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Antioxidant measurements

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IOP PUBLISHING PHYSIOLOGICAL MEASUREMENT
Physiol. Meas. 28 (2007) R41–R55 doi:10.1088/0967-3334/28/4/R01

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

Science, Budapest, Hungary

E-mail: somogyi@bel2.sote.hu

Received 24 July 2006, accepted for publication 31 January 2007

Published 7 March 2007
Online at stacks.iop.org/PM/28/R41

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.

Keywords: Oxidative stress, antioxidant, measurement, antioxidant capacity

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

Table 1. Reactive oxygen species (ROS).

Free radicals
Hydroxyl OH
.
Superoxide
.
O2−
Nitric oxide NO
.
Thiyl RS
.
Peroxyl RO2
.

Table 2. Non-radical reactive oxygen species.

Non-radicals
Peroxynitrite ONOO−
Hypochlorous acid HOCl
Hydrogen peroxide H2O2
Singlet oxygen 1
(−1O )
g 2
Ozone O3
Lipid peroxide LOOH

Table 3. Reactive nitrogen species (RNS).

Nitrous oxide N2O Nitrosyl cation NO+

Peroxynitrite OONO− Nitrogen dioxide
.
NO2
Peroxynitrous acid ONOOH Dinitrogen trioxide N2O3
Nitroxyl anion NO− Nitrous acid HNO2
Nitryl chloride NO2Cl

and production of erythropoietin, as well as playing a role in signal transduction pathways

involving membrane receptors as part of various physiological processes.
Chemical compounds capable of generating potential toxic oxygen species can be referred
to as pro-oxidants. In a normal cell, there is an appropriate pro-oxidant–antioxidant balance.
However, this balance can be shifted towards the pro-oxidants when production of oxygen
species is increased greatly (e.g. following ingestion of certain chemicals or drugs) or when
levels of antioxidants are diminished. This state is called ‘oxidative stress’.
Sies (1997) introduced the concept of oxidative stress, that is, the dissolution of the
pro-oxidant–antioxidant equilibrium.
Oxidative stress is basically caused by two mechanisms.
(1) The concentration of antioxidants is reduced (e.g., due to mutated antioxidant enzymes,
toxins, or the reduced intake of natural antioxidants).
(2) The number of oxygen/nitrogen/carbon-based reactive species derived from activated
phagocytes is increased, e.g. in the case of chronic inflammation (disease).
Following the oxidative stress, the activity of a given signal-transducing molecule is either
reduced or increased; additionally the function of the molecule may also change.
Large amounts of ROS may be generated in one of two ways: (1) by significant stimulation
of NAD(P)H oxidases or (2) from the mitochondrial respiratory chain. In the mitochondria
ROS are the unwanted byproducts of oxidative metabolism. If so-called oxidative stress occurs
under pathologic circumstances, it leads to an ‘overproduction’ of highly reactive oxygen
derived species, which can induce chemical changes in several cell organelles: alteration of
Topical Review R43

Figure 1. Reaction catalysed by SOD.

Table 4. Primary function of some endogenous and exogenous antioxidants.

Chain breaking antioxidants Preventive antioxidants

Albumin, bilirubin, uric acid
Vitamin C, vitamin E, carotenes, lipoic acid,
Coenzyme Q10, glutathione, flavonoids
Metallothionine, transferrin,
Coeruloplasmin, myoglobin, ferritin,
Selenium, flavonoids
EDTA (ethylenediamine tetraacetate)
DTPA (diethylenetriamine pentacetate)

Enzymatic antioxidants: superoxide dismutase,

catalase, glutathione peroxidase

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.

1.1. The antioxidant system

The term antioxidant can serve as a label for any substance whose presence, even at low
concentrations, delays or inhibits the oxidation of a substrate (Halliwell 1990). There
are several molecules that play a role in antioxidant defence; these are either endogenous
(internally synthesized) or exogenous (consumed). Antioxidants can be divided into two
groups depending on their mechanism of action. They can be either chain breaking antioxidants
or preventive antioxidants (table 4).
Preventive antioxidants reduce the rate of chain initiation, and chain breaking antioxidants
interfere with chain propagation. Preventive antioxidants include enzymes such as catalases
and other peroxidases that react with ROOH (lipid hydroperoxide) and chelators of metal ions
such as EDTA (ethylenediamine tetraacetate) and DTPA (diethylenetriamine pentacetate).
Such antioxidant systems prevent the uncontrolled formation of free radicals and activated
oxygen species, or inhibit their reactions with biological structures (Riley 1994). In vivo, the
principal preventive antioxidants are superoxide dismutases (SOD) (figure 1), which act in the
aqueous phase to trap superoxide free radicals.
Intracellular antioxidants include low molecular weight scavengers of oxidizing species
and enzymes which degrade superoxide and hydroperoxides. Their chemical characters define
their localization in the cells. Hydrophilic chain breakers are found in cytosolic, mitochondrial
and nuclear compartments. Hydrophobic chain breaking antioxidants are found in cell
membranes where they inhibit or interrupt chain reactions of lipid peroxidation (Chaudiere and
R44 Topical Review

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

Flavonol Flavone Isoflavone

OH

O O +
O

OH

Flavan-3-ol Flavanone O Anthocyanidin

Figure 2. Structure of some important antioxidant compounds.

