Tert -butylhydroquinone as a spectroscopic probe for the superoxide radical scavenging activity assay of biological samples

Article abstract of DOI:10.1021/ac200788m

As a more convenient and less costly alternative to electron spin resonance (ESR) and nonspecific nitroblue tetrazolium (NBT) and cytochrome c assays of superoxide radical (SR, O₂^?-) detection, a novel probe, tert-butylhydroquinone (TBHQ), is introduced for SR nonenzymatically generated in the phenazine methosulfate-β-nicotinamide adenine dinucleotide (PMS-NADH) system. SR attacks both TBHQ and SR scavengers incubated in solution for 30 min where scavengers compete with TBHQ for the O₂ ^?- produced. TBHQ, but not its O₂^?- oxidation product, tert-butyl-1,4-benzoquinone (TBBQ), is responsive to the CUPRAC (cupric reducing antioxidant capacity) spectrophotometric assay. The CUPRAC absorbance of the ethyl acetate extract of the incubation solution arising from the reduction of Cu(II)-neocuproine reagent by the remaining TBHQ was higher in the presence of O₂^?- scavengers (due to less conversion to TBBQ), the difference being correlated to the SR scavenging activity (SRSA) of the analytes. With the use of this reaction, a kinetic approach was adopted to assess the SRSA of amino acids, vitamins, and plasma and thiol antioxidants. This assay, applicable to small-molecule antioxidants and tissue homogenates, proved to be efficient for cysteine, uric acid, and bilirubin, for which the widely used NBT test is nonresponsive. Thus, conventional problems of NBT assay arising from formazan insolubility and direct reduction of NBT by tested scavengers were overcome.

Full text of DOI:10.1021/ac200788m

ARTICLE
pubs.acs.org/ac
tert-Butylhydroquinone as a Spectroscopic Probe for the Superoxide
Radical Scavenging Activity Assay of Biological Samples
€
Burcu Bekde -s er, Mustafa Ozy €u rek, Kubilay G €u c- l €u , and Re -s at Apak*
Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul, Turkey
S Supporting Information
b
ABSTRACT: As a more convenient and less costly alternative to
electron spin resonance (ESR) and nonspecific nitroblue tetrazolium
NBT) and cytochrome c assays of superoxide radical (SR, O2•
ꢀ
)
(
detection, a novel probe, tert-butylhydroquinone (TBHQ), is intro-
duced for SR nonenzymatically generated in the phenazine meth-
osulfateꢀβ-nicotinamide adenine dinucleotide (PMSꢀNADH)
system. SR attacks both TBHQ and SR scavengers incubated in
solution for 30 min where scavengers compete with TBHQ for the O2• produced. TBHQ, but not its O2 oxidation product,
tert-butyl-1,4-benzoquinone (TBBQ), is responsive to the CUPRAC (cupric reducing antioxidant capacity) spectrophotometric
assay. The CUPRAC absorbance of the ethyl acetate extract of the incubation solution arising from the reduction of
ꢀ
•ꢀ
•
ꢀ
Cu(II)ꢀneocuproine reagent by the remaining TBHQ was higher in the presence of O2 scavengers (due to less conversion
to TBBQ), the difference being correlated to the SR scavenging activity (SRSA) of the analytes. With the use of this reaction, a
kinetic approach was adopted to assess the SRSA of amino acids, vitamins, and plasma and thiol antioxidants. This assay, applicable
to small-molecule antioxidants and tissue homogenates, proved to be efficient for cysteine, uric acid, and bilirubin, for which the
widely used NBT test is nonresponsive. Thus, conventional problems of NBT assay arising from formazan insolubility and direct
reduction of NBT by tested scavengers were overcome.
3
eactive oxygen species (ROS), including superoxide anion
radical (O2 ), have gained great attention due to their role
scavengers but also as XO inhibitors. To date, several none-
•ꢀ
•ꢀ
R
nzymatic methods employing other O2 -generating systems
in the progression of a number of human diseases and carcino-
have been identified, for example, a H O degradation system in
2
2
•ꢀ
genesis. So it is important to eliminate excessive O in vivo to
alkaline dimethyl sulfoxide, a riboflavin irradiation system, and a
phenazine methosulfate (PMS)ꢀβ-nicotinamide adenine dinu-
2
•ꢀ
prevent O2 -originated disease. All cells possess elaborate
antioxidant defense systems consisting of low and high molecular
weight components to defend against ROS attack. These pro-
tective systems include enzymatic and nonenzymatic antioxidants
that are both endogenous (produced in the body) and exogenous
4
•ꢀ
cleotide (NADH) oxidation system. In the present study, O
2
is generated under aerobic conditions in a PMSꢀNADH system
5,6
by oxidation of NADH in a two-step reaction (eqs 2 and 3):
+
+
NADH + H + PMS f NAD + PMSH2
ð2Þ
(
supplied through diet). Biologically important antioxidative
compounds within cells, cell membranes, and extracellular fluids
PMSH2 + 2O2 f 2O2• + 2H + PMS
ꢀ
+
ð3Þ
can be up-regulated and mobilized to neutralize excessive and
inappropriate ROS formation.
1
A large number of assays have been developed to detect O2•
all employing a “probe”, i.e., a molecule that reacts with O2
ꢀ 7
,
The superoxide radical (SR) can act both as a reducing and
oxidizing agent for a variety of compounds and is a key inter-
mediate for more active oxygen species, such as hydroxyl radical
and singlet oxygen. It has been documented that O2•ꢀ formation
occurs continuously in the cells as a consequence of both
enzymatic and nonenzymatic reactions. Xanthine oxidase (XO)
is a complex enzyme in endothelial cells that leads to the
generation of O2 . XO catalyzes the oxidation of xanthine to
uric acid yielding O2 and raises the oxidative level in an
organism. This enzymatic system is frequently used as a gen-
erator of O2 (eq 1):
•
ꢀ
producing a detectable product. The most commonly utilized
•
ꢀ
spectrophotometric detection methods for O use cytochrome
2
8
9
c
or nitroblue tetrazolium (NBT) as spectrophotometric
3+ 2+
probes. Since cytochrome c (Cyt cꢀFe f Cyt cꢀFe ; λmax:
5
50 nm) is easily reduced by reductases such as NAD(P)H
•
ꢀ
reductase and other reducing agents, it is necessary to consider
possible contaminants in the samples. The NBT method is based
on the generation of water-insoluble blue formazan dye (λmax:
•ꢀ
2
560 nm) by a reaction with O2•
. In this case, O2 acts as a
ꢀ 10
•ꢀ
•ꢀ
reducing agent toward NBT, since the standard reduction
xanthine + 2O2 + H2O f uric acid + 2O2• + 2H+
ꢀ
ð1Þ
Received: March 30, 2011
Accepted: June 1, 2011
Published: June 01, 2011
There are reports documenting that some kinds of thiols,
including dithioerythritol (DTE), acted not only as O2•
ꢀ
r 2011 American Chemical Society
5652
dx.doi.org/10.1021/ac200788m
|Anal. Chem. 2011, 83, 5652–5660

Analytical Chemistry
ARTICLE
•
ꢀ
2+
2ꢀ
potentials for the O /O2 and NBT /NBTDF (diformazan)
2
11
12
redox couples are ꢀ0.330 and ꢀ0.080 V, respectively. Con-
sequently, a nonhomogeneous suspension of mixed formazans is
created that causes problems in the reproducibility of data, and
therefore, a modified NBT assay with staining on gels was
developed due to the problems encountered when NBT is used
13
for assaying superoxide dismutase (SOD) in solution. Moreover,
NBT is reduced by various reducing agents (i.e., cellular reduc-
14
tases, fructosamines) and used for the detection of keto-amines
15
as an index of diabetic control. The chemiluminescence probes
•
ꢀ
(
i.e., lucigenin, luminol, luciferin derivative) used for O2 detec-
tion can also be applied for SR scavenging activity (SRSA) assay.
