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a r t i c l e
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Article history:
Received 23 April 2012
Received in revised form 21 May 2012
Accepted 22 May 2012
Available online xxx
Keywords:
Oxidative stress
Antioxidants
Antioxidative potency
Capacity
Assays
a b s t r a c t
Peroxidation of lipids, particularly polyunsaturated fatty acid residues (PUFA) of phospholipids and
cholesterol esters, is a process of marked implications: it shortens the shelf-life of food and drugs, it
causes fragmentation of DNA, it damages cellular membranes and it promotes the genesis of many human
diseases. Much effort is therefore devoted to a search for potent antioxidants, both synthetic and from
natural sources, mostly plants.
This, in turn, requires a reliable, simple, preferably high throughput assay of the activity of alleged
antioxidants. The most commonly used assays are based on measurements of the total antioxidant capacity (TAC) of a solution, as evaluated either by determining the rate of oxidation of the antioxidant or by
measuring the protection of an easily determined indicator against oxidation by the antioxidants.
The commonly used assays utilized for ranking antioxidants share three common problems:
(i) They usually evaluate the effects of those antioxidants that quench free radicals, which constitute
only a part of the bodys antioxidative network, in which enzymes play the central role. (ii) Both the capacity and potency of antioxidants, as obtained by various methods, do not necessarily correlate with each
other. (iii) Most estimates are based on methods conducted in solution and are therefore not necessarily
relevant to processes that occur at the lipidwater interfaces in both membranes and micro emulsions
(e.g. lipoproteins).
Given this state of art, many researchers, including us, try to develop a method based on the formation of hydroperoxides (LOOH) upon peroxidation of PUFA in lipoproteins or in model membranes, such
as liposomes. In these systems, as well as in lipoproteins, the most apparent effect of antioxidants is prolongation of the lag time preceding the propagation of a free radical chain reaction. In fact, under certain
conditions both water soluble antioxidants (e.g. vitamin C and urate) and the lipid soluble antioxidant
tocopherol (vitamin E), promote or even induce peroxidation.
Based on the published data, including our results, we conclude that terms such as antioxidative capacity or antioxidative potency are context-dependent. Furthermore, criteria of the efcacy of antioxidants
based on oxidation in solution are not necessarily relevant to the effects of antioxidants on peroxidation in
biological systems or model lipid assemblies, because the latter processes occur at water/lipid interfaces.
We think that evaluation of antioxidants requires kinetic studies of the biomarker used and that the
most relevant characteristic of oxidative stress in the biological context is the kinetics of ex vivo peroxidation of lipids. We therefore propose studying the kinetics of lipid-peroxidation in the absence of the
studied antioxidant and in its presence at different antioxidant concentrations. These protocols mean
that antioxidants are assayed by methods commonly used to evaluate oxidative stress. The advantage
of such evaluation is that it enables quantization of the antioxidants efcacy in a model of relevance to
biological systems. In view of the sensitivity of the lag time preceding peroxidation, we propose studying how much antioxidant is required to double the lag observed prior to rapid peroxidation. The latter
quantity (C2lag ) can be used to express the strength of antioxidants in the relevant system (e.g. LDL, serum
or liposomes).
2012 Elsevier Ireland Ltd. All rights reserved.
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Contents
1.
2.
3.
4.
5.
6.
7.
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.
Free radicals, ROS and antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.
Aim of the present review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.
Signicance of this review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.1.
Expectation and disappointment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.2.
The rational of using antioxidants as food supplements and the need to assay oxidative stress and antioxidative activity . . . .
1.3.3.
The role of antioxidants in prevention of pathogenic processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The most commonly used assays of antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
General comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Oxygen radical absorbance capacity (ORAC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Ferric reducing antioxidant power (FRAP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.
Trolox equivalent antioxidant capacity assay (TEAC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5.