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

The above-mentioned compounds, natural antioxidants, various enzymes and compounds,

are intricately linked to one another and function in an interrelated manner.

1.2. The antioxidant defence capability

The antioxidant defence capability of different organs varies.
Under physiological conditions the production of reactive species is counterbalanced by
the antioxidant defence system, thus maintaining redox homeostasis.
Oxidants and antioxidants are core elements of the cell’s signal transductory system;
they also play key roles in adaptive mechanisms. Oxidative damage occurs when the
pro-oxidant/antioxidant equilibrium is shifted in a way that leads to an excess of pro-
oxidant agents, which damage certain organs/organ systems. The sensibility of any
given organ or organ system to oxidative stress is to a great extent linked to the equilibrium
of oxidative stress—inducing and inhibiting agents that are present in the organ (Prechl et al
1996). Thus, according to their pathomechanism, certain diseases are in fact so-called free
radical diseases (Kahl and Hildebrandt 1986). Organ systems most susceptible to damage
are the pulmonary system, the brain, the eye, the circulatory system and the reproductive
system (Nadeem et al 2005, Somfai et al 2006, Studinger et al 2004, Stadler et al 2003). For
this reason, investigations which focus on the damage resulting from oxidative stress, on the
physiological role of the antioxidant system and on ways to influence it have moved to the
forefront of medical research (Granot and Kohen 2004).
A widespread, effective group of protective agents and defence mechanisms, collectively
referred to as the antioxidant defence system (ADS), acts to regulate oxidative reactions.
The ADS includes enzymes and non-enzyme antioxidants to prevent the start of oxidative
damage and/or control its spread.
Chain breaking antioxidants remove ROS and include the following categories.
• Small molecule antioxidants. They include both water-soluble compounds such as vitamin
C or glutathione and lipid soluble compounds such as vitamin E, carotenes, lipoic acid
and coenzyme Q10.
• Enzymatic antioxidants. They include superoxide dismutase (SOD) that detoxifies the
superoxide ion, catalase, which deals with hydrogen peroxide (H2O2) and glutathione
peroxidase (GPx) whose function is to detoxify cellular peroxides. These enzymes
counteract oxidative damage and target damaged molecules for destruction and
replacement. The enzymes must be synthesized by the cells.
The preventive antioxidants include albumin, metallothionine, transferrin, ceruloplasmin,
myoglobin and ferritin that bind ROS to protect essential proteins.
The main classes of naturally occurring antioxidants include flavonoids and polyphenolics,
phytoestrogens, vitamin A and related compounds and vitamin E.
This review summarizes general aspects of methods to measure the antioxidant defence
system all in one (total antioxidant capacity) and discusses shortly the methods which are
currently used for detection of antioxidant properties.

2. Possibilities of measuring antioxidants

Measuring plasma antioxidant capacity may help to evaluate physiological, environmental

and nutritional factors that influence the redox status in humans. There are several techniques
to determine antioxidant capacity (Cao and Prior 1998, Pellegrini et al 2003, Prior and Cao
1999, Cao et al 1995, Hagymasi and Blazovics 2004, Kocsis et al 2004, Novak et al 1990);
R46 Topical Review

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).

2.1. ORAC assay

The ORAC assay (oxygen radical absorption capacity) was originally developed by Cao and
Prior (1998). The assay measures the effectiveness of various natural antioxidants, present
in plasma or tissue homogenates, in preventing the peroxyl-radical-induced oxidation of the
fluorescent marker protein, fluorescein. This method is based on the inhibition of peroxyl-
radical-induced oxidation by antioxidants, which can be detected as loss in the fluorescence
intensity, during peroxyl-radical-induced free radical damage; thus this method is not sufficient

