Professional Documents
Culture Documents
net/publication/51379307
Antioxidant measurements
CITATIONS READS
126 622
5 authors, including:
Some of the authors of this publication are also working on these related projects:
All content following this page was uploaded by Aniko Somogyi on 20 May 2014.
Antioxidant measurements
This content has been downloaded from IOPscience. Please scroll down to see the full text.
(http://iopscience.iop.org/0967-3334/28/4/R01)
View the table of contents for this issue, or go to the journal homepage for more
Download details:
IP Address: 41.220.28.138
This content was downloaded on 08/10/2013 at 23:44
TOPICAL REVIEW
Antioxidant measurements
Anikó Somogyi1, Klára Rosta2, Péter Pusztai1, Zsolt Tulassay1,3 and
Géza Nagy1
1 2nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary
2 Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis
University, Budapest, Hungary
3 Semmelweis University, Clinical Gastroenterological Research Unit of Hungarian Academy of
E-mail: somogyi@bel2.sote.hu
Abstract
Chemical reactions, including oxidation and reduction of molecules, occur
in every cell. These reactions can lead to the production of free radicals.
Free radicals react with organic substrates such as lipids, proteins, and DNA.
Through oxidation free radicals cause damage to these molecules, disturbing
their normal function, and may therefore contribute to a variety of diseases.
The anti-oxidation system, which consists of enzymatic antioxidants and non-
enzymatic antioxidants, defends against oxidative stress. The aim of this review
is to summarize general aspects of methods to measure the antioxidant defence
system all in one (total antioxidant capacity) and discuss a number of methods
which are currently used for detection of antioxidant properties.
1. Introduction
A free radical is any chemical species capable of independent existence, possessing one or
more unpaired electrons. Biological free radicals are thus highly unstable molecules that have
electrons available to react with various organic substrates. The presence of free radicals
(table 1) and non-radical reactive molecules derived from free radicals (tables 2 and 3) at
high concentrations is dangerous to living organisms, because of their ability to damage
cell organelles. Nitrogen monoxide (NO), superoxide anions and related reactive oxygen
(ROS) and nitrogen (RNS) species, however, also play important modulating roles in certain
signal transduction pathways. Several ROS-mediated reactions protect the cell from oxidative
stress and serve to stabilize redox homeostasis. In more developed organisms NO and ROS
act as signal transducing molecules, modulating vascular tone, monitoring oxygen pressure
0967-3334/07/040041+15$30.00 © 2007 IOP Publishing Ltd Printed in the UK R41
R42 Topical Review
Free radicals
Hydroxyl OH
.
Superoxide
.
O2−
Nitric oxide NO
.
Thiyl RS
.
Peroxyl RO2
.
Non-radicals
Peroxynitrite ONOO−
Hypochlorous acid HOCl
Hydrogen peroxide H2O2
Singlet oxygen 1 (−1O )
g 2
Ozone O3
Lipid peroxide LOOH
DNA and proteins, and peroxidation of lipids (Ohshima et al 2006, Barzilai and Yamamoto
2004, Sohal 2002).
Specific markers show that oxidative damage occurred. Certain biomarkers well
characterize the cell damage in vitro. 8-Hydroxy-2 -deoxyguanosine (8-OHdG) and 8-
hydroxyadenine (Stillwell et al 1989, Ames 1989) measured in urine are characteristics
for DNA damage, lipid peroxides and other end products of the peroxidation process:
isoprostanes (e.g. 8-epi-prostaglandine, malondialdehyde (MDA)) or diene conjugation shows
lipid damage, while nitrotyrosine (Beckman et al 1994) is useful to detect protein damage.
All of these end products provide useful tools to monitor oxidative stress in human organisms.
Cells utilize enzymatic and non-enzymatic compounds—the so-called antioxidants to
defend themselves against oxidative stress.
CH3
HO
CH3 CH3 CH3
H3C O CH3
CH3
CH3 Alpha-Tocopherol
HO
O O
O H
HO
N
HN
O
HO OH NH
O N
Ascorbic Acid Uric acid
OH HO
O O
H2C H2C
H3C H3C CH3H3C
O N N N N O
H H H H
Bilirubin
B O O
O
A C
OH
OH O OH O OH O
OH
O O +
O
OH
Ferrari-Iliou 1999). Enzymatic compounds are primarily responsible for intracellular defence
(Shanker et al 2005). Non-enzymatic compounds, such as albumin, uric acid and bilirubin,
as well as a certain group of vitamins (based on their significant chain breaking capabilities)
are among the most important non-enzymatic antioxidant compounds that can be found in
blood plasma (Soriani et al 1994, Blokhina et al 2003, Fang et al 2002, Burton et al 1985).
