carcinogensto induce mutationsand monstrosities.
In the conceptof hierarchicorganization,mutations are consideredto
result from changestaking place at the gene level, with lower levels left
unaffected.Monstrositiesresult from changesat the chromosomelevel.
with mutationsand monstrositieshas led us
Comparisonof carcinogenesis
to consider that cancerouschangesbegin at levels much below those involved in mutations and monstrosities,possiblyat the nucleo-proteinlevel
or. even below. The complex cancerouscondition to which the invasive

270 /

x z s E A R c Hr N p H y s r o p A T H o L o c y

form correspondscan thus be seento be the result of a seriesof anomalies


which have taken place at differentlevelsbelow the tissular.Carcinogenesis
at the invasivephaseis conditionedby the existenceof changesat all the
lower hierarchiclevels.while they can appearas the result of the development of the organization, conceivably these changes can be hastened or
even inducedby the carcinogens.
The concept of multiple changesin carcinogenesishas caused us to
searchfor multiple factors in carcinogensthemselves.The possibilitythat
such factors might be found was suggestedby the existenceof so+alled
co-carcinogenicagents.These are substances
without carcinogenicactivity
of their own but capableof inducingsuch activity in casesin which some
carcinogensare administeredin dosestoo small to induce invasivecancers
by themselves.This peculiar intervention of co-carcinogenscan be explainedthrough the multiple factors in carcinogenesis.
It can be conceivedthat the factorspresentin a carcinogendo not have
equal activity. The differencesappear evident when the carcinogenis administeredin very sma.llamounts.While some factors still have sufficient
potency in these small amounts to accomplishtheir part in the complex
processof carcinogenesis,
others are quantitativelyinsufficientand do not
induce changes.The total eftect is an incompleteset of changes.Under
thesecircumstancesthe addition of a co-carcinogencan replacethe action
of the quantitativelyinadequatefactors,and consequentlycomplete the
plural action necessaryto produce an invasivecancer. Becauseany one
co-carcinogencan replaceonly certain factors, co-carcinogenactivity has
a certain specificity.
With the hypothesisof multiple actions in the same carcinogen,the
next step was to try to recognizethem. A study, identifyingdifferent active
energeticcenters in the structuresof carcinogens,has substantiatedthe
hypothesis.
We attempted,as a first step to systematizethe analysisof such energetic centersin carcinogens.A short resumeof this study is presentedhere.
Energetic Factors
A well known and generallyacceptedconcept tries to correlate carcinogenicactivity with the presenceof one energeticfactor. identifiedas a
"condensationof electrons,"at certain regionsof a moleculeand revealed
by the physicomathematicalapproach offered by Pulman and Dawdel.
were started
Studiesof the role of electrondistributionin carcinogenesis
by Otto Schmidt (43), which showedthat an electrondensityexceeding
0.44e/* in the meso region of the moleculeappearsnecessaryto confer

P R O B L E M Sr N C A N C E R

271

carcinogenicproperties.This conceptwas partially modified and amplified by Pulman,Dawdeland their co-workers(44) who have shown,by
quantumanalysisof variouscarcinogens,
that the densityof the a electrons
is increasedin certain preferredregionsof the molecules,the K regions.
They showedthat, whenelectrondensities
exceed1.292eat theseregions,
the substances
have carcinogenic
properties.Figure 95 showssuch a K
region.

o)

i-=
\z

\,2

CHs

9:l0 Dimethyl
| :2:7:8 Dibenzanthracene
Flc. 95. The regions K in carcinogenicmolecules.

From our point of view, a tentativelyinterestingaspctof this condensation of rr electronslies in two facts: the presencein some carcinogen
moleculesof more than one such K region, and the presenceof different
valuesfor theseK regionsin differentmolecules.It would be the presence
of more than one K region in the same molecule which would result in
interventionin more than one processand thus contributeto plural activity.
Further analyses,however,suggestedthat the condensationof z electrons in K regions would represent only one of the factors that would
induce activity in theseagents.We have identifiedanother energeticfactor
in the presenceof two atoms having the same electrical sign and being
bound togetherwithin the molecule.
Twin Formation
We have considered the existenceand importance of these "twin formations" as indicationsof energeticactivity in the course of studies on
electronicmoleculararrangements.In a molecule,an alternationof successiveatomsresultsin part from the alternatingpolarity of theseatomswithin
a molecule and in part from the opposite charactersconferred upon the

272

R E S E A R c Hr N p H y s r o p A T H o L o c y

two carbon atoms when they form acetic acid, an important precursor in
biologicalsyntheses.It is through alternatepolarity that an induction effect
of an energeticcenter in thc molecule propagatesitself along the chain.
The presenceof any energeticcenter in the moleculerepresentedby polar
groupsor a lateral chain, for instance,will enhancethis alternalepolarity.
When one or more such inductiveetTectsare propagatedthrough the chain,
two adjacentatoms may be found to possessthe same electrical sign for
their chargeor ionoid character.The twin formation which results representsa center of increasedmolccular reactivity. This reactivity can be so
intense as to lead to breaking down of the molecule, something which
occurs often in inorganic substances.
This has led Pauling to believe that
this condition,called "adjacentchargeru|e," cannot exist.
"Pauling has pointed out that the murual potential enerry of two electrical chargesof the samesign is so high that a canonical structurehaving
net residualchargesof the same sign on any adjacent atoms would have
too high an energy level to contribute appreciably to the real molecular
structure." So notes William A. Waters in "Physical Aspects of Organic
C h e m i s t r y . "( 4 5 )
The form suggestedfor nitrogenperoxide (N.On), (Fig. 96l would
appear to be impossiblebecauseof the high energy developedat the two
positive nitrogens.

NitrogenPeroxide
Frc. 96. The existencc of nitrogen peroxide molecule is prevented by the high energy developed at the two adjacent positive nitrogens.

However, the forces that exist in most of the organic molecules are
much weaker, so that the resulting"twin formations," although energetically potent. arc not strong enough to induce the breaking down of the
molecule.Consequently,they would exist and rePresentimportant energetic
centers.

P R O B L E M Sr N C A N C E R

273

we have studied a number of carcinogenicagents,seekingtwin formations. Analysisof the ionoid characterof the carbonsof the methylcholanthrene molecule revealsthe presenceof twin formations which could be
localizedat various points of the molecule.Figure 97 shows the energetic
aspectof methylcholanthrene
and the ionoid characterof its carbons.It is
the presenceof the cyclopentanegroup in the molecule that induces the
samesign in two adjacentcarbons.The presenceof the methyl group would
determine the electrical character of C2e and consequentlythe srccession
of alternate signs. On the other hand, the double bonds will determine the
probable localization of these twin formations in the molecule at the K
formation itself, that is, at C5 and C6.

Hsc

1)

20-Me
t hyI choI anthrene
Ftc. 97. The energeticaspectof methylcholanthrene.with twin formations.

Twin formations can be found in many carcinogens.It must be emphasized,however,that unequal energeticvaluescan be recognizedeasily
for difterent twin formations and would explain differencesin their activity, a fact which would confer possible plural properties upon this group
of qualitativelysimilar energeticformations.
Another aspect of the relationship between these formations and carcinogenesisappearsto be even more interesting.While no twin formations
can be found in severalagents,the formations are presentin the substances
resultingfrom metabolism of these agentsin the body. The relationship of
twin formation to carcinogenicactivity can be suspectedwhen such changes
appear simultaneouslywith carcinogenicity.
For example,no twin formation occurs in 2-naphthylamine,(Fis. 98)
whosedirect carcinogenicityis questioned.but such a formation appearsin
heterocyclic3:4:5:6 dibenzcarbazole,
one of its intermediates(46), which
is known for its carcinogenicproperties.(Fig. 98bis) This is also true for

274

nESEARcH rN pHysropATHoLooy

NHz

(a)

2-Napht
ylamine
Ftc. 98. No twin formations exist in 2-naphthylamine.

aminofluorcne,which is also relatedto 2-naphthylamine.(Fig. 99) The


existenceof a twin positive carbon group or a twin negativein the same
molecule can further explain the diversity of the tumors produced by this
carcinogenand its acetyl derivative,which has the s:uneenergeticpicture.
( 4 7 , 4 9 , 4 9 ,5 0 , 5 1 , 5 2 )

(b)

3:4:5:6 Dibenzcarbazole
F r c . g E b i s .A t w i n f o r m a t i o n a p p e a r si n t h e i n t e r m e d i a t e3 : 4 : 5 : 6 d i b e n z c a r b a z o l e .

Twin carbons can be correlated with the degree of carcinogenicity of


the sulfur isosters(53) in each of which a thiophenenucleusreplacesthe
This also appliesto the
benzoneringof 9:10 dimethyll:2 benzanthracene.
:rzo compounds with twin formation at the level of the azo bond. Figure
100 showsthe presenceof a twin nitrogen at the level of the azo bond, due
to the influence exerted by the symmetric rings.

PROBLEMS IN

CANCER

275

NHz

Z-Aninofluorene
F l c . 9 9 . A t w i n c a r b o n g r o u p i s p r e s e n ti n a m i n o f l u o r e n e .

Furthermore,it is the relationshipof twin formation to carcinogenicity


which indicatesthe need for consideringthe metabolismof various carcinogensin the organism.
Dimethylamino-azobenzene,
butter yellow, which has a twin formation and is an active carcinogen,can becomestill more active through
the metabolicchangcsoccurringin the body which lead to productswith
trvin carbons.The sanrc2:2'-azonaphthalene,
with a twin formation, becomes more active becauseof its transformationinto amines passing
throughhydrasecontpounds.2:2'-diamino-l: l'dinaphthyl, with twin car(-t

(-)

cHg

N: l'l

cHg
4

Dimethylamino
azobenzene

F l c . 1 0 0 . A t w i n f o r m a t i o n i s p r e s e n ti n 4 - d i m e t h y l a m i n o - a z o b e n z eantet h e l e v e l o f
the azo bond.

bon formation, is more active than the precursor,2:2 Azonaphthalene.


( 5 4 ) , ( F i s s .l 0 l a n d 1 0 2 )
It is possiblethat benzidinerearrangements
of the hydrazo dcrivative
determinetwin formation and thus explain its carcinogeneity.
The similarity in kinds of tumors produced by the derivativesof 4(Fig. 103), and the aminofluorene
aminostilbene
derivatives(55), makes
us think that twin formations can appear in this case through changes
occurringin the organism.

ti.,v:.Si:"Sffi&Sl

276

n E S E A R c Ht N p H y s r o p A T H o L o c y

<\
N

(+)

(+)

2:2' Azonaphthalene
Frc. l0l . 2:2' Azotaphtbalenehas only a slight activity.

Some artificial estrogensof high potency (56) diethylstilbestroland


triphenylethylenic
acid, ( 57), (Figs. 104 and 105) are known to have carcinogenicactivity. Whilc a twin carbon is present in both, such a formation is assumedto appearmore activein the latter,as the result of metabolic changesin the body.

2:2'
l: I'

Di a mni o
dinaohthyl

F r c . 1 0 2 . T h e p a s s a g eo f . 2 : 2 ' a z o n a p h t b a l e n ci n t o t h e a c t i v e 2 : 2 ' - d i a m i n o l : l ' - d i n a p h t h y l r e s u l t s i n t h e a p p e a r a n c eo f a n a c t i v e t w i n c a r b o n f o r m a t i o n d u e t o t h e


influencc excrted by the amino-group.

An interestingaspectis furnishedby urethaneand other estersof carbamic acid.Figure 106 showsthat no twin formationscan be seendirectly
or through a changein the molecule.This accordswith these substances'
lack of capacity,noted by many authors,to induce cancerouslesionsor
e v e nt u m o r s .( 5 8 , 5 9 , 6 0 ) O n ( 6 1 ) r e l a t e sl e s i o n sp r o d u c e db y c a r b a m i c

PROBLEMS tN

CANCER

277

acid esten to chronic inflammations,noting their regressionwhen treatment is discontinued,(Note l1


From analysesof the substancesable to induce invasivecancers,it can
be observed that many present a twin carbon or nitrogen formation, usually activatedby the inductionexertedby a polar group or by double bonds.
Some of these substancesoriginally without twin formation become carcinogensonly when changesoccur in the body leadingto the appearance
of a twin formation.

/C",,,

\.r. ,*,

CH: CH

4-DimethyI aminosti I bene


FIc. 103. 4-Aminostilbenederivative.

It must be emphasized,however, that according to the concept of


plural factors in carcinogenesis,
twin formation does not appear to be an
obligatorycondition for carcinogenicactivity; other factors can produce
suchactivity.
It is interestingto note that in most carcinogens,especiallyin the hydrocarbons,the twin formation is electrophobicdue to its richnessin electrons.For the present,we wish to stressonly that in substances
considered
to be activelycarcinogenic,i.e., capableof inducing invasivecancer, twin

lott

D i e t h y l si ltb e s t r o l
F r c . 1 0 4 . A t w i n f o r m a t i o n e x i s l si n d i e t h v l s t i l b e s t r o l .

278 /

x E S E A R c Hr N p H y s r o p A T H o L o c y

Triphenyl-ethylene
F r c . 1 0 5 . T h e p o s i t i o no f t h e t w i n f o r m a t i o n i n t r i p h e n y l - e t h y l e n e .

formation appearsto be an added factor which insurescomplex activity.


Intervcntionof groups of two energeticcenterswith the same character,
in carcinogens,places in a speciallight a group of agents which, under
particularcircumstances,
inducetumors.One group with alkylatingactiviry.
is formed by the nitrogenmustards,diepoxides,polyethyleneaminesand
dimethanesulfonoxyalkanes.
One of the physicochemicalcharacteristics
of this group is the presenceof two electrophiliccentersnear enough to
eachother to permit joint action. Still more important seemsto be the fact
that, through changesin all thesesubstances,
new formations may appear
which energeticallycould be ultimately consideredsimilar to twin formations. Through this character,their activity could also be parallel to that
mentionedabove.
in the carcinogens
encountered

NHe-C0-OCzHs
Urethan
F r c . 1 0 6 . U r e t h a n h a s n o t w i n f o r m a t i o n a n d a p p a r e n t l y - a c c o r d i n gt o m a n y a u t h o r s
- n o d i r e c t c a r c i n o g e n i ca c t i v i t y .

Nitrogen Mustud Derivatives


of this group are
and betterstudiedsubstances
The most representative
by the 2-halocthylamine
thc nitrogenmustard derivativcs,characterized
(Fig.
group
107) attachedto a radicalwhich can be aliphaticor aromatic.
It seemsthat it is through hydrolysisthat the compound becomesbiolog-

PRoBLEMSrN CANCER

279

ically active,and Haddow has shown that activity is presentonly if hydrolysis is sufficientlyhigh. (62) The inequalityof hydrolysisin different
memberscan be related to the influenccexertedby the radical bound to
the nitrogen.It seemsthat the presenceof a strongercnergcticcenter,as
it appears in positively or negativelycharged atoms bound to the cyclic
radical, reduces the dissociationof the chloroethyl group. Generally.
nucleophilicgloups would retard the dissociation.
Sufficientevidenceexists
to show that biologicalactivity follows the elimination of the chloride ion
and the appearanceof a carbonium ion as a reactive intermediate.A
further passageinto the ethyleneimonium
ion, consideredmore stableand

R *a"'
'cH2

cHzcl
c+z ct

NitrogenMustard
F l c . 1 0 7 . T h e n i t r o g e nm u s t a r dd e r i v a t i v e s .

thereforelessreactive,seemsto completethe transformation.Figure 108


showsthesechanges.
The haloalkyl side chains in the moleculeappar indispensable
for
biologicalactivity. (63, 64,65, 66) Thcy lead to the immediateappearanceof two positiveelectrostaticenergeticcenters.This does not represent
a minimalcondition,accordingto Landingand co-workers.( 67, 68) These
investigators
have shown that in nitrogenmustards,cytotoxicityincreases
with the number of haloalkylside chains.It is to be noted that a double
electrophiliccenter is found not only in the two original haloalkyl side
chains,but also in the later product,the ethyleneimonium
ion. In view of
the more frequentappearanceof this ion also for other agents,the analysis
of the relationshipof this group to twin formation appearsinteresting.
group, while a negativechargecan be seenat
In the ethyleneimonium
the nitrogen,a positivechargeappearsto be presentbetweenthe two CH,,,
providing a certain polarity. With two carbons positively charged and in
a relativelyfixed position,this group is similar energetically
to a positivc
tn'in carbon group. A two-step change,with the imonium group in the
first, and a carbonium in the second,can explain, as we shall see below.
the strangebiological activity of the nitrogen mustardswhich have a carcinogenicactivityonly throughchangeswhich take place in the organism.

280

xEsEARcH rN pHysropATHoLocy

zCHz-CHz-CL

C H-sN <\ - C H z - C H z - C L
I

nCHz

*ct-

CHr-r'r.(CH.

\r*,

- c H z- c t

+Hz0

J
-.CHz-CHr-oH
cHs- N< - C H . - C H r - c L

, L H r - C H ro H
/

CHs- u "(-Cn.

\l ' C H r

+ Ct-

+ HrO
/.'CHz
C H s- N . _ _^ . .

- CHu - OH r+l

-CHr -CHr - 0H F)

Dielectrophilic

'Ihc
Fto. 108.
c h a n g e sc l c c u r r i n gi n t h e n i t r o g e nm u s t a r d l e a d s t o e t h y l e n e i m m o n i u m
i n w h i c h a n e n e r g e t i ca s p e c ts i m i l a r t o r h a t o f a t w i n f o r m a t i o n i s p r e s e n t .

This agreesstrongly with the nature of the more recently studiedrelatively


activecarcinogens,the ethyleneimines,
wheresimilar centersare seen.(Flg.
i,09) The biologicaleffectof the ethyleneimine
group has been considered
to be relatedto a reactiveintermediatc.
Generally, if sufficient influence is exerted by another center in the
molecule,the imine group becomesactive.This centercan be a nitro group
as in 2:4 dinitrophenyl-ethyleneimine,
or other ethyleneiminegroups as

P R O B L E M Sr N C A N C E R

2gl

in methyleneimine
l:3:5 triazine,(Fig. l,r0l Through the influenceexerted
by these centers,the ethyleneiminegroup can have its carbons charged
sufficientlyto bccome a dielectrophilicformation. The possibilityof a reactive intermediate and a more stable electrophilic form thus appears
common to the two groups,mustardsand ethyleneimines.

;>. a=-<-;
\z
N

H,cArr,
Triethylenimine
2-4-6-Triazine
Flo. 109. Ethyleneimines are active carcinogens,probably related to their energctic
aspectwith a formation energeticallysimilar to the twin formation.

Eporide Cucinogens
A similar condition is also found in the epoxide carcinogens.carcinogenic activity has been recognizedfor substanceshaving two epoxide centers in close proximity in the molecule. The epoxide center by itself can
leadto a formationsimilar to that of carboniumion, as seenin Figure 108
and thus to the same formation found in mustards and ethyleneimines.
The analogy goes still further. The energeticcenter appears insrrfficientto
accomplishbiologicalchangeswithout an inductiveactivation.In the caseof
epoxides, this is usually brought about by another similar epoxide group
in the samemolecule.
As no carcinogenicactivity has been found in substanceswith only one
epoxidecenter or with two epoxidecentersfar apart, the inductive centers
seemto be of primary importance.The two energeticcentersforming the
epoxide g'oup, similar to those of the ethyleneimines,do not alone appear

282

,/

xEsEARcH rN pHysropATHoLocy

sufficiently reactive to induce important changes.Only when enhanced by


reciprocal induction is their reactivity adequate to induce either the appearanceof a reactive intermediateor a sufficientcharge in the ethylene
carbonsto produce biologrcalactivity. Thesechangescan be measuredby
the reaction with thiosulfate ion and consequentlycan be related to the
reciprocalpositionsof the two epoxidecenters.
The biologrcalactivity of dimethanesulfonoxyalkanes
can also be related to a similar energeticformation. Such a formation appearswhen the

NOz

i)
N

H,cAcH,
2 : 4 D i n i tr o p h e n y l
Ethylenimine
F I c . l l 0 . T h r o u g h t h e i n f l u e n c ee x e r t e db y t h e n i t r o g r o u p u p o n t h e e t h y l e n e i m i n e ,
t h e i m i n e g r o u p o f 2 : 4 d i n i t r o - p h e n y l - e t h y l e n e i m i nbee, c o m e sd i a l e c r r o p h y l i c .

moleculeis metabolized,with the differencethat the two CHr in this instance seem to come originally from other chains. (Fig. I I I ) For the
it is possiblethat a similar processoccurs during the
methylolamides,
changesthat take placein the organism.
Some corroboration can be found in the fact that two forms can be
observedin theselast groupsof carcinogens.One is electrostaticallyactive;
that is, it has a certain ionic character.The secondhas a dual electrophilic
activity which can be relatedto a twin formation with nrolecularreactivity.
Thus, twin formation,with its specialreactivity,appearscommon in
many carcinogenicagents.To be biologicallyactive,the twin formation has
to be sufficientlystrong and this is insured by an induction effect exerted

PRoBLEMS

rN

CANCER

283

by other formationsin the molecule,such as double bonds in parallel position or polar groups.A twin formation as energeticcenter in the molecule
would exert a molecular field effcct. It would thus representa center of
molecularreactivity which has to be consideredas such in the analysisof
plural activity.

Ar.N{

- . C H z - C H z 0 S0 2 ' A r

RCH2+
t

\cHr c H 2- o s o 2 .A r

-0502' Ar
R C H 2 ++

Sulfonoxyalkane

-0302'

Ar

ReactiveIntermediate

F I c . t l l . C h a n g e so c c u r r i n g i n s u l f o n o x y a l k a n c sl e a d i n g t o t w o a c t i v e C H r c e n t e r s .

SynjugatedFormations
The study of variouscarcinogenshas permittedus to recognizeand
relate to complexcarcinogenicactivityanothercnergeticinfluenceexerted
by two or more doublebondswhen prcsentin a parallelreciprocalposition
in cyclic molecules.This led us to the conceptof "synjugatedformations"
with 2, 3, 4 or more suchparalleldoublebonds.
In studyingmethylcholanthrene,
one o[ the most potent of the known
carcinogenicagenLs,the curve of its absorptionin ultraviolet light was
considered.This curve is shown in Figure | 12. The place and form of
the peakscould be interpretedin a peculiarway when conjugateddouble
bond formationswere considered.In thc curve of methylcholanthrene,
we
portions
that corrcspondto an inverseof the curvesobcould recognizr
tained from variousconjugatedpolyenes.Furthermore,the curve obtained
through the spectralanalysisof methylcholanthrene
can be consideredto
have high similaritiesto the inverscof the curveof a mixtureof conjugated
polyenes.Figure I l3 showsthe spectralanalysisof conjugatedcod liver oil
f a t t y a c i d s ,w h i l eF i g . l l 4 s h o w st h e i n v e r s ec u r v eo f m i x t u r eo f c o n j u g a t e d
fatty acidsof cod liver oil in which conjugateddi-, tri-, tetra-,penta-and
hexaenesare identified.Figure I l5 shows the comparisonbetweenthe
and the inverseof the peaksof the mixture.
curve of methylcholanthrene
We were thus led to consider the conceptualinterpretationof these
curvesin termsof the specialrelationshipthat existsbetweendoublebonds
in the same molecule.In the classicalconcept,two double bonds are consideredconjugatedif two of their carbonsare joined by a singlebond. ln
the zig-zag representationof aliphatic molecules,the conjugated double

r,iti{

284

RESEARcH rN PHYSToPATHoLocY

bondsfnlfill this condition.(Frg. 1I6a) Applying this relationshipto cyclic


what wasconsideredto correspondto conjugation,accordingto
molecules,
this criterion,did not showpropertiessimilarto conjugatedaliphaticmembers.(Frg.II6b) This madeus consider,as the conditionfor the properties
presentin conjugated
formations,anothercharacter:the reciprocalparallelismbetweendoublebondspresentas they appearin the aliphaticmole-

c
o

I
c
L

Y a v eL e n g t h( n 1 )

Fto. I 12. An interpretation ol the spectral analysesol methylcholanthrene. Curve (a)


shows the spcctral analysisin ultra-violet of methylcholanthrenc.

cule. Two or more double bonds in a cyclic molecule would thus realize a
similar kind of energeticformation when parallel, and would do so independentlyof the number of the singlebondspresentin-between.(FiS. 1I6c)
For didactic purposes,we have applied the term "synjugated" to energetic
formations resulting from parallel double bonds separatedby more than
one single bond.

- , , r - : r . $ : : , :':+--"1ar.i:i:
{1$.
:1 .i"r: i..;itri. ,

I riilL

P R O B L f : M S I N

C A N C E R

285

c
cl

; $
tn

E
o
C
.E
L

*60
br

mpp))
f i a v e L eennqqt thh ( m
F t c . I 1 3 . T h e c u r v e s h o w st h e s p e c t r a a
l n a l y s i so f r h e m i x r u r e o f c o n j u g a t e df a t t y
a c i d sw i t h m e m b e r s h a v i n g f r o m 2 t o 6 d o u b l e b o n d s . a s o b t a i n e d b y t r e a t i n g c o d
liver oil fatty acids with KOH.

Thus, in the methylcholanthrene


molecule,there exist formationscomposedof two, threeand four paralleldoublebonds(Fig. I 17), which we call
di-, tri-, and tetraenicsynjugatedformations.It is logicarto assumethat
they are important in determining the energeticaspect of this molecule

286

R E S E A R c Hr N p H y s r o p A T H o L o c y

when the relationshipof its spectral analysisto the curve corresponding


to the inverseof conjugateddi-, tri- and tetraenescan be recognized.Frorn
the point of view of its relationshipto the plurality of factors determining
the carcinogenicityof a substance,the presenccof parallel double bonds,

c
ct
o
tt

E
tt
c
(!
L

a.a

tlave Lenoth (-r)


F t c . I 1 4 . T h i s c u r v e i s t h e i n v e r s eo f t h e c u r v e o f F i g . l l 3 .

and the synjugatedformationswhich they constitute,is interesting.Theoretically,eachone of thcsesynjugatedfornrationswould by itself represent


a reactivepossibility.Althoughqualitativelysimilar,they would showmanifest quantitativedifferences.It must be noted that, while they are not presthc'yare in most active,realizingdi-, tri-, tetra- and
cnt in all carcinogens.

PRoBLEMS rN CANCER

287

evenpenta-synjugated
formations.According to the concpt of plural activity in carcinogenesis,
synjugation,while not indispensablefor carcinogeneticactivity,would representone of the factorsthat can make it possible.
Togetherwith the condensationof the electronsin the K regionsand
".
the presenceof polar groups, the twin and synjugatedformations would

?to
o
(-

-c
c

,'
,

.I'

l\
, l

Eta

|
I

-c

269

I
L -

60

l ' .

J \

c
o

'

it
234

, ' I

'izo
c
(g
L
}Q

Joo

280

250

llaveLenoth('r) for Methylcnolanthrene


F r c . l l 5 . D i r e c t c o m p a r i s o n b e t w e n t h e c u r v e o f t h e s p e c t r a l a n a l y s i so f m e t h y l c h o l a n t h r e n ea n d t h e j n v e r s eo f t h e p e a k s c h a r a c r e r i s t i fco r t h e different conjugated
f a t t y a c i d sa s s e e ni n F i g s . l 1 3 a n d l 1 4 .

288

REsEARcH tN pHysropATHoLocy

confer high plural activity upon the molecules of active carcinogens.An


energeticspectrumof a carcinogencan be establishedin which thesefactors
can be presentedsystematically.
F i g u r e l l 8 s h o w s a s p e c t r u mf o r 9 : 1 0 D i m e t h y l I : 2 : l : 8 D i b e n z anthracene.
In the light of this analysis,it appearslogical to conceive that the carcinogenicity of a chemical compound is a result of many factors, and that
the great differencesin carcinogenicpropertiesof various agentsis the re-

a
Frc. l16. Conjugation and synjugation. ln the aliphatic chain (a) the presence of
single bonds between the double bonds induce the parallel position of double bonds.
It is this parallelism, which through thc reciprocal influence cxertcd, induccs thc
energctic characteristicsof the conjugated formations. Io the benzene molecule (b)
whcre thc double bonds, although separated by single bonds, arc not paraltel, thc
lack of this parallelism explains the lack of the properties charactcristic to thc conjugated formation. Tbe parallelism when present in cyclic molecules (c) realizcs thc
"synjugated" formations.

sult of difterences in their energetic spectra. The di.fterencesare consequently qualitative as well as quantitative. From this viewpoint, it is
possiblethat the great carcinogenicactivity recognizedfor some substances
would correspond to the presencein them at once of a great number of
energeticfactors.
The study of the correlationbetweenthe presenceof various energetic
centers and carcinogenesishas been facilitated by relating carcinogenic
changesto levels of organization.Taking place at different levels, the induced processes
can be seento correspondto an entire seriesof manifestations which, while present also in invasive cancer, often can be recognized

PRoBLEMSrN cANcER

289

Tetrasynjugat
ed

p;
HS
/

.r'

Trisynjugat
ed/----/-'- J

/'

Eisynjugat
ed

ion in 20-Methylcholanthrene
Synjugat
F I c . l l 7 . T h e p a r a l l e l p o s i t i o n o f t h e e x i s t i n gd o u b l e b o n d s i n m e t h y l c h o l a n t h r e n e
c o r r e s p o n d st o a b i - , t r i - , a n d t e t r a s y n j u g a t i o n .