Table 5. Summary of methods.
Method ORAC(FL)
Measures Hydrophobic and hydrophilic
antioxidants total antioxidant
capacity (TAC)
Detected compound Fluorescein, loss in the
fluorescence intensity
Directly measures Inhibition of peroxyl-
radical-induced oxidation
Principle of the method Hydrogen atom transfer
Advantages/ Possible to separate lipophilic
disadvantages and hydrophilic antioxidant
fractions from plasma
Not sufficient to measure
a only a single antioxidant,
influenced by dietary
intake of antioxidants
TRAP Crocin-bleaching assays
TAC in plasma TAC in plasma
The decay of R-PE Carotenoid crocin
fluorescence
R-phycoerythrin (R-PE) Colorimetric measures
bleaching crocin
Hydrogen atom transfer Hydrogen atom transfer
It is an economical and
sensitive method, to measure
total antioxidant capacities
from human plasma, natural
compounds and plant extracts
Topical Review
TEAC FRAP
TAC in plasma TAC in plasma
Absorbance at 734 nm Coloured ferrous-
with respect to time tripyridyltriazine complex
ABTS radical cation Ferric to ferrous
formation ion reduction
Electron transfer Electron transfer
Peroxidases present in the Does not measure SH-group
serum can yield to higher containing antioxidants,
absorbance values. The antioxidants less relevant to chain-breaking
tested can react with the antioxidants activity
reagents, not only directly
with the free radicals. Since
there are no free radicals
introduced into the system,
there is no way of comparing
the antioxidant capacity towards
different kinds of radicals
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 ·

Figure 4. Schematic diagram of peroxyl radical produced from AAPH.

HCl NH
H2N
H3C CH3
N N
H3C
H3C NH2
HN HCl

Figure 5. The molecular structure of 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH).

to measure a only a single antioxidant. Thermo decomposition of azo-compounds, such as

2,2
-azobis(2-amidinopropane) dihidrochloride (AAPH) (figures 3–5), is used as a peroxyl
radical generator.
Topical Review R49

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).

2.2. FRAP assay

The FRAP assay (ferric reducing antioxidant power) (Benzie and Strain 1996, 1999, Ozgen
et al 2006) measures antioxidant power with the help of an oxidant, i.e., Fe3+. Ferric to ferrous
ion reduction at low pH causes the formation of a coloured ferrous–tripyridyltriazine complex.
FRAP values are obtained by comparing the absorbance change at 593 nm in test reaction
mixtures with those containing ferrous ions in known concentration. In the FRAP assay,
reductants (‘antioxidants’) in the sample reduce the Fe(III)/tripyridyltriazine complex, present
in stoichiometric excess, to the blue ferrous form, with an increase in absorbance at 593 nm.
The change in absorbance is proportional to the combined (total) ferric reducing/antioxidant
power (FRAP value) of the antioxidants in the sample (Ou et al 2002).
Absorbance changes are linear over a wide concentration range with antioxidant mixtures,
including plasma, and with solutions containing one antioxidant in purified form.
The FRAP assay is simple and inexpensive, but does have several drawbacks. Since
there are no free radicals introduced into the system, there is no way of comparing the
antioxidant capacity towards different kinds of radicals. The FRAP assay cannot measure the
antioxidant capacity of certain antioxidants accurately, which can react with iron(II) and SH
group-containing antioxidants.
Ou et al (2002) analyzed 927 freeze–dried vegetable samples using the oxygen radical
absorption capacity (ORAC) and ferric reducing antioxidant capacity (FRAP) methods. The
data show that the ORAC and FRAP values of vegetable are not only dependent on species,
but also highly dependent on geographical origin and harvest time. The two antioxidant assay
methods, ORAC and FRAP, also give different antioxidant activity trends. They concluded that
the ORAC method is chemically more relevant to chain breaking antioxidant activity, while
the FRAP assay has some drawbacks such as interference, reaction kinetics and quantitation
methods.

2.3. TEAC assay

The 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) equivalent antioxidant
capacity (TEAC) assay was first reported by Miller et al (1993), and then modified by Re et al
(1999). When 2,2
-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is incubated in
the presence of a peroxidase and hydrogen peroxide or in the presence of hydroxyl, peroxyl,
alkoxyl and inorganic radicals, ABTSˇ.+ radical cation is generated. As the ABTSˇ.+ radical
R50 Topical Review

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).

2.4. TRAP assay

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).

2.5. DCFH-DA-based assay

The dichlorofluorescin-diacetate (DCFH-DA)-based assay which also measures TRAP is the

spectrophotometric assay reported by Valkonen and Kuusi (1997). This assay uses AAPH
to generate peroxyl radicals and DCFH-DA (dichlorofluorescin-diacetate) as the oxidizable
substrate for the peroxyl radicals. The oxidation of DCFH-DA by peroxyl radicals converts
DCFH-DA to dichlorofluorescein (DCF). DCF is highly fluorescent (Ex 480 nm, Em 526 nm)
and also has absorbance at 504 nm. Therefore, the produced DCF can be monitored
either fluorometrically or spectrophotometrically (Hammes et al 2005). In this assay, DCF
fluorescence or absorbance formation contains four phases.
Topical Review R51

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.

