Inhibition of Peroxynitrite-Mediated Reactions by Vanillin

Article abstract of DOI:10.1021/jf030319d

Several neurodegenerative diseases such as Alzeimer's and Parkinson's as well as septic shock and inflammation involve formation of reactive oxygen and nitrogen species that include peroxynitrite (PON). PON can also react with endogenous antioxidants. Therefore, dietary supplementation with antioxidants may help in these diseases. An exogenous antioxidant, vanillin (4-hydroxy-3-methoxy-benzaldehyde), used widely as a food flavoring agent, was evaluated for its ability to scavenge PON and inhibit PON-mediated reactions. Nitration of tyrosine by PON was assessed by high-performance liquid chromatography (HPLC). This reaction was inhibited by vanillin. The oxidation of dihydrorhodamine 123 to fluorescent rhodamine 123 was also inhibited by vanillin. The kinetics of reaction between PON and vanillin was studied by stopped-flow technique. The products of this reaction were analyzed by HPLC, and hydroxyvanillin was identified as one of the five products with absorption at 350 nm. These data demonstrate that vanillin effectively scavenges PON in cell-free systems.

Full text of DOI:10.1021/jf030319d

J. Agric. Food Chem. 2004, 52, 139−145 139
Inhibition of Peroxynitrite-Mediated Reactions by Vanillin
S. SANTOSH KUMAR,† K. INDIRA PRIYADARSINI,§ AND KRISHNA B. SAINIS*,†
Radiation Biology and Health Sciences Division, Bioscience Group, and Radiation Chemistry and
Chemical Dynamics Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India
Several neurodegenerative diseases such as Alzeimer’s and Parkinson’s as well as septic shock
and inflammation involve formation of reactive oxygen and nitrogen species that include peroxynitrite
(PON). PON can also react with endogenous antioxidants. Therefore, dietary supplementation with
antioxidants may help in these diseases. An exogenous antioxidant, vanillin (4-hydroxy-3-methoxy-
benzaldehyde), used widely as a food flavoring agent, was evaluated for its ability to scavenge PON
and inhibit PON-mediated reactions. Nitration of tyrosine by PON was assessed by high-performance
liquid chromatography (HPLC). This reaction was inhibited by vanillin. The oxidation of dihydro-
rhodamine 123 to fluorescent rhodamine 123 was also inhibited by vanillin. The kinetics of reaction
between PON and vanillin was studied by stopped-flow technique. The products of this reaction
were analyzed by HPLC, and hydroxyvanillin was identified as one of the five products with absorp-
tion at 350 nm. These data demonstrate that vanillin effectively scavenges PON in cell-free
systems.
KEYWORDS: Vanillin; peroxynitrite; phenoxyl radicals; pulse radiolysis; stopped-flow; antioxidant
INTRODUCTION
DNA and mitochondrial membrane against oxidative stress in
vitro (24-26). Vanillin displayed antimutagenic activity in both
bacteria and mammalian cells (27, 28). It reduced chromosomal
damage induced by mitomycin C, hydrogen peroxide, and
ionizing radiation in cultured Chinese hamster ovary cells (28,
29). It also inhibited chemically induced hepatocarcinogenesis
in rat (30, 31). However, evidence for scavenging of highly
reactive nitrogen species (RNS) such as PON by vanillin has
not been reported so far.
The amino acid tyrosine has been shown to be susceptible to
the action of PON, and the stable end product, 3-nitrotyrosine,
is used as a standard oxidative marker. The ability to inhibit
nitrotyrosine formation provides a useful assay to screen
compounds for PON scavenging ability (32, 33). The other type
of reaction of PON with its substrates is oxidation. The inhibition
of PON-induced oxidation of dihydrorhodamine 123 to rhodamine
123 is also a widely used assay for antioxidants (34). The present
studies were, therefore, carried out to assess the ability of
vanillin to inhibit PON reactions and to separate and identify
the products generated during the reaction. The kinetics of the
reaction between PON and vanillin was also monitored by
different methods.
Peroxynitrite (PON) is formed in vivo by diffusion-controlled
reaction of nitric oxide (NO) with superoxide anions (O2•-) (1,
2). Stimulated macrophages, neutrophils, and endothelial cells
are known to produce PON, and its in vivo formation has been
shown during pathophysiological conditions (3-6). It has been
implicated in several neurodegenerative diseases such as Alzhe-
imer’s and Parkinson’s, in ischemia-reperfusion injury, and also
in septic shock and inflammation (7-10). Under physiological
conditions, PON reacts with various cellular macromolecules
such as DNA, lipids, and proteins, leading to strand breaks, lipid
peroxidation, and nitration of amino acids, respectively (11-
16). In addition, PON reacts with different endogenous anti-
oxidants such as ascorbate, R-tocopherol, and thiol-containing
molecules, which form the primary defense against oxidative
stress, leading to their depletion and reduced availability (17-
20). Hence, exogenous compounds, especially of dietary origin,
that are capable of scavenging reactive oxygen/nitrogen species
may play a pivotal role in preventing/controlling degenerative
diseases.
Vanillin, a plant phenol, is used widely as a food flavoring
agent in confectioneries, chocolates, butter, toppings, icings,
distilled spirits, etc. (21, 22). Its antioxidant activity has been
ascertained using diphenylpicrylhydrazyl radical (DPPH) assay
in our laboratory (23). Furthermore, it was shown to protect
MATERIALS AND METHODS
Vanillin, hydroxyvanillin, dihydrorhodamine 123 (DHR123), ty-
rosine, and nitrotyrosine were obtained from Sigma-Aldrich Chemical
Co., St. Louis, MO. Sodium azide, phosphate salts, potassium nitrite,
and potassium nitrate of the highest purity available were obtained
locally from Glaxo India Ltd., Mumbai. IOLAR grade gases were used.
Synthesis of Peroxynitrite. PON was synthesized by the ozonolysis
of alkaline sodium azide solution for 2 h at 0-4 °C as described by
* Address correspondence to this author at Associate Director, Bioscience
Group, Bhabha Atomic Research Centre, Modular Labs, Trombay, Mumbai
400 085, India (e-mail kbsainis@apsara.barc.ernet.in; fax 91-22-25505326/
25505151).
† Radiation Biology and Health Sciences Division.
§ Radiation Chemistry and Chemical Dynamics Division.
10.1021/jf030319d CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/09/2003