The lucigenin assay has been criticized, because lucigenin itself can
•
ꢀ
•ꢀ
react to produce O2 and in some cases can stimulate O2
production by intact cells. Luminol, unlike lucigenin, is oxidized by
2•ꢀ
Figure 1. Oxidation of TBHQ to TBBQ by O
PMSꢀNADH system.
generated in the
•ꢀ
O2 . This leads to a complex series of reactions among luminol,
luminol radicals, oxygen, andO2 , ultimately producinga luminol
endoperoxide, which decomposes with the release of a photon.
Since O2 is involved as both initiator and intermediate in the
reaction, objections have been raised to the use of luminol as a
quantitative measure of O2•ꢀ production. As electron spin
•ꢀ
scavengers is measured, because the incubation reaction medium
would show a greater CUPRAC absorbance when scavengers are
present (i.e., the CUPRAC-reactive TBHQ is converted to a
lesser extent into the CUPRAC-nonresponsive TBBQ in the
presence of SR scavengers, as shown in Figure 1). The tissue
homogenates (kidney and liver) were evaluated for their SRSA
using the proposed method in comparison with the high-
performance liquid chromatography (HPLC) and NBT refer-
ence methods.
•
ꢀ
7
resonance (ESR) techniques along with a spin-trapping agent
e.g., 5,5-dimethyl-1-pyrroline N-oxide) require a specialized and
4
(
•ꢀ
expensive ESR spectrometer, novel O2 detection methods are
needed with more readily available equipment. No matter which
assay is used, it is important to include proper controls in the
experimental design in order to be certain that any signal detected
is due to O2 . A positive control experiment with SOD may be
useful; i.e., if the signal is not abolished by SOD, it does not
represent O2 production.
The CUPRAC (cupric reducing antioxidant capacity) method
of antioxidant measurement, introduced by our research group to
world literature, is based on the absorbance measurement of
the CUPRAC chromophore, Cu(I)ꢀneocuproine (Nc) chelate,
formed as a result of the redox reaction of antioxidants with the
CUPRAC reagent, Cu(II)ꢀneocuproine, where absorbance is
recorded at the maximal light absorption wavelength of 450 nm.
Properly located phenolic hydroxyls are oxidized to the corre-
sponding quinones in the CUPRAC redox reaction. tert-Butyl-
hydroquinone (TBHQ) is a O
8.1 μM. Quinones shuffle electrons enzymatically or none-
nzymatically among their reduced form (hydroquinone), oxi-
dized form, and/or their semiquinone radicals to construct redox
cycles. TBHQ can be oxidatively converted to the corresponding
semiquinone radical (TBQ , one-electron oxidation product)
or TBBQ (fully oxidized by two electron loss),
potential of the TBBQ/TBQ pair being E1/2 = 0.52 V. Since
the standard reduction potential, E°, of the O /H O redox
couple in acidic medium is 0.940 V, O2 can oxidize TBHQ to
TBBQ (the latter redox couple having E° = 0.360 V). As
opposed to its role toward NBT, O2 then acts as an oxidizing
agent toward TBHQ. Thus, the idea in the present study is to use
TBHQ probe with the PMSꢀNADH nonenzymatic O2 -gen-
erating system for SRSA assay of thiol-type antioxidants (e.g.,
glutathione, cysteine), amino acids (e.g., serine, threonine),
plasma antioxidants (e.g., bilirubin, albumin), and other antiox-
idants (e.g., methionine) by measuring the CUPRAC absorbance
of the remaining TBHQ in the reaction medium (TBHQ is
CUPRAC-reactive and is isolated by ethyl acetate extraction
from other CUPRAC-reactive interferents in the aqueous
phase). As a result, the difference in the CUPRAC absorbance
of the TBHQ probe in the absence and presence of O2•
•ꢀ
’ EXPERIMENTAL SECTION
•ꢀ
Reagents and Apparatus. The following chemical sub-
stances of analytical reagent grade were supplied from the
corresponding sources: β-nicotinamide adenine dinucleotide
reduced dipotassium salt (β-NADH), L-threonine, L-leucin,
L-phenylalanine, L-serine, 1,4-dithioerythritol (DTE), bilirubin,
neocuproine (2,9-dimethyl-1,10-phenanthroline), superoxide
dismutase from bovine liver (30 KU) (SOD), tert-butylhydro-
quinone (TBHQ), R-tocopherol, uric acid, 2-tert-butyl-1,4-ben-
zoquinone (TBBQ), and nitroblue tetrazolium chloride (NBT)
were from Sigma-Aldrich (Steinheim, Germany); copper(II)
chloride dihydrate, L-tryptophan, L-glutathione reduced, L-ascor-
bic acid (AA), and albumin from bovine serum were from Merck
(Darmstadt, Germany); ammonium acetate, dimethyl sulfoxide
(DMSO), and ethyl acetate (EtAc) were from Riedel-de Haen
(Steinheim, Germany); phenazine methosulfate (PMS), L-glu-
tathione oxidized, L-cysteine, L-methionine, and N-acetyl-L-cy-
steine were from Fluka (Buchs, Switzerland).
The visible spectra and absorption measurements were re-
corded in matched quartz cuvettes using a Varian Cary Bio 100
UVꢀvis spectrophotometer (Mulgrave, Victoria, Australia).
Other related apparatus and accessories were a J. P. Selecta water
bath (Barcelona, Spain) and Telstar Cryodos freeze-dryer
(Terrassa, Spain). The chromatograph was from a Waters Breeze
2 model HPLC system (Milford, MA, U.S.A.) equipped with a
2998 photodiode array detector (Chelmsford, MA, U.S.A.), and
ACE C18 column (4.6 mm ꢁ250 mm, 5 μm particle size)
(Milford, MA, U.S.A.). Data acquisition was accomplished using
Empower PRO (Waters Associates, Milford, MA).