Non-vitamin antioxidant compounds (albumin, uric acid, bilirubin, etc) are responsible for
50–80% of the cumulative chain breaking capability possessed by blood plasma, even though
it is not their primary role (figure 2).
Other antioxidant compounds (e.g. coeruloplasmine, transferrin, thiols) only play a limited
role in antioxidant defence, since their concentration in blood plasma is low.
Topical Review R45
some of these methods are in vitro examinations (Huang et al 2005). These methods are either
based on direct interaction with reactive molecules or on free radicals reacting with metal
ions. These reactions are monitored by coupled chemical measurements. Other methods to
determine antioxidant capacity can be performed on intact cells in situ (Maalouf et al 2002,
Hochberg et al 2006, Takahashi et al 2000). However, the results of these tests are only
partially relevant for humans (Prechl et al 1996). In humans, the oxidative stress status can be
determined by measuring radical-specific DNA modifications in urine and in selected tissues
(Erhola et al 1997, Shigenaga et al 1990, Chen et al 1999).
Antioxidant activity is an ability of a compound to reduce pro-oxidants or reactive species
of pathologic significance. Most methods that have been defined as inhibition methods involve
reactive species, which are usually free radicals.
The total antioxidant capacity (TAC) (Ghiselli et al 2000) considers the cumulative action
of all the antioxidants present in plasma and body fluids, thus providing an integrated parameter
of measurable antioxidants (Prior et al 2005). The total antioxidant capacity refers to a full
spectrum of antioxidant activity against various reactive oxygen/nitrogen radicals. Among
these antioxidant molecules, bilirubin, uric acid and protein thiols are the major endogenous
antioxidants in plasma, while vitamins C and E, as well as a number of food-derived (poly)
aromatic substances (belonging to stilbens, flavonoids and phenolic acids), are the main classes
of dietary antioxidants. For this reason the capacity of known and unknown antioxidants and
their synergistic interaction can be assessed. TAC measured in vitro bears no similarity to
in vivo measurements and may not have direct implication in vivo.
Some of the methods available for the measurement of total antioxidant capacity are
described here: the chemical principles of antioxidant capacity assays depend upon the
reactions involved. The assays can be classified into two types: assays based on hydrogen
atom transfer (HAT) reactions and assays based on electron transfer (ET).
The majority of the HAT-based assays apply a competitive reaction scheme, in which
antioxidant and substrate compete for thermally generated peroxyl radicals through the
decomposition of azo-compounds. These assays include inhibition of induced low-density
lipoprotein autoxidation, oxygen radical absorbance capacity (ORAC), total radical trapping
antioxidant parameter (TRAP) and crocin bleaching assays.
The ET-based assays measure the capacity of an antioxidant in the reduction of an
oxidant, which changes colour when reduced. The degree of colour change is correlated with
the sample’s antioxidant concentrations. The ET-based assays include the total phenols assay
by Folin–Ciocalteu reagent (FCR), Trolox equivalence antioxidant capacity (TEAC), ferric ion
reducing antioxidant power (FRAP), ‘total antioxidant potential’ assay using a Cu(II) complex
as an oxidant and DPPH.
Most of the assays determine the antioxidant activity in the micromolar range, needing
minutes to hours.
We discuss a limited number of the currently used methods to measure total antioxidant
capacity in body fluids (table 5).
R47
R48 Topical Review
ROS/RNS
(ROO-, HO-, O2, ONOO-)
Fluorescent Probe
Fluorescent Probe +
+ Hydrophilic Antioxidant
Blank or
Lipophilic Antioxidant
Loss of Fluorescence
Loss of Fluorescence
integration
integration
AUCblank
AUCantioxidant
Antioxidant Capacity = AUCantioxidant- AUCblank
AUC= area under the curve OH-= Hydroxide ion ONOO- = Peroxynitrite
Figure 3. Principle of the oxygen radical absorption capacity assay. AUC = area under the curve
OH− = hydroxide ion ONOO− = peroxynitrite (reproduced with permission from Brunswick
Laboratories).