Evaluation of antioxidants using more than one criterion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Testing and comparing antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
The need for kinetic data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Characterization of lipid peroxidation based on kinetic proles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Oxidative stress and ranking of antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Oxidative stress, denition and quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Terminology used to characterize antioxidative effect(s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Problems associated with assaying OS and ranking antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
The lack of a universal criterion of OS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Experimental details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.
The time-dependence of measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.
Comparison of data given in terms of different units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5.
The difference between reactions in solutions and at interfaces (location, location, location) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ranking antioxidants on the basis of OS methods: the effects of antioxidants on ex vivo peroxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.
Inhibition of peroxidation of PUFA in serum lipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.
Evaluation of antioxidative potency in model membrane systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions (Take-home messages) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Background
1.1. Free radicals, ROS and antioxidants
An antioxidant is a molecule (or an ion, or a relatively stable
radical) that is capable of slowing or even preventing the oxidation
of other molecules. The continuing growth of the market of antioxidants reects the hope to cure the wide range of diseases that are
believed to be caused or promoted by oxidative stress (Halliwell
and Gutteridge, 2007; Niki, 2011; Dotan et al., 2009a). The latter term, commonly dened intuitively as an imbalance between
pro-oxidative and anti-oxidative factors, is often associated with
high levels of reactive oxygen and nitrogen species (ROS and RNS,
respectively) and with high levels of oxidation products, particularly hydroperoxides and DNA fragments (Sies, 1986; Dotan et al.,
2004).
Harmans discovery of such oxidative damages led to the postulation of the free-radical hypothesis of aging (Harman, 1956,
2009) and of the oxidation-stress hypothesis of atherosclerosis
(Steinberg et al., 1989). Following the latter hypotheses, oxidative
stress has been associated with the pathogenesis of many other
diseases including diabetes mellitus, atherosclerosis, neurodegenerative diseases, different malignant diseases and virus infections,
including AIDS (Halliwell and Gutteridge, 2007).
These hypotheses raised the reasonable expectations that
antioxidants should prolong the shelf life of foodstuff and drugs and
reduce the mortality and/or the morbidity of supplemented people
by neutralizing the harmful free radicals (Frankel, 2007). In fact,
antioxidants prolong the shelf-life of food and drugs, as expected
and this, in turn, helps the ongoing effort to produce more active
antioxidants. It also explains, at least partially, the very large number of publications on antioxidants. Noticeably, the results of the
commonly used assays of antioxidants are, in general, reasonable
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The methods used to assay the effect of antioxidants on peroxidation of water-soluble substrates have been reviewed extensively
(Prior et al., 2005; Bartosz, 2010). Noticeably, less than a third of
the 60,000 scientic papers published in the last 5 years with the
term antioxidant(s) in their title or abstracts appeared in medical or
pharmacological journals. More than a third appeared in food and
agriculture literature and about a third appeared in chemical and
biochemical literature. Furthermore, only a small minority of newly
developed methods appeared in medical journals, most other being
in Food chemistry, agricultural and analytical journals.
1.2. Aim of the present review
The present, non-comprehensive review reects our point of
view on the available and potential assays of antioxidants. It is
based primarily on our experience and understanding, which will
hopefully contribute to the on-going search for a reliable, biologically relevant, sensitive and easy-to-conduct assay. To achieve this
goal, we rst describe the general features of free radicals chain
reactions and the terminology used to describe the characteristic
factors. Next, we describe the scope and limitations of the commonly used assays. Most of these assays are conducted in solution,
which means that their relevance to the ability of antioxidants to
protect aggregated substrates against peroxidation of lipids (which
occurs at the lipid/water interface) is questionable.
This review leads us to the conclusion that we should evaluate
antioxidants on the basis of the methods used to evaluate another
context-dependent term, oxidative stress (OS). This possibility has
been considered more than a decade ago but rejected on the basis
of experimental difculties (Prior et al., 2005). Re-evaluation of the
idea of using OS-assays to estimate the activity of antioxidants is
presented below.