1
6
•ꢀ
scavenger with IC50 of
2
1
7
1
•ꢀ
18,19
the redox
•ꢀ
20
•ꢀ
2
2 2
•
ꢀ
21
•
ꢀ
•
ꢀ
ꢀ
3
Preparation of Solutions. The TBHQ solution at 1.0 ꢁ 10
M was prepared in EtOH/water (1:9, v/v). β-NADH (468 μM),
PMS (60 μM), NBT (300 μM), CuCl (10 mM), and ammo-
nium acetate buffer solutions (1 M, pH 7) were all prepared in
pure distilled water (Millipore Simpak1 Synergy 185, U.S.A.),
2
ꢀ
5
653
dx.doi.org/10.1021/ac200788m |Anal. Chem. 2011, 83, 5652–5660

Analytical Chemistry
ARTICLE
and neocuproine solution (7.5 mM) was prepared in absolute
ethanol. All thiol-type antioxidants and amino acids, ascorbic
acid, and SOD were freshly prepared in distilled water at 1.0 ꢁ
SRSA percentage (y) can be empirically correlated to con-
centration of scavenger (x) within an absorbance range over
which Beer’s law is valid:
ꢀ
2
ꢀ5
1
0
ꢀ5.0 ꢁ 10 M concentration, bilirubin and uric acid were
ꢀ
4
y ¼ mx + n
ð5Þ
in 0.5 M NaOH, and R-tocopherol was in EtOH at 1.0 ꢁ 10
M
concentration prior to measurement.
where m and n are the slope and intercept of this linear
correlation, respectively. IC50 can then be calculated for 50%
inhibition (y = 50) such that
Preparation of Tissue Homogenates. CD-1 mice were
obtained from the experimental animal facility of the Faculty of
Veterinary Medicine of Istanbul University. The mice were
2
housed in polycarbonate cages (450 cm area per animal),
y ¼ 50 ¼ mðIC50Þ + n
or
IC50 ¼ ð50 ꢀ nÞ=m ð6Þ
acclimatized under laboratory conditions (23 ( 2 °C, humidity
5
0ꢀ60%, 12 light/dark cycles), and fed by standard mice food.
Liver and kidney tissues were isolated after sacrifice by decapita-
tion from mice. The tissue samples were washed with 0.9% NaCl,
weighed (10%, w/v), and homogenized by adding cold 1.15%
KCl in a glass homogenizer. Homogenates were immediately
Testing of TBHQ and Reaction Products in the Presence of
AA with the UV Method. The tubes bearing the reaction
mixtures (0.5 mL of TBHQ (1.0 mM) + 2 mL of NADH
(
(
468 μM) + 1 mL of PMS (60 μM) + 0.5 mL of AA
0.1ꢀ0.5 mM) or water) were incubated for 30 min in a 25 °C
22
frozen in liquid nitrogen and kept at ꢀ80 °C until analysis.
water bath. The mixture solution was then extracted with adding
Homogenates were separated from proteins by adding EtOH,
centrifuging (4000 rpm, 5 min), and filtering through a 0.45 μm
membrane filter before analysis.
3
.0 mL of ethyl acetate and vortexed for 20 s. The UV spectrum
of the extract containing TBHQ probe and its oxidation product
(TBBQ) was recorded in the wavelength range of 260ꢀ320 nm
against EtAc (reagent blank).
Modified CUPRAC Assay (Proposed Method). Superoxide
anion radicals are generated nonenzymatically by a PMSꢀ
5
NBT Assay. The commonly used indirect reference method of
NADH system. To a test tube was added 0.5 mL of 1.0 mM
•ꢀ
10
determining O2 is the reduction of NBT to the insoluble blue
TBHQ (probe material), 0.5 mL of scavenger solution at a
suitable concentration or water (for reference), 2.0 mL of 468
μM NADH, and 1.0 mL of 60 μM PMS, rapidly in this order. The
reaction was started by adding PMS solution. The mixture in a
total volume of 4.0 mL was incubated for 30 min in a water bath
kept at 25 °C. At the end of this period, the remaining TBHQ
together with its oxidation product (TBBQ) was extracted by
adding 3.0 mL of ethyl acetate (extraction of probe) and vortexed
for 20 s. To 1.0 mL of the EtAc extract, the modified CUPRAC
formazan. SRSA was evaluated by spectrophotometric measure-
•
ꢀ
ment of formazan formed from NBT reduction by O2
•ꢀ
.
NBT2+ + 4O2 + 2H f diformazan + 4O2
+
ð7Þ
To a test tube was added (2.5 ꢀ x) mL of DMSO,
x = 0ꢀ0.5 mL of scavenger solution at a suitable concentration,
2
.0 mL of 468 μM NADH, 1.0 mL of 300 μM NBT, in this order.
The reaction was started by adding 1.0 mL of 60 μM PMS
solution to the incubation mixture. The mixture in a total volume
of 6.5 mL was incubated for 5 min in a water bath kept at 25 °C,
and the absorbance was read at 560 nm against DMSO.
Decreased absorbance of the incubation reaction mixture indi-
cated increased SRSA. The inhibition ratio of scavengers (%) was
calculated using the following formula:
1
6
method was applied in the following manner:
1
:0 mL of CuðIIÞ + 1:0 mL of Nc + 1:0 mL of NH4Ac buffer
1:0 mL of EtAc extract + 0:1 mL of EtOH ðVtotal ¼ 4:1 mLÞ
The absorbance at 450 nm of the final solution at 4.1 mL of
+
total volume was recorded 30 min later against a reagent blank.
The colors of the incubation solution in the presence and
absence of SOD (scavenger) were compared with respect to
their stabilities, the corresponding CUPRAC absorbances being
recorded between 0 and 35 min.
The IC50 (50% inhibitive concentration) values of the scaven-
gers were determined with the use of the NADHꢀPMS system by
means of a linear plot of inhibition percent as a function of
inhibition ratio ð%Þ ¼ 100½ðA ꢀ AÞ=A ꢂ
where A and A are the absorbances of the incubation reaction
mixture in the absence and presence of scavenger, respectively.
HPLC Assay. A 2 mL aliquot of EtAc extract was freeze-dried
for 10 min. The remaining residues were dissolved with 2 mL of
ð8Þ
o
o
o
1:1 (v/v) EtOHꢀH O mixture. The analyses were carried out
2
using a reversed-phase Agilent C18 column (4.6 mm ꢁ250 mm,
5 μm particle size) (Milford, MA, U.S.A.). The mobile phase
consisted of two solvents, i.e., methanol (A) and bidistilled water
(B). The following parameters and gradient were used for the
Cscavenger, where A is the initial CUPRAC absorbance of the
o
original TBHQ probe solution, A and A are those of the TBHQ
1
2
probe subjected to PMSꢀNADH action in the absence and
presence of SR scavenger, respectively, and C is the molar
concentration of relevant scavenger. The IC50 values were then
analysis of EtAc extracts: (V
= 20 μL; flow rate = 1.0 mL
sample
ꢀ
1
min ; λTBHQ = 290 nm; λTBBQ = 252 nm): 1 min 60% A to 40%
B (slope 1.0); 4 min 70% A to 30% B (slope 1.0); 10 min 80% A
to 20% B (slope 1.0); 15 min 60% A to 40% B (slope 1.0). The
capability of O2• scavenging was calculated using a modified
version of eq 4:
10
compared with those found with the NBT method. The CU-
PRAC absorbance of the ethyl acetate extract of the incubation
solution arising from the reduction of Cu(II)ꢀneocuproine re-
agent by the remaining TBHQ was higher in the presence of O2•
scavengers (due to less conversion to TBBQ); therefore, increased
absorbance of the reaction mixture indicated increased SRSA.