Tl l n ai
.a--

\
-\

cHs

\ i
il-l
r ll
\ / l

{
I
f-\ I

,
t

.
/

t
l -

lt /

'l r l r

-'\
t

T w o K R e g i o n s+ .
T r oT r i n F o r m a t i o n s
[tneTrisynjugated
Bond
Tro Tetrasynjugated
Bonds

L'{r H :
/

Tetra

-V

Tetra

9 : 1 0D i m e t h y ll : 2 : 7 : 8 - D i b e n z a n t h r a c e n e
F r o . l l 8 . ' f h e e n c r g e t i cp i c t u r e o f 9 : 1 0 d i m e t h y l . l : 2 : 7 : 8 d i b e n z a n t h r a c e nseh, o w s
t h e p r e s e n c eo f t w o K r e g i o n s ,t w o t w i n n e g a t i v ef o r m a t i o n s ,o n e t r i s y n j u g a t e db o n d
and two tetrasynjugatcdbonds.

29O /

n E s E A R c Hr N p H y s r o p A T H o L o c y

in casesin which an invasivecanceris not induced.Followingthis view, it


can be expectedthat carcinogenesis
is the summationof a wholeseriesof
actionsinducedin the organism,someexogenous
and othersendogenous.
Consideration
of thepluralityof the factorswhichintervenein chemical
carcinogenesis
and makeit a complexprocessleadsus to considerviruses
in the etiologyand pathogenesis
of cancerin a similarlight.
VIRUSES AND CANCER
More than fifty years ago, Borrel presentedhis hypothesisof viral origin
of cancer basedprimarily on analogies,Sincethen, although an enormous
amount of material on this subjecthas accumulated,much of it has been
contradictory and it has appearedto be impossible to arrive at any clearcut conceptof the role of virusesin the pathogenesis
of tumors. However,
it seemedthat an attempt to correlatemost of the data with information
furnishedby the study of chemicalcarcinogenesis
in the light of the concept
of cancer as a complex hierarchicdiseasemight be of some value for an
initial simplificationof the problem.(293, 312) (Note 2)
Some theoreticalconsiderationshave helped in systematizingthe data
and in indicatingthe probablelimits of viral interventionin carcinogenesis.
Just as with chemicalcarcinogens,it could be assumedthat virus intervention may bring to bear multiple factors.An analysisof the processeswhich
occur under viral influenceindicatesthat this hypothesisis plausible.
Even more than chemical carcinogens,virusesare able to act only at
certain levels of organization,Their intracytoplasmaticand often intranuclear developmentconditionsthe interventionof theseagentsat theselevels.
Fundamentaldifferencesin carcinogeniceffectcould be expectedif vinrses
are able to influencethe subnuclearlevels,or the nucleus,or, on the other
hand if their activity is limited to the cytoplasm.This view has permitted
us to understandthe striking differencein influence exerted by various
viruseswhich, although recognizedby any worker in the field, has not been
the subjectof any specialconsideration.
Two Types ol Carcinogenic Eflects
The differencc lies in the time neededfor a virus to produce carcinogenic effects.Inoculation of fowls with purified Rous sarcoma virus, for
instance,has been seen to produce a clear<ut, immediate eftect. Changes
have been recognizcdwithin 48 hours at the site of inoculation.They take
place in the nuclei of fibrocytesand consist of swelling, appearanceof a
more distinctnuclearmembrane,clearednucleoplasm,marginationof chro-

PRoBLEMS

tN

CANCER

2gl

matin and one or more enlargednucleoli,In as few as onc or two days,


cytoplasmicchangesare also cvident.Thcre is manifestbasophiliawith
swellingof the cell which becomesgreatly enlarged.Concomitantwith
thesechanges,the abnormal cells invade the fibrillar tissue.The tumor
whichdevelopshasthe characterof the classicalspindlecell Rous sarcoma.
Thus a canceroustumor in the invasivestage,with typical nuclear and
cytoplasmiccell characters,is induccdin only a ferv days at the site of
inoculation.This is charactcristicof one type of viral carcinogenesis,
the
extremelyactiveone. Integratedin the conceptof complexcarcinogenesis
presentedabove, it would mean that the cntirc seriesof changes-from
thoseat the lowestlevel which determinethe cancerouscharacterto the
cytoplasmicchangeswhich producethe prolifcrative,invasives6ngs1-ha5
been achievedby the virus in this short time. ln fact, this tumor grows
rapidly, is palpable even at the f,fth day and fatal in rwo to three weeks.
Almost diametrica.lly
opposedto this type of carcinogcnesis
are tumors
which representanothertype of virusintervcntion,
suchascertainmammary
cancersin mice. viruses that producesuch tumors can be obtainedfrom
variousorgans,even from thoseof animalswithout apparenttumors.They
inducethe appearance
of tumorsbut only undervery characteristic
circumstances.Preferablyintroducedin the first days of life-subcutaneously,
intraperitoneallyor even orally-they will producetheir effect only aftcr
many monthsor even afterone or two years,as tumorsof a specificorgan,
such as the mammary gJand,for example.However,such tumors appear
almostonly in femaleswho havehad onc or more prcgnancies.
In this case,
the virus acts only upon highly differentiated
cells and acts independently
of the site of inoculation.The extremelylong period without manifestation,
the fact that the virus can be found to someextentin variousorganswhich
show no change,and the specificlocalizationin a highly differentiated
organ such as the mammarygJand,would indicatethat the carcinogenic
interventionof the virus is highly relatedto a specificcharacterof thesecells,
their parriculardifferentiation.This would place virus interventionat the
cytoplasmiclevel where differentiationoccurs.
Under this interpretation,the length of time necessaryfor tumor apparancewould be relatedto the time neededfor a natural evolutionof
the mammary cells to the point where they arc sufficientlydifferentiated.
It appearsprobablethat this lengthof time corresponds
to that neededby
abnormal hierarchicentitiesof the mammarycells to have arrived,independentlyof the virus, at a state correspondingto that of precanceror
noninvasivecancer.Interventionof the virus at the cytoplasmiclevcl would
then transform the relativelvadvancedbut still noninvasivecancer cells

292

xEsEARcH rN pHysropATHoLocy

into invasivecancer cells. Viruses would act, in this case, as cytoplasmic


carcinogens.
These two types representthe extremesof carcinogenesisin which
virusesplay a role. They help to rnterpretmany of the other data furnished
by experiments.For didactic purposes,we sha.llregard as "broad-scale"
viruses those which act from very low to high levels of the organism, and
as "cytoplasmatic"thosewhich act at the higher cellular level only.
some of the rapidly acting broad-scaleviruses will induce evolving
tumors in a much shorter time than any known chemical or physical carcinogen,a fact that can be interpretedas meaning that these viruses are
more capable of inducing not only the cytoplasmaticcarcinogenicchanges
but also the entire scale of preparatory changesleading to invasive cancer.
Viruses differ from the usua.lactive chemicalcarcinogensin their special
capacity to induce changeseasily at the cytoplasmiclevel where they are
particularly capable of multiplying and acting. This would contrast with
most chemicalagentswhich generallyhave low carcinogenicactivity at the
ceUularlevel. Chemicaland virus carcinogenic
activity would complement
each other. This is in accord with experimentsof Russianscientists,which
have shown that culturesof cells treatedwith methylcholanthrene
in vitro
becomehighly carcinogenicwhen inoculatedin animals if a cancerousvims
is also added.
This view of the activity of cytoplasmaticvirusesat the higher cellular
level, as contrastedwith many chemicalcarcinogensusually more active
at lower levels, appearsalso to be in agreementwith the experimentsof
Rous and Kidd (69, 70) which demonstratedthe capacity of coal tar
extractsto localize the Shope papilloma virus. The high cytotropic character of this virus is well known. It would easily act upon cells already
transformedfrom normai into noninvasiveform by chemicalagentswhich
are active at the lower levels. These chemical agentsthus "localize" the
vira-lactivity. This is in accord with the ability of chemical agentsin the
Rous and Kidd experimentsto increasethe percentageof invading carcinomas as compared to thc papillomaspresent. According to the view
presentedabove,the papillomaas a benigntumor would representchanges
similar to those seenin cancerbut limited exclusivelyto the higher levels,
without cancerousentitiesat the lowcr levels.The addition of an agent
with a broad-scaleof carcinogenicactivity, that is, acting also at the lower
levels,suchas the chemicalagent,would give the resultinglesionthe entire
canceroussca-le,
that is, the characterof nralignancy.
The integrationof viral carcinogenesis
in the concept of cancer as a
complexcondition and recognitionof the two extremetypes of viral car-

PRoBLEMS lN

cANcER

293

cinogenicactivity permits us to understandthc reservc of most authors


overthe viral etiologyof cancer.For instance,many have refusedto accept
as a carcinogenicfactor the virus shown by Bittner ( 7l ) to be presentin
maternalmilk and to influencethe appearanccof mammary carcinoma in
mice.The refusalis basedupon comparisonof this virus with that of the
first typ seen in fowl tumors. With the systematizationpresentedabove
and the concept of broad-scaleand cytoplasmaticcarcinogenicviruses,
this reason is not vaiid. Furthermore,a carcinogenicvirus should not be
consideredto be the indispensable
factor able to induce proliferativecancer
in animals which usually have a virus cancer.This would explain why, in
certainbreastcarcinomasin mice, a viral agentcould never be found. (72)
The specificcapacity of a virus to act upon a dilrerentiatedcytoplasm
explainsthe fact that a virus may be widely distributedamongorgansbut
does not induce tumors except in spccial cells. previous prepzuatory
changesseemnecessaryfor the cytoplasmaticvirus to intervene.This is in
accordwith a low incidenceof tumors in certain strainsof mice despitean
abundantpresenceof the "milk factor" virus. ( 73)
These facts shed a new light on the entire problem of the relationship
between viruses and tumors. Viruses can multiply in organismswithout
inducingcancer.The virus of mammarycarcinomain mice can be transmitted to femalesthrough spermatozoaand can be found in large amounts
throughoutthe organism.The virus is presenta long time before any cancerouslesionsare seenand is presentin organsthat will neverhave tumors
and even in animals that never develop cancer.The developmentof this
virus,like all viruses,takesplace in the cell cytoplasmwhich doesnot necessarilymean the induction of carcinogenesis
as long as other factors are
not present.No tumors appearas long as the cell has not undergonethe
prior changesrequired if the cytoplasmaticcarcinogeniceffect is to take
place.without the previouschanges,the virus will not influencethe cell
any more than many other noncarcinogenic
viruses.It is only in the presenceof an advancedcellularchangethat the virus will produccan invasive
cancer.
Plural Activiry
The capacityof a broad-scalevirus to inducean invasivetumor in a
short time through plural activity at different levels has been related to
its richnessin lipids. Its analysismakes us suspectthe presenceof several parts in the virus, each one able to act at a diflerent level, as in the
caseof activechemicalcarcinogens.
A similar plural influencecan be seen

294 /

B , E s E A R c Hr N p H y s r o p A T H o L o c y

exertedby virusesother than those with carcinogenicactivity. The study


of bacterialviruseshas shown the existenceof such plural parts. (Note I )
Luria (74) has shown, after irradiating a bacteriophagewith ultraviolet Iight, that if lytic activity can no longer be obtainedby the intervention of a singleone of thcseparticlcs,it can be inducedwith two or more
of them. They act as though severalparts, which usually are presentin the
virus but which were unequally inactivatedby the irradiation, would be
necessaryin order to induce the processof lysis. This agreeswith the experimentsof Debruck and Hershey (75), which have shown that new
typesof viruseswith new propertiescan be obtainedwhen units of the same
phagestrain or related strainsare mixcd together.The new propertiesare
combinations
of thoseof the mixedunirs.(298)
Similarchangesin thc plural constitutionof the viruseswould explain
other peculiaritiesobservedin bacterialphages.Bacteria can carry phages
for generations
beforeany lytic activityoccurs.The lysogenicstrains(76)
of bacteriaare examples.It is possiblethat a virus may undergotemporary
changesunder certain circumstances;this would explain the frequent impossibilityof finding a virus immediatelyafter it infects a bacterium, ild
the "disappearance"of somevirusesin animalsimmediatelyafter infection.
Since, in both cases,the virus is found later, a change which makes it
unable to act and therebybe dctectedis plausible.The "masked" virus
would be one with only some of its plural propcrties present.The possibility of recoveringa lost property was demonstratedin the Berry-Dietrich
phenomenon,when a heat-inactivatedmyxoma virus recoveredits lethal
capacityif inoculatedalong with a libroma virus.
This conceptof plural activity finds further applicationin the explanation of many phenomenaobservedin virusesin generaland in variations
in carcinogenesis.
The "self-sterilizationof the neuro-infections"described
by Levaditi has to be regardedrather as partial inactivationof the viruses
especiallyif the virusescan be reactivated.This occurrencemust be separated from caseswhere a total destructionof the virus can be supposedto
have taken place.
The lethal infection induced in mice injected intraperitoneally with
salivary gland virusesof certain strains is an example of the latter. The
presenceof thc inclusionbodiesin liver and other organs,and the total
inabilityto produce the diseasein other mice (77) can be interpretedas
a signof a destructionof thc virusesin the organism.The inclusionbodies
can be interpretedas resultingfrom an agglutinationof the virusesthemselvesas shown by Nicolau in herpes.(78) In other cases,such as protracted herpesinfectionin rabbits (79), or vaccinalinfection in rabbits

PROBLEMS lN

CANCER

295

(80), only partial inactivation can be consideredto occur since electrophoresis,repeatedpassage,or even dilution restorespathogenicity,The
restorativefactor can be of varied nature.Casesin which pathogenicityis
restoredby a nonviral agent-activation of the virus of swine influenzain
the presenceof Hemophilusinfluenza(81), for example-are most revealing.
Virus and the Host
This conceptof plural activity cxplainsthe rclation betweentumorgenesisand destructioninduced by viruses.Often "neoplastic" infection
and "destructive"infectionare inducedby the samevirus. (82)
The herpes virus thus induces necrotic lesions in the chick embryo
whenintroducedin early stages,but if the cmbryo is more developed,the
samevirus producesproliferativechanges.(83) The myxomavirus induces
more proliferative lesions if attentuatedthan does the unchangedvirus.
(84) Under specialcircumstances,
such as in oldcr animals,shecp pox
virus inducespapilloma insteadof pustular infection (85). It must be
remarkedthat thesedifferentresultsare not limited to viruses;they occur
with radiationor even with other infectiousagents.(86) Bartonellabacilliformis,which inducesoften-lcthaloroya fevcr, seemsto be the causeof
"verruga peruviana," a fibroangiomatoustumor often seen in subjects
recoveringfrom the acute disease.
The differencesin activity of thc same virus appear to be related to
the age of the host. Generally,youth of the host increasesthe virus' capacityfor acting at more levels.The virus can produce lethal destructive
disease
in young animalsbut only a neoplasticrcsponsein adults,as seen
for the fibroma virus in rabbits.Furthermorc,the neoplasticresponsealso
occursin young animalsbut only if a small amountof virus is inoculated,
or if an attenuatedvirus, such as a long-storedone, is used. (87) This is
clearin the caseof the Rous sarcomaand other chickcntumors.
When injected into very young animals. Rous sarcoma and other
chickentumor virusesproduce a hemorrhagiclesion (88) but they will
inducetumors in adult animals.The dcstructivccffect can bc repeated
with repeatedpassages
of the virus in very young animalsbut in adults
eachpassageproducesthe ncoplasticresponsc.This is also true for some
strainsof lymphomatosisvirus (89) which induce tumor formation in
adultsand necrotizingprocesses
in young animalsor embryos.It is also
true for the virus of neurolymphomatosis
(90), and of gliomas. (91)
Theseviruses,althoughselectivefor thc nervoussystem,induceinflammatory or neoplasticlesionsaccordingto the age of the infcctedanimal.

;1,,,,i1#ff$ii#ffiffi,($

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REsEARcH rN pHystopATHoLocy

In a generalway, it has beenpostulatedthat for viruses,as for bacteria,


the young animal represcntsa favorableterrain, while a certain resistance
is encounteredin the adult. waters and Bywaters (92) have shown that
the filtrable agentisolatedby Prickett and Belding (93) is not transmitted
spontaneouslyif the animal is older than 40 days. It is transmittedthrough
the eggs, although months elapse before there are manifestations.(94)
The problem cannot bc limitcd to the host, since,by passingthe virus
through young or old animalssomeof its propertiescan be changed.Gross
(95) has shown that a cell-freeextract obtainedfrom leukemicmice of the
AK strain, would induce the condition in c3H mice, provided the inoculation is given within a few hours after birth. The resultspublishedby Gross
and the inability of other authors ro reproducethem (96) could be explained by differencesin the virus strainsused. (97)
This was shown in the experimentof F. Duran-Reynals(98), in
which the Rous sarcoma virus undergoeschangesduring passagein the
adult chicken, which make it adaptableto another species,namely ducks.
The virus growing in young chicks seemedunable to induce the disease
when inoculatedin ducklingsor in older animals.Tumors obtained even
through cell suspensions,some of them very large tumors, could not be
transmittedfor more than one or two generations.However, filtrates of
tumors from older chickens,when injectedinto ducklingsno more than a
few days old, induced tumors which easily could be passedto young as
well as adult ducks. This changein the virus was strictly conditioned by
the age of the chicken; it occurred only if the animal was between three
and ten monthsold. If the animalwas more than 19-20 monthsold, injection of the filtrateswas alwaysunsuccessful,
and injection of the cells only
rarely induced tumors.
These changesin the virus arc explainedby mutation. Among various
resonanceforms which appear on a purely statisticalbasis, one different
from those previouslypredominantfinds favorableconditionsfor its development---+onditions that are not favorable for the predominant forms.
These experimentshave permittedus to further correlatethe intervention
of viruseswith the influenceexcrtedby severalchemicalfactors upon the
complextumor pathogenesis.
Virus and Lipoids
In studying the influenceexcrted by lipoids upon viral activity. we
could show that the presenceof free fatty acids, especiallypolyunsaturated,inducedchangesoppositeto thoseinducedby the anti-fatty acids.
In rabbits, administrationof various preparationsof fatty acids, espe-

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cially polyunsaturated,induced an unusual degreeof resistance.Animals


previouslygiven subcutaneousinjectionsof fatty acid preparationsshowed
a reduced generalresponseto chicken pox inoculation as compared with
controls, and practically no responsein the skin at the site of the fatty
acids injection. on the other hand, adminisrrationof insaponifiablelipid
fractions obtainedfrom tissuesof receptivespecieswas followed by manifest responseslocalized in the zone of injection, even in speciesotherwise
refractiveto viral infection. It is under this specialinfluenceof lipoids that
we have further investigatedthe interventionof the virusesin carcinogenesis.
cells vary in their content of lipids. we could see that richnessin
sterolsof a group of cellsincreascs
their receptivityro, and favorsthe development of, viruses in general,while richnessin fatty acids, especially
polyunsaturated,has an opposite effect. The local increasein a tissue's
richnessin sterolsmakes it more susceptiblcto the localizationand development of a virus, as is shown in thc followingexperimcnt.
In rabbits, intracutaneousor subcutaneous
injectionsof a colloidal
suspensionof cholesterol were made on epilated skin at several sites.
Twenty-fourhours later, the animal was injectedintravenouslywith suspension of smallpox vaccine.Characteristiclcsions were observedto develop at the sitesof the cholesterolinjections.
The generaleffectof sterolsupon receptivityto viruses,noted in many
experimentsin animals, was also recognizedin humans. The following
observationappearsinteresting.Mrs. D. R. had alwaysappearedrefractory
to smallpox vaccines.Until the age of 40, repeatedinoculationsproduced
constantlynegativeresponses.
She was treatedat that timc with a cholesterol preparationfor precordialpain, receivingdaily 2-3 cc. of a 2.5vo
solutionof cholesterolintramuscularly.
After threeweeksof this treatment,
shewas obligedto go abroad and it was nccessaryfor hcr to have the routine smallpoxvaccination.For the first time in her life, a characteristic
positiveresult was obtained.
The relationshipbetweensterolsand viruses,which would explainthe
affinityof most virusesfor the nervoussystcmand skin, sinccboth are of
exodermicorigin and particularlyrich in free sterols,would also explain
why young cellssimilarlyricher in sterolsare more susceptible
to viruses,
and the facility with which almostall virusesdevelopin embryos,such as
in chicken embryos.
Changesin richnessin lipids wereobservedundernaturalcircumstances
other than thoserelatedto age.Thus seasonal
changescould be noted,the
cold seasonleading to an increaseof fatty acids while the summer season

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brought an increaseof sterols,This would help to explain the seasonal


changesusua.llyobservedin naturally occurring viral infections.(99)
The epidemiologyof poliomyelitismay be related to the organism's
richness in sterols in the summer, particularly on hotter days. Seasonal
changeswere noted in naturally occurring tumors in which a viral etiology is seen. A certain resistanceappears in the fall and increasesin the
winter in the caseof leukosesand possiblyin other natural viral tumors.
( 100) This would explain the manifest seasonalchangesobservedby us
in the transplantedWalker tumor in rats or in grafted tumors in mice in
general,and in the induction of tumors through carcinogens.Similarly, the
induction of teratomas in testes through local admini5jlsllsn of zinc
chloride was noted to be influencedby the seasons.( l0l )
The influenccexertedby sterolswould explain the fact that virusesable
to act only at a higher level, as in the cytoplasm,tend to developin animal
cells abnormallyrich in sterols.It is highly probably that once it has penetrated, a virus will develop within a cell only under favorable conditions,
and these are hsured by the presenceof sterols. The virus will persist,
interferinglittle with the fate of the cell until other changesoccru at lower
levels.These othcr changestake many months or even years to be completed,and only then would the influenceof the virus be apparentthrough
its activation of the noninvasiveabnormal cell. Activation can occur regardlessof seasonalchangesin sterol richness.It seemssuperfluousto note
that this relationshipholds more for cytoplasmaticviruses than for those
with broad-scaleactivity.The latter are also more active in young animals.
changes in age of the host and other circumstancescan modify the
characterof viral carcinogenesis,
leading to rapid or very slow develop
ment, or even to completclack of rcsponse.This was often noted for Rous
sarcoma.In young animals,small amountsinducedrapidly growing tumors
with multiple hemorrhagicmetastases
rich in fi.ltrablevirus. In adult animals, despitethe largc amount of virus necessary,tumors took months to
appear,seldom metastasized,
and could be transmittedwith difficulty, or
not at all, by f,ltratesor evenby cells. (102)
The relationshipbetweenviral carcinogenesis
and lipids has been the
basisfor a group of experimcntsin which we tried to influencethe carcino.
genic activity of a virus by administrationof stcrols. Experimentsstill in
progress,using sterolsobtained from chicken embryos, seem to indicate
that lipids can strongly changeviral carcinogenicactivity. In general,they
inducean increascdresponseto viruses.
Many other peculiaritiesof the relationshipbetweenvirusesand carcinogenesishave been analyzedin terms of intervention of lipids as an

PROBLEMS IN

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intermediaryfactor. The capacityof a virus to induce tumors in different


organs-as seenfor leukemictissuecell-freeextractsin mice,which induce
peculiar salivarygland tumors ( 103), or tumors in the adrenalgland or in
the subcutaneoustissues (104)---can be explainedby certain peculiar
affinitiesof the virusesfor differentiatedtissues,possiblyrelatedto certain
specificlipids found in thesetissues.A similar affinity for the salivarygland
is seenfor rabies virus. There are also the affinitiesshown by the neuroand dermatotropicviruses,and by the neoplasticvirusesfor mammary
piland,lymphatictissue,ncuroglia,etc. while affinityfor the adrenalgland
could be related to its richnessin sterols,afffrnityto others sites could be
relatedto other lipids.
The plurality of localizationsof virusesalso can be relatedto affinities
of mutatedagentsfor differentcells.The experiments
of Gross(105), which
show the possibilityof separatingout of the samefiltrate the agentsresponsible for salivary gland tumors and for leukemic changes,would indicate
that a changein the virus must be also considered.However, the change
can be interpretedas a mutation and can be relatedto the influenceexerted
by lipids upon the virus. Treatmentof virus with lipids has shown the possibility of inducing changesin its behavior.Data showing the influence
upon tissularreceptivityof such changcswill appearin future publications.
with this concept of the role of virusesin the pathogenesis
of cancer,
it seemspossibleto explain other peculiaritiesthat have led to confusion
in this field.
It has been noted that viruses act as factors determining the change
to a cancerousentity which, once induced,can continueto developwithout
need of further interventionof the virus. This posesthe problem of the
relationshipbetweenviral carcinogenesis
and developmentof the virus in
the tumor itself. Even in a tumor, the multiplicationof the virus has to be
separatedfrom that of the $owth of the tumor. Although often interrelated,
they must be consideredas two different processes.The growth and even
direct transmissibilityof the tumor can continue,independentof the presence of the agentthat originallyinducedit. when tumors have been induced by a chemicalcarcinogen,they can be transmittedin continuous
generationsover many years, producing large tumors each time, a fact
which would precludeany possibledirect interventionof the agentin these
later tumon. Similarly,tumors once inducedby virus can be further transmitted by cells,the virus no longer being apparentin the tumors. A tumor
inducedby a virus often servesas a medium for the multiplication of the
virus.However,even while the tumor can continucto grow, it can become
an adversemedium for the further multiplicationof the virus.

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REsEARcH rN pHysropATHoLocy

This explainsthe peculiar facr that tumors induced by a virus can be


rich in or can lack an appreciableamount of virus, as often seen in a
s h o p e p a p i l l o m a( 1 0 6 ) , o r e v e n i n t u m o r s i n f o w l ( 1 0 7 ) , w h i c h p a s s
through periods when transmissionthrough cell-freefiltrates becomesimpossible,while transmission
throughthe transplantof tumor cells still continues, The virus multiplication capacitycan vary not only with the host
but with the virus itself, thus explainingthe changesnoted above.
The resultsof the interestingstudiesof Bryan, Galman and Maloney
(108) who have investigated
the relationshipbetweenrichnessin virus of
an induced Rous sarcomaand the percentageof positive resultsin induction easily can be interpretedunder this view. The chancesof inducing
tumors increasein casesin which the host is also a favorable medium for
the multiplication of the virus, and vice versa. This would explain why
theseauthorsfound little or no activevirus in casesin which the injected
material producedless than 5oo/o positiveresults,but casesoriginated by
a material that induced a large proportion of positive resultswere rich in
active virus. The capacity to multiply after inoculation in the host itself
thus increasesthe ability of the virus to act as a carcinogenicagent, which
seemslogical. The relative independence
of the two processes-the multiplicationof the virus and the inductionof [sp615-sppeared clearerin the
casesmentioned above, where the virus developsin the entire body of
mice, even in successive
generations,
without inducingtumors.
The presenceof virusesin the organism,even without inducingtumors.
helps to explain the rathcr puzzlingexperimentin which a transmission
through filtrates,consideredcharacteristic
for viruses,was seen to occur
for tumors inducedby chemicalcarcinogens.
Carrel has claimed to have
transmittedthrough filtrate passages
tumors induced by arsenic,tar preparationsand even indoles.Thesetumorswere of the Rous type obtainedin
fowl. More recently,similar tumors transmittedthrough filtrates were obs e r v e db y M c l n t o s ha n d S e l b i e( 1 0 9 ) , M a i s i n ,H a d d o na n d H a a g e n( l l 0 ) ,
and oberling and Guerin ( I I I ) after injection of methylcholanthrene.
especiallyin fowl, The considerationspresentedabove furnish a logical
explanationfor theseobservations.
A first factorto consideris the presence,
in animalsregardedas normal.
of a virus able to interveneto produce a neoplasticeftect under spe.cial
circumstances.
we have noted that such a virus can be presentwithout inducingtumors,Fowl appearto be especiallysusceptible
to viruses(l l2),
statisticsshowingthat viral lymphomatosesare responsiblefor SoVo of the
malignancies
in chickens.Even while some speciesdisplay an inborn reto viral infection,othersare higily receptive,as seenfor the viruses
sistance

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of sarcomas(l13) and lymphomatosis.


( I 14) viruseshavebeenfound in
as matryas l0%o and even20Vo of.chickens,accordingto somereports.