-N2 2O2
37°C
RN=NR [R·N2·R] 2R · 2ROO ·
HCl NH
H2N
H3C CH3
N N
H3C
H3C NH2
HN HCl
This method was limited to hydrophilic antioxidants due to the aqueous environment.
In 2002 Huang et al expanded the ORAC(FL) assay to lipophilic antioxidants. Randomly
methylated beta-cyclodextrin (RMCD) was introduced as the water solubility enhancer for
lipophilic antioxidants. Nowadays the ORAC assay alters peroxyl-radical-induced oxidation
so that measures of both lipophilic and hydrophilic antioxidants can be obtained by using
the same peroxyl radical generator (figure 3). These methods provide, for the first time, the
ability to obtain a measure of ‘total antioxidant capacity’ in the protein-free plasma or tissue
homogenates using the same peroxyl radical generator for both lipophilic and hydrophilic
antioxidants. Separation of the lipophilic and hydrophilic antioxidant fractions from plasma
was accomplished by extracting with hexane after adding water and ethanol to the plasma
(hexane/plasma/ethanol/water, 4:1:2:1, v/v). Lipophilic antioxidants represented 33.1 ± 1.5
and 38.2 ± 1.9% of the total antioxidant capacity of the protein-free plasma in two independent
studies of six and ten subjects, respectively (Miller et al 2006, Kinnunen et al 2005, Prior
et al 2003, Freeman et al 2005, Huang et al 2002b, Sofic et al 2005).
cation begins to form, the absorbance increases. When antioxidants are added before the
addition of hydrogen peroxide, the antioxidants scavenge the radicals formed by the hydrogen
peroxide, delaying the formation of the ABTSˇ.+ radical cation, thus inducing an increase in
the percentage of inhibition of the absorbance. The TEAC assay is based on the inhibition by
antioxidants of the absorbance of the radical cation of ABTS, which has a characteristic long
wavelength absorption spectrum showing maxima at 660, 734 and 820 nm. The ABTS radical
cation based reaction has been commercialized by Randox Laboratories Ltd (Crumlin, UK)
(McAnalley et al 2003).
The original total radical trapping antioxidant parameter (TRAP) method was developed
by Wayner et al (1985). Their test is based on the measure of oxygen consumption during a
controlled lipid peroxidation reaction induced by thermal decomposition of an azo-compound.
It was the most widely used method for measuring total antioxidant capacity of plasma or
serum during the last decade (Alho et al 1998, Leinonen et al 1998).
The TRAP assay uses peroxyl radicals generated from 2,2-azobis(2-amidinopropane)
dihydrochloride (AAPH) (figures 4 and 5) as initiator of free radical generation.
After adding AAPH to the plasma, the oxidation of the oxidizable materials is monitored
by measuring the oxygen consumed during the reaction. During an induction period, this
oxidation is inhibited by the antioxidants in the plasma. An earlier detection method used
the principle that peroxyl radicals produced from AAPH oxidize luminol, which led to the
formation of luminol radicals that emitted light. The emitted light used to be detected by a
luminometer. Under normal circumstances this reaction produces low intensity light emission
that may decay rapidly. The characteristics of the reaction can be altered substantially by the
addition of the enhancer para-iodophenol, giving a more intense, prolonged and stable light
emission. The light emission is sensitive to interference by antioxidants. The length of the
induction period (lag phase) is compared to that of an internal standard, Trolox C (6-hydroxyl-
2, 5, 7, 8,-tetramethylchroman-2-carboxylic acid), a water-soluble vitamin E analogue, and
then quantitatively related to the antioxidant capacity of the plasma.
There were a few modifications made to the method, so that plasma TRAP avoids
interference with plasma proteins or lipids and the TRAP values are proportional to plasma
dilution (Ghiselli et al 1995). This newer, modified version of TRAP uses the fluorescing
properties of R-phycoerythrin (R-PE) and the ability of the plasma to protect R-PE from
peroxyl radical attack produced by AAPH instead of measuring the rate of lipid peroxidation.
The original procedure used the commercial reagent kits as ECL Antioxidant Detection
Pack NK8989 from Amersham International (Buckingshamshire, UK).