The SRSA of various scavengers were calculated using the
following equation:
ꢀ
ꢀ
inhibition ratio ð%Þ ¼ 100½ðA2 ꢀ A1Þ=ðAo ꢀ A1Þꢂ
ð9Þ
where A and A are the peak areas of the TBHQ probe in the
1
2
absence and presence of SR scavenger, respectively; A is the
0
peak area of the TBHQ probe at initial concentration in the
reaction mixture.
SRSA ð%Þ ¼ 100½ðA2 ꢀ A1Þ=ðAo ꢀ A1Þꢂ
ð4Þ
5
654
dx.doi.org/10.1021/ac200788m |Anal. Chem. 2011, 83, 5652–5660

Analytical Chemistry
ARTICLE
Figure 2. HPLC chromatograms for standard (original) and remaining TBHQ after PMSꢀNADH reaction in the presence and absence (reference) of
AA (λ = 290 nm): (a) TBHQ standard; (b) reference (0.5 mL of TBHQ (1.0 mM) + 2 mL of NADH (468 μM) + 1 mL of PMS (60 μM) + 0.5 mL of
water); (c) 0.5 mL of TBHQ (1.0 mM) + 2 mL of NADH (468 μM) + 1 mL of PMS (60 μM) + 0.5 mL of AA (0.3 mM).
Statistical Analysis. Descriptive statistical analyses were
performed using Excel software (Microsoft Office 2002) for
calculating the means and the standard error of the mean. Results
were expressed as the mean ( standard deviation (SD). Using
SPSS software for Windows (version 13), the data were evaluated
within which the relative standard deviation (RSD) was 3.4%.
The limit of detection (LOD) and limit of quantification (LOQ)
were calculated using the equations LOD = 3s /m and LOQ = 10
bl
s /m, respectively, where s is the standard deviation of a blank
bl
bl
and m is the slope of the calibration line. The LOD and LOQ for
TBHQ were found as 0.2 and 0.67 μM, respectively.
23
by two-way analysis of variance (ANOVA).
Conversion of the TBHQ probe to TBBQ and inhibition of
this reaction with a SR scavenger (i.e., SOD) were followed by
measuring the CUPRAC absorbance of the mixture as a function
of time (Figure 4). Inhibition of this reaction proceeded slowly in
the presence of SOD, and an optimal measurement time of
30 min was chosen (Figure 4). This optimal period is sufficient to
achieve an absorbance difference between TBHQ and TBBQ.
Thus, due to the high conversion yield of the selected probe,
SRSA of the studied compounds could be rapidly and precisely
determined by recording the relative absorbances within 30 min.
For comparison, time-dependent absorbance changes were
recorded using TBHQ standard solutions incubated under
identical conditions. There was no noteworthy change in absor-
bance for TBHQ within the 0ꢀ30 min time interval (Figure 4),
and autoxidation reaction (i.e., without PMSꢀNADH) did not
occur in this system that would otherwise cause interference.
Thus, the short-lived intermediate of TBHQ oxidation (i.e.,
’
RESULTS AND DISCUSSION
Optimization of the Method. A modified CUPRAC method
was applied to assess the SRSA of amino acids (i.e., L-serine,
L-threonine, L-leucin, L-phenylalanine, L-tryptophan), thiol-type
antioxidants (i.e., L-cysteine, N-acetyl-L-cysteine, L-glutathione
reduced, and L-glutathione oxidized), and plasma antioxidants
(
i.e., bilirubin, uric acid, albumin, R-tocopherol, ascorbic acid, and
SOD). CUPRAC absorbance arises from the reduction of the
16
Cu(II)ꢀNc reagent to the Cu(I) chelate by the TBHQ probe
2+
+
+
(i.e., TBHQ + 2Cu(Nc)2 f TBBQ + 2Cu(Nc)2 + 2H ),
whereas the quinonic product of the (TBHQ + NADHꢀPMS)
incubation system, TBBQ, had a negligible original CUPRAC
absorbance. A scheme of conversion of TBHQ to TBBQ by O2•
is shown in Figure 1. The chromatograms for TBHQ conversion
upon O2 attack—in the absence and presence of the SR
scavenger (AA)—are provided in Figures 2 and 3.
TBHQ, by virtue of its two-electron oxidation to TBBQ, acts
as a reductant toward the CUPRAC reagent, Cu(II)ꢀNc. The
molar absorptivity for TBHQ in the CUPRAC method was
ꢀ
•ꢀ
24
semiquinone radical) was not detected. Li et al. have demon-
strated that no semiquinone radical was detectable by ESR spin
trapping when 1.0 mM TBHQ was incubated in phosphate buffer
saline (PBS) in the absence of added Cu(II), whereas the
incubation system of this work, {PMSꢀNADH + TBHQ}, did
not contain a Cu(II) catalyst, and TBHQ may possibly be
4
ꢀ1
ꢀ1
ε = 1.57 ꢁ 10 M cm , and the linear concentration range was:
ꢀ7
ꢀ5
5
.09 ꢁ 10 ꢀ7.60 ꢁ 10 M (correlation coefficient: r = 0.998),
5
655
dx.doi.org/10.1021/ac200788m |Anal. Chem. 2011, 83, 5652–5660

Analytical Chemistry
ARTICLE
Figure 3. HPLC chromatograms for TBBQ (λ = 252 nm) formed from TBHQ in the PMSꢀNADH system in the presence and absence (reference) of
AA: (a) reference (0.5 mL of TBHQ (1.0 mM) + 2 mL of NADH (468 μM) + 1 mL of PMS (60 μM) + 0.5 mL of water); (b) 0.5 mL of TBHQ (1.0 mM)
+
2 mL of NADH (468 μM) + 1 mL of PMS (60 μM) + 0.5 mL of AA (0.3 mM).
24
results are confirmed in a study by Li et al., examining the
Cu(II)-mediated redox-dependent activation of TBHQ. Upon
mixing 1 mM TBHQ with O2• generated by the PMSꢀNADH
system in the presence of AA at concentrations 0.1ꢀ0.5 mM, the
absorbance of TBHQ at 290 nm decreased with a corresponding
increase in absorbance at 252 nm (Figure 5), indicating that the
reaction of TBHQ with O2• results in its oxidation to TBBQ.
Thus, there is a proportional increase of 290 nm peak heights
ꢀ
ꢀ
(
Figure 5) with increasing concentration of scavenger (AA).
The effects of NADH and PMS, individually and simulta-
neously, on the CUPRAC absorbance of the TBHQ probe (in
the absence and presence of scavenger, AA) were studied under
the same experimental conditions, where neither compound in
the EtAc phase gave a 450 nm absorbance (Table S-1, Supporting
Information). In other words, the only constituent giving rise to
an absorbance in the system is TBHQ, i.e., the CUPRAC-reactive
substance (CUPRAC-rs) mentioned in Figure 1. PMSꢀNADH
can produce O2• , and these radicals are manifested by the
ꢀ
decrease in absorbance of the CUPRAC chromophore (A
0
=
4
50
.065) as a result of TBHQ oxidation with O2• to TBBQ,
ꢀ
whereas the absorbance due to the remaining TBHQ probe
increases upon competition with AA (A450 = 0.490) (Table S-1,
Supporting Information). The inhibition ratio (%) leading to
SRSA estimation is calculated from the relative decrease of
CUPRAC absorbances of the EtAc extracts using eq 4. The
competition with AA of the TBHQ probe for O2 can be
followed simply by observing the changes in the concentration of
the EtAc-extracted TBHQ probe without interference from
water-soluble SR scavengers and other system constituents
(i.e., PMS and NADH).