(2e4)

The number of animalswith virusesand no tumors must bc considered


still higher when presenceof viruses is revealed by antibodies.DuranReynals, in collaborationwith the East Lansing Agricultural Experiment
station (115), has shown that, while not one of 23 chickenskept isolated
and free of lymphomatosisshowedantibodiesin rhe blood, hundrcdsof
chickenstaken at random did have the antibodies.I his facr makesit highly
probablethat the presenceof thc virusesin the chickensused for carcinogenic studieswas independentof the adnrinistrationof the chemical carcinogen. Furthermore, the role of chemical agents acting alone as
carcinogenscan be discountedbccausctheir elfectsdiffer widely. In the
seriesof Mclntosh, the tumors appcarcdfar from the site of injection which
is very unusualfor methylcholanthrene.
Furthermore,thc agentsuscd by
Carrel,excepttar extracts,gencrallyhavelittlc or no carcinogenicactivity.
The two hypotheses----one,
that chemicalcarcinogensalone can induce
tiltrabletumors;the second,that this is only coincidenceand the tumor is
entirelyof viral origin----canbe rcconciledunder the conceptof plural intervention.Thus the chemicalcarcinogenwould induceonly part of the proccss; the remainder,at highcr levels,would result from viral intervention.
The changethat occurs in thc cells through the influenceof the chemical
carcinogencould also favor the changein the virus, making it not only
more activebut alsoo[ neoplasticcharacter.'l'he conccptof plural changes
neededto induce active carcinogenesis
permits us not only to integratethe
interventiono[ virusesin the conceptof carcinogencsis
in general,as presentedabove,but also to considerthis intervcntionin relation to other
[actors.
Hormones can play a part; they are neededto induce the degreeof
differentiation that is a condition for viral co-carcinogenicintervention.
Inoculated intraperitoncally,the Bittner milk factor, although active, will
rarely induce mammary tumors in virgin femalesalthoughthe virus can be
proved to be in the body. The hormonalchangesrelatedto pregnancyand
lactation influencethe mammary gland and cause a differentiation.Since
this diftercntiation representsa condition for viral neoptasticactivity in
these cases,hormonal interventioncan be integratedas an added factor
importantfor the viral carcinogenicactivity.Thc hormonal factor would
appearto be an indirect co-carcinogen,and it is under this aspectthat its
role in carcinogenesis
has to be studied.
The concept of plural co-carcinogenicinterventionpermits us not only

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n EsEARcH rN pHysropATHoLocy

to relatethe diftercntpathogenicfactorsinvolvedin carcinogenesis;


in addition, by relating these factors to various levels of organization,it allows
us to obtain new insight into the geneticfactor.

GENETICS AND CARCINOGENESIS


The geneticfactor in carcinogenesis
can be understoodin terms of phylogenetichierarchicdevelopment.Such developmentresultsfrom a seriesof
progressivechangeswhich lead to successive
hierarchiclevels of organization. As we have seen,for each level of the organizationa seriesof different solutionsare availablewhen a new hierarchicentity is to be realized.
This results first from the fact that various numbers of entities take part
in the constitutionof the principal parts. Since different constituentscan
form the secondaryparts of these new hierarchicentities,the number of
solutions is increased.The resulting solutions can be considered on a
statisticalbasis. Many of these new entities will die immediately; others
will subsistas such; still otherswill progess. Their fate resultslargely from
their interrelationshipand the conditions present in the environment in
which they find themselves.
The striking similarity to the resonanceprocessstudied in the lowest
lovelsof organization,such as atoms or molecules,has led us to consider
that changesat higher levels are of the same fundamentalnature. of all
the resonanceforms that occur at each of the levcls, there are some that,
on a statisticalbasis,persistand develop.Thesepersistingresonance
forms
make up the normal organism. The favored resonanceforms are determined by heredity and also by environmentalconditions.while the resonanceforms apPearon a statisticalbasis,the environmentalcondition can
vary and new resonanceforms will mark the intervention of external
factors. As a normal entity is composedof the pcrsistentresonantforms,
abnormalityoccurswhen suchan entity persists.The characteristics
of any
individual are provided by the resonanceforms which have developed
phylogeneticallyand also ontogenetically.These predominant forms are
"isotropic."For didacticpurposes,we callcdthe others"allotropic."
Allotropic ResonanceF orms
It must be acceptedthat, originally,it was the interventionof allotropic
resonanceforms which permittedthe appearanceof new forms able to respondwell to the environmental
changes.The phylogenetic
developmentof
different phylae, species,strains and even individuals, can be seen as
resultingfrom suchdifferentsolutionsfor the sameproblems.when, horv-

PRoBLEMS rN cANcER

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ever, an allotmpic resonanceform appears,during ontogeneticdevelop_


ment, it resultsin an anomaly, At the level of genesor chromosomes,
it
producesa mutation or monstrosity.At a still lower level, such allotropic
resonanceforms may rcsult, not in monstrositiesor mutations, but in
cancerousentities. The concept of cancer as a hierarchically organized
diseaseaccordswith this view of allotropic resonanceforms. A first cancerous entity would, therefore, develop when an allotropic resonanceform
occurs at a low level of the organization.Undcr favorable cond.itions,the
allotropic entity would develop hierarchically,passingon through the different levelsof the organization,and realizingallotropic chromosomes,nuclei. cells and tissues.
In order to have an invasivecancer, it would thus be necessarythat
an entire successionof favorableconditionsbe presentinsuring the development of a continuousline of hierarchiccancerousentities.Thesefavorable conditionscan occur spontaneouslyat each level, and both cancerous
and normal entitiesmay have many allotropicforms.Carcinogenesis
would
correspond to the creation o[ these favorable conditions, and, as seen
above, it can take place at the diffcrent levels of the organization.The
result is a hierarchic successionof the persistingforms of the allotropic
seriesof respectivecancerousentities.
The long time usually necessaryfor a cancerouscondition to appear
accordswith this mechanism.The interventionof any external factor consideredcapableof inducing, by itself, the developmentof cancerousallotropic forms, has to be regardedas favoring the conditions necessaryfor
the persistenceand developmentof the successionof allotropic cancerous
cntities.(304)
This would also explain the relationshipbetween hereditary factors
and carcinogenesis.
Just as the individualhas the capacityto realizethe
successiveallotropic forms, so a strain or even a speciescan inherit the
tcndencyto developsuch allotropicforms.This explainsthe persistence
of
mutationforms. It appliesto the devclopmento[ strainswith high or low
incidenceof spontaneoustumors. It would also explain the vast differences
among different speciesand strainsin their responseto carcinogens.
The conceptof cancer as correspondingto a seriesof allotropic resonanceforms at the successive
hierarchiclevelsis of importancein terms of
the intervention of external factors. Such environmentalinfluencescan
establishconditions favorable for the developmentof ailotropic forms at
successivelevels, permitting the progressionof the allotropic line. The
inequalityof their action at differentlevelsaccountsfor the big differences

304

x E S E A R C Hr N p H y s t o p A T H o L o c y

seen betweenthe various carcinogens.It is under this specialaspectthat


we saw above thc important problem of induced carcinogenesis.
The relationshipbetween carcinogenesisand the defensemechanism
can be understoodin terms of the differencesin defensecapacitiesof allotropic and normal entities.From the study of cancerousand normal subjects, it appearsthat the latter have the capacityfor defenseresponses.It
apPearshighly possiblethat the characteristicof the "normal" resonance
forms residesin their ability to resolvenoxious interventions.The allotropic forms lack this ability. The lossof the defensemechanismat various
levels,which is characteristicof cancer,can be regarded,up to a certain
point, as being the result of the interventionof allotropic resonancforms,
which would appear to be fundamentallyinadequateto oppose the hierarchic progressionof cancer.Incapableof respondingwith the full defense
mechanism when confronted by continuous noxious interventions,cancerousentitiesuse the simpler, primitive defenseforms, and especiallythe
lipidic prolongcd form. This lipidic predominancerepresentsthe principat
factor in producingthe actual manifestationsof cancer,with their dualistic
nature.
The interventionof noxious factors in carcinogenesisis well known.
Traumaand microbialviral infectionsin particularare suchfactors.Co-carcinogenssuch as croton oil, and somesolventssuch as benzeneand toluene.
can be considerednoxiousfactors. (295)
This view of a plurality of factors interveningtogether to realize the
hierarchicallycomplex conditionof cancerhas anothervalue. It helps to
reconcilevariouscxplanations
of the pathogcnesis
of cancer,eachattributed
to a different etiologicalfactor, and each basedon incontestableevidence.
Accordingto our view, with the possibleexceptionof the broad-scalevirus.
which leadsto the appearance
of cancercells in a couple of days. in all
other casesa number of factorsof differentnature intervene.To resonance
changes,would be added chemical,viral, metabolic,hormonal or defense
influences,at the sameor at differentlevels,in order to provide the necessary circumstancesfor cancerdevelopment,The fact that, regardlessof the
nature of agentsused to induce them, cancers,once induced, differ very
little or not at all makesplausiblethe hypothesisof plural exogenousfactors
actingto bring about the necessaryfavorableconditions.
The above presentation-a resumeof our 1sss21sh-muststill be consideredto be a working hypothesis.
Other aspectsof the cancer problem have been analyzedanew in the
light of the conceptof pathogenesis
discussed
above.

P R O B L E M S

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Lipids and Cucinogenesis


As we have seenabove,the recognitionof the fact that a seriesof successivechangestake place in carcinogenesis
has invalidatedthe concept
that in order to considcra substanceactivein this field, it has to induce
the entire seriesof changesby itself.includingthe passageinto the phase
of invasivecancer.An agentcan be consideredactiveif it inducesonly a
part of the successive
scriesof changcs.Didactically,we can thus consider
changesconcerningthe subnuclear,nuclear,cellular and metazoiclevels.
We have investigated
the interventicln
of lipids in carcinogenesis
from this
specificpoint of vicw.
The coordinationof datafrom variousobservations
and experiments
has
indicatedthat some of the lipids would act especiailyat the cellular and
tissuelevels.
Statisticaldata have indicatcda greaterproportion of cancer of the
cervix in non-Jewishwomen as comparedto Jewish women. This was
tentativelyrelated to the circumcisionof the respectivemales and this
correlationwas confirmedby statisticaldata concerningother groups of
populationpracticingcircumcision,such as the Moslems.The probable
role of smegmawas seenin experiments
in animalsmadeby differentworkers. It was reportedthat in mice, smegma,sterilizedor non-sterilized,
introducedin the vaginaof mice followedby the sutureof the vagina,would
inducepapilomatousand cancerousIesionsof the cervix,
Statisticalstudies(324) showeda similar correlationbetweencancer
of the prostateand circumcision,with a lower proportion of cancer in
circumcisedindividualsthan in those not circumcised.Entirely different
resultswere obtained by other workers. Studiesmade by the group of
Memorial Hospitalin Cleveland,concerningcanccr-in-situ,
showedno differencesbetweenJewishand non-Jewishwomen.Similarly,severalworkers
have reportedthat the prostatesin individualsovc'r 40 ycars of age, who
diedof conditionsotherthan canccr,havepresentin high proportioncancerin-situcells.No correlationwith circumcision
could bc found in thesecases,
the same proportionbeing found in all the ethnicgroupsexamined.(325)
We tried to interpretthesetotally discordantconclusionsof the two
groupsof statistics
and found thc explanationin thc conceptof plural interIn the lirst group of statisticsin the casesof both
ventionof carcinogens.
femalesand males,the cancerousprocesses
considercdwere those in the
invasivecellular stage,while in the secondgroup of statistics,the noninvasivecancerphasewas considered.It appearsthus quite clear that the
interventionof the circumcisionand respectively
of the smegmaexistsbut

306

xESEARcH rN pHysropATHoLocy

has to be placedat a precisepoint in the progressive


changesin the evolution of the cancerouscondition,at the passagefrom noninvasiveinto invasivecancer.without any influenceupon the appearancc
of the cancer-insitu,the smegmawould act manifestlyby changingthe proportionof active
cancerpresent.Its influenceappearsthusto be exertedat the cellularlevel,
where the occurring changesresult in the passageof the noninvasiveinto
the invasivecancer,and at the tissuelcvcl wherethe lossof the capacityto
defend itself againstthe cancercells permits their invasion.This led us
to the hypothesisthat the cancerousprocessin the cervix and prostate,
evolvesindependently
of circumcisionuntil the cancer-in-situ
step,but will
make the next step toward invasivecancerpredominantlyunder the influenceof the smegmaof noncircumcisedmales.Without such intervention
at this stage,sucha changemay occur but in an impressively
lower proportion. The invasivecancerof the cervix is thus almostentirelynon-existent
in virgin nuns. This consideration
and the richnessof smegmain positive
lipids has permittedus to go farther and considerthe problem of the role
playedby thesclipids in carcinogenesis.
We have tried for a long time, but with litle success,
to inducecancer
in animalsthroughthe administration
of unsaponifiablc
fractionsalone.The
several positive cases obtained in mice with repeated injections of the
unsaponifiable
fractionsof placenta,chickencmbryos,eggsor butter,have
not appearedsufficientlyconsistentto warrantany conclusions.
In our attemptto investigatcan interventionof the positivelipids for
the specificchange,corresponding
to the passageof the noninvasiveto the
invasivecancer, we carried out the following experiment.we selected
femalemice with at leasttwo previouspregnancies.
After a third pregnancy,
when in lactation,the mammaryglandwas injectcdwith small amountsof
fractions.The resultsseemto indicate
the abovementionedunsaponifiable
a higherproportionof mammarycarcinomain thcscmice than in controls.
Similarly,we have tried to influencecellsof the cervix in mice. through
the introductionof the samedifferentpreparationsof unsaponifiable
fractions in the vagina of ex-breedermice, followed by suture of the vagina.
The first resultshave shown a high proportionof malignanttumors. The
has led us
conceptof the interventionof abnormallipids in carcinogenesis
to utilize in similar expcriments,insteadof the above preparations,unto be heterogenized
by beingheatedabove
fractionsconsidered
saponifiable
which
are in progressseem to
320"C. At the moment,theseexperiments
indicatethe existenceof such an influence.
It is of even greaterinterestto note the influenceexertedby the unfractionsupon animalsthat had receivedurethane,according
saponifiable

PRoBLEMS tN C^NcER

3O7

to the experimentsof Berenblum.By combiningthree factors, urethane


for the first changesin the amino-acids,
severalpregnancies
for the changes
until the noninvasivephaseand unsaponifiable
fractionsespeciallyheterogenized for the passageinto the invasivephase,a high proportion of invasive mammary and ccrvical canccrssecrnsto bc induced in the first
experiments.
Of importanceappearsthe factor that a certainlapseof time
is necessarybetween each two factors which are applied in the above
mentionedsuccession.
This concordswith the conceptof plural successive
changesin carcinogenesis
discussedabove.
CarcinogenicActivity ol Urethane
The interestingresearchof Berenblumhas brought an importantcontribution not only for the largelydebatedrole of urethaneas carcinogen,
but also for the problem of carcinogenesis
in general.The fact that croton
oil, applied to the skin, inducesthe appearanceof malignanttumors in
animalspreviouslyfed with urethane,concordslargely with the conceptof
plural changestaking place in carcinogenesis.
The ana.lysis
of the influence
exertedby carbamicacid upon amino-acidswould place the interventionof
tbis agentat the first membersof the biologicalrealm. It can thus be seen
that the bond betweenthe amino-acidgroup and the carboxyl and amine
Sroupsof carbamicacid occur in a way similar to that which occursbetween
two amino-acidswith the big differencethat in the first caseit would result
in tbe appearanceof the CNCN formation. (Fig. 201) As mentioned
above,this CNCN formation representsthe group which characterizesthe
first biologicalentity. The placeof this CNCN group, not at the end of the
moleculeopposedto the carboxyl as in the alkalineamino-acids,but as correspondingto the bond which rcsultsin polymers,representsthe anomaly,
which accordingto the work hypothesis
we advance,would correspondto
-[he
the first cancerousentity.
fact that thc specific activity of urethane
takes place at the lowest levelsof organization,explainsthe necessitythat
a certaintime separatesits interventionfrom that of croton oil, which would
act only at the higher levels, probably inducing the passagefrom noninvasive to invasive phase. This time is necessaryfor the first cancerous
changesto build up the seriesof canceroushierarchicentitiessince the
cocarcinogen,
croton oil, would act only in thosemore evolvedcancerous
entities.In experimentsin progress,the passageof the urethane-induced
noninvasive
cancerousentitiesinto invasivecancer,is successfully
obtained
by treatmentwith preparationsof unsaponifiable
fractionsof placentaor
eggs.

,,,giw'*ffi,

308

R E S F - A R c Hr N p H y s r o p A T H o L o o y

INTERVENTION OF PSYCHOLOGICAL FACTORS


IN CANCER PATHOGENESIS
The role of psychologicalfactors in the pathogenesisof cancer, although
still obscure,has beenof increasinginterestin recentyears.Various theoretical considerations(i.e. the relationshipbetweenthe known eftect of
emotionsupon hormonal and biochemicalbalancesand the possibleeftect,
in turn, of the latter upon neoplasrn^s),
as well as a number of clinical reports and experimentaland statisticalstudies,point toward psychological
influencein cancerpathogenesis,
but little is actuallyknown in this area.
In order to explore this matter further, a researchprogram has been
carriedon in our Instirutesince 1952 by Dr. L. LeShan.This study has
includcd the evaluationof projectivepersonalitytests given to over 300
c a n c e rp a t i e n t s i;n t c r v i e r vos f 2 t o 8 h o u r s e a c h w i t h o v e r 1 5 0 p a t i e n t s :
and extensivecxplorarorypsychorhcrapy
(of from 60 to 400 hours) for 25
patients.Control groups werc also included in sach category.For patients
undergoingpsychotherapy,regular comparisonswere made between the
personalitypicturcand variousbiochemicalactivitiesreflectedin blood and
urine analyses.
A "back-and-forth"methodbetweenthe threetechniquesof personality
evaluationhas bccn employcd.Hypotheses
formulatedfrom data obtained
with one techniquehavebeencvaluated,refinedand clarifiedby data from
the others.whcn a hypothesiswas consistently
supportedby all three approaches,an attempt was made to formulate it in tenns permitting it to be
subjectedto critical test by experimentalor statisticaltechnique.
As an examplc,an hypothesiswas dcvelopedthat the cancer patient,
more often than chancewou.ldallow, had lost a major emotionalrelationship, and had beenunableto find a satisfactorysubstitute,sometime before
the first apparentsympromof cancer.This hypothesisappearedto be validated by data from all three techniques.It was then formulated in terms
by which it could be tested.If the hypothesiswere true, then certain social
groups which, a priori. had known higher rates of such lossesshould also
have a highcr cancermortality rate. Thus, for example,if marital status
were taken as the only variable,then, after age was cancelledout, we
shouldexpectthe highestcancermortalityrate in the "widowed," the next
in the "divorced,"the next in the "married" and the next in the "single."
Publisheddata, such as censusmaterial,could be used to exptorethe accuracy of this prediction.various predictionsof this type-all based on
the hypoths5i5-ws1smade.When testedagainstpublishedstatisticaldata,
a l l w e r e d e m o n s t r a t etdo b e v a l i d . ( l l 6 , I l 7 )

P R O B L E M Sr N C ^ N C E R

309

At this point of the research,one general.cmotionalpattern has been


found in over 50o/cof the 300 studiedcancerpatientsand in approximately
lOVo of the equatedcontrols:An early life historywith much self-doubting
and someanxietyover relatingto others;the establishment
of one personal
relationshipthat affordeda high dcgreeof satisfaction,
meaningand validity to the individualand providedhim with a "raison d'tre"; and the loss
of this relationship,followcd by inabilityto find a substitute,and a period
of intense(if often concealcd)dcpression.
This has beenelaboratedupon,
and casehistoriespresentedin variouspublications.(118, l19, lzo, lzl,

t22\
In summingup this researchin a recentpapr (123), the following
conclusions
couldbe reachedby LcShan:
l. There seernsto bc a correlationbctweenthe existenceof neoplasticdiseaseand the persistence
of certaintypesof psychological
situations.
2. The most consistentlyrepnrted.relevantpsychologicalstate has
beenthe lossof a major cmotionalrelationship.Often the psychic
state resulting from this loss could be traced to a period shortly
beforethe first notedsymptomsof cancer.
3. There appearsto be somerelationshipbetweenpersonalityorganization and the evolutionof the cancerouscondition.
4. There may be some relationshipbetweenpersonalityorganization
and the type or location of a cancer.
It would seemas if future researchin this area,to bc as usefulas possible,must focusupon the chemicophysiological
changeswhich resultfrom
variationsin psychic statesin general.It is highly probable that these
changesare mediatedthrough the endocrinesystem.Through the linking
of psychic stateswith hormonal changes,we may be able not only to
integratepsychologicalfactors with the many other factors influencingdevelopmentof cancer,but also to relatcthcm to certainlevelsof organization. It may be possibleto establishthe relationshipof psychologicalfactors
to other influenceswhich favor or even inducepassageof cancerfrom one
phase to another. This may prmit a more complete understandingof
cancer and help in finding new points at which some therapeuticvalue
might be expectedfrom psychological
intervenrion.
The relationshipbetweenthe adrenalsand psychicstateson the one
hand,and betweenthe adrenalsand the lymphaticsystemon the other hand.
couldexplainwhy the inlluenceof a psychologically
unresolvedproblem is
most evident,among all cancerousconditions.in lymphomas.LeShanhas
been able, by analyzinga sizablenumber of these lymphoma cases,to

310

./

nEsEARcH rN pHysropATHoLocy

recognizemore clearly and more consistentlythan in other conditionsthe


existenceof a patternof psychological
changesoccurringprior to clinical
illness.Recurrences
of symptoms,or periodsof exacerbation,
also could
be connectedto eventswhich had deep repercussions
upon the psychologicalstate.
Researchalong theselines, seekinginformationon psychicfactors in
the pathogenic
mechanism
of cancer,is beingpursuedactivelyby LeShan
andhisco-workers
at our Institute.

C H A P T ELR2
PHARN,IACODYNAIUIC]
ACTIVITY
TT

we have investigated
l[* ou* REsEARCH,
the pharmacodynamicactivity
of a seriesof substances
in terms of thc physiopathological
conceptsdiscussedabove: the level of organizationat which thcy act, the dualistic
natureof their activity,their relationshipto body constituents,
especiallyto
lipids, and the changesthcy induce in the dcfensemechanism.We will
limit ourselvesin this presentation
only to thoscof the substances
investigated which are of therapeuticinterest.
The dualistic concept has pcrmitted didactic separationof agentsinto
two groupswith antagonisticproperties.In the last analysis,this separation
could be related to the two fundamentaltendenciesin nature, homotropy
or heterotropy.For inorganic agents,this criterion appearsto be directly
relatedto the elementspresent.However, for organic agents,especiallyfor
lipids and lipid-like substanccs,
this simplecritcrionis lessvalid. Another,
the positive and negativeelectricalcharacterof their active polar groups
could be used. It must be emphasized,however,that some of these polar
goups show either positivc or negative properties depcnding upon the
medium in which they work. An alcohol,for cxample,can act as an acid
under special circumstances,in which case it forms metal alcoholates;or
it can act as an alcoholforming esterswith acids.For this reason,in conwe havc limited the sphereof changesstudiedto
sideringthesesubstances,
thosecorrespondingto the conditionspresentin biologicalentities.
Since, as seen above, substanceswith negativc polar groups actively
while those with positivepolar groups conintervenein various processes,
trol tbe activity of the first, we will start this presentationwith the former.
3ll

312

xESE^RCHrN pHysropA't-HoLoGy

AGENTS WITH ACTIVE NEGATIVE POLAR GROUPS


Among agents which have negative polar groups, a further division
can be made accordingto the natureof thesegroups.Under biologicalconditions,the most important of negativepolar groups is the carboxyl. Losing
a proton when dissociated,the carboxyl confers acidic property to tbe
moleculeinto whose structure it enters.We will consider,first, such carboxylic acids. Among such agents,the individual difterencesseen in biological activitieshave to be related to their nonpolar parts.
In the frame of our research,with organic acids, we were especially
interestedin those with a predominanceof the nonpolar group; that is,
those having lipoidic proprtics.They are, principally, fatty acids, which,
accordingto the presenceof one or more double or triple bonds in the
nonpolar part, can be separatedinto saturatedand unsaturated.
F"t$

Acids

SaturatedFatty Acids
The lipoidic character in this homologousseries starts with the five
carbon valeric acid, although caproic acid is the first lipoidic member
found in animals.The role of the saturatedfatty acids in organizationis
largely related to the activity, as caloric metabolites,of the memberswith
even carbon numbers.These saturatedfatty acids are absorbed,circulated
and stored as triglycerides,and it is also this bond to glycerol which ap
parentlyfavorstheir biologicalrole as caloricmetabolites.
As previously noted, only the memberswith a relatively short chain,
lcss than 12 carbons, can be directly metabolizedthrough Knoop beta
oxidation.Longer chain saturatedfatty acids undergo a breakdown before
further metabolic degradation.Members with l6 and more carbons also
play a limited constitutional role, usually together with an unsaturated
member, when bound to glycerophosphoricacid to form phospholipids.
Even when frec-that is, whcn their polar group is not neutralized-they
do not have a manifestfunctional role and, consequently,show no important pharmacodynamicactivity.
The higher fatty acids exert no dcmonstrableinfluenceupon microbes
nor upon viruses such as bacteriophages.
No influencehas been observed
upon monocellularorganisms,cells or tissuesin vitro. No changeswere
seenin the respirationof liver slicesor of Sa 180 ascitescancerouscells in
the Warburgmicrorespironreter.
Similarly,no influencecan be seenon red
blood cells nor on leucocytestreated in vitro. No changesin the chloride

P H A R M A C o D Y N A M T CA C r r v t T Y

3 1 3

contentof tumorsor woundsare inducedby administrationof theseacids.


The fact that no changccould be induccdin the secondday wound crust
pH explainsthe lack of any effectupon pain.
Even largc doses,such as 20 cc. of a l0% solutionof theseacids in
sesameoil, do not changethc intensityof pain of alkalineor acid pattern
in humans.No influencehas been noted upon tumor evolution, even with
the direct techniqueof dipping tumor transplantsin the agent, repeated
for successive
transplants.Existingedemaor ulcerationis not influenced.
At the organic level, no changescan be seen in the function of various
organs or upon liver regenerationrate. These fatty acids do not influence
convulsionsinduced by thiamine in rats even when administeredin very
large doses,such as 5 cc. or more of l}o/o solutionsfor 100 gr. of body
weight. The same lack of influenceis seen on systemicmetabolicmanifestations,as recognizedthrough variousanalyses.Except for palmitic acid,
which showsan adrenaldefenseindex of 12, the index for the other members of this seriesis below 6, indicatingonly a certain participationof the
adrenalsin the defensemechanismagainsttheseacids.
No changescan be found in the number or characterof leucocytesin
animalstreatedfor a few days with theseacids.No changein the sodium
or potassiumcontent of the blood or in urinary analysesis induced by
thesesubstances
even in subjectswhoseanalysesrevealabnormalpatterns.
No action upon body temperatureand no influenceupon the evolutionof
the defensemechanismcan be observed.
It is interestinghowever,to note the effectof the sodiumsaltsof these
acids,especiallyon in vitro lysisof differentcells.(Note I )
UnsaturatedF atty Acids
Of the unsaturatedfatty acids,oleic acid is the most widely encountered
in nature.As previouslynoted,ntonoethenoids
have a group of 9 carbons
either toward the carboxyl or mcthyl end of the molecule.with the double
bond betweencs and cro in a moleculeof 18 carbons,oleic acid appears
to satisfyboth tendencies,which could accountfor the ubiquity of this acid.
oleic acid circulates,is depositedas reserve,and is used as caloric
metabolite especially when bound to glycerol. In considerably lesser
amounts,when bound to glycerophosphoric
acid, it takes part as a phospholipid in the formation of the lipidic systemof the organism.It is only
slightlyactive in oxidation processes.
For this reason,large dosesare necessary to influenceabnormal processcsat differentlcvels. Even then, only
limited changesare induced.In in vitro studies,oleic acid producesno
changesin bacteriophages.
A certain influenceis noted for receptivityof

;,lr1[arr,
Fil..:'S

314

,/

xESEARcHlN

pHysropATHoLocy

dermotropeviruses.Injected subcutaneously
in rabbits, oleic acid induces
in the skin, at the site of injection a zone of low receptivity for smallpox
virus. Oleic acid has a reducedeffect upon microbes,causingthe appearance of some gram negativeindividual forms of Bac. subtilis, for instance.
Mixed directly with blood, oleic acid induceshemolysis.when plasmais
treatedwith this acid, and then mixed with red cells.the influenceexerted
upon red cells becauseof the small amount of acid fixed upon plasma, is
reduced. Although olcic acid may influence the pH of the second day
wound crust, causingan increasedlocal alkalosis(Fig. II9), its influence
0leic actd

F t c . l l 9 . T h e s e c o n dd a y w o u n d c r u s t p H f o r o l e i c a c i d
s h o w st h e c o n s t a n tp r e s e n c eo f a c h a n g et o w a r d m o r e a l k a l i n ev a l u e s .

upon pain is almostnil. Little or no changeis scen in standardizedradiation woundsin animalstreatedwith the acid. A limited influencecan be
observedon tumor grorvthby using thc techniqueof dipping transplants
gencrations.Usc of a 1094.solutionof oleic acid in tricap
for succcssive
gcncrations,
impairsgrowth of
rylin bcforegrafting,rcpeatcdfor successivc
after thc sixth or sevcnthgenerationin
Ehrlich mouse adenocarcinonra
some experiments,even later in othcrs.Negativepassagetakes place betweenthe cighth and tenth graft. Under the samecondition,very little or
no changesare noted in other tumors,and no changesare seenin tumors
in animalstreatedwith this acid even thoughchangeshave been reported
of cells of differentorgans,treated
by some authors, (124) Suspcnsions
of oleic acid and injectedin animalsof
in vitro with colloidalsuspcnsion