1.3. Signicance of this review
1.3.1. Expectation and disappointment
For many years, peroxidation of lipids, particularly lipoproteins was considered responsible for CVD and for many other
diseases. Antioxidants have been therefore expected to reduce the
frequency of ROS-dependent diseases. Unfortunately, several large
clinical trials show that indiscriminate supplementation of vitamin E increases the mortality (both cardiovascular and all-cause) of
the vitamin E-treated subjects (Bjelakovic et al., 2007; Miller et al.,
2005). The results of our recent decision analysis supported these
meta-analyses (Dotan et al., 2009b).
The mechanism responsible for these unexpected results may
indicate that free radical chain reactions are not the major cause of
the pathogenesis of arthrosclerosis. Alternatively, the latter results
may be attributed to vitamin E-induced inhibition of the production
of antioxidative enzyme(s) and/or for reducing the concentration
of ROS beyond the level of being sufcient to function normally. In
addition, the unexpected results may reect experimental errors.
Regardless of the mechanism responsible for the unexpected
results, the latter ndings stress the importance of appropriate
analysis of results obtained by methods used to evaluate OS and
antioxidants. Only such methods can enable selective antioxidant
treatment. Meanwhile, several supporters of vitamin E supplementation think that indiscriminate supplementation is justied
because it reduces the risk of many diseases (Pryor, 2000). Other
authors argue that the results of the meta-analyses are questionable, due to serious aws in both the experiments and statistic
analyses (Blumberg and Frei, 2007). The latter view accords with
that of the producers of antioxidants, who still encourage indiscriminate use of their products. This view can explain the popularity
of antioxidants expressed by the presence of antioxidants in most
homes in developed countries. For many years the general attitude
was that if antioxidants do not help, at least they will not harm. The
more recent studies invalidate this approach. We think that many
people may benet from supplementation of antioxidants, including vitamin E (Witztum, 1998; Milman et al., 2008). The challenge
is to establish criteria to predict who is likely to gain from such
supplementation (Niki, 2011).
1.3.2. The rational of using antioxidants as food supplements and
the need to assay oxidative stress and antioxidative activity
As stated above, many investigators, particularly R&D experts
in industrial companies, devote much time, money and effort to
test the possibility that some virtuous antioxidants may have
greater antioxidative potency than the commonly used preparations, containing mostly vitamin E and C. Such a possibility may
be attainable either because new antioxidants may be designed to
be more efcient reducing agents and/or more efcient quenchers
of free radicals or/and due to preferred organ distribution and/or
other pharmacokinetic reasons. Given the expected market for efcient antioxidants, both the industry and university researchers
are looking for safe and potent antioxidants, both synthetic and
extracted from natural sources. This search, of course, requires an
assay that will be easy, reproducible, relatively cheap, preferably
high throughput and, most importantly, closely reect the effect of
the studied antioxidant in vivo.
1.3.3. The role of antioxidants in prevention of pathogenic
processes
A measure of the antioxidative potency and/or capacity can be
used to evaluate the involvement of free radicals in the pathogenesis of a given disease. Specically, if free radicals are involved
in a given pathogenesis, we could expect a correlation between
the efciency of the antioxidants to inhibit peroxidation and their
pharmacological effects. As an example, it is commonly believed
that Alzheimer disease (AD) is associated with aggregation of the
polypeptide Amyloid (A) into typical brils. Several antioxidants inhibit this process and many of them cause dissociation of
pre-formed brils (Shoval et al., 2007).
This does not necessarily mean that oxidative stress plays a
role either in the brilization of A or/and in the dissociation of the
brils. One way to test the question of whether the latter nding is
causal is to test the correlation between the capability of different
antioxidants to inhibit the formation of A brils and their antioxidative activity. This again, requires a reliable way of evaluating
the latter factor. In our previous study, we found that the antioxidative potency of 7 polyphenols, as determined by kinetic studies,
correlates with the potency of the studied polyphenols to cause dissociation of the amyloid brils (Shoval et al., 2007; Bartosz, 2010).