Figure 4. CUPRAC absorbance vs incubation time curves of TBHQ
alone and TBHQ subjected to the PMSꢀNADH reaction in the
•ꢀ
2
ꢀ1
presence (6.4 U mL SOD) and absence (reference) of a O
scavenger.
•ꢀ
oxidized by O •ꢀ directly to TBBQ with the concomitant
2
formation of H O .
2
2
TBHQ and TBBQ exhibit UV-absorbance spectra with peak
absorbances at 290 and 252 nm, respectively (Figure 5). These
Superoxide and H O can serve as substrates for the formation
2
2
of hydroxyl radicals ( OH) having a potential to interfere with
3
5
656
dx.doi.org/10.1021/ac200788m |Anal. Chem. 2011, 83, 5652–5660

Analytical Chemistry
ARTICLE
Figure 5. UV spectra of the remaining TBHQ and its oxidation product, TBBQ, as a result of PMSꢀNADH incubation in the presence of ascorbic acid
AA) at various concentrations: (a) 0 mM (reference, without scavenger), (b) 0.1 mM, (c) 0.3 mM, (d) 0.4 mM, (e) 0.5 mM AA, and (f) 0 mM (TBHQ
standard).
(
2
5
the proposed assay only in a peroxidase- or transition metal
HPLC value, the peak areas were used to calculate the inhibition
% of samples with respect to eq 9. The considerably larger IC50
value of SOD found with the use of the NBT method (Table S-2,
Supporting Information) compared to those found by other
methods may arise from numerous pitfalls met in assaying SOD
by NBT conversion to formazan such as the SOD-noninhibitable
26
ion-catalyzed reaction, whereas in this study, such catalyzers
were absent. H O is normally present at minute concentrations
2
2
27
in leaf homogenates (at 40ꢀ120 nmol/g fresh weight) and
28
rat liver homogenate (at a concentration level of 0.1 μM).
When the possible effect of H O at 0.1 and 1.0 μM levels was
2
2
13
investigated in the nonenzymatic PMSꢀNADH incubation system
portion of NBT reduction. Likewise, it has earlier been
reported that in many tissues where assay interferences were
noted, no Mn-SOD activity could be detected using the NBT
ꢀ
1
with and without 721 U mL catalase used as H O scavenger
2
2
(
table not shown), it was seen that the added H O did not
2 2
29
interfere with the modified CUPRAC assay, producing A450
values in the EtAc phase between 0.058 and 0.072 absorbance
units, i.e., not distinguishable from reference values. Besides
added H O , any new formed H O as a result of O oxidation
of the TBHQ probe was naturally eliminated by ethyl acetate
assay. In the presence of potent scavengers such as AA, the
conversion ratio of the probe was considerably smaller, as is
apparent from the significant lowering of the peak heights of
•
ꢀ
5
2
2
2
2
2
TBBQ, e.g., the peak area for TBBQ as 6.48 ꢁ 10 (reference)
5
(Figure 3a) decreased to 3.61 ꢁ 10 with AA (Figure 3b). It has
extraction (i.e., H O remained in the aqueous phase) followed
by CUPRAC measurement in the organic phase.
been reported that AA protects against oxygen poisoning with its
2
2
role as a potential scavenger of O2•
. Nishikimi has found the
ꢀ 30
•
ꢀ
Comparison of CUPRAC and HPLC Findings. In the chro-
matograms of this work (Figures 2 and 3), the retention times for
TBHQ (detected at 290 nm) and its lone oxidation product
TBBQ (detected at 252 nm) were 6.00ꢀ6.05 and 9.75 min,
respectively. The amounts of TBHQ was found with the aid of
the calibration curve drawn as peak area versus concentration,
and the HPLC-found concentration of TBHQ multiplied by its
absorptivity gave the experimentally measured CUPRAC absor-
bance. Concentrations of TBHQ (in μg mL ) remaining after
PMSꢀNADH reaction in the presence of scavenger (AA) using
the modified CUPRAC and HPLC methods were 2.18 ( 0.03
and 2.26 ( 0.05, respectively (initial concn of TBHQ was
second-order rate constant for the reaction of AA with O to be
2
5
ꢀ1 ꢀ1 30
2.7 ꢁ 10 M
s
,
in accordance with the value found in
5
ꢀ1 ꢀ1
the present study (3.81 ꢁ 10 M
s ). The calculations for the
•ꢀ
second-order rate constant of O2 scavenging by AA are
illustrated in Table S-3 and Figure S-1 (Supporting Information).
In order to allow comparison among assays, it is useful to
compare the obtained inhibition of the scavenger analyte with
that obtained by the SOD enzyme, or by standard antioxidants,
such as AA. Since the SRSA measurements were made under
nonequilibrium conditions where O2• was generated continu-
ꢀ
1
ꢀ
3
1
ously, the results should be interpreted with caution. SOD-
equivalent SRSA activities of AA, L-serine, and NAC (at 0.5 mM
scavenger concentration) calculated with CUPRAC, HPLC, UV,
and NBT methods are presented in Table 1. The findings of
modified CUPRAC and HPLC methods agreed among them-
selves with tolerable error (Table 1) (Fexp = 0.059, Fcrit = 18.51,
Fexp < Fcrit at P = 0.05). The problems encountered in observing
very high SOD-equivalent SRSA values for N-acetyl cysteine
(NAC) in the NBT method (Table 1) is thought to arise from
2
7.76 ( 0.14, Figure 2a), and both methods correctly reflected
the relative decrease in probe (TBHQ) conversion into TBBQ in
the presence of AA (Figure 2, parts b and c).
SRSA activities of SOD (as IC ) were calculated with
50
spectrophotometric methods (i.e., modified CUPRAC, UV,
and NBT methods) using eqs 4 and 8, as well as with HPLC.
CUPRAC and HPLC assay results agreed among themselves
with tolerable error (Table S-2, Supporting Information) (IC50
values of SOD with respect to the CUPRAC and HPLC methods
13
the pitfalls met in assaying SOD with this method, and not from
the NAC assay itself, as the IC50 values of NAC found with the
modified CUPRAC and NBT methods were alike (Table 2). By
ꢀ1
were 8.66 ( 0.34 and 8.58 ( 0.68 U mL , respectively). For the
5
657
dx.doi.org/10.1021/ac200788m |Anal. Chem. 2011, 83, 5652–5660

Analytical Chemistry
ARTICLE
Table 1. SOD-Equivalent SRSA of Certain Scavengers Calculated with Respect to the Modified CUPRAC, HPLC, UV, and NBT
a
Methods (N = 4 or 5 Data Points)
ꢀ1
ꢀ1
ꢀ1
ꢀ1
SR scavengers
SRSACUPRAC (U mL
)
SRSAHPLC (U mL
)
SRSAUV (U mL
)
SRSANBT (U mL )
ascorbic acid
N-acetyl cysteine
serine
10.56 ( 0.42
11.51 ( 0.69
1.70 ( 0.03
10.57 ( 0.31
9.94 ( 0.39
2.71 ( 0.13
12.65 ( 0.63
13.25 ( 0.66
5.65 ( 0.05
13.58 ( 0.95
44.21 ( 3.85
25.82 ( 1.54
a
SOD equivalent was calculated for a 0.5 mM scavenger concentration. (The calculation of SOD-equivalent SRSA is given in the Supporting
Information.)