The first lag phase is due to the antioxidants in the sample. After the consumption of
antioxidants by peroxyl radicals, the reaction proceeds to the first propagation phase. The
second lag phase, which interrupts the first propagation, is due to Trolox (the internal standard)
added during the first propagation phase. The second propagation of the reaction follows the
consumption of Trolox. The first lag phase is compared to the second lag phase and, thus,
related to the antioxidant capacity of the sample.
in absorbance, while the FRAP assay measured the increase in absorbance due to formation
of ferrous ions. According to their results, it is strongly recommended to use at least two
methods when measuring total antioxidant capacity due to the differences between the test
systems investigated (Schlesier et al 2002).
According to the First International Congress on Antioxidant Methods in 2004 (Prior
et al 2005, Liu and Finley 2005) and according to data in the literature three assays were
proposed to consider for standardization: the oxygen radical absorbance capacity (ORAC)
assay, the Folin–Ciocalteu method and possibly the Trolox equivalent antioxidant capacity
(TEAC) assay. Other assays should be developed and considered in the future as more is
learned about some of the other radical sources and their importance to human biology.
4. Summary
A great variety of methods have been proposed for the assay of total antioxidant activity or
capacity of serum or plasma. Antioxidant capacity, on the other hand, is the measure of
moles of a given free radical scavenged by a test solution, independently of the capacity of
any one antioxidant present in the mixture (Ozgen et al 2006). Therefore, for plasma being
a heterogeneous solution of diverse antioxidants, the antioxidant status is better reflected
by antioxidant capacity rather than activity. Determining plasma AC may help to identify
conditions affecting oxidative status in vivo (e.g., exposure to reactive oxygen species and
antioxidant supplementation). Moreover, changes in the plasma AC after supplementation
with galenic antioxidants or with antioxidant-rich foods may provide information on the
absorption and bioavailability of nutritional compounds.
Indeed, there are situations in which knowledge of the individual levels of specific
antioxidant components might be more useful than the total antioxidant potency of the medium
Topical Review R53
concerned. Such situations might occur when investigating the structure–activity relationships
of pure antioxidant compounds, or when determining the antioxidant contributions of specific
dietary components and their relationships to antioxidant composition and activities of
the individual constituents, or during studies of decreased plasma antioxidant activity in
individuals under oxidative stress in specific disease states. Assays for total antioxidant
capacity in plasma differ in their type of oxidation source, target and measurement used to
detect the oxidized product. These assays give a wide range of results, should never be used
in isolation, and results should be interpreted with caution.
The interpretation of the changes in plasma or serum antioxidant capacity depends upon
not only the method used in detecting these changes, but also the condition under which the
plasma or serum antioxidant capacity is determined. A true measure and a golden standard of
total antioxidant capacity (hydrophilic and lipophilic AOC) are not yet available.
Acknowledgment
This study was supported by the Ministry of Welfare (448/2006, ETT), Hungary.
References
Alho H, Leinonen J S, Erhola M, Lonnrot K and Aejmelaeus R 1998 Assay of antioxidant capacity of human plasma
and CSF in aging and disease Restor. Neurol. Neurosci. 12 159–65
Ames B N 1989 Endogenous DNA damage as related to cancer and aging Mutat. Res. 21 441–6
Barzilai A and Yamamoto K 2004 DNA damage responses to oxidative stress DNA Repair (Amst) 3 1109–15
Beckman J S, Chen J, Ischiropoulos H and Crow J P 1994 Oxidative chemistry of peroxynitrite Methods Enzymol.
233 229–40
Benzie I F and Strain J J 1996 The ferric reducing ability of plasma (FRAP) as a measure of ‘antioxidant power’: the
FRAP assay Anal. Biochem. 239 70–6
Benzie I F F and Strain J J 1999 Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity
of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic
acid concentration Method Enzymol. 299 15–27
Blokhina O, Virolainen E and Fagerstedt K V 2003 Antioxidants, oxidative damage and oxygen deprivation stress: a
review Ann. Botany 91 179-94
Burton G W, Foster D O, Perly B, Slater T F, Smith I C and Ingold K U 1985 Biological antioxidants Phil. Trans. R.