The latter nding does not prove that antioxidants cause dissociation of brils by reducing the concentration of free radicals, but it
strengthens the latter hypothesis
2. The most commonly used assays of antioxidants
2.1. General comments
Many assays have been developed to test and rank antioxidants.
Some of these assays are modications of same basic methods.
Yet, only a small number of methods were considered for standardization in The First International Congress on Antioxidants
Methods. These methods also happened to be the most commonly
used methods (Prior et al., 2005).
The most commonly used assays are not necessarily better, easier, more relevant or more accurate than other assays. Nonetheless,
widely used assays, particularly those conducted with commercial
kits, are less likely than other assays to suffer from irreproducibility due to trivial technical problems. Nonetheless, in the absence
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GSH) and albumin but does include reductants that do not act as
antioxidants.
2.4. Trolox equivalent antioxidant capacity assay (TEAC)
The TEAC assay is based on the reduction of the ABTS radicalcation in the presence of ferry myoglobin, activated by hydrogen
peroxide. A newer version is based on decolorization of a preprepared ABTS radical cation, which has a long shelf life (about 2
days). The results have been compared to those observed for Trolox
in similar conditions of concentration and time. The strong points
of this assay are that it can be applied for both water-soluble and
lipid soluble antioxidants. This assay is the basis for more elaborate
assays, which add more time points, more samples and more complex calculations. It has been adapted for clinical studies, based on
more complex conditions (Re et al., 1999).
2.5. Evaluation of antioxidants using more than one criterion
Given the context-dependence of both terms OS and antioxidative capability, it is important to choose the assay of antioxidative
potency and/or capacity according to the aim of the study. Specically, different assays may have to be considered when the goal is to
prevent lipoprotein oxidation or to search for antioxidants that will
maximize the shelf life of a given drug and/or foodstuff. In short,
there are advantages of using several methods, each representing
a different criterion, to rank antioxidants.
Nevertheless, the very large number of assays used by different groups constitutes a major difculty in the search for potent
antioxidants because it interferes with the possibility of comparing results of different laboratories. Even when different assays are
used to evaluate the same criterion, the result may vary considerably. The long list of abbreviations given above is merely a partial
list of the many methods. Several abbreviations denote a group
of methods that often differ from each other in important details
either with respect to the general experimental approach, or the
reagents used in the actual assay and/or the units used to describe
the results. Notably, all the methods listed above measure criteria that are based on the total capacity of antioxidants to reduce
free radicals or to inhibit peroxidation of easily detectable probes
developed to assess the potency of antioxidants in terms of more
specic criteria.
In an attempt to overcome the problem of having too many
methods, several leading scientists in this eld organized in 2004
a meeting entitled The rst International Congress on Antioxidant
Methods. Similar to many other consensus conferences, The First
International Congress on Antioxidants Methods did not yield a
universal assay. Instead, it yielded a consensus to recommend
shortening the list of assays to the three most commonly used
methods. According to the general agreement on this resolution,
using one or more of the three chosen methods covers the whole
spectrum of different antioxidants, from water-soluble and lipidsoluble reducing agents to the level of ROS and the concentrations
of inhibitors of oxidation of uorescent biomarker by ROS.
In retrospect, the First Conference on Antioxidant Methods
(2004) did not stop the effort to develop improved new assays of
antioxidative potency. Furthermore, to the best of our understanding; the agreement on a short list did not prevent the development
of many more methods because in the last 5 years scientists felt that
they can develop better assays. As a result, it appears that The First
International Conference of Antioxidant Methods was also the last
such conference. A Second Conference on Antioxidant Methods
never convened and may never be held in the future (Prior et al.,
2005).
