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34 Part 1: Principles/Food Analysisthese two reaction mechanisms, the descriptions of the various
assays, and their comparisons have been published extensively
over the past 20 years (Halliwell 1994, Prior and Cao 1999,
Huang et al. 2005, Prior et al. 2005, Apak et al. 2007, Phipps et
al. 2007, Moon and Shibamoto 2009). HAT mechanisms mea-
sure the ability of antioxidants to quench free radicals by do-
nating a hydrogen atom. This ability is critical as the transfer of
a hydrogen atom is a critical step in preventing/stopping radi-
cal chain reactions (Huang et al. 2005). HAT-based assays uses
a synthetic free-radical generator, an oxidizable probe (usually
fluorescent), and the antioxidant (Huang et al. 2005), monitor-
ing the competitive reaction kinetics with final quantification
derived from kinetic curves. ET-based assays, on the other hand,
are colorimetric assays that measure the change in color as the
oxidant is reduced. The mechanism itself involves only one re-
dox reaction, with the result being a quantifiable measurement
of the antioxidant’s reducing capacity (Huang et al. 2005).
Common HAT assays include oxygen radical absorbance ca-
pacity (ORAC), total radical trapping antioxidant parameter
(TRAP; and some of its variants), and total oxidant scavenging
capacity (TOSC). ORAC assays are the most widely used in re-
search, clinical, and food laboratories for antioxidant quantifica-
tion. This assay represents the classical antioxidant mechanism
by hydrogen transfer and can measure both in vivo hydrophilic
and lipophilic antioxidants, the latter by using selective combi-
nations of solvents with relatively small polarities. It is widely
used to measure the antioxidant capacity (AOC) of food sam-
ples ranging from pure compounds (e.g., melatonin) to complex
matrices such as fruits and vegetables (Prior and Cao 1999).
Peroxyl radicals generated with an azo-initiator compound are
added to microplate wells along with a fluorescent probe and
the extracted antioxidant. As the reaction progresses, fewer and
fewer of the antioxidants are available to donate hydrogen atoms
to the peroxyl radicals, which lead to radicals combining with
the fluorescent probe and forming a nonfluorescent molecule.
The reaction is quantified using a fluorometer over a period of
35 minutes, which is the time taken for the free radical action to
complete. The length of the ORAC assay also minimizes the pos-
sibility of underestimating the total antioxidant concentration as
some secondary antioxidant products have slower reaction ki-
netics. The effectiveness of the antioxidant is calculated from the
area between two curves generated when the assay is performed
with a sample and a blank, thereby combining both length of
inhibition and percentage inhibition into a single quantity (Cao
et al. 1995), with the results expressed as Trolox (a vitamin E
analogue) equivalents, that is,μM Trolox equivalents (TE) per
gram. One of the great advantages of the ORAC method is other
radical sources can be used (Prior et al. 2005) and has also been
developed for automation, which reduces the traditional time-
consuming analysis as well as human reaction time and other
sources of human error. In 2007, the United States Department
of Agriculture published a report entitled “Oxygen Radical Ab-
sorbance Capacity (ORAC) of Selected Foods—2007” (USDA
2007), which lists the ORAC values (hydrophilic and lipophilic)
as well as ferric-ion reducing antioxidant power (FRAP) and
Trolox equivalence antioxidant capacity (TEAC) assays for 59
different foods in the American diet.The second HAT mechanism is the TRAP assay, which mea-
sures the ability of an antioxidant to interfere with the reaction
between a hydrophilic peroxyl radical generator such as 2,2′-
azobis(2-amidinopropane) dihydrocholride and a target probe.
The effectiveness of the antioxidant to interfere in this reaction
is measured by the consumption of oxygen during the reaction
with the oxidation of the probe followed optically or by fluores-
cence (Prior et al. 2005). In the initial lag phase, the oxidation
is inhibited by the antioxidants and is compared to the internal
Trolox standard. TRAP values are reported as a lag time or reac-
tion time compared to the corresponding times for Trolox (Prior
et al. 2005). The TRAP antioxidant assay is most useful for
measuring serum or plasma AOC, that is, in vivo nonenzymatic
antioxidants such as glutathione and nutritive antioxidant (pro-)
vitamins such as vitamin A (β-carotene), C (ascorbic acid), and
E(α-tocopherol). This assay is also sensitive to all antioxidants
that are capable of breaking the reaction generating peroxyl
radicals. The major disadvantage is that the assay assumes all
antioxidants have a lag phase, but this is untrue. The total AOC
is underestimated as the antioxidant value contributed after the
lag phase (if one is present) is not taken into account and also
assumes that the lag phase is proportional to AOC.
The third assay to measure the HAT mechanism
is TOSC, which quantifies the absorbance capacity of
three oxidants—hydroxyl radicals, peroxyl radicals, and
peroxynitrite—thereby making it applicable to evaluate antiox-
idants from different biological sources (Prior et al. 2005). The
substrate isα-keto-γ-methiolbutyric acid, which forms ethylene
when oxidized and is measured over time by headspace analysis
by GC. The AOC of the antioxidant is determined by its ability
to inhibit ethylene formation. Like the ORAC, this assay is de-
termined by the area between the curve generated by the sample
and that of the control. The major advantage is that it can quantify
the effects of three common radicals of interest, but it is time-
consuming as the GC analysis can take 300 minutes, requiring
multiple injections of ethylene that has been collected from a sin-
gle sample into the GC. Additionally, the percentage antioxidant
inhibition and its concentration are not linear, which requires a
graph generated by various dilution factors with the DT 50 cal-
culated by the first derivation of this dose–response curve.
The only major assays measuring ET mechanisms (reducing
power) is the FRAP, which was initially developed to measure
the OAC of plasma but has been adapted for samples of botan-
ical origins (Prior et al. 2005). The low pH reaction measures
the reduction of ferric 2,4,6-tripyridyl-s-triazine by antioxidants
in a redox-linked colorimetric method with a redox potential
<0.7 V (Phipps et al. 2007). The bright blue color caused
by the presence of the ferrous ion is measured spectrophoto-
metrically at 593 nm. The major advantage is that the assay is
inexpensive, the results are producible, and the assay is short,
leading to automated or semi-automated high throughput. The
major drawback with the FRAP assay is that a chemical reduc-
tant and a biological antioxidant are treated identically, but the
assay neither directly use a pro-oxidant nor use an oxidizable
substrate (Prior and Cao 1999). The ferric ion (Fe^3 +) is not nec-
essarily a pro-oxidant such as the ferrous ion (Fe^2 +) that reacts
with hydrogen peroxide (a peroxyl free radical). Additionally,