Soc. B 17 565–78
Campanella L, Favero G and Tomassetti M 1997 A modified amperometric electrode for the determination of free
radicals Sensors Actuators B 44 559–65
Cao G and Prior R L 1998 Comparison of different analytical methods for assessing total antioxidant capacity of
human serum Clin. Chem. 44 1309–15
Cao G, Verdon C P, Wu A H, Wang H and Prior R L 1995 Automated assay of oxygen radical absorbance capacity
with the COBAS FARA II Clin. Chem. 41 1738–44
Chaudiere J and Ferrari-Iliou R 1999 Intracellular antioxidants: from chemical to biochemical mechanisms Food
Chem. Toxicol. 37 949–62
Chen L, Bowen P E, Berzy D, Aryee F, Stacewicz-Sapuntzakis M and Riley R E 1999 Diet modification affects DNA
oxidative damage in healthy humans Free Radic. Biol. Med. 26 695–703
Erhola M, Toyokuni S, Okada K, Tanaka T, Jiai H and Ochi H 1997 Biomarker evidence of DNA oxidation in lung
cancer patients: association of urinary 8-hydroxy-2-deoxyguanosine excretion with radiotherapy, chemotherapy,
and response to treatment FEBS Lett. 409 287–91
Fang Y Z, Yang S and Wu G 2002 Free radicals, antioxidants, and nutrition Nutrition 18 872–9
Freeman L M, Rush J E, Milbury P E and Blumberg J B 2005 Antioxidant status and biomarkers of oxidative stress
in dogs with congestive heart failure J. Vet. Intern. Med. 19 537–41
Ghiselli A, Serafini M, Maiani G, Azzini E and Ferro-Luzzi A 1995 A fluorescence-based method for measuring total
plasma antioxidant capability Free Radic. Biol. Med. 1 29–36
Ghiselli A, Serafini M, Natella F and Scaccini C 2000 Total antioxidant capacity as a tool to assess redox status:
critical view and experimental data Free Radic. Biol. Med. 29 1106–14
Granot E and Kohen R 2004 Oxidative stress in childhood—in health and disease states Clin. Nutr. 23 3–11
Hagymasi K and Blazovics A 2004 Antioxidants in liver protection Orv. Hetil. 145 1421–5
R54 Topical Review
Halliwell B 1990 How to characterize a biological antioxidant? Free Radic. Res. Commun. 9 1–32
Hammes E, Hoffmann A, Plieth C and Hansen U P 2005 Light-induced decrease in DCF fluorescence of wheat leaves
in the presence of salicyl hydroxamate Protoplasma 227 11–5
Hochberg M, Kohen R and Enk C D 2006 Role of antioxidants in prevention of pyrimidine dimer formation in UVB
irradiated human HaCaT keratinocytes Biomed. Pharmacother. 60 233–7
Huang D, Ou B, Hampsch-Woodill M, Flanagan J A and Deemer E K 2002a Development and validation of oxygen
radical absorbance capacity assay for lipophilic antioxidants using randomly methylated beta-cyclodextrin as
the solubility enhancer J. Agric. Food Chem. 50 1815–21
Huang D, Ou B, Hampsch-Woodill M, Flanagan J A and Prior R L 2002b High-throughput assay of oxygen
radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate
fluorescence reader in 96-well format J. Agric. Food Chem. 50 4437–44
Huang D, Ou B and Prior R L 2005 The chemistry behind antioxidant capacity assays J. Agric. Food Chem. 53 1841–56
Kahl R and Hildebrandt A G 1986 Methodology for studying antioxidant activity and mechanisms of action of
antioxidants Food Chem. Toxicol. 24 1007–14
Kampa M, Nistikaki A, Tsaousis V, Maliaraki N, Notas G and Castanas E 2002 A new automated method for the
determination of the total antioxidant capacity (TAC) of human plasma, based on the crocin bleaching assay
BMC Clin. Pathol. 2 3
Kinnunen S, Hyyppa S, Lehmuskero A, Oksala N, Maenpaa P, Hanninen O and Atalay M 2005 Oxygen radical
absorbance capacity (ORAC) and exercise-induced oxidative stress in trotters Eur. J. Appl. Physiol. 95 550–6
Kocsis I, Blazovics A, Pallai Z and Feher J 2004 Methods of monitoring oxidation–reduction balance and its potential