33
method due to its high sensitivity. NBT is known to be four-
electron-reduced by O2 to the water-insoluble blue-colored
Table 2. SRSA of Various Scavengers Using the Modified
•ꢀ
CUPRAC Method in Comparison with the NBT Method
formazan (λ : 560 nm) with a high molar absorptivity (i.e., ε >
a
max
ꢀ1
(IC50 with Respect to eq 5, N = 4 or 5 Data Points)
4
ꢀ1
ꢀ
1
0 M cm ). Any added SR scavenger capable of reacting
•
with O2 inhibits the production of formazan. SRSA can there-
fore be expressed as the “percentage inhibition of NBT reduc-
tion”. Since formazan is easily detected spectrophotometrically
over a wide pH range, NBT has been used in clinical determina-
IC50 value with respect to IC50 value with respect to
SR scavengers
CUPRAC method (mM)
NBT method (mM)
Thiol-Type Antioxidants
23
tion of SOD and NADH or NADH-dependent enzyme
glutathione
0.51 ( 0.02
0.41 ( 0.03
0.38 ( 0.02
0.37 ( 0.02
34
(
dehydrogenase) activities. However, the mechanism of reduc-
(GSH)
35
tion of NBT is rather complicated, and its half-reduced (two-
electron-reduced) insoluble species (mono- and diformazans)
may often lead to erratic absorbance readings in aqueous
solution. In this study, DMSO was used as solvent for solubilizing
glutathione
0.48 ( 0.01
0.36 ( 0.03
oxidized (GSSG)
N-acetyl cysteine
3
6
(
NAC)
cysteine
,4-
the formazan compounds formed in the reference NBT assay.
b
0.28 ( 0.01
0.29 ( 0.02
N.D.
Although the reduction of 1 mol of NBT to 1 mol of formazan is
ꢀ 9
1
0.63 ( 0.04
assumed to theoretically require 4 mol of O2• , the redox cycling
•+
dithioerythritol
of the intermediary reduction product of NBT (i.e., NBT radical
37
cation) with molecular oxygen, combined with the generation of
S-Containing Antioxidants
two different formazan species from NBT at the same redox
methionine
3.55 ( 0.21
4.37 ( 0.30
35
potential and with the existence of superoxide-independent
38
Amino Acids
4.74 ( 0.19
4.48 ( 0.27
0.16 ( 0.01
0.13 ( 0.01
0.35 ( 0.02
pathways for the reduction of NBT, are limiting factors to
•ꢀ
quantitative conversion of NBT to formazan as a probe for O2
serine
51.00 ( 3.00
52.00 ( 2.08
4.39 ( 0.28
2.45 ( 0.17
0.47 ( 0.02
estimation. The NBT test producing the insoluble formazan
product was reported not to permit a reliable quantitative photo-
threonine
leucin
metricdeterminationofO2• in solution. Onthe otherhand, the
conversion efficiency of TBHQ probe to TBBQ without any
scavenger (reference) was >99%. As a result of the PMSꢀNADH
reaction, product conversion varied with respect to the nature of
scavenger, e.g., the conversion ratio of the original probe (TBHQ)
to its only oxidation product (TBBQ) in the presence of 0.1 mM
AA was approximately 90ꢀ92%, whereas with 0.3 mM AA, this
ratio decreased to 64ꢀ66% (Figures 2 and 3).
ꢀ
39
tryptophan
phenylalanine
Plasma Antioxidants
c
c
albumin
6.09 ( 0.31
21.82 ( 1.74
d
R-tocopherol
uric acid
0.24 ( 0.01
24.8 ( 0.96
0.41 ( 0.02
0.11 ( 0.01
N.D.
b
N.D.
ascorbic acid
bilirubin
0.61 ( 0.04
Thiols like glutathione, N-acetyl cysteine, and cysteine were
effective SR scavengers (Table 2) in accordance with literature
d
N.D.
e
e
SOD
8.66 ( 0.34
26.36 ( 2.37
40ꢀ42
41
reports.
Winterbourn and Metodiewa examined the reac-
a
•ꢀ
P = 0.05, Fexp = 2.5499, Fcrit(table) = 4.965, Fexp < Fcrit (table)). Data
tion of thiol compounds with O2 as demonstrated by the loss
of thiol groups and enhanced oxygen uptake in the xanthine
oxidase incubation system. Among the biologically important
thiol compounds tested, cysteine showed a high reactivity (i.e.,
exhibiting the highest superoxide-dependent thiol loss upon
b
presented as (mean ( SD), N = 3. Interference (reduction of NBT).
c
ꢀ1 d
Unit of IC50 value is mg mL . N.D.: SRSA at the studied concentra-
e
ꢀ1
tion level could not be detected. Unit of IC50 value is U mL
.
similar reasoning, it can be understood that the IC50 values in
Table 2 for SOD calculated by using the CUPRAC and NBT
methods were quite different. The agreement between modified
CUPRAC and NBT in IC50 calculation of NAC (Table 2) is in
41
incubation) in compliance with our finding (IC (mM):
5
0
43
0
.28 ( 0.04) (Table 2). Okada and Okada also reported a
ꢀ1
ꢀ1
SOD-equivalent SRSA activity value of (2.73 ( 0.12 units mL
)
for 100 μM cysteine (SRSA
: 4.73 ( 0.12 units mL ),
CUPRAC
32
conformation with the findings of Aruoma et al. who noted that
there was no observable direct reduction of NBT with NAC, but
at concentrations of 3 mM, NAC decreased the rate of NBT
reduction by 13ꢀ15%.
whereas NBT assay was nonresponsive to cysteine, probably due
to the direct reduction of NBT with cysteine. The overall
equation for the reaction of cysteine with O2 is given by eq 10:
•ꢀ
Comparison of the Modified CUPRAC and NBT Methods
for SRSA Assay. Under in vitro conditions, O2•ꢀ has been widely
measured spectrophotometrically by means of the NBT
cysteineðRSHÞ + 2O2•ꢀ + 2H f cystineðRSSRÞ + 2H O
+
2
2
2
ð10Þ
5
658
dx.doi.org/10.1021/ac200788m |Anal. Chem. 2011, 83, 5652–5660

Analytical Chemistry
ARTICLE
4
4
Nishikimi et al. demonstrated that R-tocopherol (R-TOC)
•
ꢀ
was oxidized to R-tocopheryl quinone by O2 generated in the
xanthineꢀxanthine oxidase system, and IC of R-TOC was
5
0
ꢀ
1
found at the SOD-equivalent concentration of 0.19 μg mL
(
the approximate IC50 value of R-TOC was 0.2 mM) in
compliance with our finding (IC50 (mM): 0.24 ( 0.02)
(
studied concentration level could not be detected.