The association between pre-diagnostic serum levels of several oxidative stress indicators and Colorectal Cancer (CRC) was
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the initial rate of inhibited oxidation and the lag preceding rapid
peroxidation are the basic experimental factors that may be sufcient for comparing antioxidants in meaningful terms, as described
below.
No Antioxidant
Antioxidant A
Antioxidant B
20
40
60
80
100
120
Time
Fig. 1. Schematic description of the accumulation of peroxidation product during
peroxidation in the absence of antioxidants (no antioxidant) and in the presence of
equal concentrations of antioxidants A and B under same conditions (both time and
concentration are given in arbitrary units).
Denition of the effects of antioxidants in meaningful, quantitative terms under specic conditions can be achieved on the basis of
the respective kinetic proles (Denisov, 2000). In empirical terms,
antioxidants affect the kinetics of peroxidation, such that in its
presence (i) the initial rate of lipid peroxidation is lower and (ii)
the lag preceding propagation is longer. Analysis of these factors,
when analyzed as described below, can yield characterization of
antioxidants in terms of two basic factors.
In the presence of an antioxidant AH, an external free radical
X , produced at a constant rate (e.g. during peroxidation induced
by free radical generator such as AAPH) reacts with both AH and a
lipid LH either dispersed or dissolved in the liquid media
AH + X A + HX
(r.1)
LH + X + [O2 ] LO2 + HX
(r.2)
(r.3)
(r.4)
A + LO2 A-LO2
Hence, each molecule of an antioxidant quenches two free radicals X and/or LO2
The search for improved antioxidants also requires a quantitative terminology of antioxidative activity. To be able to compare
antioxidants, we need to develop clear denitions of the meaning
of the experimentally observable factors. The following discussion
indicates that to achieve this goal we need kinetic data.
Quite often it is assumed that if studied under the same conditions, in the presence of equal concentrations, antioxidants can be
compared on the basis of the concentration of peroxidation products, as observed at a given (arbitrarily chosen) time. This may be
very misleading because the concentration of peroxidation products, as measured at a given time point, depends on the time of the
assay, as demonstrated in Fig. 1.
In this gure we see that at any time point prior to the lag
observed in the presence of compound A (e.g. at 30 time units),
the concentration of peroxidized lipids is higher in the presence of
compound B than in the presence of A, which means that A protects
the lipid against peroxidation more effectively than B. The opposite
result (B is more efcient than A) is apparent at later time points
(e.g. at 80 time units), exhibiting the misleading nature of one time
point measurements. Subsequently, after all the antioxidants are
consumed, peroxidation occurs rapidly. Finally (after about 120
time units), all the oxidizable lipids became peroxidized, both in
the presence of any antioxidant and in its absence. Measurements
conducted later may lead to the erroneous conclusion that neither
A nor B is an antioxidant.
Measurements of area under the curve (AUC), as proposed for
evaluation of antioxidative effects in several assays (Re et al., 1999),
constitute a partial solution to the ambiguity, because it is based on
continuous recording of the formation of oxidation products. Both
AH + 2X HX + AX
(r.5)
(r.1 + r.4)
(r.3 + r.5)
(r.6)
(r.7)
(r.8)
(2 r.4 + r.8)
(r.9)
or LO2 radicals. When the antioxidant nears complete consumption, inhibited peroxidation becomes un-inhibited. This time-point,
commonly denoted the lag time (tlag ) is given by the established
expression:
tlag = f n [AH]/Ri
(1)
(r.10)
(2)
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on measurements of the concentrations of specic products, conducted under well dened conditions with respect to the rate of
production of free radicals, the temperature, the viscosity and the
concentrations of oxidizable lipids and antioxidants. In most assayprotocols, the characteristic measured factor is expressed as a ratio
between the measured characteristic value and the value that characterizes the effect of an arbitrarily chosen inhibitor under the same
conditions.