role in diagnostics Orv. Hetil. 145 761–7
Leinonen J, Rantalaiho V, Lehtimaki T, Koivula T, Wirta O, Pasternack A and Alho H 1998 The association between
the total antioxidant potential of plasma and the presence of coronary heart disease and renal dysfunction in
patients with NIDDM Free Radic. Res. 29 273–81
Liu R H and Finley J 2005 Potential cell culture models for antioxidant research J. Agric. Food Chem. 18 4311–4
Lussignoli S, Fraccaroli M, Andrioli G, Brocco G and Bellavite P 1999 A microplate-based colorimetric assay of the
total peroxyl radical trapping capability of human plasma Anal. Biochem. 269 38–44
Maalouf S, El-Sabban M, Darwiche N and Gali-Muhtasib H 2002 Protective effect of vitamin E on ultraviolet B
light-induced damage in keratinocytes Mol. Carcinog. 34 121–30
McAnalley S, Koepke C M, Le L, Vennum E and McAnalley B 2003 In vitro methods for testing antioxidant potential:
a review GlycoSci. Nutr. 4 1–9
McNeil C J, Smith K A, Bellavite P and Bannister J V 1989 Application of the electrochemistry of cytochrome c to
the measurement of superoxide radical production Free. Radic. Res. Commun. 7 89–96
Miller N J, Rice-Evans C, Davies M J, Gopinathan V and Milner A 1993 A novel method for measuring antioxidant
capacity and its application to monitoring the antioxidant status in premature neonates Clin. Sci. 84 407–12
Miller K B, Stuart D A, Smith N L, Lee C Y, McHale N L, Flanagan J A, Ou B and Hurst W J 2006 Antioxidant activity
and polyphenol and procyanidin contents of selected commercially available cocoa-containing and chocolate
products in the United States J. Agric. Food Chem. 31 4062–8
Nadeem A, Raj H G and Chhabra S K 2005 Increased oxidative stress and altered levels of antioxidants in chronic
obstructive pulmonary disease Inflammation 29 23–32
Notas G, Miliaraki N, Kampa M, Dimoulios F, Matrella E, Hatzidakis A, Castanas E and Kouroumalis E 2005
Patients with primary biliary cirrhosis have increased serum total antioxidant capacity measured with the crocin
bleaching assay World J. Gastroenterol. 11 4194–8
Novak Z, Varga S I, Pataki L and Matkovics B 1990 Links Simple method for the measurement of antioxidants Clin.
Chim. Acta 194 115–9
Ohshima H, Sawa T and Akaike T 2006 8-Nitroguanine, a product of nitrative DNA damage caused by reactive
nitrogen species: formation, occurrence, and implications in inflammation and carcinogenesis Antioxid. Redox
Signal 8 1033–45
Ou B, Huang D, Hampsch-Woodill M, Flanagan J A and Deemer E K 2002 Analysis of antioxidant activities of
common vegetables employing oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant
power (FRAP) assays: a comparative study J. Agric. Food Chem. 50 3122–8
Ozgen M, Reese R N, Tulio A Z Jr, Scheerens J C and Miller A R 2006 Modified 2,2-azino-bis-3-ethylbenzothiazoline-
6-sulfonic acid (abts) method to measure antioxidant capacity of selected small fruits and comparison to
ferric reducing antioxidant power (FRAP) and 2,2 -diphenyl-1-picrylhydrazyl (DPPH) methods J. Agric. Food
Chem. 54 1151–7
Pellegrini N, Serafini M, Colombi B, Del Rio D, Salvatore S, Bianchi M and Brighenti F 2003 Total antioxidant
capacity of plant foods, beverages and oils consumed in Italy assessed by three different in vitro assays J. Nutr.
133 2812–9
Topical Review R55
Prechl J, Somogyi A, Pusztai P, Kocsis I, Blázovics A, Boros I and Fehér J 1996 Szabadgyökös reakciók vizsgálata
streptozotocinnal kezelt fiatal patkányokon Orv. Hetil. 137 979–82
Prior R L and Cao G 1999 In vivo total antioxidant capacity: comparison of different analytical methods Free Radic.