P H A R M A C O D Y N A M T CA C T T V T T Y

315

the same specics,induced no changcswith a single injection.Repeated


injectionsaftcr 3 weeksinducedlcsionsin the respcctivcorgans.
At the organic level, some effectscan be obtaincd by using high doses
of oleic acid. For example,to preventconvulsionsinduced by thiamine
chloride,dosesas high as severalhundredmilligramspcr 100 gr. of body
weight are required and even then tbis eflect is not constantlyseen.Systemic changes,rccognizedthrough blood and urine analyses,are almost
ni-I,even with large dosesof oleic acid. The compoundhowever,does prolong liver regenerationtimc.
'fhe
Among the diethenoics,we studied linolcic acid.
caloric activity
of the uncombinedfatty acid is rcduccdwhile constitutionaland functional
activitiesare increased.Linoleic and linolcnic acid appear to be organizational rather than functional fatty acids becauseof their preferredbond
to glycerophosphoric
acid. They are absorbedfrom the intcstines,mainly
'I'he
as phospholipids.No effectsupon phage can be seen.
refractivity to
smallpoxvirus inducedon rabbit skin is more manifcstthan for oleic acid.
However, the effect upon microbes,such as subtilis,is less evident than
for oleic acid,Crenelatedred cells,with increased
tendcncyto conglutinate
and increasedsedimentation
ratc, are found altcr linoleictreatmentof the
blood in vitro. To avoid hemolysis,the acid is not added directly to the
blood but to the plasmawhich is then reunitedwith thc cclls. Crenelated
cells also appear in vivo in rats injectcd intraperitoncallywith large
amountsof this acid, such as l0 cc. of IO9h in oil. A definiteshift toward
alkalosisis found in the secondday wound crust pH, which explainsthe
pain of an alkalinepattern
influenceseenupon pain. Linoleicacid increascs
pain of an acid pattern,though only slightly.Only in relaand decreases
t i v e l y l a r g ed o s c s( 2 - 4 0 0 m g . / 1 0 0 g r . a n i m a l ) , d o c s i t p r c v e n tt h e c o n vulsionsinducedby thiamine.
In lesionsinduced by radiation,the administrationof smarlldosesof
this fatty acid often producesa favorablehcaling effect.This effect can be
related to the growth-stimulating
action of essentialfatty acids in small
just
quantities.The effectis
the rcverseof the unfavorableinfluenceon healing observedwhen largerdoscsare administered.
An effectupon tumors in
be
achieved
only
through
animals can
repeatedtreatment of successive
generations
transplantsor through the treatmentof successive
of the host.
The effect of repeatedinjectionsof tissuecells treated in vitro with a suspensionof linoleic acid was more manifestthan that obtainedwith oleic
acid. The effect upon the systemiclevel, althoughmore manifestthan for
oleicacid, still is limited,even undcr abnormalconditions.Blood and urinary analysesare only slightly and briefly changedtoward the D type of

,-t.f

316

R E s E , ^ R c l rr N p H y s r o p A T u o L o c y

offbalanceeven when large amountsarc administered.Blood eosinophiles


are decreasedand potassiumcontentincreased,both only slightly. Body
temperatureis slightly depressed.
Preparations
especiallyrich in tricthcnoidlinolenit'oc'idwere used and
no differencesbetween their bioloeical activitv and that of linoleic acid
could be noted.
We obtained preparationscspeciiilly rich in arachidonic rcid f.rom
salmon oi.l. The caloric contributionof this acid can be consideredalmost
nil comparedto its functional role. It is absorbed,circulated and stored,
principally as esterifyingsterols.Although this acid is presentin the body
in relativelysmall amounts,it reprcscntsmore than 25Vo of.the fatty acids
of the adrenals.In view of the highly functionalrole of the adrenals,it is
logicalto supposethat arachidonicacid'sabundancein the glands is not
merely coincidental.A libcrationof this acid, togetherwith other higher
polyunsaturatedfatty acids from the adrenalsapparsto take place during
the first phaseof the diphasicdefensephenomenon.
In the first minutesfollowing a noxiousinterventioninducingshock,a depletionof fatty acids in
the adrena.ls
occurs,coincidingwith increasedamountsin blood.
Besidesthcir role in the dcfensemechanism,the adrenal fatty acids
seem to intervene in normal physiology.Successiveliberations seem to
occur, alternatingwith liberationsof sterols,as weli as corticosterones.
Together,theseproducc the diphasicoscillationswhich characterizethe physiologic dynamic balance.
Preparationsrich in arachidonicacid secm to act at di-fferentlevels.
No manifestinfluenceupon phagesis observed.The influenceupon smallpox virusesand upon microbesis similar to that of the linoleic preparations.
The in vitro and in vivo effectsupon red blood cells, such as crenelation,
conglutinationand increasedsedimentationrate, are more manifest than
for linoleic acid. Leucopeniaalso is seen.The effcctsupon the pH of the
second day wound crust and upon pain iue, however, the same as for
linoleic acid. The intensity of acid pain is reduced while that of alkaline
pain is increased.In dosesmuch smallerthan for linoleic acid, arachidonic
acid preparationsare able to preventconvulsionsinducedby thiamine. But
they do not seem to changethe evolution of radiation lesions.The influence upon tumors is very similar to that of linolcic acid, and only limited
changes are observed after treatment of successivegenerations,either
through the dipping of transplantsor through treatment of successive
of this fatty acid were
hosts.Organ cells treatedin vitro with suspcnsion
organs,if injectedtwice at 3 weeks
seento inducechangesin the respective
interval.Systemicinfluenceis not manifesteven under abnormalconditions.

PHARMACoDYNAMTC ACrlvrTY

317

Blood and urine analysesare slightly and temporarilychangedtoward the


panern of fatty acid predominancc.
Continuing thesc studies,wc have investigatedpolyunsaturated
latty
acids with more than four double bonds, particularlyclupanodonicacids
from cod liver and sardineoils. Most of the studieswere made by using
the fractionswhich when brominatedarc solublein acetoneat a low tempera t u r e ,i . e . ,a r o u n d - 6 3 " C , T h e d i l T e r e nf tr a c t i o n so b t a i n e dw e r e i d e n t i f i e d
t h r o u g hi o d i n cn u m b e r ,n c u t r a l i z a t i ovn: r l u ca n d s p c c t r aal n a l y s i sa f t e rc o n j u g a t i o nt h r o u g ht r c a t m e n w
t ith KOH
All the biologicaleffectsupon viruses,microbcs,cells, etc., are more
accentuated
than for linoleicor evenarachidonicacid.Changesin red cells
and leucocytcsare much more apparcnt.At this point we must emphasize
the preferenceof thesefatty acids for red blood cells over plasma both in
vitro and in vivo treatmcnts.(Note 2)Thcy have pircatereffectsupon pain
than do linoleicand arachidonicacids,reducingpain of an acid patternand
exacerbatingpain of an a.lkalinepattern. 'fhe local pH, as determinedby
secondday wound crust measurements,
also is shifted toward increased
alkalinity.Convulsionsinducedby thiamineare influencedby much smaller
dosesthan those required with othcr unsaturatedfatty acids. For some
preparationswith iodinc indicesaround 350, dosesas low as 35 mg./100
gr. of body wcight arc sufficientto preventconvulsions.Changesin the
evolutionof tumors are more striking with thesepreparations,especially
when the transplantsof succcssivc
gcncrationsarc dipped in the preparat i o n . W i t h t h i s t e c h n i q u en, e s a t i v er c s u l t sa r e o b t a i n e de v e n a t t h e t h i r d
transplantfor Ehrlich mammary adenocarcinoma.
An obvious effect is
noted on radiation-induced
lesions,with ulcerationincreasedand healing
slowed.The influcnccupon cholcsterollevelsin the blood and upon hypertension is greater than for arachidonicacid. Thc effectsupon systemic
changes,obscrvedthroughanalyses,
are tcmporaryand no greaterthan for
linoleic and arachidonicacids.
Acid Lipidic Fractions
Bearing in mind the role of acid lipidic constituentsin the physiology
of variousbiologicalentities,preparationscontainingthesefractionswere
obtained.Tissues,organs,organisms,and organicproductswere saponified
and acid fractionssoluble in ether were isolated.we called them "acid
lipidic fractions," or "lipoacidic fractions." Their analysesrevealed,in addition to variousfatty acids,othcr substances
with lipidic and acid character.Some were identifiedas porphvrinicacids.
Significantdifferencesrelatedto the sourcesof thesepreparationscould

rfll:'-.
Rli

.ji

$ffi
;,**?#S1.'i,.

318

;''

RESEARcH IN

PHysIopATHoLocy

be recognizedin biologicaleffectsat differentlevels.Preparationsobtained


from intestine,for instance,had no obviouseffectson any of the manilestations; there were no systemicor organicchanges,no effectsupon pain, red
cells or leucocytesin vitro, and no influenceon tissue respiration.Treatment of successivetransplantsshowedno apparenteffects,even after ten
passagegenerations.No effectsupon organsthrough repeatedinjectionsof
cellstreatedin vivo by thesepreparationswere seen.

:6
a
c
,a

fn
aa

lave Lengthfnp.;

F l c . 1 2 0 . S p e c t r a la n a l y s i so f r a t l i v e r f a t t y a c i d sa f t e r c h e m i c a li s o m e r i z a t i o ns h o w s
t h e p r e s e n c eo f f a t t y a c i d s w i t h 3 a n d 4 d o u b l e b o n d s . ( 0 . 0 0 2 % i n e t h y l a l c o h o l )

On the other hand, other prcparationsfrom placenta,liver, blood, etc.


did show activity upon all manifestations,
including pain. They induced
negativeresultson tumor growth after transplantdipping for just two or
three gcnerations.
The differcncesin activity could bc relatcd to the richnessof these
preparationsin polyunsaturated
fatty acids.It could be shown that it was
not the total numberof doublebondspresent,as determinedby the iodine
number,that was significantbut rather the relativeabundanceof higher

;illl:

PHARMACODYN^MIC ^CTIVITY

3I9

c
o
g
c
L
ira

lave Lemth6n*;
F t o . l 2 l . S p e c t r a la n a l y s i so f t h e i n t e s t i n a lf a t t y a c i d so f r a t s a f t e r c h e m i c a l i s o m e r i z a t i o n s h o w s m i n i m a l a m o u n t so f m e m b e r sw i t h 3 . 4 o r m o r e d o u b l e b o n d s .( 0 . 0 0 2 V o
in ethyl alcohol)

unsaturatedmembers, as rccognizedby special analysis after adequate


c h e m i c acl o n j u g a t i o nF. i g u r e s1 2 0 , l 2 l a n d 1 2 2 s h o w s u c ha n a l y s e s .
Abnormal Fatty Acids
Becauseof the role of abnormal fatty acids in the pathogenesis
of the
offbalancetypc D, particularlyrelatedto radiation,a study was made of
preparationsof acid lipids obtainedfrom abnormaltissues,organsand organisms.In an initial group of researches,
animalsthat had died of radiation sickness,shock, acute infections or after adrenalectomywere used.
Guinea pigs infectedwith B. anthracisand mice infectedwith strep hemolyticus were used as sourcesof abnormal lipids in a large number of experiments.Acid lipids obtainedfrom autolysatesof tissueswere employed.
We also used the fatty acids of cod and sardineoil, which may be consideredto correspondnot to natural but to alteredfatty acids sincethey were
obtainedafter autolysisof cod liver and whole sardinebodies.
After having determinedthat conjugationof double bonds is the basis

.ffi{r',
ffii,j

. $qi- jj![t

320

RESEARCH IN

PHYSIOPATHOLOCY

"l
;

*1
I

i*t
t

I rol
I

I
6u

fb

8c

f a v e L e n g t h( n p )
Frc. 122. Spectral analysisof the fatty acids of the entire bo<ly of rats, after chemical isomerization shows the presenceof di- and triethenic members, and littlc of
m e m b e r sw i t h m o r e d o u b l e b o n d s . ( 0 . 0 0 2 V oi n e t h y l a l c o h o l )

of abnormality in pathogenicfatty acids, conjugatedisomers of different


fatty acicisw'erepreparedand studied.Eleostearicacid obtainedfrom tung
oil was used extensivelyin animal and clinical research.Parinaric acid
was obtained from nuts of parinarium laurinum and was used to a lesser
degrce.various conjugatedfatty acid isomers,recognizedthrough spectral
analysisand oxalic indices, were obtained in a higher proportion from

ffi,-N

PH^RMAcoDYNAMTcAcrrvrTY

321

different lipoacidrcpreparationsby using a modificationof classicalmcthr.. (Note 3) Nlany mixtures of naturally occurring fafty
ods of conjugar..c
acids found in sapcnifiablefractionsu'cre conjugatedby the same method.
Conjugated di, tri, tetra, penta-and hexaenic fatty acids werc further
separatedfrom tl::sc itii:turesand studied,The unexpectcdrelationshipof
t h e s ec o n j u g a t c Jf , , l : ' , ' a c i d tso o x y g e nr v a so f i n t e r e s tW
. hilethe treatment
of linoleic acid at 37"C with oxygcn lcads ro progressiveincreasein the
amountof peroxidespresent,this doesnot occur for the conjugatedisomer
(FiS. 123) apparentlybecauseof repeateddestructionsof the peroxides
formed.
In general,the effectsproduced by conjugatedfatty acids at difterent
levels of the organizationare more intensiveand persistlonger than those

t2

(a)

to
cl
vf

de
z
o

8
e
OJ

>.

d
?o4
tr
,'

'-

--/t

,)

\---__-.,

,n,

\tv.,"-.

(b)

""

6
l 'i me ( hour s)
Frc. 123. Curves of peroxides of samples of linoleic acid (a) and its conjugated
isomer (b) induced tbrough the passageof oxygen ( 100 ml per minute for 30 cc of
sample) at 37'C. While peroxides are progressivelyincreasing i n t h e l i n o l e i c a c i d
prcparation,they do not change in the conjugated isomer,

322

n E S E , A R c Hr N p H y s r o p A T H o L o c y

of the nonconjugatedisomers.It is interestingto mention here the in-fluence


exertedby thesepreparationsupon viruses.Although there are no changes
for bacteriophages,
a marked influenceis seenin vivo upon receptivity of
the organism to viruses.Subcutaneousinjection of conjugatedfatty acids
in rabbits establishesa zone of refractivity toward inoculationof smallpox
vaccine in the skin, which is greater and more persistentthan that observedfor nonconjugatedisomers.The effect upon microbesalso is clear.
Gram negativestrainswith manifestmorphologicalchangeswere obtained
from B. anthracis,and persistedas such for 6-15 passages
beforethe old
morphologicaland tinctorial charactersreturned.The effect upon red cells
and leucocytesin vitro is more apparentthan for correspondingnonconjugated isomers.This is also true for the influenceupon the respiration in
vitro of tissuesor ascitescells.
The difference in the effects of conjugated and nonconjugatedfatty
acids is particularlystriking in certain manifestations.
For example,pain
with an acid patternis more easilychangedto alkalineby treatmentwith
conjugatedfatty acids than with nonconjugatedisomers.Once the alkaline
pattern appears,it persistsfor a long time. A manifesteffect is seen upon
lesionsinducedby radiation.Standardized
radiatedwoundsin rats, which
heal in about four weeksin control animalsand require six to eight weeks
to heal when treatedwith nonconjugated
fatty acid preparations,fail to
heal at all when treatedwith correspondingdosesof conjugatedisomersof
the samefatty acids.
The isomers of fatty acids also differ in their effect upon animals in
shock. When anaesthetizcd
animalsare scaldedby immersionup to the
level of the xiphoid in water at 90oC, immediatefatal shock occursif the
immersionlastsfor more than four scconds.With a three secondscalding
the animalsdie after severalhours.The administrationof fatty acid preparations markedly reducesthe survival time, and this effect is more manifest
for conjugatcdmembers,especiallyfor eleostearicacid. In general,animals
in shockinducedby any means,suchas by the Noble-Collipdrum, show a
specialsensitivitytoward conjugatcdfatty acid preparations.
The effectof conjugatedfatty acids at the systemiclevel, as recognized
by blood and urine analyses,is in the same direction as that for the nonconjugatedbut is more manifest.
There are significantdifferences
betweenthe effectsof conjugatedand
nonconjugatedfatty acidsupon the evolutionof transplantedand spontaneous tumors. In a small proportion of mic, a mixture of nonconjugated
fatty acidspreparedfrom cod livcr oil or sardineoil preventsthe appearance of methylcholanthrene-induced
tumors, The conjugated fatty acids

PHARMACODYNAMIC ACTTVITY

//

323

obtainedthroughthe treatmentof thesepreparationsshow this preventivc


effectin a large proportion of mice.Theseexperimentsarc interestingfrom
severalstandpointsand are presentedin somedetailin Note 4.
Conjugatedfatty acids producean incrciuc in the chloridecontentof
woundsin animals.Values are 407o highcr than in untreatedlcsions.The
same increaseof chloridesoccurs in tumors. When Dba mice with adenocarcinoma were treatcd for ten days with conjugatedfatty acid preparations, and tumors were removedand chloridecontentdetermined,values
were up to l80o/ohigherthan for controlswith untreatedtumors.(Note 5)
An interestingeffectwas noted in two rat tumors.For years,passages
of
Guerin'srat tumor and of sarcomainducedin our laboratoryby the injection of benzpyrene
have showna peculiarcharactcr.Althoughthey grow to
large size,the tumors have no necroticzones.After treatmentwith conjugated fatty acids,large necroticzonesappeared,leadingto ulcerationand
death.The appearance
of thesezoncsof necrosiscorresponds
to changesin
the fundamentalcharacterof the tumor. Transplantsof fragmcntsof thesc
treatedtumors,althoughtakenfrom nonulcerated
rcgions.or youngtumors,
resultedin tumors showingearly central ulceration.This charactcrpcr'l'he
sistedwithout further treatmentin succceding
gcncrations.
ulcerating
tumors appearto be a mutant of the originaland the nrutativechangecan
be relatcd to interventionof conjugatedfatty acids.This was confirmedby
the fact that similar changeswere constantlyobtainedin the same tumors
with thc sameagens. A similar but lessmanifestand less constanteffect
is obtainedwith preparationsof cod liver oil fatty acids administercdrepeatedlyin largeamounts.The samceffectwas obtainedwith the injection
of the fatty acidsdirectly in thc tumor itself.
The secondday wound crust pH showsmarkedchangcstoward alkalinity under the influenceof conjugatedfatty acids.The eflect upon regeneration time of liver is also manifest;cells full of fatty dropletsdo not
appear at all or appear much later than in untreatedcontrols. In rats
weighing200 grams,with a largeenoughdose,such as 2 cc. of l0% solufatty acidsobtainedfrom cod liver oil and repeated
tion of polyconjugated
for two days,the adrenalsshow completedepletionof fats. They become
small and red in color and contain no sudanophilmaterial.The liver rcgenerates
with smallcellswith compactcytoplasmand almostentirelybare
of fatty droplets.
The effectof conjugatedfatty acidsupon the lymphaticsystemis manifest.A marked involutionof thymus,spleenand lymphaticgland follows
particularlyof a mixthe injectionof conjugatedfatty acid preparations,
tureof conjugatedcod liver oil fatty acids.The effectupon tumorsis irregu-

324

xESEARcH

IN

PHYSToPATHoLoGY

lar. In some animals a marked retardationoccurs,in others no effect can


be observed.In convulsions,no greatereffect is seen than that produced
a color darkerthan when
by nonconjugated
isomers.Blood in vitro assumes
other fatty acidsare used.Eosinophilcs
arc markcdly rcducedafter administrationof conjugatedfatty acid preparations.Important changesin organs
were obtained with repeatedinjectionsof the respectivecells treated in
vitro with suspensions
of theseacids.Changesin the analyticalvaluesof
urine, however,are not greatly differentfrom those obtained with nonconjugatedisomcrs.With sufficientamountsof conjugatedfatty acids,a frank
hypothermiais obtained.
Bixine
In the samegroup of fatty acidswe can place bixine, a member of the
polyterpene
family with 9 conjugated
doublebondswhich we obtainthrough
saponificationof the seedsof Bixa orellana and have studied widely. The
changesproduced in microbesare similar to those with other polyconjugated fatty acids. Changesin connectivetissuesin animals appar to be
particularly interesting.The first reaction to subcutaneousinjection of an
oily solutionof I Vo bixtne in rats or mice is an inflamntatoryprocess,with
the injected material dividing into hundredsof tiny droplers.Some, however, melt away and the unabsorbedinjectedmaterial again forms one or
two big drops. The wall containingthe drops is very thin and transparent
and appearsto be made up of very few conxectivecells which have extremely long fibrils, rcpresentingthc highestdcgreeof differentiationof
thesecells.
The effect upon pain is similar to that of conjugatedfatty acids. The
secondday wound crust pH showsa manifestchangetoward alkalosis.The
effectupon radiationwoundsis the sameas for conjugatedtrienes.In animals injccted with convulsantdosesof thiamine, only a few milligrams of
the bixine preparationare requiredto preventconvulsions.Of all the fatty
acid preparationsused, bixine apparsto be the most efficientin its anticonvulsivantaction.The iodinenumberof 430, found in our preparations.
confirms,thus, in this case too. the correlationseenbetweenanticonvulsivant effectand richnessin double bondsof the fatty acids.
The distributionof bixine followingadministrationis interesting.Chromatographicstudyof blood constituents
after hydrolysisshowsthat almost
all the bixine is in the red cells,with minimal quantitiesin plasma.Lesions
such as wounds or tumors, after the administrationof bixine, become particularly rich in this substancein comparisonto normal tissues.Changesin
evolution of tumors also are manifest.The administration of this agent

P H A R M A C O D Y N ^ M I C ]^ I ' T I V I T Y

325

often leadsto rapid necrosisald cdenra.In aninralsand huurans,we saw


massivetumors becomeulceratcdin a fcw days after use of only a few
milligramsof the substance.
The ulceratingcffectof conjugatedfatty acids
u p o n t u m o r sr e a c h e si t s m a x i m u n rw i t h b i x i n e .l n a n i m a lt u m o r sw h i c h ,i n
s u c c e s s i vt er a n s p l a n t sh,a d n c v c rs h o w ns p o n t a n e o uusl c e r a t i o n st h, e i n j e c t i o n i n t o t h e h o s t o f o n l y a f e w m i l l i g r a m so f b i x i n e i n o i l y s o l u t i o n
produced,in addition to ulceration,a changein the tumor which can be
c o n s i d e r e dt o c o r r e s p o n dt o i l m u t a t i o n .F u r t h e r t r a n s p l a n t cs o n s i s t e n t l y
developedulceratingtumors and the ulcerativecharacterpersistedin succccdinggcnerations.
Massivedegencrating
changeswereobtainedin organs
after repeatedsubcutaneousinjcctionsof suspcnsionsof cclls obtained
frorn theseorgansand treatedin vitro with bixine.
The changcstoward oflbalanceD induccdin varioussystcmicpatterns
in humans by bixine are sinrilar to those producedby conjugatedfatty
acids.However,once induccd,thcscchangesare very persistent,
and often
remain uninfluenced
by anti-fattyacids.It is this characteristic
of resistance
t o f u r t h e rc h a n g e sw h i c h r c p r e s e n tas c e r t a i nh a n d i c a pf o r t h e r a p e u t iuc s e
of this agent.
Fudittg Response
I n c o n t r a s t o t h c r e l a t i v e l yp e r s i s t e ncth a n g e si n d u c c db y b i x i n e ,t h c
s t r i k i n gc h a r a c t e or f t h e e f f c c t su p o n p a i n o r s y s t e m i cn r a n i f e s t a t i o no sb tained with fatty acid preparations.and especiallywith the conjugated
i s o m e r s ,i s t h e i r s h o r t d u r a t i o n .F u r t h e r m o r c a, t t h e b e g i n n i n go f t h e i r
administration,theseagents,evcn in relativelysmall doses,exert intense
eflects,but such eflectscannot bc obtainedlatcr without continuouslyincreasingthe amountused.Aftcr a certaintime. cvcn largedoseshave very
little effect.
An explanationof the fadingcharacterof the resultsobtainedwith these
and many other agentscan be found in thc fact that the organismdefends
itself againstany factor able to inlluenceits balancc.In the caseof conjugatedfatty acids,this defenseseemsto bc providedmainly by the adrenal
glandsE
. . F . T a s k i c rh a ss t u d i e dt h i s a s p e c ot f a d r c n a d
l c f e n s ci n o u r l a b oratory and this researchis presentedin Note 17, Chapter 6. An adrenalectomizedanimal is usually less rcsistanttoward the administrationof
animal.This drop in resistmany agentsthan a normal or sham-operated
ance is expressedas an adrenaldefenseindex, as the ratio betweenthe
minimal lethal dose for the normal and for the adrenalectomized
animal.
preparations.
this index is betweentwo and three,and
For most fatty acid
greater
conjugated
for
the
fatty acids.Fatty acidswith three conbecomes

326

R E S E A R c Hr N P H Y S T o P A T H o L o c Y

jugated doublc bonds, however,have an adrenal defenseindex of 120.


Eleostearicacid is 120 times lesstoxic for sham-operatedcontrols than for
adrenalectomizedanimals, indicating a specific adrenal defense against
these acids. Progressivelyincreasedintervention of the adrenals would
explain the fading effect mentionedabove.
Alpha Hydroxy Fatty Acids
Alpha hydroxy fatty acids were obtained by fixing an OH at the carbon
adjacentto the carboxyl. Some of these acids exist in nature-in significantamountsin the brain and skin, and in vcry small amountsin the
kidneys. In our research, they were originally prepared from natural
sources,such as animal brain and skin. Most of the studieshowever,were
made with syntheticalpha-hydroxy-fatty
acids.For experimentalpurposes
in animals and humans,we principally used pure syntheticalpha hydroxy
fatty acids.Mixtures of thesemembersobtained through the treatment of
acid lipidic preparationsalso were cmployed.
In animalsand humans,alpha hydroxy fatty acids induce lesssystemic,
organic, tissue and cellular changesthan do thc correspondinguntreated
acids. However, we would like to mention a striking exception: the responseof lymphosarcoma
6C3HED in mice to the administrationof alpha
hydroxy-caprylic
acid. Althoughthis tunlor uniformly kills control animals
within 10-12 days, it disappearsin ovcr 8O7oof animalstreatedwith alpha
hydroxy caprillic acid, even if treatmentis institutedlate, that is, when the
tumor has alreadygrown to I cm. in diameter,a size usually rcached 2 or
3 days beforedeath.In the few animalsin which the tumor does not disappear,its growth is so sloweddown that survival time is extendedto three
or more weeks.(Note 6)
Other alpha hydroxy fatty acidscloseto caprylic acid, such as alphahydroxy-caproicand capric acids,show no influenceupon evolution of this
tumor. We could not obtain similar effectswith any of the other saturated
alpha hydroxy fatty acids that have chains with 4-20 carbons, nor with
alpha hydroxy-olcicor linolcic acids.Nor did alpha hydroxy-caprylicacid
or any of its homologuesappear to have any influenceupon other transplanted tumors in mice, the Walker tumor in rats or, spontaneousmammary tumors in mice.
Other Fatty Acids
The fading eftect secnwith naturally occurringfatty acids is so great a
handicap for therapeutic use of these substancesthat we searched for
fatty acids which the organismdoes not normally encounter
heterogeneous

PHARMACoDYNAMIC ACTtVTTY

327

and against which it would not be prepared to defend itself. This brought
us to the study of fatty acids having different nonpolar groups than those
of the normal and abnormal constituents.while most of these were prepared syntheticallyin the laboratory, we utilized on a substantiallylarge
scale two natural fatty acids which exist in plants and are sufficientlyheterogeneous,ricinoleic and crotonic acids.
Ricinoleic acid has a double bond between9 and l0 and a hydroxyl at
carbon 12 insteadof the seconddoublebond found in linoleicand linolenic
acids. As a result of the induction effect propagatedfrom the carboxyl
through the chain, the crr is a positive carbon. The positive characteris
enhancedby the adjaccntdouble bond betweence and c1e, and by the
hydroxyl bound to c12.C,, thusis very stronglypositive.we relatedthe intenselocal alkalosiswith consequent
watcr cxcretioncorresponding
to the
a l k a l i n ew a t e r yd i a r r h e at o t h c c f f e c to f r i c i n o l c i ca c i d l i b c r a t c di n t h e i n testine,and have consideredit to correspondto a local organicoffbalance
similar to that induccd in tissuesby unsaturatcdfatty acids.This would
explainthe intensivelaxativecflectof castoroil.
We uti.lizedricinoleic acid parenterallywith the aim of obtaining a
similar effcct in abnormalcellularand tissuelesions.The oily solutionof
ricinoleicacid has low toxicity when administeredparcnterally.However,
no manifesteffcct upon tumors or at differentlevelsof organizationwas
obtained.Crotonicacid did not showthe expectedinfluenceat theselevels.
Other hcterogeneous
agents,polyhydroxy fatty acids, were studied.
They were preparedby adding one or morc OH groupsto the nonpolar
groups at the double bonds of unsaturatedfatty acids.9, l0 dihydroxy
and 9, lO, 12, l3 tetrahydroxyfatty acidswere no differentthan thc corresponding unsaturatedfatty acids in their effects upon pain or systemic
analysesin humans,or upon tumor growth in animalsand humans.
Peroxide Fatty Acids
Peroxidesand epoxidesof fatty acidswcre preparedand studied.They
showedmore manifesteflectsupon virusesand bactcriain vitro and in vivo
than the other fatty acids. Investigationsof the effectsof these fatty acids
upon higher levels havc been limited until now. It seemsthat the effects
upon systemicand organic manifestationsare somewhatdifferent than
those obtained with use of the fatty acids from which the peroxidesand
epoxideswere derived. Influenceupon pain and upon tumors was grcater
for the correspondingunsaturatedfatty acids. This research---cspecially
with polyepoxidefatty acids-is still in progress.