Table 2). SRSA of R-TOC using the NBT method at the
•
ꢀ
The second-order rate constant for NBT reduction by O
2
4
ꢀ1 ꢀ1 45
(
k = 6.0 ꢁ 10 M
s
) is ∼4 orders of magnitude lower than
9 ꢀ1 ꢀ1 46
that of SOD (k = 2.0 ꢁ 10 M
s ), and this may lead to
•
ꢀ
underestimation of O2 as a result of scavenging in some
biological samples containing SOD. Compared to NBT reac-
•ꢀ
tion, the rate constant calculated for TBHQ oxidation by O
(
order of magnitude higher, and this is believed to cause less
interference to the SRSA measurement of biological samples.
Moreover, Spitz and Oberley reported extremely prevalent tissue
specific interferences, where spontaneous tissue-mediated NBT
reduction occurred in assay mixtures without the initiation of
2
5
ꢀ1 ꢀ1
kTBHQ = 4.7 ꢁ 10 M
s ) (Supporting Information) was an
Figure 6. SRSA (%) of some tissue homogenates calculated with the
modified CUPRAC method in comparison to NBT and HPLC
methods.
48
quenching. Likewise, Logan et al. indicated that SRSA showed
a linear correlation with the volume of red wine up to a limited
volume, and exceeding this critical volume resulted in some
saturation resulting in a curved correlation. The SRSA values
measured with the proposed CUPRAC and reference methods
are comparatively depicted in a bar diagram (Figure 6); the
percentage inhibitions of identical tissue homogenates found
with CUPRAC were almost equal to those measured with HPLC.
In future work, the proposed assay may be extended to the
SRSA measurement of phenolic superoxide scavengers. Cos
•
ꢀ
29
O2 generation.
Following the general reasoning of competition kinetics, when
inhibition percentage (y) was correlated to micromolar concen-
tration of inhibitor antioxidant (x), the resulting linear equations
conforming to eq 5 enabled the calculation of IC50 values of
inhibitors using eq 6 (RSD: 6ꢀ7%). The precision of IC50
measurements was reflected in the r values of the competition
plots which varied between 0.935 and 0.998 for both modified
CUPRAC and NBT methods in the optimal concentration range
of the tested SR scavenger (Figure S-2, Supporting Information).
The ANOVA comparison by the aid of F-test of the mean-
squares of “between treatments” (i.e., IC50 values of different
samples with respect to the modified CUPRAC and NBT
49
et al. have reported that flavonoids exhibit SRSA at micromolar
concentration levels (e.g., the IC50 values of quercetin, apigenin,
and myricetin were 1.63, 1.33, and 0.33 μM, respectively). Thus,
it is required that the phenolics in a test sample would not show
an initial CUPRAC absorbance, and yet, they would react with
23
methods depicted in Table 2) and of residuals for a number
of real samples (consisting of 11 SR scavengers) enabled us to
conclude that there was no significant difference between treat-
ments. In other words, the experimentally found CUPRAC
results and NBT results were statistically alike at the 95%
confidence level (Fexp = 2.549, Fcrit = 4.965, Fexp < Fcrit at
P = 0.05) (by exclusion of the values for compounds with highest
IC variability, i.e., SOD, albumin, threonine, serine, leucin, and
•ꢀ
O2 generated by a PMSꢀNADH reaction. After the necessary
incubation, dilution, and extraction, the phenolics themselves
would not give an appreciable absorbance (i.e., A450 e 0.02
absorbance units using a 1 cm optical cell). In that case, the
measured CUPRAC absorbance at the end of the proposed assay
utilizing the TBHQ probe at millimolar levels would definitely
come from TBHQ remaining after PMSꢀNADH reaction, and
50
•ꢀ
not from the consumed micromolar levels of phenolic O
2
tryptophan; Fexp = 1.252, Fcrit = 5.987, Fexp < Fcrit at P = 0.05).
Thus, the proposed methodology was validated against the
classical NBT method.
scavengers.
’
CONCLUSIONS
Application of the Method to Certain Tissue Homoge-
nates and Possibility of Extension to Further Studies. The
SRSA of tissue homogenates was determined by the modified
CUPRAC, HPLC, and NBT methods as IC50 values in the
PMSꢀNADH reaction mixture. The liver homogenates were
generally shown to exhibit higher scavenging activity than kidney
The proposed method renders the relatively specific detection
of O2• and is applied to SRSA estimation of a rich variety of
biologically active compounds (i.e., amino acids, thiols, plasma
antioxidants, and vitamins) and tissue homogenates. Thus,
conventional problems of the widely used NBT method arising
from the insolubility of formazan and from direct reduction of
ꢀ
47
homogenates. Similarly, Nandi and Chatterjee found that rat
liver tissue homogenates (total SOD activity: 1700 ( 100 units
per g tissue) are stronger SR scavengers than kidney homo-
genates (total SOD activity: 750 ( 80 units per g tissue). The
SRSA of kidney homogenates as inhibition % versus homogenate
volume is shown as a curve in Figure S-3 (Supporting In-
•
ꢀ
NBT by tested scavengers without involvement of O2 were
overcome. The modified CUPRAC results were close or compar-
able to those found by the conventional NBT and HPLC
methods. This alternative cost-effective method emerges as a
promising tool to better understand the role of O2• in biology.
ꢀ
•ꢀ
formation). Generally, in the study of O2 scavenging activity
of complex plant extracts and biological fluids and homogenates,
a plateau region of inhibition (or scavenging) percentage as a
’
ASSOCIATED CONTENT
47
function of concentration was reported. This plateau probably
arises from the fact that, above a limiting concentration, scaven-
gers may not effectively compete with the probe for O2•
S
Supporting Information. Additional information as
b
noted in text. This material is available free of charge via the
Internet at http://pubs.acs.org.
ꢀ
5
659
dx.doi.org/10.1021/ac200788m |Anal. Chem. 2011, 83, 5652–5660

Analytical Chemistry
ARTICLE
’
AUTHOR INFORMATION
(25) Chen, S.-X.; Schopfer, P. Eur. J. Biochem. 1999, 260, 726–735.
(
26) Pasternack, R. F.; Halliwell, B. J. Am. Chem. Soc. 1979,
Corresponding Author
1
01, 1026–1031.
*Fax: +90 212 473 7180. E-mail: rapak@istanbul.edu.tr.
(27) Veljovic-Jovanovic, S.; Noctor, G.; Foyer, C. H. Plant Physiol.
Biochem. 2002, 40, 501–507.
28) Gonzalez-Flecha, B.; Cutrin, J. C.; Boveris, A. J. Clin. Invest.
1993, 91, 456–454.
(
’
ACKNOWLEDGMENT
(
(
29) Spitz, D. R.; Oberley, L. W. Anal. Biochem. 1989, 179, 8–18.