The choice of an experimental factor to be used for characterization of the effects of inhibitors, as well as the choice of terms to
quantitate the inhibitory effect of antioxidants, is not trivial. At least
ve different terms have been used in relating to the protection
of various molecules against oxidation. In a literature search (ISIS
WOS database) for antioxidant* , we found that the term capacity has been used many more times (6035) than the other terms:
potency (1828), strength (775), effective (625) or active (133).
Quite often, the difference between two antioxidants is
described by ambiguous statements. For example, in relating to the
difference between the inhibitory effect of two antioxidants A and
B, as observed in a given experiment, the results are often described
in ambiguous terms such as A is a better antioxidant than B or A
is a stronger (or a more powerful) antioxidant than B or that A is
more effective than B or that the strength (or efciency, or capability, capacity or potency) of A is higher than that of B. In other
words, no universally accepted criterion has been established to
enable quantitative comparison between antioxidants.
In Fig. 1 described above, antioxidant A prolongs the lag less than
antioxidant B, indicating that A is a weaker antioxidant. Hence,
at the time that A is completely consumed, considerable fraction of
B is left, so that the phase of inhibited peroxidation is longer. Thus,
it can be concluded that the antioxidative capacity of B is higher.
By apparent contrast, accumulation of hydroperoxides during the
lag time is smaller in the presence of A than in the presence of B,
namely, based on the initial rate of lipid peroxidation during the
phase of inhibited peroxidation, A is a more potent antioxidant
than B or the antioxidative potency of A is higher than that of B.
5. Problems associated with assaying OS and ranking
antioxidants
As discussed above, the capacity of antioxidants is contextdependent. The biomedical relevance of the commonly used
methods of evaluating antioxidants is questionable because of the
following considerations.
5.1. The lack of a universal criterion of OS
The use of kinetic assays of OS to evaluate antioxidants is limited
by the lack of a universal criterion of OS. Accordingly, we should rst
consider the major problem of determining the oxidative stress,
which is the lack of a universal criterion. The three most commonly
used assays for the determination of total antioxidant capacity of
human serum (ORAC, TEAC and FRAP) have been compared. The
results demonstrated that there was a weak but signicant linear
correlation between serum ORAC and serum FRAP and no correlation either between serum ORAC and serum TEAC or between
serum FRAP and serum TEAC (Cao and Prior, 1998). Furthermore,
the authors describe the differences between the scope and limitations of the different assays and recall that different extraction
techniques in the ORAC assay also make a gross differentiation
between aqueous and lipid-soluble antioxidants. Since then, many
studies indicated that OS, as assayed by different methods, do nor
correlate with each other and we had reasons to propose (Dotan
et al., 2004) that there are different types of oxidative stress. Nevertheless, the use of the term OS without specifying the method
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rarely instructed to conduct concentration-dependent measurements. Hence, in spite of their convenience and ease of utilization,
OS kits, particularly HTS assays, should be conducted with caution.
In conclusion, evaluation of both terms oxidative stress and
antioxidative capacity is questionable. These general statements
are based on their context-dependent nature, the large number
of different criteria, measured by methods of relatively different
factors, the sensitivity of the measured criteria to experimental
details including the time of measurements relative to the onset
of the reaction and the consequent inability of comparing results
from different studies. The study of capacity of antioxidants in
solution is further complicated by the difference between the reactions in solution and at interface. In view of these difculties, we
propose assaying the inhibition of lipids peroxidation by different
antioxidants in the given system. Specically, we propose dening
antioxidant criteria on the basis of the concentration dependence
of their inhibition of the substrate peroxidation in the relevant system. This is what we have done in our recent study (Pinchuk et al.,
2011), as summarized below.
At the present time, the whole concept of the good antioxidants
protect us against the bad ROS is being questioned. Specically,
ROS plays important physiological roles, which means that lowering its concentration below a critical value is harmful (Birringer and
Ristow, 2012; Ristow and Schmeisser, 2011).
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