328

R E S E A R C H

I N

P H Y S T O P A T H O L O G Y

Halogenic Compounds ol Fatty Acids


The study of changesin lesionswhereabnormalfatty acidsare present
has shown the importanccof fixation of chloride ions in thesesubstances.
Consideringits place in the periodicchart, chlorine is an elementwith heterotropic character.consequently,it could be conceivedof as being antagonisticto fatty acids,counterbalancing
their homotropiccharacter.This
was confirmed by pharmocologrcalstudy of fatty acids to which chloride
ions were added at the double bonds. We were particularly interestedin
conjugatedfatty acids in which effectsof treatmentwith chlorine could bc
followed through spectralanalysis.
when a mixture of conjugated fatty acids from cod liver oir was
treated with chlorine, the peaks in spectral analysis progressivelydisap
peared.(Fie. I2a) This did not lead,as expected,
to increasedtoxicity.Thus
far, in early trials, the differentpreparations,from 9-10 dichloro-stearic
acid to the polychlorinatedmixture of fatty acids from cod liver oil, have
not shown effects greatly different from those of conjugated fatty acids
upon tumor evolution,systemicanalyses,and pain. No differencehas been
noted betweenthe derivativesand their correspondingfatty acids in animal

t
!
c
o
L

h a y e L e n o t h( b N

F r c . 1 2 4 . I n f l u e n c ee x e r t e db y c h l o r i n e8 , a su p o n t h e c o n j u g a l e df a t t y a c i d s .A p a r a l l e l
d e c r e a s ei n t h e a m o u n t o f a l l t h e m e m b e r si s s e e n .( 0 . 0 0 2 % i n e t h y l a l c o h o l )

P H A R M A C o D Y N A M T c^ C r l v l r Y

3 2 9

cxperimentsor in rescarchin hunrans.Experiencewith these products,


howcver. indicatesthat they may inducc milliar gastriculcerationswhich
u'e considerto rcsult from the inllucnceexertedby the fatty acids upon
the gastricmucousmembranewherethey are broughtby the chlorideions
to which they arc strongiybound. Researchin this directionconfirmsthe
part which thesefatty acids,solidly bound to chloride ions, take in the
pathogenesis
of the stateof shock.
An over-allanalysisof the pharmacological
activity of the fatty acids
mentionedabove showsa similarity in fundamentaleffectsobtainedwith
most of thesepreparations.
This can be interpretedas resultingprincipally
f r o m t h e f a c t t h a t a l l h a v ei n c o m m o n ,t h e i r l i p i d i cc h a r a c t ear n d t h e s a m e
polar groupwith acid properties-the carboxyl.As a result,thesesubstances
are fixed in the samepositionin abnormalentities,a fact which seemsto
representthe most importantfactorin their pharmacodynamic
activity.The
further biologicaldifferenccs
secnbetweenthe inlluenceexertedby the various membersstudiedwould be rclatedto a secondaryeffectof thesesubstancesresuJting
from the interventionof the nonpolargroups.This finding
-that the fundamentalpharmacological
activityof fatty acidsis connected
rvith the sitc of activity which is detcrmrnedby the lipoidic characterand
the polar group presentwhilc' the proper pharmacological
activity is due
to the interventionof the nonpolar group-has bcen of capital importance
not only for undcrstandingthc activity of thesesubstances
but also for
determiningthe path of our further research.Becauseof this, we investig a t e d ,i n a s e c o n ds t e p ,l i p o i d sw i t h o t h c r n e g a t i v ep o l a r g r o u p s .

LIPOIDS WITII OTHER NEGATIVE POLAR GROUPS


Lipoaldehydes.'
With a carbonylas polar group, the lipoidic properties
appearwith propanalin the homologoussericsof the aliphaticaldehydes.
with the carbonyllessdissociated
than the carboxyl,the lipoaldehydes
rep
resentnegotivelipoidsableto act for a longertime than the respective
acids.
We were especiallyinterestedin threegroupsof aldehydes.In one, with a
nonsaturatedshort nonpolar chain, we searcheda conjugatedformation
betweenthe double bond of thc oxygenof the carbonyl and the double
bond of the nonpolar chain. Another goup of the lipoaldehydescorrespondedto long chain fatty acids,while the third was formed b1'saturated
s h o r tc h a i n a l d e h y d ew
s i t h a n o d d n u m b e ro f c a r b o n s F
. rom the energetic
point of view, there were the first and especiallythe third groups which
appearedas the most interesting.ln the last group the opposite influ-

330

nESEARcHrN pHysropATHoLocy

ence exerted by the carbonyl and methyl groups upon the intennediary
carbonsof the chain givesthe entire moleculean especiallyhigh reactivity.
This oppositeinfluenceis seenat its maximum in propanal,where cL suffers the influenceencrgeticauyoppositeof the carbonyl and methyl group.
The fact that, due to its relativelyhigh solubility in water, propanalwhich is a lipoid--{an be administeredin aqueoussolutionsand still act
upon the lipidic system-makes of it an especiallyinterestingagent.
we have investigatedthesegroups of lipoaldehydesfrom the point of
view of their influenceexertedupon the two offbalances.In the group with
unsaturatedshort chains, we studicd acrolein and crotonic and malcic
aldehydewithout seeingany specialeffect upon the other levels than the
cellular one, where a vacuolationwas obtained.Furthermore their toxicity
has represented
an handicap.More interestinghas appearcdthe group of
the saturatedshort moleculeswith odd number of carbons. while with
heptanalwe havc obtainedbesidesan influenceupon pain, also a manifest
inhibitoryeffect upon thc growth of expcrimentaltumors, it was propion
aldehydewhich has shown the most interestingcffectsupon pain.
This was secn for the group of aldehydeswith aliphatic saturated
chain such as propionit' and heptylic aldehl,tlesor with cyclic, as .rclicy'lic
aldehydes.Inadequatedoscs-f rom ,urocc. to 2 cc. of the l0% solutionof
propionicaldehyde,or of the I % solutionof the heptylicor salicylicaldchydes-a manifcstinlluencewas obtainedupon the systemiccondition as
well as upon pain. Patientsin offbalanceA with pain and generaldiscomfort, wcre seento have a decrease
of the symptomsaftcr the administration
of propionicaldehyde.The eflectupon tumorswas reducedand propionic
aldehydedid not changethe cvolutionof the tumorsin spiteof the marked
improvementof thc gencralconditionand even of the ccssationof pain.
Lipoids n'ith Thiol Groups
Mercaptans: According to thc systematizationof lipoids presented
above, a thiol group, acting as a polar group, will form a lipoid when
prepondcrantaliphaticor cyclic nonpolargroup.
bound to an cnergetically
In thc homologousrncrcaptanseries,evcn thc lowestmembersare lipoids
forcesof the thiol group.
becauscof the weak electrostatic
Although methyl mercaptanis a lipoid accordingto our classification,
this substanceis too volatilc to be used.Therefore,the first low membcr
was ethyl mercaptan.
of this homologoussericsto be investigated
The effectsof ethyl mcrcaptanupon microbeswere more limited than
those seen for fatty acids. To determinethe effectsat the different levels

PHAR,M^CODYN^MIC ACTIVITY

331

of the organization,ethyl mercaptanhad to be administeredparenterally.


As for all other membersof this scries,we utilizedethyl mercaptanin 5 or
lOVo oily solution in cottonseedoil. In acute toxicity tests,the lethal dose
was found to be 145 mgs./100grams of body weight for mice, and 153
mgs./100 gramsfor rats. The immediateeffectsupon nuclei were similar
to, but lessintensethan thoseof conjugatedfatty acid preparationsleading
to caryorrhexisor pycnosisin abnormal cells. No abnormal mitosis was
seen in organswith high mitotic activity.such as the intestinalmucosaor
bone marrow, although appreciablechangeswere observedin mitosis in
animal tumors. A secondaryeffect,an exaggerationof aging processes,

7&,
7U
782
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77,!8
776
7.7a
712

dd"

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-t-1

-t-

770
7&
76
7e.
7.62
7eo

F l c . 1 2 5 . S e c o n dd a y w o u n d c r u s t p H s h o w sa c o n s t a n tc h a n g et o w a r d m o r e a l k a l i n c
v a l u e s f o r a l l t h e s u b s t a n c e sh a v i n g s u l f u r i n t h e i r p o l a r g r o u p .

was first recognized,severaldays after administrationin the changesin


granulocytesat the site of injection.The averagenumber of nuclear lobes
of the leucocyteswas often very high, even above seven.This was also
true for leucocytesin circulating blood when the product was injected
intraperitoneallyor even subcutaneouslyin rats. The immediateeffect of
such injectionswas a prolongedleucopenia,especiallya lymphopenia.
The chloride content of tumors and wounds was especiallyincreased
through treatment with mercaptans.At the tissular lcvel, local pH of the
second day wound crust was increased,an effect characteristicfor all
l i p o i d sw i t h s u l f h y d r yal s t h ep o l a rg r o u p .( f i 9 . ; , 2 5 ) T h i sc x p l a i n st h e d i r c c t
effectsof ethyl mercaptanupon pain and othcr alkaline or acid symptoms.
Theseeffectsare qualitativelysimilar to thoseseenfor the polyunsaturated
fatty acids but much slower to appear.While placentaacid lipid prepara-

332

nESEARcHIN PHYStoPATHoLocY

tions, for instance,produce an effect upon pain-an increasein intensity


for an alkaline pattern and a decreasefor an acid pattern----venwithin
5 to 8 minutes after parenteraladministration,the effect of mercaptansis
reducedand appean after half an hour, or later.
The eftect upon tumors in animals was especiallymanifest upon a rat
sarcomaoriginally induced by benzpyrenein our laboratoriesand passed
through successivetransplantsover a period of many years. Throughout
this period,this tumor showedl00Vo positivetakes,characteristicallygrowing to huge sizr, at times as large as the rest of the animal, but without
ever showing either spontaneousregressionor necrotic areas.When ethyl
mercaptanwas injected subcutaneously
in daily doscsof t/z cc. of a SVc
solution in cottonseedoil into animals with these tumors, interesting
changeswere observed.If the tumors were alreadylarge, above 6 cm. in
diameterfor instance,only a few regresscd(5 /20 for tumors of 6 cm. in
diameter).In tumors that did not regress,large necroticareasdeveloped
and were followedby ulceration.Most becameinfected,leadingto death.
Tumors smaller than 6 cm. in diameter frequently regressedrapidly and
then disappeared(between9/20 and 20/20 in difterentexperiments).No
such striking results were observedin any of the other tumor strains in
rats or mice treated with ethyl mercaptan,although in several,growth arrest occurredor necrotic zonesappeared.Ethyl mercaptaninjected in the
tumors themselvesinduced the same necrotic changesin most animals.
( F i s .1 2 6 )
The effect upon lymphaticorganswas manifest.Spleen,thymus and
lymph nodes were involuted in animalstreated for a few days with mercaptans.The eftectupon convulsionswas irregular.Even with small doses,
convulsionswere preventedin some casesbut, in general, the effect was
lessconstantthan with fatty acids.Eosinophilesdecreasedrapidly in animalsor humanstreatedwith this substance.
All urinary analyticaldata were
influencedby administrationof mercaptan,with changestoward the patterns of type D offbalance.
Becauseof the very disagreeableodor of ethyl mercaptan,we were
obligedto discontinueits useso that effectsof this agentupon some analyses,suchas surfacetension,sulfhydryland calciumexcretion.could not be
studied in humans.
Other Mercaptans:Superiorhomologuesof the mercaptanserieswere
used.They were divided into three groups.The first includedpropyl, butyl,
amyl and hexyl mercaptans;the second,heptyl and allyl mercaptans;the
third, memberswith more than l0 carbons.The first group producedmuch
the sameeffects,which tendedto diminish as the number of carbonsin-

P tt A R N{AC ()t)Y N A N{| ('

AC T I V I TY

1 1 1
-t .1 .1

creascd.For examplc.cflectswerc considerablyrcducedfor hexyl mcrcilptan as comparedwith ethyl mercaptan.Thc secondgroup-thc heptyl and
'I
allyl mercaptans-procJucedmorc intensivceffccts. his was espcciallytrue
for allyl mercaptan.Membersof the third group-rvith longercarbon chains
such as dodecyland hexadccylmcrcaptans-producedcflccts so slight that
ttrey seemedalmost nonexistent,exceptupon pH of the secondday wountl
crust, which showed valucs far abovc the rangc of thc controls, just as
with other membersof this homologousscrics.
Yesks

Control

ooo
Start treatoent

Treated
rl
ethyl
ercaptan

Treatedrith
ethyl
mcaptan

OOOI
Start treatnenl
U l s e r arto n

e O - .
i;.

F t c . l l 6 . l n f l u c n c e c x c r t e d h 1 ' c t h r ' l n r c r c a p t i u l u p ( ) n l \ : r r c o n l i r r n d t r e c t lb l , h e n z pyrene. For biggcr lumor\ jt induccs constltntly ln ulccration, while for rmall tun)ori,
thcir disappearance.

With the idea of usingthiol as a polar group and havingrnothcr ccntcr


in the molecule &s a sccondirrycenter, we studiecla seriesof othcr sub(the B A L preparation) oftcn used
stances.One was dimercapto-propanol
for heavy metal poisoning. [t proved to be conrplctcly rvithout cffect on
pain, tumor grorvth and systcmicchanges.It had lcss activity than the
highermercaPtans,
*'hich theniselves
rvcrclessactivethan prlyunsaturated
and abnormalfatty acids.
The difficultiesencountercdin adnrinisleringmcrcaplans,nrainly relatcd to offensiveodor of lou'er mcnrbcrsand inactivityof thc lessobnoxiousmembers,lcd to a scarchfclrothcr chemicalagentsthat rnightbc activc
without being evil smelling.Extensivestudy n'asmade of variousprcpara-

334

RESEARcHrN pHysropATHoLocy

tions that appearedto have a bivalent sulfur bond at the nonpolar group.
We investigatedcolloidal sulfur which, if introduced in the organism,
seemedto undergo changessimrlar to those of bivalent sulfur. We found,
thus, that the sulfur absorbedaftcr being administcredin colloidal form,
was almost entirely eliminatedafter oxidation in the form o[ sulfates.In
animals,no pharmacodynamicinfluenceswere observed.OnIy the pH of
the secondday wound crust was increasedwhen thesepreparationswcre
administeredparenterally in suspensionor were given orally mixed rvith
food, in proportions up to 4%. There was no evident change in tumor
evolution in animalsor humans.
Hydro-Persulfides
Another sulfur compound,so-called"sulfurizedoil," in which sulfur
and fatty acidsappearto form a hydropersulfide,
(Note 7) was tested.This
hydropersulfidepreparation,althoughit exhibitedno influenceupon viruses
in vitro, did inducea good degreeof local resistanceof the skin to smallpox
infection.The effectsupon microbeswere reduced.There was little direct
influenceexerted upon cells. The preparationswith 0.5 to lVo srtfur
bound to cottonseedoil were well toleratedlocallv when administeredintramuscularlyor intraperitoneally.
The effect of parentera.land oral hydropcrsulfrdeupon pain was slow
to appear,in contrast to the effect of fatty acids and even of mcrcaptans.
However, it persistedfor a long time. Pain of the acid pattern was eased;
pain of alkaline pattern was exacerbated.The influenceupon the second
day wound crust pH was marked.The local pH increasedto valueseven
higherthan 7.80. In radiationlesions,the dimensionof ulcerationincreased
and healingwas retardedor even prevented.In some tumors in animals,
the rate of groMh was slorved.This latter effect was not uniform in the
different types of tumors tested and even in the same type of tumor in
different groups o[ animals. systemic changesalso varied. Doses correspondingto 5 mgr. of sulfur were not toxic for 30 grn. mice in a single injection.Nor were l5 mgr. dosesin 200 gr. rats. chronic toxicity studies
showedthat 0.2 mgr. daily injectionsin mice and 5 mgr. injectionsin rats
for as long as three monthsdid not inducepathologicalchanges.High doses
suchas I to 2 cc. of a lVo preparationinjectedseveraltimes a day in humanswas almost uniformly followed by a rise in temperature,usually after
a few davs.

PH^RM^CODYN^MIC

ACTIVTTY

335

Other Compoundswith a Thiol Group


The resultsobtained with hydropersulfidepreparationsled us to seek
other compoundswith sulfur bound to fatty acids insteadof triglycerides
as in those mentioned above.
Sulfur compounds were prepared from various conjugated fatty acids
such as conjugatedlinoleic acid and eleostearicacids and from mixtures of
conjugatedfatty acidsobtainedfrom cod liver oil, fish oil, human placenta,
blood and various organs.while active in smalleramounts,they were not
qualitatively difterent from hydropersulfideprcparations obtained from
cottonseedoil, producing the same pharmacologicaleffectsin most tests,
especiallyupon pain, systemicmanifestationand evolution of cxperimental
tumors.
The fact that sulfur bound to the nonpolargroup,as in hydropersulfides,
produced lcss manifest results in animals and humans than mercaptans,
which have a thiol group as a polar group, led to the study of other substancesin which thiol radicalsinsteadof sulfur were added in similar positions,and consequently
wereconsideredto act as secondaryenergeticgroups.
preparations,
A seriesof
in which one or more thiol groupswere fixed at the
double bonded carbons in various conjugatedor nonconjugatedpolyunsaturatedfatty acids,were obtained.Thesesubstances
differ fundamentally
from the fatty acids mentionedabovein which sulfur atoms were fixed not
at the carbons bound by double bonds but at the carbon adjacent to
the double bond. 9-10, dithiostearicacid, 9, 10, 12, 13, tetrathiostearic
acid, as well as polyunsaturatedand conjugatedfatty acids with thiol
groupsfixed at their double bonds, were obtained.In general,they showed
no marked biologicaleffectson animals,no influenceupon pain or systemic
patternssimilxl to those observedfor the other lipidic productswith bivalent sulfur.
The hydropersulfidegoup was addedto soaps.Sodium and ammonium
soaps were obtained through saponificationof the triglyceridesof fatty
acids on which sulfur was already fixed. Effects at the different levels of
organizationwere markedly reduced.There was no influenceupon pain,
organic or systemicmanifcstations.However, a striking eftect was noted
on many microbes.Growth of Bac. anthraciswas preventedin someexperiments,even with dilutions of. | /2,000,000. For staphylococcusaureusand
streptococcushemolyticus, a similar effect was obtained with dilutions
higber than l/200,000. In animals,even oral administrationin drinking
water in a dilution of 1/500 and, in someexprimentseven l/1000, con-

336

p . E S E A R c Hr N p H y s t o p A T H o L o c y

trolled infectioncausedby thesemicrobes.The antibiotic activity appeared


to be similar to that of penicillin.(Note 8)
7'etrahydronaphthalene
Persulfides(SulfurizedTetraline)
we utilized the known marked tendencyof tetrahydronaphthalene
ro
fix oxygen with the resultingexplosiveperoxides,to fix sulfur in similar
combinations.Persulfidesof this substancewere thus obtained and their
pharmacologicalactivitystudied.white only a limited effectwas noted upon
viruses and microbes, the influence upon tetrahymena pyriformis approached that seen with active polyunsaturatedfatty acids. The producr
with 5 gm. sulfur fixed for 100 gramsof tetrahydronaphthalene,
showeda
relativelylow toxicity in normal animals,T5 mgr./100 gr. in mice and
125 mgr./100 gr. in rats beingwcll toleratedin intraperitoneal
administration. The influenceupon wound healingin animals,upon pain, and upon
the systemicanalysesin humans,was similar to that seenfor the mercaptans.Although the immediateeffecton pain was limited, prolongedadministration was effective.The analyticalchangesof the urinary surfacetension
and blood potassiumwere the most manifest.The toxicity was highly in,
c r e a s e df o r a n i m a l sw i t h a s c i t e st u m o r s ( S a 1 8 0 , E h r l i c h a n d K r e b s ) .
However,the influenceexertedupon transplantedand spontaneoustumors
in animalswas one of the most favorableones, compared to the effect of
other testedagents.
Thiosullates
We investigatedthiosulfates with the intention of studying agents
which, in addition to a manifestreductioneffect,would act througb bivalent sulfur liberated in the body. The elimination of part of the sulfur of
thiosulfatesas mercapturicacid has led to the suppositionthat the bivalent
sulfur ion, separatedfrom the thiosulfateion, would act through combinationssimilar to those found in the metabolismof thiolipoids.It is for
this indirect action that although hydrosoluble,without any direct connection to lipoids, we investigatedthe biological activity of thiosulfate
together with and under the same specific aspect as the lipoidic sulfur
compoundsconsideredabove.
There was a very limited effect upon microbesand viruses,no effect
upon phages,and no changein the receptivityof rabbits to smallpox virus.
With either oral or parenteraladministration,the immediate effect upon
pain was manifest.Oral administrationof 1/z cc. of a lOVo solution of
sodium thiosulfate in water, or intramuscularinjection of. l5 cc. of the
4o/o solution,was usually followed by a definite effect upon pain in less

PH^RMACODYNAMTC^CTlvtTY

33'1

than | 0 minutes.As this was found to be oppositefor the two patternsof


pain, sodiumthiosulfatewas usedcven for diagnosisof pain pattern.Pain
of an alkaline pattern increasedwhile a decreaseoccurred in pain of an
acid pattern. The second day wound crust pH increasedmanifestly with
the use of this substance.
Cellular and nuclear changesfollowing administrationof thiosulfate
preparationswere similar to those produced by mercaptans.The effects
upon the lymphaticsystemand on liver regcneration,
however,wcre minimal. Convulsionsinduced by thiamine were controlled well by thiosulfate
in dosesof 120 mgr. per 100 gram of body weight. Injectedsimultaneously
with administration
of thiamine,thiosulfatepreventedconvulsionsin a high
proportionof animals(17/20).
The effect upon radiation lesionswas lcss manifest.An increasein
wound size and prolongedulcerationoccurredonly with use of relatively
large amounts of sodium thiosulfatcdaily. Dosesabove 4O mg./100 grn.
of body weight were needed to obtain thesc efTccts.The hcaling of a
simplewound was retardedonly with largc doses,around50 mg./100 gm.
of body weight.On the other hand, when very small doseswere administered, such as 5 mg./100 grn., the healing effect was cnhanced.Effects
upon tumorswere lessmanifestin animals.Slightand inconsistent
changes
were seenin graftedtumors. Very often in the sameexperimentalgroup,
tumors disappearedin some animalswhile in others the pgowthrate was
only slowedor remainedunchangcd.The erratic resultson tumors in animals producedby thiosulfateswcre similar to thoseseenfor many of the
sulfur preparations,and appearedas characteristicfor this group. Repeated
injectionsof thiosulfatesin tumors were seento induce the disappearance
of the tumor if growth was slow cnough to permit such injections for
severalweeks.At the systemiclevel,the most markedeffect,other than that
on sulfhydryl index, was on surface tension which usually dropped with
the administrationof a sufficientlylarge amount.
Most of the researchon thiosulfatewas done with sodium salts.In a
few cases,very high dosage,such as 6-10 grams daily, produced moonfaceand slightleg edema,apparcntlyrelatcdto sodiumretention.This disappearedwith cessationof the medication.
Changing the cation of the thiosulfatc from sodium to magnesium
appearedto increase,sometimesmarkedly,the resultsobtainedin our experiments.Potassiumthiosulfate seemedto be more effective,especially
againstpain. Its use however, has been limited by disadvantages.When
administeredparenterally,it causesconsiderablelocal pain at the site of
injection as most potassiumsalts do. lf administeredto patients having

338

xESEARcH IN pHYSropATHoLocy

pain or other symptoms of an alkaline pattern or systemic manilestations


correspondingto type D, a more markedincreasein intensityof symptoms
occursthan for the sodium or magnesiumsalts.
We also investigatedsodium tetrathionate.Except for lower dosagerequirements,no other advantageswere found in its use. Its relative instability is a handicap.
Alpha-T hio-F atty Acids
In other studies,we tried to introducesulfur into the fatty acid molecule,this time changingthe polar group itself. With the sulfhydryl replacing
the hydroxyl of the carboxyl group, a bivalent negative sulfur was introduced, thus realizing a thionic group. (R-COSH)
We prepared severalmembersof this thionic acid series corresponding
to various saturated,polyunsaturatedand even conjugatedfatty acids. we
studied in particular the efiect of hexylthionic acid, correspondingto
caproic acid. The resultsobservedwcre essentiallythe same as those seen
with the other bivalent sulfur containinglipoids mentioned previously.In
addition to influencingpain and systemicchanges,hexylthionic acid produced some interestingeffects upon cxperimental tumors, reducing the
growth of a few of them. However, there were no important differences
from the effectsof the other sulfolipoids.
Another entire seriesof productswas prepared by introducing a thiol
goup at the Cz, or alpha position, of various fatty acids, with the intention
of creating a more complex polar group similar to that present in alpha hydroxy or alpha amino compounds.Alpha-thio-fatty acids were thus obtained for the entire homologousseriesof saturated,and for many of the
nonsaturated,fatty acids. Some membersof this seriesof alpha-thio-fatty
acids,such as caproic,caprylic and myristic, were studiedextensivelyboth
in animals and humans.From the biologicalpoint of view, however,they
showedno manifestdifferencesover the thiolipoidspreviouslydiscussed.
Thioglycolic Series
All these researcheswith limited biological results brought us to the
study of lipoids in which the thiol representsa polar group but in which a
secondarypolar center is present in the molecule. Many such synthetic
thiolipoidswere preparedin our laboratorywith the hope that they would
prove biologically more effective and would have alkylating activity as
well. Two seriesappearedto be interesting,since they were being active
particularly at lower levels oi organization.This led us to utilize them also
on a larger scale in clinical work, While consistentresults were obtained

PH^RM^CODYN^MtC

^CTIVITY

339

on pain and systemicchanges,the influenceupon animal tumors was erratic


and no different from that of other preparationswith thiol groups or the
sulfur compounds mentioned above. There were marked effects in some
animals with tumors; in others with the same tumor treated identically,
there were no effects at all. In humans the eftectson pain, tissular, organic
and systemiclevels were similar to those of many other sulfolipoids.
Starting with these substances,derivativeswere prepared. One group
comprisedderivativeswith a specialcharacter.In order to have only one
active polar group, one of the two polar groups had to be blocked. For
thioglycolic acid which we studied,either the thiol or the carboryl group
could be blocked,leavingthe uncombinedradical as the active polar group.
Since we were interestedin substanceshaving the thiol group as active
polar radical, the carboxyl group was blocked by replacing its hydrogen
with a methyl group. Methylthioglycolatehas been thoroughly studied in
our laboratory. Its pharmocologicalactivity is similar to that of the other
thiol preparationsmentionedabove.
other thioglycolateesterswith ethyl, propyl or butyl insteadof methyl.
were preparedand studiedbut showedno advantageover the methyl ester.
we tried to obtain the allyl esterin order to have a more potent secondary
centerbut we were unableto synthesizeit.
In the same group of agentswe studied another substance,beta mercaptopropanoicacid, having a thiol and a carboxyl as polar groups.uscd
uncombined,it could be seenthat here again,as with thioglycolicacid, it
is the carboxyl that acts as active polar group while the thiol acts as a
secondaryenergeticcenter at the nonpolar group. This acid is very toxic
in animals,producingas a peculiareffect,manifestmuscularspasms.The
compound also produced abnormal muscular rigidity. seen immediately
after death. In nontoxic doses,it showeda marked influenceupon tumor
growth. Many tumors disappeared;in many others,a reduction in size occurred. Impressiveresultswere obtained in spontaneousmammary carcinoma in mice where a fairly high proportionof tumors disappeared(2g/
40). Repeatedinjectionsinto theseanimalsgavegood resultsif the gowth
was slow enoughto pcrmit treatmentfor a periodof at leasta month. The
preparation,however,showedtoxic effcctsin animalswith tumors, producing weight loss similar to that producedby the thioglycolicseries.To
change the polar group and have the thiol act as such, we blocked the
carboxyl with a methyl in some experimentsand with an allyl group in
others. But the influenceupon tumors in animals was reducedthroueh
thesechanges.