30) Nishikimi, M. Biochem. Biophys. Res. Commun. 1975,
One of the authors (Burcu Bekde -s er) thanks the Istanbul
University Research Fund, Bilimsel Arastirma Projeleri (BAP)
Yurutucu Sekreterligi, for the support given to her Ph.D. thesis
project T-5761 and to Istanbul University, Institute of Pure and
Applied Sciences (I.U. Fen Bilimleri Enstit €u s €u ), for the support
given to her Ph.D. thesis work with the title “Development of
Spectrophotometric Methods for Measurement of Reactive
Oxygen Species Scavenging Activity in Biological Samples”.
The authors also extend their gratitude to TUBITAK (Turkish
Scientific and Technical Research Council) for the Research
Project 110T725.
6
3, 463–468.
31) Frankel, E. N.; Meyer, A. S. J. Sci. Food Agric. 2000,
0, 1925–1941.
32) Aruoma, O. I.; Halliwell, B.; Hoey, B. M.; Butler, J. Free Radical
(
8
(
Biol. Med. 1989, 6, 593–597.
(33) Beauchamp, C.; Fridovich, I. Anal. Biochem. 1971, 44, 276–287.
(34) Ishiyama, M.; Miyazono, Y.; Shiga, M.; Sasamoto, K.; Ohkura,
Y.; Ueno, K. Anal. Sci. 1996, 12, 515–519.
(
(
35) Umemoto, K. Bull. Chem. Soc. Jpn. 1989, 62, 3783–3789.
36) Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.;
Tierney, S.; Nofziger, T. H.; Currens, M. J.; Seniff, D.; Boyd, M. R.
Cancer Res. 1988, 48, 4827–4833.
’
REFERENCES
(37) Tarpey, M. M.; Fridovich, I. Circ. Res. 2001, 89, 224–236.
(
1) Nijveldt, R. J.; van Nood, E.; van Hoorn, D. E. C.; Boelens, P. G.;
(38) Thayer, W. S. Arch. Biochem. Biophys. 1990, 276, 139–145.
(39) Frahry, G.; Schopfer, P. Planta 2001, 212, 175–183.
(40) Winterbourn, C. C.; Metodiewa, D. Free Radical Biol. Med.
1999, 27, 322–328.
(41) Winterbourn, C. C.; Metodiewa, D. Arch. Biochem. Biophys.
1994, 314, 284–290.
(42) Benrahmoune, M.; Therond, P.; Abedinzadeh, Z. Free Radical
Biol. Med. 2000, 29, 775–782.
(43) Okada, Y.; Okada, M. J. Agric. Food Chem. 1998, 46, 401–406.
(44) Nishikimi, M.; Yamada, H.; Yagi, K. Biochem. Biophys. Res.
Commun. 1980, 627, 101–108.
(45) Bielski, B. H. J.; Richter, H. W. J. Am. Chem. Soc. 1977,
99, 3019–3023.
(46) Fridovich, I. Annu. Rev. Pharmacol. Toxicol. 1983, 23, 239–257.
(47) Nandi, A.; Chatterjee, I. B. J. Biosci. 1988, 13, 305–315.
(48) Logan, B. A.; Hammond, M. P.; Storno, B. M. Biochem. Mol.
Biol. Educ. 2008, 36, 39–42.
(49) Cos, P.; Ying, L.; Calomme, M.; Hu, J. P.; Cimanga, K.; Poel,
B. V.; Pieters, L.; Vlietinck, A. J.; Berghe, D. V. J. Nat. Prod. 1998,
61, 71–76.
van Norren, K.; van Leeuwen, P. A. M. Am. J. Clin. Nutr. 2001,
7
4, 418–425.
2) Terada, L. S.; Leff, J. A.; Repine, J. E. Methods Enzymol. 1990,
86, 651–656.
(
1
(
(
3) Corte, E. D.; Stirpe, F. Biochem. J. 1972, 126, 739–745.
4) Unno, T.; Yayabe, F.; Hayakawa, T.; Tsuge, H. Food Chem. 2002,
7
6, 259–265.
5) Nishikimi, M.; Rao, N. A.; Yagi, K. Biochem. Biophys. Res.
Commun. 1972, 46, 849–854.
6) Robak, J.; Grylewski, R. J. Biochem. Pharmacol. 1988,
7, 837–841.
(
(
3
8
(
(
(
7) Bartosz, G. Clin. Chim. Acta 2006, 368, 53–76.
8) Cohen, G.; Heikkila, R. E. J. Biol. Chem. 1974, 249, 2447–2452.
9) Bielski, B. H. J.; Shiue, G. G.; Bajuk, S. J. Phys. Chem. 1980,
4, 830–833.
10) Yu, H.; Liu, X.; Xing, R.; Liu, S.; Li, C.; Li, P. Bioorg. Med. Chem.
Lett. 2005, 15, 2659–2664.
11) Lee, J.; Koo, N.; Min, D. B. Compr. Rev. Food Sci. Food Saf. 2006,
, 21–33.
12) Machado, R. A.; Vargas, F.; Arce, A. J.; Rivillo, D.; De Sanctis,
Y.; Jorge, G.; Martinez, J. D.; D’Ornelas, L. Ciencia 2008, 16, 234–240.
(
(
3
(
(13) Beyer, W. F.; Fridovich, I. Anal. Biochem. 1987, 161, 559–566.
(14) Sutherland, M. W.; Learmonth, B. A. Free Radical Res. 1997,
2
7, 283–289.
15) Johnson, R. N.; Metcalf, P. A.; Baker, J. R. Clin. Chim. Acta
983, 127, 87–95.
16) Apak, R.; G €u c- l €u , K.; Ozy €u rek, M.; Karademir, S. E. J. Agric. Food
Chem. 2004, 52, 7970–7981.
17) Badary, O. A.; Taha, R. A.; Gamal El-Din, A. M.; Abdel-Wahab,
M. H. Drug Chem. Toxicol. 2003, 26, 87–98.
18) Kim, M. C.; Pratt, D. E. In Phenolic Compounds in Food and
(
1
€
(
(
(
Their Effects of Health (II); Ho, C. T., Lee, C. Y., Huang, M. T., Eds.;
American Chemical Society: Washington, DC, 1992; pp 200ꢀ218.
(19) Bergmann, B.; Dohrmann, J. K.; Kahl, R. Toxicology 1992,
7
4, 127–133.
20) Pinkus, R.; Weiner, L. M.; Daniel, V. J. Biol. Chem. 1996,
71, 13422–13429.
21) Van Ommen, B.; Koster, A.; Verhagen, H.; Van Bladeren, P. J.
Biochem. Biophys. Res. Commun. 1992, 189, 309–314.
22) A lꢀi a, M.; Horcajo, C.; Bravo, L.; Goya, L. Nutr. Res. (N. Y.)
003, 23, 1251–1267.
23) Miller, J. C.; Miller, J. N. Statistics for Analytical Chemists, 3rd
ed.; Ellis Horwood and Prentice Hall: New York and London, 1993.
24) Li, Y.; Seacat, A.; Kuppusamy, P.; Zweier, J. L.; Yager, J. D.;
Trush, M. A. Mutat. Res. 2002, 518, 123–133.
(
2
(
(
2
(
(
5
660
dx.doi.org/10.1021/ac200788m |Anal. Chem. 2011, 83, 5652–5660