2.6. TOSC assay

The total oxidant scavenging capacity (TOSC) assay (Winston et al 1998) permits easy
quantification of the absorbance capacity of antioxidants towards three potent oxidants,
i.e. hydroxyl radicals, peroxyl radicals and peroxynitrite. These oxidants were generated
by the iron plus ascorbate-driven Fenton reaction, thermal homolysis of AAPH and 3-
morpholinosydnonimine N-ethylcarbamide (SIN-1), respectively. These oxidants react with
alpha-keto-gamma-methiolbutyric acid (KMBA), which is oxidized and yields ethylene. The
antioxidant capacity of the compounds tested is quantified from their ability to inhibit ethylene
formation relative to a control reaction. Thus, the relative efficiency of various antioxidants
could be compared under conditions of quantitatively similar KMBA oxidizing capability
by the three oxidants. The TOSC assay is useful and robust in distinguishing between the
reactivities of various oxidants and the relative capacities of antioxidants to scavenge these
oxidants (Regoli and Winston 1999, Winston et al 1998).

2.7. Carotenoid (crocin) bleaching-based assay

Lussignoli et al (1998) developed a colorimetric assay. In the crocin assay, the extent of
bleaching of crocin, a carotenoid from saffron, by peroxyl radicals is generated by thermal
decomposition of azo-initiator. In detail, the test is based on a measure, in 96-well microplates
at 450 nm, of the bleaching of carotenoid crocin by peroxyl radicals generated during thermal
decomposition of AAPH. The inhibition of this bleaching is a function of the antioxidant
power of substances added to the incubation mixture. This method was adapted on a modern
autoanalyzer by Kampa et al (2002).
Chatterjee et al compared Indian saffron and ‘Sigma’ saffron in 2005. They concluded
that the crocin assay using the Indian saffron is an economical and sensitive method for
measurement of total antioxidant capacities from human plasma as well as natural compounds
and plant extracts.
Several research groups intended to compare the results, advantages and disadvantages
of the above-mentioned antioxidant measurement methods. Schlesier et al (2002)
used six methods: trolox equivalent antioxidant capacity assay (TEAC I-III assay),
total radical-trapping antioxidant parameter assay (TRAP assay), 2,2-diphenyl-l-
picrylhydrazyl assay (DPPH assay), N,N-dimethyl-p-phenylendiamine assay (DMPD assay),
photochemiluminescence assay (PCL assay) and ferric reducing ability of plasma assay (FRAP
assay) to determine the delay of radical generation as well as the ability to scavenge the radical.
The antioxidants measured included gallic acid representing the group of polyphenols, uric
acid as the main antioxidant in human plasma, ascorbic acid as a vitamin widely spread in
fruits and Trolox as water-soluble vitamin E analogue. The six methods presented can be
divided into two groups depending on the oxidizing reagent. Five methods use organic radical
producers (TEAC I–III, TRAP, DPPH, DMPD, PCL) and one method works with metal ions
for oxidation (FRAP). Another difference between these tests was the reaction procedure.
The tests determining radical reduction used preformed radicals and measured the decrease
R52 Topical Review

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.

3. Measurements of antioxidant enzymes and biomarkers

Antioxidant enzymes are located in subcellular organelles or in the cytosol of eukaryotic

cells.
Superoxide dismutase activity is usually measured by indirect methods. Chemically or
enzymatically generated superoxide radicals react with a detector molecule, which scavenge
the radical. It is also possible to detect superoxide radicals using electrochemical techniques
(McNeil et al 1989). Campanella et al (1997) introduced a system that is based on the
reduction of cytochrome c (induced by superoxide radicals)—a true carbon paste electrode is
used, having cytochrome c as mediator and Fe(III)–protoporphyrin as promoter for continuous
superoxide radical determination working at constant applied potential—the reduced form of
the cytochrome is electrochemically detected using cyclic voltammetry.
Glutathione peroxidase activity can be measured by polarography or fluorimetric
microassay.
Catalase activity is determined by the liberation of oxygen with an oxygen electrode or
by polarography.
Nowadays these assays are available in test kits to determine these enzymes.
Tocopherols and carotenoids are naturally occurring lipophilic micronutrients, suggested
to play a role in the prevention of several degenerative diseases. Thus, methods for the
quantification of these nutrients in human samples have been developed during recent years.
Our group (Somogyi et al 1996, 2000) has elaborated a new technique to store and to measure
tocopherols and carotenoids in human samples using chromatographic analysis.

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.

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