Biol. Med. 27 1173–81
Prior R L, Hoang H, Gu L, Wu X, Bacchiocca M, Howard L, Hampsch-Woodill M, Huang D, Ou B and Jacob R 2003
Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORACFL)) of
plasma and other biological and food samples J. Agric. Food Chem. 51 3273–79
Prior R L, Wu X and Schaich K 2005 Standardized methods for the determination of antioxidant capacity and
phenolics in foods and dietary supplements J. Agric. Food Chem. 18 4290–302
Re R, Pellegrini N, Proteggente A, Pannala A, Yang M and Rice-Evans C 1999 Antioxidant activity applying an
improved ABTS radical cation decolorization assay Free Radic. Biol. Med. 26 1231–37
Regoli F and Winston G W 1999 Quantification of total oxidant scavenging capacity of antioxidants for peroxynitrite,
peroxyl radicals, and hydroxyl radicals Toxicol. Appl. Pharmacol. 15 96–105
Riley P A 1994 Free radicals in biology: oxidative stress and the effects of ionizing radiation Int. J. Radiat. Biol. 65
27–33
Schlesier K, Harwat M, Bohm V and Bitsch R 2002 Assessment of antioxidant activity by using different in vitro
methods Free Radic. Res. 36 177–87
Shanker G, Syversen T, Aschner J L and Aschner M 2005 Modulatory effect of glutathione status and antioxidants on
methylmercury-induced free radical formation in primary cultures of cerebral astrocytes Brain Res. Mol. Brain.
Res. 13 11–22
Shigenaga M, Park J and Cundy K 1990 In vivo oxidative DNA damage: measurement of: 8-hydroxy-2 -
deoxyguanosine electrochemical detection Method Enzymol 186 521–30
Sies H 1997 Oxidative stress: oxidants and antioxidants Exp. Physiol. 82 291–5
Sofic E, Rimpapa Z, Kundurovic Z, Sapcanin A, Tahirovic I, Rustembegovic A and Cao G 2005 Antioxidant capacity
of the neurohormone melatonin J. Neural Transm. 112 349–58
Sohal R S 2002 Role of oxidative stress and protein oxidation in the aging process Free Radic. Biol. Med. 33 37–44
Somfai G M, Knippel B, Ruzicska E, Stadler K, Tóth M, Salacz G, Magyar M and Somogyi A 2006 Soluble
semicarbazide-sensitive amine oxidase (SSAO) activity is related to oxidative stress and subchronic inflammation
in streptozotocin-induced diabetic rats Neurochem. Int. 48 746–52
Somogyi A, Herold M, Blázovics A, Szaleczky E, Pusztai P and Rosta A 1996 Vitamin A and E determination in
blood plasma by isocratic high pressure liquid chromatography ACH Models in Chem. 133 545–51
Somogyi A, Rosta K, Herold M and Tulassay Zs 2000 Effect of low temperature storage on the alpha-tocopherol
(vitamin E) content of human lipoproteins determined by high-performance liquid chromatography ACH Models
in Chem. 137 807–15
Soriani M, Pietraforte D and Minetti M 1994 Antioxidant potential of anaerobic human plasma: role of serum albumin
and thiols as scavengers of carbon radicals Arch. Biochem. Biophys. 312 180–8
Stadler K, Jenei V, Bölcsházy von G, Somogyi A and Jakus J 2003 Increased nitric oxide levels as an early sign of
premature aging in diabetes Free Radic. Biol. Med. 35 1240–51
Stillwell W G, Xu H X, Adkins J A, Wishnok J S and Tannenbaum S R 1989 Analysis of methylated and oxidized
purines in urine by capillary gas chromatography–mass spectrometry Chem. Res. Toxicol. 2 94–9
Studinger P, Mersich B, Lénárd Zs, Somogyi A and Kollai M 2004 Effect of vitamin E on carotid artery elasticity
and baroreflex gain in young, healthy adults Auton. Neurosci. 113 63–70
Takahashi H, Hashimoto Y, Aoki N, Kinouchi M, Ishida-Yamamoto A and Iizuka H 2000 Copper, zinc-superoxide
dismutase protects from ultraviolet B-induced apoptosis of SV40-transformed human keratinocytes: the
protection is associated with the increased levels of antioxidant enzymes J. Dermatol. Sci. 23 12–21
Valkonen M and Kuusi T 1997 Spectrophotometric assay for total peroxyl radical-trapping antioxidant potential in
human serum J. Lipid Res. 38 823–33
Wayner D D M, Burton G W, Ingold K U and Locke S 1985 Quantitative measurement of the total, peroxyl radical-
trapping antioxidant capability of human blood plasma by controlled peroxidation. The important contribution
made by plasma proteins FEBS Lett. 18 33–7
Winston G W, Regoli F, Dugas A J Jr, Fong J H and Blanchard K A 1998 A rapid gas chromatographic
assay for determining oxyradical scavenging capacity of antioxidants and biological fluids Free Radic. Biol.
Med. 24 480–93