340

nEsEARcH rN pHysropATHoLocy

Relationship to Sullur Metabolism


The study of lipoids with sulfur posedthe problem of the relationship
betweentheir structure and biological activity. we mention this here because it not only explains the influenceexerted by these agentsbut also
becauseit indicatesthe manner in which this researchhas had to be developed. We have seenthat, accordingto the systematization
of the elements,
sulfur representsa nonmeta.lanti A element,active especiallyat the metazoic level.It is its actionas an isolatedelementthat appearsinteresting,in
additionto the metabolicchangeswhich it inducesin the organismwhen
presentin a negativelipoid.
It seemsthat the organismgenerallymetabolizesbivalent negativesulfur by changingit ultimatelyto the hexavalentform. Ferguson and du
Vigneaudhave studiedthe metabolismof methionineand cysteinewhich
are the principalsourcesof sulfur in the organism.(125, 126) While the
evidencewe have on this subject is too limited to provide more than a
working hypothesis,it would indicate that other compounds with lipidic
.h313s191-1hs thiol-containing lipids-h3vs more important biological
activity.
The metabolismof thesecompoundsvaries among individuals,especially thosewith abnormalconditions.The study of the excrerionof sulfhydryls through the urine, expressedas the sulfhydryl index, has servedus as
a guide in studying the metabolismof sulfur up to a point. Thc high excretion of the thiol group seemsto be related not only to low oxidation
but to an abnormal form, as mentionedabove, since with the exaggerated
excretion,the thiol level in the blood is reduced.This form is to be consideredas an excrementalone. This seemsto be confirmedby the fact that
the administration
of bivalentsulfur,evenin a largeamount,is not followed
consistentlyby its eliminationin the form of thiols in the urine. In some
subjects,an impressivelyhigh proportion of the thiols administeredappearsin the urine, and this is true even for relativelysmall dosesof thiols
or of bivalentsulfur as in thiosulfates.
In other subjects,on the contrary,
even when larger amountsare administered,
the increasein eliminationis
minimal or does not occur at all. The abnormalityin sulfur metabolism,
which appearsto be a limited capacityto oxidize it to the hexavalentpositive form, also means an exaggerated
interventionof the thiol group as
such in the economyof the organism.This occurs along with symptoms
and signs, previously noted, correspondingto an exaggeratedoxidative
interventionof fatty acids.in which processes
the thiols probablytake part.
We tried to study the capacityof the organismto fully oxidize thiolic

PHARMACODYNAMIC
ACTtvrrY

341

sulphur by following the responseto the administrationof a known amount


of sulfur in bivalentnegativeform. Thc changeof the sulfhydrylindex of
urine would serve as a tolerancetest for thiol metabolism.
After injectionsof 80 mg. of sodium thiosulfate,the difterencesin the
capacity of various organismsto metabolizeit could be seen and related
to pathological conditions. This concept of thiol metabolismcan be the
basisfor understandingan abnormal form of thiolic sulfur which may be
involved in the pathogenesisof abnormal conditions.Substancescontaining a thiol group,suchas methionine,cysteineand particularlyglutathione,
are presentin sizableamounts in the organism,but it is not this form of
thiol that intervenesin the abnormalmetabolism.A large amount of the
normal form of thiol is presentin the blood of subjectswith a low urinary
sulfhydrylexcretion.when another form, the abnormalone, intervenes,
it is
excretedin the urine. The organismeliminatesthis "abnormal" compound
with sulfhydryls.It seemsquite probablethat this abnormal thiol compound is in a lipidic form since the sulfhydryl-containingcompound is
readily extractedby ether from the urine. Its affinity for the lipidic system
would explain the influence exerted upon fatty acids and the oxidative
processes
occurring in thc lipidic system.The thiolipoidsintervenecatalytically in the oxidation of the fatty acids,as seenin experimentsin vitro.
Thus, the thiol group in lipoids containingbivalent sulfur rather than
metabolizedsulfate would increasecatabolic metabolism. Although the
thiol in this abnormal form is largely eliminatedby the urine, apparently
as a defensemechanismagainstits pathologicalactivity, someof it is probably retained in the cellular lipids where it continuesits activity. Circulation of sulfur in thiolic lipidic form, with consequentimpairment of its
changefrom the bivalent negativesulfur into the hexavalentpositivesulfur,
would thus appearas the fundamentalsourceof the participationof sulfur
in the abnormal pattern. The influenceexertedby administrationof thiosulfatesupon the sulfhydryl index can serve as an indication for these
specific changes.
Sulfur is an anti-A elementand it is activeas such in all the forms in
which it existsin the organism,althoughthe intensityof its action varies
at differenthierarchiclevels.The activityof thiol as an anti-A factor can
be relatedto the influenceexertedby carcinogensor other agentsupon the
biological activity of this radical, especiallywhen it is taking part in the
formation of enzymes.This relationshipexplainsthe resultsobtainedby
repeatedinjections of organ or tumor cells treated in vitro with agents
having a thiol as polar group. The heterogenizationinduced leads to the
of severechangesresultingfrom the allergicreaction,
appearance

342

xESEARcH rN pHysropATHoLocy

It is interestingto note that sulfur has an anti-A tendencyeven in the


hexavalentpositiveform in which it appearsas sulfate.The sulfateion has
a capacityin the organismto inactivatelipoids of a positivecharacter.The
sulfate becomesbound to such substances,
thereby tending also to facilitate their excretion in urine. Many of these substancesare eliminated in
combinationwith the sulfuric radicalin the forms called "sulfo<onjugated."
In view of this, the effectof sulfateions appearsto parallel that of the
fatty acids.Both opposesubstances
having a positive polar character;that
is, both are biologically antagonisticto anti-fatty acids. In the sensethat
they opposeantifattyacids,both the bivalentnegativeand hexavalentpositive sulfur havc anti-A activity, the first directly and the last indirectly.
The characteristic influence exerted by sulfur in its different forms is
basedon its action as an agent inducing changestoward increasedhomotropy, by acting at levels above the cellular. This is a typical example of
the relationship between an element'sactivity and its place in the periodic
chart. Sulfur is a member of the serieswith homotropic action; it belongs
to the period which correspondsto the metazoic compartment and thus
acts above the cellular level.

Selenium Lipoids
The influence exerted by bivalent sulfur upon oxidation processesin
which fatty acids participate,servedas a guide for further research.Seeking agentsthat would act at a still lower levcl of the organization,even
below the cclls,we consideredother substances
which also affectoxidation
processes.Theoretically,at least, it appearedpossibleto induce changes
at a compartmentbelow the metazoic,at which sulfhydryl-containingcompoundsact.
We have discussedpreviouslythe systematization
of the biologicalactivity of elements,their fundamentalanti-A and anti-D influence,their distribution among the various levels of the organism, all related to their
atomic structureand their place in the periodic chart.
All of this led us to investigateseleniumwhich is the nonmetal element next to sulfur in the sixth seriesof Mendeleeffsperiodic table-the
serieswith an anti-A characterin which oxygenis the first member.According to its period, seleniumbelongsto the cellular compartment.
The first problem was the nature of the compound in which it would
be active.We wcre particularly interestedin using bivalent negativeselenium becauseof the activity of bivalent negativesulfur. However, we did
investigatethe selenicand seleniousacids.Theseacidsor their sodium salts

PHARMACODYN^MIC

^CTIVITY

343

have limited effect upon viruses and microbes. An interesting effect is


seen in Tetrahymenapyriformis,where a manifestcellular vacuolization
is induced.The influenceupon tumors, pain, organic and systemiclevelsis
less manifest and toxic effects are great. Thereforc, we prcpared lipoid
compoundswith a predominantnonpolar group and with a negativebivalent selenium.We utilized, on a larger scale,hexyl and heptyl diselenides
synthesizedin our laboratoriesby M. Bier,
Hexyl and Heptyl Diselenides
Studieshave shown that hexyl and heptyl diselenidesare lower in acute
and chronic toxicity than selenicand seleniousacidsand their sodium salts.
In wounds and tumors, these selenium preparationsinduce a relatively
limited fixation of chlorides,the increaseabove controls b"ing only about
l6Vo. In only a very few casescould any direct effect upon pain be observed within a few hours. However, the long range effectsafter several
daysof administration,seemedto be superiorto thoseof various fatty acid
preparations.This was true both for the decreasein intensityof pain with
an acid pattern and the increaseof pain with an alkaline pattern. The
eftectspersistedfor many days. In animal tumor experiments,there were
relativelyslight changesin growth or survival time,
No manifest influence was seen at the organic level. with relatively
large amountsof theseagents,an involutionof the lymphaticsystemwas
obtained. Thymus, lymph glands and spleen were markedly reduced in
sizein animalsdying after acutetoxicity tests,and adrenalswere small and
appearedto be depletedof their sudanophiliccontent. In rats, a frank
lymphopeniafollowed administrationof largesdosesof thescpreparations,
and eosinophilopenia
also was uniformly seen.Changcsin urine analyses
also were obtainedwith high doses.
It is noteworthythat administration
of disclenide
to a subjectwith a type
A pattern inducesthe appcaranceof oxidizingsubstanccs
in the urine, as
first
one of the
changes.
Effects at the cellular level are seen even with microgram dosages.
Vacuolizationoccursin the cellularcytoplasm.It is interestingto note that,
despitethe cellular vacuolization,pericellularedemaoccurs.The fact that
theseselcniumcompoundsare active in smail dosesmay be an indication
that they act entirely at the cellular and not the metazoiclevel. This effect
at the cellular level is confirmedby the fact that almost constantlythe administrationof seleniumif in sufficientamount is followed by a manifest
increasein serum potassiumvalues,and a decreasein the amount in rcd

344

RESEARcHIN PHYSIoPATHoLocY

cells.This changein serum potassiumis apparentbefore any other change,


and is generallyobtainedwith relativelyvery low dosesof selenium.
The effectsupon cells of another lipoidic selenium preparation, with
seleniumthis time as the polar group, warrant mention. The preparation,
synthesizedin our laboratory, is hexylselenoicacid in which the hydroxyl
of the carboxyl group has been replaced by a SeH radical. A manifest
eftect is produced by this agent in animals with ascitestumors. Intraperitoneal injection leads almost constantly to the disappearanceof such
turnors,even if the compound is used after ascitesis already present. We
usedthis product to bind seleniumin vitro to cancerouscells as will be seen
below.
Tetrahydronapht halene Perseleni de
The fact that in the first phasesof the defensemechanismthe organism
usesfatty acids acting largely through their productsof oxidation, has directedus-as we have seenabove-to searchin the therapeuticapproach
for agentshaving as pharmacodynamic
activity an interventionof peroxides.
parallel
peroxides,
a
further
we investigatedsimilar prodIn
step,
to lipidic
ucts in which, insteadof peroxides,persulfideswere present,sulfur being
the elementimmediatelyabove oxygenin the Vlth seriesof elements.We
studied thus the persulfides,among which the tetrahydronaphthalene
persulfide has been an interestingcompound. Its activity was explained, according to the biological systematization
of the elementsin which oxygen
correspondsto the organismlevel, while sulfur representsa metazoic element.Following the sameline, we searchedsimilar compoundsfor selenium
-3n slsrnentstill higher in the VIth series,which correspondsto the cellular level.We thus preparedand studiedperselenides
by boundingselenium
to tetralinein the sameway as was done for oxygen and sulfur.
The eftectsof the perselenideson microbes or animals were similar
to thoseof the other seleniumpreparationdiscussedabove. Tetraline perselenideshowedlow toxicity in animals,rA cc. of the lOVo solution of the
product obtained having 25 mg. seleniumVo, was not toxic in intraperitoneal injections in mice. Administeredorally in humans, in doses from
1/ru-2cc. of the solutioncontaining25mg. of seleniumper I cc., repeated
even severaltimes a day, did not show toxic effects.The influenceexerted
on pain and systemicchangestook some time to appar as with the other
seleniumpreparations.The influenceexertedupon the growth of experimental tumors in animals was more manifestthan for the other selenium
of naphpreparation.Similar resultsrvereobtainedwith the perselenides
thaleneand other aromatichvdrocarbons.

PHARMAcoDYNAMTC
ACrrvtTY

345

An investigationof the influenceexerted by the immediately heavier


member of this VIth series,tellurium, is in progress.
The foregoingdata on lipoids with negativecharacterindicatethat their
activity generallyis related to changesin processesin which ultimately an
interventionof oxygen takes place. This brings us to the first member of
the sixth series,oxygen,a nonmetalwith D inducing biological activity.
we studiedthe eftectsof ionic oxygen,usingcompoundswhich liberate
oxygen readily. These includedhydrogenperoxideas well as peracidsand
their salts, such as perchloric,perboric,persurfuricand periodic.
The changesinduced by thesesubstancesupon microbes,viruses and
cells are similar to those obtainedwith polyunsaturatedfatty acids and all
are cataloguedas radiomimetic.This fact tends to confirm the importance
of oxidation changesin the pharmacodynamyof fatty acids. The effects
upon pain and at organic and systemiclevelsalso were similar to those of
polyunsaturatedfatty acids. It is interestingto note in the same frame of
activity the appearanceof oxidizing substancesin the urine following the
oral administrationof theseagentsin higher doses.
we investigatedthe effectsof turpentineoil which is known to induce
the appearanceof peroxidein vitro. Highly oxidizedthrough treatmentwith
oxygen or especiallybound to sulfur, turpentineoil has shown interesting
pharmacologicalactivity. An old therapeuticdevice was parenteral administrationof turpentineoil to stimulatethe defensemechanismin cases
of septicemia.However, we saw no such stimulating effect in the fight
againstcancerouscells.The influenceexertedupon the cellular level of the
organizationwas quite reduced.The action of atomic oxygen appearsto
be difterent from that of the molecular.as we will seelater.

ALKYLATING

AGENTS

We investigated,as compounds with negative character, certain alkylating agents,choosing from the large number availablethose which also
showed lipidic properties.We were especiallyinterestedin two members,
sulfur mustardand epichlorohydrine.
Sulfur mustardcontains,along with
one active polar chloroethyl group, a secondrepresentedby the bivalent
sulfur polar group. It has the effect at different levels of organizationof
producing an offbalancewith predominanceof the acid lipids. We will
discussbriefly here some of the experimentsin which this influenceupon
the body lipids has been observed.

346 /

x E s E A R c Hr N P H Y s r o P A T H o L o o y

Su$ur Mustard
In studying sulfur mustard,we were first interestedin its effectsupon
body lipids and, through them, upon the lipidic systemof organisms.In inducing sulfur mustard'scharacteristicskin lesions,an interestingrelationship wasobserved.Pure sulfur mustardwas appliedto mechanicallyepilated
skin of rats. If a suffficientamount-2 to 3 drops-was used and spread on
one squarecm., the animal died. However,the time of death varied. If the
lesionshoweda massivenecrosis,followed by deep ulceration,similar to a
burn of the third degree,the animal died in about three weeks. However,
if the lesion was only erythematous,similar to a burn of the first degree,
the animal died in only 3 to 4 days. It seemedas if the lesion itself intervened secondarilyin the pathogenesisof changesleading to death. Sick
but still living cells appearto have an activity which is highly detrimental.
The abnormal cells apparentlyproduce substanceswhich are responsiblefor
rapid death of the animal. In widely necrotic tissues,these abnormal but
still living cells are limited in number; in an erythematous lesion, they
form the lesion itself. This correlation of toxicity with local Iesion was confirmed by the fact that excision of the lesion itself, il performed in time,
preventeddeath in some animals.The administrationof ferrous sulfate to
rats having sulfur mustard applied to their skin was seen to induce the
erythematousform of the lesions,with death in 3-4 days.
The similarity between the influence exerted by mustard burns and
caloric burns with a sufficientlyextensivefirst degreeburn producing more
rapid death than a third degreeburn, was of interest.
Analysis of the body of an animsl killed by a mustard burn reveals
abnormal amounts of unsaturatedfatty acids and reduced amounts of
sterols. In some cases,where death occurred after more than three weeks,
body sterols were found to be almost completelylacking. In these cases,
almost no insaponifiablefraction could be found. The lesion itself, especially an erythematousand edematousone, was very rich in unsaturated
fatty acids.Histologicalstudy of theseskin lesionsrevealedchangessimilar
to those obtained through the intradermic injection of concentratedsolutions of body acid lipidic fractions.The study of these lesionsfurther revealedthat the lesionsthemselveswere separatedfrom the organism by a
barrier of adipouscells,the resultof an exaggerationin number of the cells
of the subdermicfatty layer.
We studied these important changesfrom severalpoints of view. We
could show that an exaggerationof the adipouslayer underneaththe skin
occurs wben lipoids with negativecharacter,such as polyunsaturatedfatty

PHARMACODYNAMTC ACTMTY

347

acids,thiolipoids, etc. act upon the skin. Thus, this subcutaneousadipous


formation appearsto be a defenseweapon,designedto keep such lipoids
from passinginto the organism. The defenseappearsto be unequal for
males and females,as shown in the following study.
In collaborationwith the late Prof. R, Leroux of the Faculty of Medicine in Paris, we studiedhistologicalchangesin the ears of rats after local
applicationto the skin of a small amount of pure sulfur mustard.Normally
there are no adipouscells in the pavillion itself except at its base. Twenty
minutes after the application of sulfur mustard on the skin of the ear, 2
or 3 layersof adipouscells were seenin the connectivetissuebetweenskin
and cartilage.(Chapter 6, Note 22 )
curiously enough, the rapid appearanceof adipous ceils 20 minutes
after application occurred in femalesand not in males. The spaying of
femalesor castrationof males did not changethis responseeven after a
lapseof months.The administrationof male sexhormonesto female ratsspayed or not---{r of fcmale sex hormones to males----castratedor not
-also produced no change.It was only by the administrationto males,
ovcr a period of days, of a sufficiently large amount of the insaponifiable
fraction obtainedfrom the bodies of rats that this rapid responsewas induced. The administrationto femalesof the acid lipid fraction obtained
from rat bodies was seen to prevent the rapid adipous response.This differencein responsebetwecnmalesand females,can be relatedto the differencesin the amounts of membersof the two groups of lipids ordinarily
found in malesand females,as mentionedabove.
we studiedsulfur mustardfrom the point of view of pharmacological
activity.Dosesof 100 mcgr./100 gr. of body weight 1of a O.7Vosolution
of sulfur mustardin oil) were nontoxic in rats and micc. Except for an intensivelocal reactionat the injection site, no important immediatechanges
were obtainedin humanswhen I to 3 cc, of the 0.2% solution was injected
intramuscularly.The influenceupon pain-a decreasein the intensity of
acid pattern and an increasefor the alkaline pattern-was only temporary.
The influenceon tumor evolution was not sufficientto warrant clinical use
of this agent, especiallyin view of the persistentand intensivesystemic
changestoward an offbalance of the type D which appeared after a few
days. Through its anti-A action, which is the most intensiveof all agents
tested,sulfur mustard remains one of the most interestingsubstancesfor
experimental studies, especiallyfor the eftects exerted upon the anti-fatty
acids.

348

xESEARcH rN pHysropATHoLocy

Epichlorohydrine
The importance of the relationship betweenthe energeticcenters present in alkylating agentsand their ability to produce type D offbalance, led
us to study a group of these substanceswhich, at once, have both short
molecules and two polar groups in close proximity. The desire to have
such an agent with lipoidic property as well led us to study epichlorohydrine which correspondsto propane and has an epoxy group binding C2
and C3, while cr binds a chlorine. Solublein neutral solvents,epichlorohydrine becomes soluble in water only after hydrolysis. Its biological
activity differs from other chlorohydrinessuch as chloropropanediolor
trichloropropane,both of which can be consideredto be closely related to
the substanceproducedby hydrolysisof epichlorohydrine.The acute toxic
dose of epichlorohydrinewas found to be 6 mgr./30 gr. for mice and 25
mgr./100gr. for rats by intraperitoneal
administration;22and 35 mg./100
for mice and rats by subcutaneous
injection.In testsfor chronic toxicity, it
was apparentthat dosesof 5 and L5 mgr. injecteddaily were well tolerated
respectivelyby rats and mice. with higherdoses,the animalsbecamerapidly
emaciatedbeforedying. Used orally in drinking warer,a solutionof l/3000
was well toleratedby rats and mice even for months.With the use of solutions of l/2000, only a few animalsdid not lose weight,while a solution of
l/1000 invariablyinducedwcight loss.
There were no effectsobservedupon microbesor bacteriophage.
It appearsthat epichlorohydrine,actingbelow the morphologicallevels,
induceschangessimilar to thoseseenfor other alkylating agents.However,
it is not upon the desoxyribo-nucleic
acidspresentthat an important action
is seen but in the lipidic system at these lower levels. Epichlorohydrine
seemsto act also at other levels.The influenceupon pain-an increasefor
alkaline pattern, a decreasefor acid-was more noticeablethan for sulfur
compounds.Delaycd eftects,however,were more obvious than immediate
ones. The influence upon wound healing was similar to that of polyunsaturatedfatty acids. Cancerouscells, such as those from mouse ascites,
were destroyedin vitro by a 0.5 solutionof epichlorohydrine.The effect in
vivo upon sarcoma 180 or Ehrlich ascitestumors was most interesting.
Administeredby subcutaneous
or intramuscularinjection,epichlorohydrine
had no effecton the tumor even in dosesas high as 2,5 mgr. daily. However,
when administeredin drinking water in a 1/1000 solution,it prevented
the developmentof ascitesin 19/20 animals.But the toxicity was too high.
A L/2000 solution,usedas drinking water,controlledthe condition in morc
than 50o/aof the animals, while a I /3000 solution showed favorable re-

P H A R M A C o D Y N A M T CA C ' r l v t r Y

349

sults in only a few animals. Under the same conditions,there was no


apparent effect upon solid tumors in mice, even those induced by subcutaneousinjectionof ascitestumor cells.
In humans,all effectsupon the tumors were interestingand wilr be
discussedbelow. The influenceupon systemicpatterns was relatively reducedexceptfor a markedeffectupon the eliminationof calciumin urine,
obtainedeven with small doseswhich producedno other changes.Repeated
injectionsof organ cells treated in vitro with epichlorohydrinewere able
to induce severedegenerative
changesin the respectiveorgans.Experiments
with tumor cells treated and administeredin the same manner are stitl in
progress.
THE ELEMENTS
We have discussedpreviouslythe method used to classifythe elementsin
accordancewith their predominantbiologicalintcrvention.The place of
the elementsin the periodic chart, which establishes
the relationshipbetween their structureservedas a further basisfor this systematization.
The
capacity to induce changestowards an offbalanceof the same type was
found to be :r common property for clementsin the same series in the
periodic table. The series could be separatedinto two groups, one Ht
(from heterotropic)inducingan A and the other Hm (from homotropic)
inducinga D offbalance.Elementssystematizndas different periods in the
chart have been found to have predominantactivity in various compartments or groups of levels which form the hierarchicorganizationof complex organisms.we have related each element to a compartment (or
sometimeseven to a level), which didactica.llyis called the compartment
(or the level) of the element.We have tried to utilize this svstematization
in the study of the pharmacologicalactivity of elements.
From the beginning,severalbasicfacts about biologicalactivity became
apparent. Often the elements,used as such, do not induce the changes
which characterizetheir physiologicalactivity. Basicallyelementsact in
normal physiologythrough specificcompoundssuited to the compartment
or level to which they belong.In general,knowledgeof the level of an element permits us to identify also the proper compoundand its activity. The
interventionof an elementat levelsof the organizationother than its own
can be understoodonly in terms of the relationshipbetween the element
with its characteristic
compoundthe proper leveland the compoundspresent at other levels.
Two factorsare fundamentalin determiningthe activityof an element:
a) its availabilityand b) the possibilityto enter into its proper combination.

350

xEsE^RcH rN pHysropATHoLocy

The nature of any abnormality in activity of an element can be recngoizrl


only by relatingit to thesetwo factors.The amount of the elementavailable
and the capacity of entities of the level to which the element belonp to
manufacturethe proper compound,ultimately governsthe amount of the
elementat its own and at the other levels.
Under normal conditions, the entities of the proper level utilize only'
the amount of the elementneededto maintain the normal constants.The
rest of the elementusuallyis eliminated.An excessof the elementthus does
not induce a permanentexcessat the proper or higher levelsunder normal
conditions.Such an excessnormally is only temporary.A persistentexcess
of the elementat the proper level and at thc higher level indicatesan abnormal general availability. A persistentexcessat the higher level only
correspondsto a qualitativedeficiencyat the proper level. The organism
maintainsan excessof the elementat the higher level in an effort to compensatefor the qualitativedeficiencyat the proper level. Thus, an excess
of an element at the higher level indicatesa qualitative deficiencyat the
proper level, only if the value of this level is found low.
When there are low valuesat higher levels,we also need to know the
amount present at the proper level in order to interpret the abnormality.
Low valuesof the elementat higherlevelswith a low amount of the proper
level indicates a quantitative general deficiency,while a high value at
the proper level permits us to recognizea qualitativeexcessiveutilization
of the elementat the proper level.For example,in cancer,copper is low in
tumor cells and liver, but abnormallyhigh in blood. When a high amount
of copper appears in blood, the diagnosisof a qualitative deficiency in
utilization in cells or an abnormal generalamount can be made by investigating the amount in cells. A low amount of copper as found in tumors
and liver cells in subjectswith cancer make the qualitative nature of this
deficiencyevident.The trouble lies not in too Iittle copper availablein the
body but in the lack of the capacityof the cancerouscells to manufacture
the compoundthroughwhich copperbecomesactive,in this case,catalase.
A normal amount of an elementat its proper level reflectsnormal utilization.The pathologicalamountcan resultfrom a quantitativeor qualitative
abnormality,Any anomaly in an elementnot only means an inadequate
amount of it at its own proper level but in other levelsas well. A quantitative dcficiencyresultsin an insufficientamount at its proper level. A qualitativc deficiency-that is, incapacity of the entities to manufacture the
proper compound-also leads to a reduced amount of the element at its
own level.
Thus, in both cases,quantitativeand qualitativedeficiency,the amount

PHARM^CODYNAMTC ACTTvTTY

351

of the element present at its own level is low. However, in quantitative


defrciencythere is also a low amount of the element in the hierarchically
superiorlevel, while an increasedamount at this superiorlevel occurswhen
there is a qualitativedeficiencyat the proper level.
The same oppositevariationsbetweenthe amountsat the two levels is
seenin the caseof an excessiveutilizationof the elementat its proper level.
The amount of the elementat the superiorlevel is also high, if a quantitative excessis presentbut this amount at the superiorlevel is reduced as a
meansof controlling the excessiveutilization at the proper level if a qualitative anomaly occurs.This makesit possibleto recognizethe quantitative
or qualitative nature of the abnormalityin utilization of an element at its
proper level by determiningits amount both at this level and at the next
superior level. Too much of the elementpresentat the proper level indicates either cxcessivczmount present or excessiveutilization, while too
little at the proper level can be due either to a qualitativeor to a quantitative deficiency.A low amount at the higherlevel indicateseither a quantitative generaldeficiencyor a qua.litativeexcessiveutilization at the proper
level. An excessamount at the higher tevel indicateseither a quantitative
generalexcessor a qualitativedeficiencyat the proper level.
This relationship,which is also critical for the understandingof the
pharmacologyof the elements,can be summarizedin the fouowins table:
Amount of Element
at the ProperLevel

Amount of Element
at the Higher Level

high
Iow
high

high
high
low

low

low

Interpretation
: Occurrence
at the Proper Level
quantitativeexcess
qualitativeinsufficiency
qualitativeexcessive
utilization
quantitativedeficiency

From a practical point of view, we must have information on the


amount of the elementboth at the proper level and at a higher level. we
found that for the elementsproper to the cellular level, such information
can be obtained by comparing the amounts in plasma and red cells (or
total blood). It is not the ratio betweentheseynlus5-25 often supposedwhich is important, but rather the valuesthemselves.For changesat the
systemiclevel, the comparisoncan be made betweenblood and urine. the
latter corresponding to the level above the systemic.
The importanceof this conceptcan be seenin the following examples.
Potassiumis a cellular level element. In cancer, in offbalance type A,
potassium is present in abnormally higb quantity in proliferating cells. It

352

xEsEARcH tN pHystop^THoLocy

is also found in high amountsin blood red cells. In thesecases,potassium


is found in low valuesin the hierarchicallyhigher level in the blood plasma
or serum. The abnormality does not reside in a simple hypokaliemia,but
in excessiveutilization of potassiumat the cellular level, a low amount of
potassiumin red cells also would indicate a potassium deficiency. A high
amount of potassium in serum and red cells can be interpreted, as mentioned above, as correspondingto a quantitativeexcess.The reduction in
the quantity of potassiumin red cells, togetherwith an excessin the serum,
indicatesa qualitativedeficiencyat the proper level.
we have the true picture of the situation if we considerthat .,qualitative" excessor deficiencyis determinedby the ability to form the proper
compounds.While it is the elementas such which has to be administered
in order to correct a quantitative deficiency,other factors must be changed
to overcome qualitatively deficient utilization, excessiveutilization, and
quantitativeexcess.(FiS. 127)

trr

Excess

E
+

v,
E
:'
L

o)

(t)

TotalBloodK+rtq
F r c . 1 2 7 . T h e r e l a t j o n s h i pb e t w e e nt h e a m o u n t o f p o t a s s i u mi n s e r u m a n d i n t o t a l
b l o o d p e r m i t s t o i n d i c a t et h e e x i s t i n gc o n d i t i o n a s b e i n g i n n o r m a l l i m i t s , i n q u a n r i tative deficiencyor excess,or in an offbalancetype A or D.

PHARMACODYNAMIC ACTIVITY

,,/ 353

Although these relationshipsrepresentthe most important aspect of


the pharmacodynamyof the elements,still others must be considered.An
excessor deficiencyof an elementat a superiorlevel.even if it servesas a
biological defensemeans to combat an abnormality at a lower level, representsa problem by itself for the supcrior level.The fact that the element
doesnot belongto this levelgivesa noxiouscharacterto its influence.The
influenceexerted upon sensitiveorgansoften inducesimportant abnormal
manifestations.
Hyperkalemia,evenoriginatedby an abnormallylow utilization of potassiumat the cellularlevel,can lead to serioustroublesin the
function of the nervoussystemor of the heart.
Another importantaspctof the reactivityof an elementis its influence
upon levelsbelow its proper level. An elementacting at a lower level usually has a biologicallyoppositeeffectto what it has at the proper level and
thereforeis a noxious influence.At the lower level, the A or D type of
activity of the elementis reversed.
Sodium, for instance,which is an agent of the A type of the metazoic
compartment,producesan offbalanceof type D at the cellular level, which
is hierarchicallyinferior to its own. Similarly, Mg, which is a D agent at
its own metazoiclevel,has an A inducingactivityat the cellularlevel.
Analysisof the pharmacologyof elementsin terms of their A or D inducing activity, the level at which they belong and the compoundsthrough
which they act, is still only in its early stagesalthough it representsa program of great promise. ln the presentationwhich follows, we will try to
interpret the data concerningthe elementsin terms of A and D inducing
activity. We start with Hm elementshaving a D inducing activity. They
parallel in their action the lipoids, with a negative polar group. Tear-E
XVII lists Hm elementsor the D seriesand relatesthem through their
periodsto the organizationalcompartments.
T,rsI-e XVII
Hr'r Elevexrs
Compartments
Organic
Metazoic
Cellular
Nuclear
Submorphol.
Primary Biol.
Submolecular

Metals
Be
Mg
Ca Sc V
S r Y Nb
Ta
Ba
Ce N d
Ra Th U

Non-Metals

c
si
Mn
Tc
Re
Sm
Pu

Co C u
Rh Ag
Ir
Au
C d Dy
Cm Cf

Ge
Sn
Pb
Er

o
s
Se
Te

Yb

We have alreadydiscussedthe pharmacologicalactivity of sulphur and


seleniumthrough the compoundsin which they enter and will not discuss

354

REsEARcH rN pHysropATHoLocy

them again here. Before analyzing the other elements,it is of interqst to


emphasizeagain a principal characterof their activity. As most of the elements act through specific compounds, the factors which determine the
entry of elements into specific biological combinations appear to be of
capital importance. Availability of the element alone is only one factor in
its pharmacodynamy.with this in mind, we have investigatedsome of the
elementsof this Hm group.
we know little about any influenceof berryllium as a metal upon the
organismas an entity. Its toxic effectsare due to abnormal amountsactive
at lower levels.In the same period of the chart of elements,we have two
nonmetals,c and o, for which the organismrepresentsthe proper level.
The general pharmacologicalnature of oxygen is indicated by the role of
oxidation in metabolic changes.Oxidation representsthe first step toward
catabolic,homotropic changes.The respiratoryphasein the metabolismof
carbohydrates,the oxidative fissionof fatty acids, and the oxidative desaminationof amino acids representexamplesof the fundamentalhomotropic interventionof oxygen.
Acting at the systemic level, immediately inferior to its own level,
oxygen has a different action. According to the rule mentioned above,
oxygen,a D agentat its proper level, will have an A activity for the blood,
which we will study below togetherwith the other A agents.The homotropic relationshipof oxygcn to fatty acidswas discussedabove with the
study of the biological role of thesesubstances.
The relationshipbetweenCo2 and fatty acids also is interesting.Large
amounts of free fatty acids in the blood were seen to allow better fixation
of COz to hemoglobin,just as large amountsof sterolsdo for oxygen. We
have investigatedthis correlationbetweenCo2 and fatty acids by keeping
a fatty acid, such as linoleic acid, in an atmosphereof Co2 connectedto a
manometer.A manifest negativepressureresults.The venous blood, rich
in CO:, which also shows a predominanceof fatty acids, loses CO2 and
fatty acids during passagethrough the lungs.
Magnesium
Among offbalanceD inducing elementsof the II A seriesof the periodic
chart, we first studied magnesium,which belongs to the metazoic compartment and thus, is related to the sea as original environment.Much of
the activity of this element can be interpreted as D inducing activity. Mg
in many respectsis antagonisticto Na, the cation of the samecompartment,
a memberof a serieswith an oppositeA inducingcharacter.High valuesof

P H A R M A C O D Y N ^ M I CA C T I V I T Y

,/

355

Mg found in blood were related to adrenalinsufficiency,(127) while low


It'lglevelsusually occur when blood cholesterolis high. (128)
Excessof Mg was seento induce adrenaldeficiency.we explainedthis
actionthrough the antagonism betweenMg and Na. We could thus counteractthe salutary effect of NaCl in adrenalectomizedrats through administrationof magnesiumsulfate parenterally.A similar effect was obtained
evenwith oral use of magnesiumthiosulf.ate.(Note 9)
The relationshipof magnesiumto the defensemechanismis of special
interest.
Mg seemsto intervenein the lytic effectof sera upon ascitescancercells (extensivelystudied in our laboratory by R. Willheim, p. Fluss
andM. Auber) (129) which, as we have seen,representsa characteristic
featureof D inducing activity. Similarly, magnesiumpreventsthrombosis,
actingas an antithrombocyticagent.Not only does it prevent the appearanceof fibrin, partly preventing the destructionof thrombocytes(130);
but it also favors the lysis of already existing thrombi. ( l3l ) It appears,therefore, to be a valuable agent in the treatment of thrombosis.
( 132) Its concomitantaction againstcholesterolhas led to its use in the
preventionand treatmentof coronary thrombosis.
Magnesiumappearsto be part of anotherdefensemechanism,the nonspecificone, representedby the properdin system.Properdin is active only
in the presenceof magnesium;neitherca nor Na can replaceit. (133)
Higheramountsof magnesiumincreaseproperdin activity.
Magnesiumsometimesis seento parallel the action of cu, another D
inducingelement. In animals fed milk too long, leading to a type A offbalance,the amount of magnesiumfalls along with the amount of cu. The
quantityof magnesiumin the blood is low in humans with convulsion.
(134) Mg appearsto be espcciallyeffectivein the prevention and treatmentof "grass tetany" in animals, which often follows feeding on grass
withhigh potassiumcontent.( 135)
Magnesiumhas a similar antagonismtoward K and this can explain
its activity in cancer. Lower than normal valuesof magnesiumare found
in cancer,as opposedto high amountsof potassiumwhich is an A inducing element, and the low Mg seems to favor cancer growth. Moisture
increases
the amount of K and lowers the amount of Mg in plants, a fact
relatedto cancer frequency in various geographicregions. (136) The
difference
between the preventiveand curative actionsof magnesiumis of
specialinterest. Administered after a carcinogenhas becn applied, magnesiumreduces the percentageof cancers induced. (137, 138) It has
minimal influence, however, once the tumor has appearedor upon trans-

356

R E s E A R C Hl N

pHystopATHoLocy

planted or spontaneoustumors. We will discuss this occurrence below


togetherwith the effect of other elements.
It is necessaryto bear in mind, when we have to choose nonspecific
combinationsin which to administer it, that the D inducing activity of
magnesiumis particularly manifestedat the metazoic level. Magnesium
sulfate appears to be suitable for parenteral use, while the thiosulfate
appearsto be suitablefor oral administration.In theseforms, Mg has been
found to induce marked local alkalosisin the secondday wound crust pH
and to produce salutary effectsupon pain of the acid pattern. A marked
influenceupon thiamine-inducedconvulsionsin rats and mice has led us to
usemagnesiumthiosulfateas a tool not only in tetany but in the treatment
of convulsions.It appearsto be effectivein preventingepileptic seizures
and valuableeven in casesof statusepilepticus.We have utilized the same
preparationsuccessfullyin cancer when pain and preterminal conditions
correspondingto an A offbalancewere present.Less important effectsare
seenfor magnesiumsulfate and magnesiumthiosulfateat lower levels.
Calcium
The biological activity of calcium, another member of the II A series
and a D inducingelementbelongingto the cellular level, also is of interest.
In its absorptionby grass from the soil, Ca parallels Mg, another D
inducingelement,but opposesK, an A inducingelement.Grasstetany is
thus induced by high K and low Ca and Mg values.Ca, which is another
D inducing elementlike copper is antagonisticto zinc, an A inducing element. It has been observedthat cancr is less frequent in the so-called
calcerousclay regionswhere the soil is formed by limestone.(139) Together with other minerals, an optimum of calcium in soil may help to
prevent cancer. While SiO,,favors cancer,Ca appearsto prevent it. Calcium also is an antagonistto zinc which, in high doses,seemsto favor the
developmentof cancer. (140) The relationshipbetweenCa and K has
permitted us to be more precise about the role of Ca in cancer pathogenesis.As opposedto K, which increasesby as much as 60Vo in tumors,
the contentof calciumdecreases
by 44Vo. ( l4l -147)
Confronted with K and Ca changes,it appearedinterestingto see to
which element we could directly attribute the increasein malignancy. In
the regenerationof liver cells, where rapid growth without malignancy
takes place, potassium is increasedwhile the amount of calcium is unaltered.Similarly in other rapidly growing but normal cells, calcium is not
diminishedwhile K is increased.Potassiumthus appearsto be related to
the processof cellular growth and multiplicationwhich representsalso an

P H A R M ^ C o D Y N A M t C A C r l v r T Y

3 5 7

addedfactor in transformingnoninvasiveinto invasivccancer.However,


potassiumis not directly related to the cancerouscharacter of the cells.
on the other hand, reduced amountsof calcium appear to be peculiar to
the cancerousprocess
. (147 ) The reducedcalcium in cancer is not due to
a lack of the elementin the organismsince calcium is not only available
but even apparently present in excessat the systemiclevel. As we have
shown,a high urinary calcium index, indicating exaggeratedexcretion, is
presentin the type A offbalance.The anomaly residesin the low capacity
of the cancerouscells to fix and properly utilize calcium. As calcium acts
at the surfaceof the cell and its deficiencyreducescellular adhesiveness,
(148, 149) lack of cellularcalciumcan be seento increasethe invasivenessof cancercells and the tendencyto metastases.
Deficiencyof calcium
in cells appearsrelated to the characterof youth while excessseemsto
resultin rapid agng.
The anomaly induced by the qualitativedeficiencyin calcium thus appearsto be at the cellular surface.Relatedto it also are manifestationsat
the tissular level.
Administrationof any calciumsalt inducesa manifestincreasein local
alkalosis(Fis. i,28)of the secondday wound crust pH. It appearedinterestingthat in bone lesions,especiallyin bone cancermetastases,
the offbalancetype A is characterizedby an osteolyticprocess,the D type by an
osteoplasticone. The local acidosispresentin lesionswith an A type of
offbalanceexplainsthe mobilizationof calciumin theseosteolyticprocesses.
ca is depositedin important amountsin metastases
with a type D offbalance,a fact which can be related to the local alkalosisresultingfrom the
abnormal metabolism.This alkalosisrepresentsa condition favoring the
precipitationof calcium. Indirectly, the deposit of calcium in bone metastasesappearsto corrcspondto the D pattern of tissular abnormality.
Calciumhas a D inducingactivityevenin this case.
with calcium excreted in excessthrough the urine, the problem of
calcium pharmacologyin the A type of cancer is related to the form in
which it acts at the cellular level, which appean qualitativelyimpaired. As
the quantitativedecreasemust be considercdto be a consequenceof
qualitativeinsufficiencyand not a generalquantitativedeficiency,the problem is not to provide calcium but to find a way to insure better utilization
at the cellular level. It is for this reasonthat administrationof most calcium sals does not influencethe evolutionof experimentalor clinical cancer, but has a preventive effect upon the induction of tumors through
carcinogens.Administered after the injection of the carcinogen,calcium

358

xESEARcH rN pHysropATHoLocy

appearsto reduce the percentageof positive results.Administered after the


tumors have appeared,the influenceis minimal or nil.
As we have mentioned above, an excessivecalcium excretion is, in
itself,sufficientto indicatethe cxistenceof a deficiencyin calcium utilization at the cellular level without a calcium deficiencyin the organism.The
therapeuticindication is for agentsable to influencethe fixation of calcium
at the cellular level. Fatty acids which changethe cellular metabolismso
t o
>
t
o
i6
|
Fo
l r
6 C
I
r L
6 0
|
t ! l u o
-- l
L
t
O V

G
($

F r c . 1 2 8 . T h e i n f l u e n c ee x e r t e d b y t w o e l e m e n t s ,p o t a s s i u ma n d c a l c i u m , u p o n t h e
second day wound crust pH shows a frank tendency toward acidification for potassium and alkalinization for calcium. For diflerent salts, the differences result from
t h e u n e q u a l i n f l u e n c ee x e r t e d b y t h e a n i o n w h i c h w o r k s a d d i t i v e t y t o t h a t o f t h e
cation.

as to induce local a.lkalosis,have appearedto be the most active agents.


Testosterone,and calciferolhave appearedhelpful but not nearly as active
as fatty acids.
while high urinary excretionof Ca indicatesa type A oftbalance,low
or no urinary excretioncan result either from an excessivecellular utilization of calcium or from a type D oftbalanceat the cellular level. other
analysescan be used to indicate the probable occurrence.With calcium
excretion low and other analysesindicating type D offbalance, all the
chancesare that the low excretionis part of the D offbalance.If, on the
contrary, only the ca excretionis low and the other values correspondto

358

r.ESEARcHrN pHysropATHoLocy

appearsto reducethe percentageof positiveresults.Administeredafter the


tumors have appeared,the influenceis minimal or nil.
As we have mentioned above, an excessivecalcium excretion is, in
itself,sufficientto indicatethe cxistenceof a deficiencyin calcium utilization at the cellular level without a calcium deficiencyin the organism.The
therapeuticindication is for agentsable to influencethe fixation of calcium
at the cellular level. Fatty acids which changethe cellular metabolismso
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i
|
o C
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o o
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F r c , 1 2 8 . T h e i n f l u e n c ec x c r t e d b y t w o e l e m e n t s ,p o t a s s i u ma n d c a l c i u m , u p o n t h e
second day wound crust pH shows a frank tendency toward acidification for potassium and alkalinization for calcium. For diflerent salts, the differences result from
t h e u n e q u a l i n f l u e n c ee x e r t e d b y t h e a n i o n w h i c h w o r k s a d d i t i v e l y t o t h a t o f t h e
cation.

as to induce local alkalosis,have appearedto be the most active agents.


Testosterone,and calciferolhave appearedhelpful but not nearly as active
as fatty acids,
While high urinary excretionof Ca indicatesa typ A offbalance,low
or no urinary excretioncan result either from an excessivecellular utilization of calcium or from a typ D offbalanceat the cellular level. Other
analysescan be used to indicate the probable occurrence.With calcium
excretion low and other analysesindicating type D offbalance, all the
chancesare that the low excretionis part of the D offbalance.lf, on the
contrary, only the Ca excretionis low and the other values correspondto

PH^RM^CODYNAMIC ACTIVITY

359

A type of offbalance,the probability is a quantitativelack of calcium in


the organism. This can be corrected by the administration of calcium
parenterallyor orally in any absorbableform. Low excretion as well as
somesymptoms can be overcome in a short time through the adminl563tion of sufficient calcium. In the opposite type of case, with metabolic
calciumretention,administrailonof calcium will induce an increasein the
intensityof symptoms.
Excessof calcium in the urine thus correspondsto lower values at the
cellularlevel and need for fixation of calcium. This indicatesagain that
the problem is not the amount of calcium presentbut the deficiencyin its
utilization.
Copper
copper, from the lB seriesand another anti-A elementof the cellular
level,appearsindispensablefor the synthesisof heme of catalase.A deficiencyof copper results in reducedactivity of this enzyme.Similarly, the
synthesisof hemoglobinis possibleonly in the presenceof copper. cytochrome oxidase contains Fe and Cu. Cu deficiencyreduces liver cytoc h r o m eo x i d a s e .( 1 5 0 , l 5 l , 1 5 2 ) c u i s p r e s e n it n b l o o d s e r u mb o u n d t o
a proteinto form ceruloplasminwhich also acts as an oxidase.(153) Cu,
while favoring the synthesisof these substances,intervenesultimately in
the processeswhich lead to the active catabolicintcrventionof oxygen. In
this way, cu acts as a D inducing agent. cu intervenesactively in the
metabolismof sulfur. (154, 155) The transformarionof sulfhydryl to
disulphideis slow and incomplctewhen there is a copper deficiency.The
samedeficiencyreducesthe formation of phospholipidsas seenin rat liver.
(156) Indirectly,Cu favorsanti-A activities.
The influenceof cu upon ca metabolismalso is indirect.we have seen
that a local depositof calcium in bonescorrespondsto a local D pattern.
Deficiencyin copper, a D inducing agent,permits the appearanceof local
A conditions,which in the case of bone, will result in a lack of calcium,
theoppositeof what is seenin local D offbalance.This correlationexplains
why, in spite of sufficientP and Ca, the lack of coppr inducesan osteomalaciawith bone fracture and symptomsof rickets, as seen in animals
with an indirect Cu deficiencycausedby an excessof molybdenum, ( 157,
158, 159) an A inducing agent.The administrationof copper helps to
repairthesefractures.(157) Parallelreductionsin Cu and Mg occur in
milk-fedcalves. (160) A richnessof zinc, like molybdenum,can provokea deficiencyin Cu and Ca. With copper deficiencyand low catalase,
resistance
to infectionsis lowered,In brucellosis,
a deficicncyof Cu and Ca

360

nEsEARcH rN pHystopATHoLocy

coincideswith reduced concentrationsof Mn and Co in the blood and


p i t u i t a r yg l a n d s .( 1 6 1 , 1 6 2 , 1 6 3 ) C u , C a , M n a n d C o a r e a l l m e t a l so f t h e
D inducinggroup.
In cancer,a qualitativedeficiencyof copper is found. A frank reduction of the catalasecontentis seenin cancerouscellsas well as in the liver
of canceroussubjects,( I 64, 165) on the other hand, cu contentis considerablyincreasedin the blood of thesesubjects,with valueseven three
times greater than normal, often encountered.(166. 167, 168) These
valuesreturn to normal if the canceroustumor disappears.(169)
In cancer,copper deficiencyat the level of the cells is manifest,shown
not only by reducedcatalasebut also by upsetsin the cytochromeoxidase
system,the heme system,the sH metabolites,
and the phospholipids.The
deficiency,however, is only local and consequent.lyqualitative since an
excessof Cu is found at the immediatelysuperiorlevel, the blood.
A local abnormalityresidingin inadequatecapacityto utilize copper
thus appearsrelatedto cancer.In normal animals,coppr in excesscan be
utilizedand is able to prevcntthe appearance
of tumors.This explainswhy
Cu, which protectsrats againstcarcinogenetic
azodyes,(170, l7l, 172)
does not influencethe tumorsonce they have been induced,i.e., once the
qualitative insufficiencyis present.The recognitionof this difterence between the form in which copperis utilizedby the normal animal and the
deficiencyin the cancerousentitics,hasbeenthe basisfor a seriesof studies
concerningthis key problemin the pathogenesis
of cancer.The therapeutic
useof copper-and of other elemcntswhich we will discussbelow-is not
a quantitativebut a qualitativeproblem.
Manganeseand Cobalt
Two other elementsof the cellularcompartmenthave appearedinteresting.Manganeseby interveningas a catalystin processes
resultingin an
activationof oxygen,indirectlymanifestsD inducingcharacter.Its presence
in smaller amounts in tumors or cancerousorgans than in controls, has
beenconsidered.Just as with copper,no effectsare seenin treating tumors
with manganesecompoundsalthougha certain degreeof preventiveaction
is obtainedin tumors inducedby carcinogens.Similarly,with cobalt we
have obtained a certain degreeof preventionagainsttumor induction by
carcinogensbut no effect upon the evolution of tumors once they have
appeared.No effectshave beenseenin transplantedtumors.

r
.i::

-ia$^

, :, ;

. a:ia ii,*:.tj1l\!.rl
n:!ii:
iil\r*#iiini

a
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P H A R M A C O D YN ^ M I C

A C T I V I T Y

361

Heavier Elements
we have studiedthe activity of elementscorrespondingto the lower
lgvsl5-5ugh as strontium and tin for the nuclear;barium, gold and lead
for the submorphologic;and cesium for rhe primary biological compartments. The lower the level of the element,the greater appearsto be the
preventiveeftect againstinduction o[ tumors by carcinogens.But the minimal, or completeabsenceof effect upon alreadyexistingtumor cells remains unchanged.
As mentionedabove,we conncctedthis paradoxicalactivityto the fact
that, while normalcellsare able to manufacturecompoundsthroughwhich
the appearanceof a cancerousentity can be prcvented,these compounds
are no longerformed if an cntity is alreadycancerous.
These have been the considerations
forming the basis for an entire
seriesof studiesof the role of variouselementsin the pathogenesis
of
cancer.
Elements in the Pathogene,sis
ol Canc'er
Investigationshave been made of the amount and form of the element presentin the normal animal as contparedto the cancerousanimal.
The quantitativeand especiallyqualitativedifferenceshave been seen to
indicatethe site of the abnormalitylargelyresponsible
for the lack of influenceexertedby the element.The resultsexplainwhy the administration
of the elementalone is unable to influencethe cvolution of an a-lready
existingcancerousprocess.More interesting,
they show what compoundof
the elementcould have an influence.This research,which is in progress,
opens the door for possibletherapeuticapplications.It is through such
compounds,presentin the normal and lackingin the cancer-stricken
animal, that attemptsare beingmadeto influencethe evolutionof cancer.An
important step has been the finding that suitablecompoundscan be obtained by the treatmentof fresh organsin vitro with some of the elements.
Their study may make possiblesyntheticpreparationof suitable compounds.The few resultsalreadyobtainedin experiments
with animalsconfirm that D-inducing activity representsa factor which the promising elementsshare.

C H A P T EI R5
PHARMACODYNAMIC
ACTTVTTY(PART TW O)
ANTI.FATTY ACID GROUP

ID
ll- nnellrL

To INVEsTTcATIoN
oF AcENTScapable of correcting offbal-

ancesof type A, attentionwas directedto agentsthat miglrt influencethe


oppositeoffbalance,type D. Since fatty acids are involved in the pathogenesisof type D offbalance,agentswith anti-fatty acid propertieshad to
be sought as correctives.Some of these are natural constituentsused by
the body to control normal and abnormalinterventionof fatty acids. They
were consequentlyisolated and studied. Synthetic agents also were obtained and studied,their choice largely inspiredby the control mechanism
used by the body.
Anti-Fatty Acids Constituents
We have seenthat a free fatty acid losesmost of its biological activity
rvhenits polar group is bound to anotherradical. This led us to inveswhich naturally are bound to fatty acids. It could be
tigate substances
shown further that each major group of fatty acids is bound in the organism to specificconstituents.Thc saturatedfatty acids are principdly bound
acidsas lecithins,
to glycerol,the low unsaturatedacidsto glycerophosphoric
and the high unsaturatedmembersto sterols.The conjugatedfatty acids,
found in abnormal conditions,appcaredto be opposedby neoglucogenic
corticoids.The constituents'were
conceivedof as being naturally occurring
first effort was to study how they interacid
substances
and
our
anti-fatty
vene to balance the activity of fatty acids, especiallywhen the latter act
as pathogenicfactors.

p H A R M A C o D y N A M T cA c r t v t r y

(p,rnr rwo)

363

one, a relatively
wereinvestigatcd:
In this study,two typesof influences
direct effect induced through a neutralizationof the energeticcenters resulting in a more or less advanceddegreeof inactivationof the fatty acid;
the second,an indirect effect achievedthrough changcsin the metabolic
processes
in which fatty acidsintervene,In a differentkind of intervention,
the anti-fatty acid to which a fatty acid is bound governs its ultimate
biological fate. For example,the bond to glycerol favors caloric metabolism. The bond to glycerophosphoricion converts a fatty acid, saturated
as well as unsaturated,into an organizationalconstituent.The bond to
sterolsfavors a functional role, even for monoethenoids.
We started the study of the naturally occurring anti-fatty acids with
those agents known to be bound to fatty acids in the organism. The
simplestsuch agentis glycerol.
Glycerol
Glycerolis the most ubiquitousfatty acid-bindingsubstancein nature.
We attempt to explain this fact on the basisof glycerol'sstructureand the
specialbiological role it confers.We have seen that fatty acid molecules
take reciprocal parallel positions when they form monomolecularlayers.
In their bond to glycerol,thesefatty acid moleculesconservethis recipro(Fig. 129)Thiscould cxplainwhy, in the body, the bond of
cal relationship.
being
fatty acid and glycerolalwaysis a triglyceride,mono and digJycerides
(?)

CD

(-)

c)

(-)
(J
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O
c(\.1) o
- t -: E
a

(?)

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cf)
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cf)
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(-)

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-(J

F t c . 1 2 9 . T h e f a t t y a c i d r a d i c a l st a k e i n t h e t r i g l y c c r i d em o l e c u l e sa s i m i l a r p a r a l l e l
position as when they form a monomolecular la1'erat the surface of water.

364

nESEARcHrN PHYsropATHoLocY

only intermediatesteps.The samefatty acid has different biological activities if administeredas free acid or as triglyceride.The combinationseems
to serveas an energy-furnishingmetabolite.Bound to glycerol,fatty acids
with long or short chains, saturated,monounsaturated,polyunsaturated,
and even conjugated,seem to representenergeticreserveswhich are utilized as caloric metabolites,especiallyin those specieswhich are able to
storethem.
With this relationshipin mind. we administeredglycerolwith two objectives: I ) to obtain, as an immediateeffect,the inactivationof the free
fatty acids presentin abnormalconditionsthrough the neutralizationof
their polar groups, and 2) to eliminatethesefatty acids by turning them
into caloric metabolites.
Studying the activity of glycerol at different levels, we could see no
influence upon phages. However, an indirect effect was observed upon
viruses.Glycerol is widely used as a specialmedium for the preservation
of virusesin tissucs.Its preservative
value can be correlated,at least in
part, with its influenceupon fatty acids.We have seenthat fatty acids have
a noxious effect upon viruses,leading to their disappearancein various
organs. The treatment with purc glycerol reduces autolysis of organs
through a dehydrationeffect.Curtailingthe lytic activity of the enrymes
active in autolysis,glycerol reduccsthe amount of fatty acids liberated
through such autolysis,and thus preventsthe destructionof the virus.
Glycerolmay also preservevirusesby acting antagonistically
to any fatty
acids which still manageto appear.
Glyccrol has a bacteriostaticeffectupon only a few speciesof microbes
and only when appliedin high concentrations.
A minimal influenceupon cells was seenfor glycerolin Tetrahymena
pyriformis and ascitestumor cells. To study its action at higher levels,
glycerol was administeredorally or parenterallyto animals or humans.
Solutionsof ZOVoglycerol were well tolerated when injected subcutaneously or intramuscularly.It should be noted that when glycerol was administeredto complcx organisms,it was largely absorbedand circulated
without alteration,a fact which would explain the effectiveness
of relatively small amounts.At the tissuelevel.glycerolinduceda changeof the
local pH of a lesiontoward the acid side,as seenin the secondday wound
crust. Figure 130 illustratesthis. The changeexplainsglycerol'saction in
increasingintensityof acid pattern pain and decreasingintensityof the
alkaline.This influenceupon pain was obtainedconstantlywith very small
amounts,permittingthe use of glycerolevenas a testfor diagnosisof pain
pattern.Intramuscularinjectionof t/z cc. of a 2Q% solutionor oral ad-

p H A R M A c o D y N A MAt c r r v r r y ( p , r n r r w o )

365

minisuation of. Vz cc. of a 5oo/osolution in water has been used for this
purpose. However, later, when other agcntswere found to produce even
more overt responses,we stoppedusing gJycerolas a routine test.
Glycerol has almost no beneficialinfluenceupon the healingof wounds
or radiationlesions.Healingwas evenretardedin someexperiments.
Various changesin the evolution of tumors occur when host or transplant are
trcated with g.lycerol.In some, these changesare minimal; in others an
obvious reduction in grofih occurs. In a high proportion (12/20), a
marked involution has been noted for Walker tumors in rats. Ascites Sa
180 in mice, after repcatedintraperitoneal
injectionsof a solution of.ZVo

GI ycerol

F r c . l l 0 . C l y c e r o l i n d u c e sa l o w e r i n go f t h e s e c o n dd a y
wound crust pH.

aoaa
aa
.O

glycerol, disappearedin TOVoof the cass.A lesser effect was seen in


Ehrlich and Krebsascitestumors and still lessin the solid tumorsobtained
with these ascitescells. (Fig. I3 I ) One of the most interestingeffectsof
glycerolwas that seenupon the tumorsin humanswherea manifestinvolution was obtainedin casesin which an offbalanceof type D was present.
This important effect will be discussedbelow with the therapeuticuse of
glycerol.Glycerol administrationhad an interestingeffectupon the amount
of cholesterolin the blood in a few subjects.When ten drops of glycerol
weregivenorally threetimesa day for a month or more,cholesterolvalues

366 /

RESEARCH IN

PHYSIOPATTIOLOOY

OO

Control

Start t reatnent

Ireatedrith
Glycero
I
rith
Treated
Glycerol
Ireatedrith
Glycerol

. l
. l
. 1

I -,.

F t c . l 3 l . C h a n g e si n d u c e di n W a l k e r t u r n o r si n r a t s by the treatnlcntsof the aninrals


with glycerol (daily subculaneousinjections with /: cc of 57a glycerol solution in
saline isotonic solution).

In somepatients,with no changein diet and no medicationother


decreased.
glycerol,
valuesoriginally above300 mgr./100 cc. serum fell to below
than
170 mgr. If thcsepaticntsalso were hypertensive,long-term administration
of glycerol produceda reductionof blood prcssure.
An impressivehcmorrhagiparcilect was noted, frequently ulccrated
lesionsstarting to bleed shortly after administrationof even a few drops of
glycerol. The relationship of hemorrhagingto glycerol was clear when in
the same subjects, repeated administration of this agent invariably was
followed by bleeding. The bleeding usually was arterial; only occasionally
was an oozing hemorrhagesecn.
Many years ago we became interested in studying, in a group of
severelyburned subjccts,the rolc of fatty acids in the pathogenesisof burn
complications.Glycerol was administercdto thess patients with good effects
upon pain. Before the use of antibiotics, one of the principal manifestations in burn patients with widely infectcd wounds was repeated chills.
These chills also were influencedby glycerol. (Note // A direct action upon
the parasympatheticsystemcould be attributed to glycerol and could explain the effect upon chills. This view has been confumed by studying the
eflects upon cardiac rhythm produced by intravenousadministration of
glycerol in rabbits. (Note 2)
Convulsions could be induced by glycerol in animals and also were
seen to occur in humans. (Note 3/ They could be induced, with much

pHARMAcoDyNAMtc

(p,rnr rwo)

^crtvrry

,/

367

smaller dosesin animalswhen, along with glycerol,an otherwiseharmless


dose of deoxycorticosterone
acetatewas administered.Injection of 0.1
mg. of this hormone in mice weighing25 to 30 grams, followed by an
injection of.Vc cc. of a 57o solutionof glycerol,inducedconvulsionswhich
r+'ereusually lethal. In terminal cancer patients,too, concomitantadministration of the cortical hormone and glycerol for a few days has produced
convulsiveseizures,which proved to be lethal in one subjectin whom no

E , *rr
?1

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2
27 to5//l

- J T J J J J I J I J J I ,

S p .l6
Grav.
, 6o
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62
pHf.1

Cll.a
/

S . T61.
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J
L

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v,
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+
><
F r c . 1 3 2 . T h e a d m i n i s t r a t i o no f I c c g l y c e r o l d a i l y i n d u c e sa c h a n g eo f t h e u r i n a r y
pH toward thc type A, before changesin the other analysestake place.

368

REsEARcHrN pHysropATHoLocy

previous abnormal cerebral manifestationshad been observed, and who


had receivedthesetwo medicationsfor only a few days.
Of the different systemic manifestationsinfluenced by glycerol, the
eftect upon acid-basebalance is most striking. Even before any other effect
upon systemicanalysesbecomesapparent,an immediatechangeof urinary
pH from acid to alkaline values is induced by glycerol administeredin
sufficientquantity.(Fig. 132)
Glucose
The pharmacologicalactivity of glycerolhas raisedthe questionof the
relationshipbetweenthis substanceand glucosesince some of the manifestationsinduced by glycerol could be obtained with glucose.The same
eftect is seenon local pH in the secondday wound crust. Glucoseadministeredfor a few days prior to, and immediatelyfollowing, wound induction
shifts local pH toward more acid values.A similar effect is obtained on
the pH of tumors.(173) Glucosedecreases
intensityof pain of the alkaline
pattern and increasespain of acid pattcrn. We were able to induce convulsionsin rats by injecting20 to 25 c*. of an isotonicsolution of glucose
subcutaneouslytwice daily. After 4 to 6 days, convulsionsappearedand
often led to death. [n some terminal cancerpatientswith brain metastases,
who had had previousconvulsions,intravenousadministrationof glucoseas
a therapeuticprocedureproduced convulsions.We have even seen lethal
convulsionsin a patient after a few days of intravenousadministrationof
glucosein salinesolution in conjunctionwith intramusculardosesof I mg.
twice daily of deoxycorticosteronc.
Although rarer than for glycerol, hemorrhagesoccurred after each
glucoseadministrationin some patientswith previouslybleedingulcerated
lesions.Bleeding stopped when glucoseadministrationwas discontinued.
Renewedadministrationwas followed each time by renewedbleeding.We
want to emphasizethis relationshipof glucoseto hemorrhagebecauseof
its clinical importance.
It seemspossiblethat glyceric aldehydeand glyceric acid, which appear during glucosemetabolism,play a role in the manifestationsmentioned
above.
GlycerophosphoricA cid
The ability to relatethe pharmacologicalactivity of glycerolto its bond
with fatty acids,has led us to consideranothersubstanceable to bind fatty
acids,glycerophosphoricacid. The bond of this acid with quaternarybases
such as choline or ethanolamine,and the fatty acids results in phospho-

p H A R M A c o D y N A M T c^ c r t v t r y

( plnr

rwo)

369

lipids which take part rn the formation of boundaries and separating


membranes.Although variousfatty acids,both saturatedand especiallyunsaturated,enter into these phospholipids,resulting in a variety of compounds, their generalbiological behavior appearsto be the same. Certain
fatty acids, the di- and tri-ethenic,however, are preferentiallybound as
phospholipidswhen they passfrom the intestineinto the circulation.
In investigatingits influenceas an anti-fattyacid agent,we administered
glycerophosphoric
acid, 50 cc. of a n/10 solutiondiluted in 1000 cc. of
isotonic saline,glucoseor other solutions,intravenouslyor subcutaneously.
It had salutary effectsupon pain of an alkaline pattern and upon corresponding lesions. A manifest influence was exerted upon the systemic
acid-basebalance,especiallyin caseswith high urinary pH and with all
other analysesshowing an offbalanceof type D. No influencewas observed
upon evolution of tumors in animalsor humans.
The increasedbasal metabolismand a marked increasein work capacity, which are observed in subjectstaking sodium glycerophosphatefor
long periods,has made us suspecta possibleeffect upon thyroid secretion.
The appearanceof thyrotoxicosisin a subjectwho had inadvertentlytaken
seemedto confirm this view.
a large doseof sodiumg.lycerophosphate
Sterols
A third group of natural constituents,which act as anti-fatty acid
agentsis composedof sterols,which are absorbedand circulatedbound to
polyunsaturatedfatty acids. Except in brain and red cells where only free
sterolsare encountered,the sterolsare found both as estersand free substancesin aU cells,tissuesand organs.
Cholesterol,phytosteroland a few sterolswere utilized in pure form in
our research.In addition,sterolswere obtainedand used as mixtures,as
in the insaponifiablefractions of tissues,organs,organismsand biological
products.In some studies,thesefractionswere further separatedinto conWe usedcholesterolin
stituents,largelythroughcolumn chromatography.
(Note 4)[Natery or gum cellulosesuspensions
were
differentpreparations.
used for in vitro and in vivo studies.
Cholesterolwas seento inducea changein the shapeof some bacilli,
such as B. subtillis, B. megatheriumand B. anthracis,turning them into
irregularround formations.At the sametime, their Gram positivestaining
became abnormally intense. The agar cultures had a creamy asPect.
The influenceupon Gram positivity explains the fact that Gram positive
individualscould be obtainedin culturesof variousGram negativemicrobes
such as Esch. coli or Eb. typhy after repeatedtreatmentwith colloidal

370

nESEARcH rN PHYSToPATHoLocY

cholesterolpreparationsaddedto broth. The Gram positiveforms, however,


could not be isolated.
Cholesterolwas seento influencered cellsin form, shape,volume, sedimentation, velocity, and oxygen-combiningcapacity. A vermillion color,
which persistedfor a long time, was obtained through in vitro treatment
of blood with cholesterolor through intravenousinjectionsin animals.Although such injections were lethal, they induced the abnormal vermillion
color. Cholesterolproduceda manifestchangetoward lessalkalinevaluesin
the secondday wound crust pH. A favorableeffectupon rabbit skin wounds
was obtained,with abnormallyintensiveproliferationof the epithelium,
this healing effect, howevcr, was less manifest for irradiated wounds.
In subjectswith ulceratedlesions,prolongedadministrationof cholesterol
was frequently observedto induce hemorrhages,especiallyof an arterial
character.However, in patientswith coronary occlusionor endarterialobliterations,the administrationof cholesterolwas followed by an increaseof
symptomsapparently related to exaggerationof the degree of occlusion.
This effect upon blood vesselsalso could be seen in animal tumors. Administrationof cholesterolinduced zonesof necrosisin the tumors which
could be related to proliferation of the cndarterialcells leading to thrombosis and ischemicinfarct. The portions of tumor correspondingto these
ischemic infarcts showed characteristicnecrosiswith unaltered structure
but without the normal staining.
In animals injected with cholesteroland then submitted to trauma in
the Noble-Collipdrum, shockwas prevented.Injectionof cholestcrolprior
to the experiment reduced mortality to zero while in untreated controls
mortality was high. A similar but less constanteffect was obtained when
cholesterolwas administeredimmediatelyafter trauma. It is noteworthy
that in animals injected with cholesterolbefore being placed in the drum.
the blood not only did not becomeabnormallyblack but the usualbleeding
from the nose, mouth and paws (if not taped during the trauma) was
abnormally bright red.
The effectupon cxprimentaltumors was investigatedthrough the dipgenerations.
ping techniquerepeatedin successive
Changesin the evolution
of certain tumors. such as mammary adenocarcinomain mice, were induced. The effect of cholesterolwas similar to that of insaponifiablefracin more detail later.
tion but was lessmanifestand will be discussed
Cholesterol'seffect upon the ccntral nervous system was interesting.
Often, immcdiatelyafter administration,both in animals and humans,
uansitory somnolencewas observed.However. repeatedadministration
inducedconvulsions.(Note 5) Exophthalmiawas seenin mice after injec-

p H A R M A c o D y N A M I cA C r t v r T y ( n r n r

rwo)

371

tion of cholesteroland was most manifest24 hours thereafter.It contrasted


with normals and especiallywith animalsinjectedwith fatty acids or similar lipoids, who showcdenophthalmia.
An ether-oil cholesterolsolution induced paraplegiawith early foot
ulcerationsin rats and rabbits. This occurred particularly in females and
was related to a predominanceof stcrolsin this sex. Castrationor administration of sex hormonesdid not changethis specialsusceptibilityof the
nervous systemof femalesto cholestcrol.However, the administrationof
fatty acidsor of acid lipid preparationsof organsor tissuesdid suppressit.
(Note 21, Chapter VI)Changes in systcmicanalyseswere generallynot
obvious and, when prescnt,were slow in appearing.They correspondedto
changestoward offbalanceA.
UnsaponifiableFractions (lnsaponifiable or Non-saponifiable)
When insaponifiablefractions wcre preparedfrom various tissuesand
organs, big differencescould be seen in the quantity and the number of
sterol compoundsnaturally present.However, a certain specificityrelated
to the origin of these insaponifiablefractions apparedmost interesting.
The insaponifiablefractions of various materials were prepared by the
usual methods.Most of the fractionsare solublein oil in higher proportion
than cholesterol,with some of them even misciblewith oil. More concentrated solutionsin sesameoil could be preparedthan for cholestcrol.In
most of the experiments,5 or lOVo solutionswere used. Colloidal suspensionsalso were preparedin the samemanner as for cholesterol.
In spite of the extremevariationsin sourcesof the insaponifiablefractions, almost all have some propertiesin common. Some are similar to
cholesterol in their eflects particularly at the lower levels. The marked
differences
appearat higher levels.Thcy inducehemorrhages
in the adrenals betweenthe fascicularand reticularzones.
On the healing process,especiallyof radiation wounds, insaponifiable
fractions of placenta,embryos and buttcr-matcrials related to growthshow impressivelygreater activity than cholesterolor preparationsof insaponifiablefraction of other origin. They induce healing processesevcn
in standardized
radiationlesionswherc cholestcrolhas a weak effect.
In their influenceupon tumors, the preparationsof insaponifiablefractions of different organs differ markedly. No changesat all were obtained
with some preparationssuch as from pig intestine,for example,while
interesting results were obtained with others. The differencesappeared
especiallyevidentin experimentsin which a direct influenceupon the tumor

372

nEsEARcH rN PHYSropATHoLocy

was exerted. Transplants of Ehrlich mammary adenocarcinomain mice


were dipped in insaponifiablepreparationsand grafted. In general,no immediatevisible effectswere seenwith this techniquefor the first transplant
generation.By repeatingthe sameprocedurefor following transplant generations,changeswere obtained which varied with the preparationsused.
The insaponifiablefraction of human placenta, for instance, produced
a marked increasein malignancy,together with morphological changes,
the tumor changingfrom an adenocarcinoma
to an encephaloid.Further
treatmentof the transplantsled to still greatermalignancywith a sarcomatoid transformation.Thereafter,negativcresults were obtained with new
treatmentof the transplants.(Note 6/ With this procedure,plancentapreparations showeda manifestinfluenceeven at the third transplantgeneration,
and they were negativepassages
for the fifth to sixth transplantgenerations.
With pig intestinepreparations,even after ten successivepassages,malignancy was unchanged.
The specificityaccordingto origin was also seenin other experiments,
such as in the influenceexerted by thesepreparationsupon the development of specificlesionsproducedby smallpoxvirus in low-reactingspecies
such as mice or rats. Preparationsfrom receptiveanimals, and especially
from organs sensitiveto the virus, were more capable of inducing local
receptivitythan were preparationsfrom refractory animals. For instance,
positiveeffectswere obtained with vacciniavirus in mice and rats previously injectedsubcutaneously
with the insaponifiablefraction of rabbit skin
or brain, while no such effectswere seenwhen the insaponifiablefractions
of pig or hen intestineswere used.
Differenceswere observedbetweenthe insaponifiablefractions of different organsfor conditionsprincipallymanifestedat the organ level. Conditions affecting mainly one organ were treated with the insaponifiable
fraction correspondingto that organ. These preparationsoften appeared
much more activethan thosefrom other organs.We investigatedthe effects
of a heart insaponifiablefraction on patientswith myocardial insufficiency.
especiallywhen responsesto other therapeuticagentscould no longer be
obtained. In like manner, we used the insaponifiablefraction of liver for
manifestliver insufficiency.The rate of liver regenerationin rats after subtotal resectionwas found most acceleratedby liver insaponifiablefractions.
The good effectsobtained in the treatmentof intractablediarrhea with insaponifiablefractions from pig and hen intestineswill be discussedbelow.
We investigatedpreparationsobtained from lymph nodes and spleen for
the treatmentof shock, particularlyin its acute form. Similarly we also
used adrenal insaponifiablefraction to influence induced adrenal insufi-

p H A R M A c o D y N ^ M l c A c T - r v r T y( p , r n r r w o )

373

ciency,and brain insaponifiable


fraction in an attemptto influenceinsomnia. The resultsof thesestudieswill be discussed
in the sectiondealingwith
therapy. The changesobtainedwith the respectivepreparationsindicate
that they have a specificitywhich rcprescntsan importantfactor in normal
and abnormalphysiology.
In a secondgroup of researches,
constituents
of the rough preparations
of insaponifiablefractions from various sourceswere separatedby different methods.The ketonicand nonketonicconstitucntswere obtainedand,
when testedin animals,showedseveraldificrencesin biologicalproperties.
Further researchof specificitywas madeusingseparations
through the
chromatographic
column method.Most of thcsepreparations
are still under
laboratoryinvestigation.Experimentsare being conductedwith difterent
organ preparations,
somefractionsobtaincdbcing identifiedas commonto
all organs,while othersarc specificto onc organ or to a goup of organs.
Theseexperimentsalreadyhaverevealeda markedpluralityof constituents
for the insaponifiablcfractionsof organswhich has to be related to the
pluralityof constituents
found in the acid lipid fractionsof the sameorgans
and which was discussed
above.The specificityseenfor organswould thus
greatly concerntheir lipidic constitucnrswhich form the acid and the insaponifiablefractions.It is especiallyin rerms of specificitythat the acid
lipidic and insaponifiable
fractionsof variousorgansare being investigated
in researchnow in progress.(Note 7)
Corticoids
The study of the defenseof the organismagainstfatty acids focusses
attentiononce againon the adrenalswhoseconstituents
appearto be part
of the naturaldefensemechanism.To date,around 30 differentcrystallizable compoundshave been isolatedfrom less than a third of the total
cortical extract. The amorphouspart, biologicallymore active than the
crystallizedpart, would containother importantcompounds.Even if some
of them are intermediarycompoundsor artefacts,adrenalinterventionstill
is characterizedby plurality of its active agcnts.Furthermore,severalopposite tendenciesare recognizedbctween groups of adrenal compounds.
While all the corticoidsshow a certain antagonisticaction toward fatty
acids,mineralocorticoids
are, from severalpoints of view, antagonisticto
neoglucogenics.
With theseconsiderations
in mind, and recognizingthe adrenalsas one
of the principalmeansfor relativelyrapid defenseagainstnoxiousagents,
we have investigated
the relationshipof the adrenalsto lipids.
We have alreadVnoted the strikins richnessof the adrenalsin arachi-

374

x E S E A R C I {r N p H y s r o p A r H o L o c y

donicacid.About 25Vo of the total fatty acid contentof the glandsis made
up of this acid which is found only in smallquantitiesin other organs.We
alsofound pentaencs
and hcxaenes
prcscntin greateramountsthan in other
organs.These fatty acids nrust have a biological purpose and two hypothesescan be advanced.Accordingto one mcntionedpreviously,(Note I I,
Chapter VI), the corticoidswould be synthesized
from arachidonicacid
throughcyclization.Accordingto thc sccondhypothesis,arachidonicacid,
as well as other high fatty acidsprcsentin the adrenals,would be used as
activefunctionalfatty acids.Secreted
by the adrenals,they would passinto
the circulation and intcrvene as nccded by the organism, especiallyfor
immediatedefcnsepurposcs.
We have seenthat an interventionof polyunsaturatedfatty acids occurs
in the first defcnsercsponscof an organismto a noxious agent.These acids
for the exaggeratcd
are responsible
through which the
oxidationprocesses
organismattacksthe noxiousagentsthemselves
or the heterogenized
constituentsresultingfrom their action. Wc considerthat some of the fatty
acidsinterveningin this defensemcchanismare liberatedlocally,especially
if they appearin responscto a conditionlimited to a lower entity. In this
case,they would come from changesinduccd in the constituentsof the
entity itself. Thc local interventionof lipolytic enzymeswould lead to a
liberationof free fatty acids.In a defcnseresponsefor the organismas the
highestentity, thc activcly intervcningfatty acids appear in the general
circulationin the first phaseof the diphasicphenomenon.Some of these
fatty acids would be of adrcnal origin, liberatedat these moments.In the
secondphaseof thc defensemechanism,
a further liberationof steroidsby
the adrenalswould occur, aimed at counteractingthe effectsof fatty acids.
The diphasicsystemicprocesswhich rcsultscan be consideredto represent
which occur alternatelyand which, through
an exaggcrationof the proccsses
normal oscillations,insurethe dynamic systemicba.lance.
The adrenalsconccivablycontrol abnormalfatty acid activityby their
quantitativelyexaggcrated
interventionand by releaseof qualitativelyabnormal products that would pass into the circulation and result in offbalances.The activity of the adrcnirlsin counteractingthe influenceexercisedby fatty acids has been made the subjectof a specialinvestigation
in our laboratoriesby E. F. Taskier.
By comparingthe doscsof an agentrcquiredto kill normal and adrenalectomizedanimals, it has been possiblenot only to identify this intervention but to judge thc degreeof this specificdefensemechanism.The
"Adrenal DefenseIndex" for an agent-the ratio betweenthe minimal
animals represents
lethal dose in normal animalsand in adrenalectomized

p H A R M ^ c o D y N A MA
r cc r r v r r y ( r l n r

rwo)

375

a numerical estimatcof this rcsponse.It could bc shown that for certain


fatty acids, such as conjugatcdtricncs,uhich are related to trauma, or
alpha hydroxy fatty acidswhich are rclatedto microbialinvasion,a highly
effectiveinterventionof thc adrcnalsoccurs,through releaseof neoglucogeniccorticoids.The administrationof neoglucogenic
corticoidsmanifestly
increases
the resistance
of the organismto the noxiouseffectsof fatty acids.
This influenceis reducedfor thc mineralcrcorticoids
and is nil for sodium
chloride, otherwisean important factor related to adrenal intervention,
(Note 17, Chapter V I )
We will discusslatcr an inrportantdillcrcncc,evcn an antagonism,between thesetwo groupsof corticoidswhcn their influcnceis exertedconcomitantly with that of other agcnts.

Synthetic Anti-FaW Acids


Analysisof the natural anti-fattyacid agcntshas revealedthe importance
of their positivepolar groups.And this has guidedus in attemptsto obtain
synthetic agentswith anti-fatty acid cflccts.
An important step was a study of alcoholswith lipoidic properties,the
lipoalcohols, starting with the primary mono-alcoholhomologousseries.
This study alsohas pcrmittcdus to rccognizcthc importanceof the lipoidic
propertiesfor their biologicalactivit)'.We startedwith butanol which is
the first memberof thc homologoussericsof aJiphaticalcoholswith lipoid
characteristics.
Butanol
Butanol has a spccialplaceamongthc alcoholsthat have been utilized
as anti-fatty acid agents,not only by virtue of its physico-chemicaland
biological proprtiesbut also bccauseof intcrestingtherapeuticresults
obtainedin animalsand humans.
Extensivestudieswith butanolhavehelpedconsiderably
in definingthe
physico-chemicaland biologicaldiffcrenccsbctwccnlipoids and nonlipoids.
Accordingto the conceptadvanccdpreviously,lipoids and nonlipoidscan
be distinguishedby solubility charactcristicswhich are dcterminedby the
energeticrelationshipbctweentheir polar and nonpolargroups.The nonpolar group is predominantin a lipoid; thc polar group is predominantin
a hydroid.Lipoids have grcatersolubilityin neutralsolvcntsthan in water,
and this providesa simplecriterionfor their recognition.
Methyl, ethyl and propyl alcoholsare all equally ntore solublein water
than in neutral solventsand thereforeare rccognizedas nonlipoids.Butanol,
lp!{d}i}r!}-qrt'i:i.".:'/?:-,:ry?:r

tY"1,:'

i,*:',i

jgf;r-J{.9!1:;'.';.r

i}.\-'ifl..il'::r.:;;

?5sJJf,;.ftrtalfg'.?aG,E-*r

if

'-S

l'*
, i ;:16',"1'";

l?6

RDSIJA RCI{

I N

PI"IYSIOPA']'}IOLOGY

t{gc-9-ry
H. Butyl
Alcohol

S e c .B u t y l TertiaryButyl
Alcohol
AIcohoI

Q rch
\IH
I sobutyl
Alcohol

F l c . 1 3 3 . S c h e m r r t i cr c p r e s c n t a t i o no f t h e n r o l e c u l l r s u r f a c e so f t h e . l i s o m c r s t r f
b u t a n o l . T h e c o n s t a n t b o f t h c v a n t l e r W a a l s f o r c e s r c l a t c d t o t h e i r s u r f a c e sa r e
u n e q u a l .A n l i n i m u n r v a l t r c i s s e c n f o r a l n r o s ts p h e r i c n r o l c c u l eo f l c r t i a r y b u t a n o l ,
a f a c t w h i c h e x p l a i n st h e n o n p r c d o m i n a n c co f t h e p o l a r g r o u p i n t h i s m o l e c u l e .r e s p e c t i v e l yi t s n o n - l i p o i d i cc h a r a c t c r .

howcvcr.differs from the lowcr membcrsof the aliphatic alcohol scriesby


being a lipoid, nlorc solublcin ncutral solvcntsthan in water. This. horvcver. is truc only ior thrcc of thc four isonrcrsof butanol.n-Butanol,secb u t a n o la n t i i s o - b u t : t n oal r c a l l n r o r c s o l u b l ci n n c u t r a l s o l v e n t st h a n i n
watcr, r.vhcrclstcrt-butanolis equally solublein both. According to our
c r i t e r i o nt h c r c f o r eu. ' h i l ct h c f i r s tt h r c c : r r cl i p o i d s ,t c r t - b u t a n oi ls n o t .
Thcsc considerations
havc enablcdus to corrclatclipoidal propertics

o
g

(tt
=

co
I
c

o
g

(It

ct

I
(J
OJ

v,

m
I

(f,
.Fl

c)
c

rO
3

m
I

o,

F t c ; . l 3 l . T h c t l i f l c r c n c c sb r t w c c n t h c r o u n d r h a p c r . ' l ft e n i a r v b u t a n o l a n d the longcr


fttrm of thc othcr iserrrcr. i s e v i d e n t* ' i t h r r r o d c l so f n r o l c c u l e s .

p H A R . M A c o D y N A M t cA c r r v t r y

(p,lnr .rwo)

3jj

with one more preciseintermolecularfactor, the prcdominanceof one of


the van der Waalscohesionforces.Comparativeanalysisof the structural
f o r m u l ao f t h e f o u r i s o m e r so f b u t a n o l( F i S . 1 3 3 ) ,r e v e a l st h e i m p o r t a n c e
of forcesrelatedto the surfaceof the moleculesin determiningdiffcrences
i n t h e i r s o l u b i l i t y I. n c o n t r a s t o t h e t h r e c l i p o i d i s o m e r st,h e m o l e c u l eo f
tertiarybutanolis rounderand hencehas a smallersurface.The difference
betweentert-butanoland the other three isomersis apparentlydue to the
cohesionforcesrelatedto the surfaceareaof the moleculc.of the van der
Waalsforces,thosedescribedas relatedto the surfaceof the molecules.or

n - B u t y l s e c - t s u t tyel n t - B u t ylls o b u t y r

F r c . 1 3 5 . W h i l e t h e 3 i s o m e r so f b u t a n o lw h i c h a r e l i p o i d si n f l u e n c et h e s e c o n dd a y
w o u n dc r u s t p H . l o w e r i n gi t s v a l u e s .t c r t i a r y b u t l n o l w h i c h i s n o t a l i p o i d , d o e s n o t
i n f l u e n c ei t .

378

RESE^RcH tN pHysropATHoLocy

as the constant b of the cohesionforces, thus appearedto be most importantin determining


(FiS.I 3a)
lipoidicproperties.
study of the four isomersof butanol has confirmed the importanceof
lipoidic propertiesfor biological activity. Like the lower members of the
homologous series of aliphatic alcohols which arc not lipoids, tertiary
butanol does not influencepH of the secondday crust of a wound, while
the three other isomers,all with lipoidic characters,lower the pH as the
h i g h e rm e m b e r so f t h i ss e r i e sd o .( F i g . 1 3 5)

29o

Contro I s
Butanol

Agein reeks
F t c . 1 3 6 . E . f f e cot f 0 . 5 9 ' bs o l u t i o n o f .n - b u ! a n o la d m i n i s t e r e di n s t e a do f d r i n k i n g w a t e r
u p o n t h c i n c r e a s ei n w e i g h t o f y o u n g r a t s . T h e v a l u c s r e p r c s e n tt h e a v e r a g ef o r 2 0
f e m a l e s ( . . . . ) . N o d i f f e r e n c e sa r e s e e n f r o m n o n t r e a t e dc o n t r o l s ( - ) .

The fact that a saturatedwater solutionat 20oC still contains7.9Vo


n-butanol is of grcat practical importance.Becauseof its degreeof solubility in water, n-butanolcould be utilized in aqucoussolutionsin suffistudiesand could be used
ciently high concentrationfor pharnracological
as a therapeuticagcntin this forrn withoutnecd for an oily solventvehicle.
The acute toxicity dose for butanol correspondsto the narcotic dose
for the respiratorycenterswhich is relatedto interferencewith the aerobiotic life of thesecells.
The minimal lethal dose of n-butanoladministeredsubcutaneouslywas
f o u n dt o b e 4 . 6 - 6 . 4g m . , / K g n rf .o r m i c e ,3 . 7 - 5 . 9g m . / K g m .f o r r a t sa n d 3 . 3 5 , 6 g m . / K g m . f o r r a b b i t s ,g u i n c ap i g s a n d h a m s t e r sT. h e s ev a l u e sc l o s e l y
approximatethe findingsof other workers.The minimal lethal dose of
is very closeto that for subcutaneous
n-butanolinjectedintraperitoneally

p H A R M A C o D y N A M T cA c r t v r r y

(p,rnr rwo)

3'79

and intramuscularadministration,indicatingthat absorptionfrom the tissuesis almostas rapid as from scrouscavities.


W e h a v e a d m i n i s t e r ebdu t a n o li n l a r g ed o s e st o h u m a n subjects,and
theseclinical studieshavc confirmedthe laboratoryfindingsthat the toxic
effectis especiallymanifestthroughthe narcoticeffectand is a t t a i n e do n l y
with the use of very large doses.(ltlote 8)

Controls
Butanol

/6

20

24

29

J2

Ageln t|eeks

F r c . 1 3 7 .T h e s a m c d a i l y c h a n g es c e ni n m a l e r a t s

Long term usc of n-butanolhas virtually no influenceupon normal


physiologyin animals.Administercdcontinuouslyin the drinking water
of young animals,it had no effcct on growth (f iS. 136) It also did not
affect reproductioncapabilitiesof mature animalsor inliuencetheir offsprings.
n-Butanolshows a definiteinfluenceon white blood cells in rats. The
leucocytecount is increasedin adult rats receivingdaily injectionsof a
saturatedsolutionof n-butanol.(Note 9)
The influenceexcrtedby smalldosesof n-butanol,as for other lipoids,
appearsto be almostentirelyconfinedto abnormaltissuesand cells.This
is evident in the influenceupon the pH of experimentallyinduced wounds
in animals.Administcrcdbeforc wound inductions.n-butanolshowedno
influenceupon normal tissue,no differenceswere observedbetweenpH of
their normal tissuesin trcatcdand untreatedanimals.During the first day

380

./

RESEARCH IN

pHystopATltoLocy

following wound inductions,pH of the lesion in trcated animalswas no


differentfrom pH in un