140 J. Agric. Food Chem., Vol. 52, No. 1, 2004
Kumar et al.
Pryor et al. (35). The PON solution thus prepared was stored at -20
°C and used within 3-4 weeks. The concentration of PON was
determined by measuring its absorbance at 302 nm, using an extinction
coefficient of 1670 M-1 cm-1. Solutions were prepared in Nanopure
water obtained from a Millipore system.
Tyrosine Nitration Assay. A 50 µL aliquot of PON (500 µM) was
added to a solution containing tyrosine (100 µM) in the absence or
presence of various concentrations of vanillin in 0.2 M phosphate buffer,
pH 7.0, in a final volume of 1 mL (33). A high concentration of buffer
was used to ensure that the samples remained at pH 7.0 even after the
addition of alkaline PON solution. The samples were then analyzed
by HPLC using a C18 column. The mobile phase used was 50 mM
phosphate buffer, pH 7.0, in 5% acetonitrile at a flow rate of 0.5 mL/
min. The amount of 3-nitrotyrosine formed was determined from the
HPLC profile of a standard monitored at 430 nm.
Assay of Peroxynitrite-Mediated Oxidation of Dihydrorhodamine
123. The PON-induced oxidation of DHR123 was performed as
described by Kooy et al. (36). Briefly, PON (final concentration ) 10
µM) was added to 50 µM DHR123 in the absence or presence of
different concentrations of vanillin in 0.1 M phosphate buffer containing
0.1 mM diethylenetriaminepentaacetic acid (DTPA), pH 7.3, at room
temperature. The fluorescence of the oxidized product was measured
in a spectrofluorometer (Hitachi F4010) with excitation and emission
wavelengths of 500 and 536 nm, respectively. At this wavelength
interference from vanillin was not observed.
Kinetic Studies. Stopped-flow experiments were carried out using
an SX-18 MV multimixing stopped-flow reaction analyzer from Applied
Photo Physics Ltd. It was used in the single mixing mode, and the
reaction was monitored using absorption detection. In this, syringe I
contained PON in 0.01 M Na2HPO4 buffer, pH 8.5 (solution A), and
syringe II contained 0.1 M phosphate buffer, pH 7.0, with or without
vanillin (solution B). The higher concentration of buffer in syringe II
ensured that the pH did not exceed 7.0 after the mixing of solutions A
and B. All of the experiments were carried out at room temperature
(25 °C). At least three independent runs were used to determine the
observed rates at any particular concentration.
HPLC Analysis. PON (final concentration ) 0.5 mM) was added
to reaction mixtures containing various concentrations of vanillin in
phosphate buffer with rapid vortex mixing, and the final pH of the
solution was 7.0. The reaction was complete within several seconds
after the addition of PON. Standard incubation time was 5 min. This
reaction mixture was filtered through a 0.2 µm filter and was separated
on a 3.9 × 150 mm reverse phase HPLC column (Nucleosil, C18)
using Waters model 515 pumps and an automated gradient controller.
Separations were carried out under isocratic conditions using 0.1 M
citric acid, pH 2.0, and 15% acetonitrile at a flow rate of 0.5 mL/min
with UV detection at 350 nm.
Pulse Radiolysis. Pulse radiolysis experiments were performed using
7 MeV electrons from a linear accelerator. All of the experiments were
carried out using a 50 ns pulse with an absorbed dose of 11-12 Gy
(37, 38). The absorbed dose was measured using aerated thiocyanate
Figure 1. (a) Inhibition of PON-induced formation of 3-nitrotyrosine by
vanillin. Percent decrease was calculated on the basis of the area under
the 3-nitrotyrosine peak detected at 430 nm using HPLC. (b) Percent
inhibition of PON-mediated oxidation of dihydrorhodamine 123 by vanillin.
Percent inhibition was calculated on the basis of fluorescence intensity
of rhodamine 123 in the absence of vanillin.
•-
dosimeter monitoring (SCN)2 at 500 nm (37). Radiolysis of water
generates reactive radicals such as e-aq, •OH, and •H and less reactive
molecular species such as HO2• and H2O2 (38). Reactions of hydroxyl
radical were studied by irradiating N2O-saturated aqueous solutions,
where eaq was quantitatively converted to OH radical (e- + N2O
-
•
aq
free and protein-bound tyrosine (39). Nitration of proteins can
potentially affect many key cellular processes such as the
phosphorylation of proteins by protein tyrosine kinases crucial
for cell proliferation and differentiation (40).
f
-OH + •OH + N2). NO2• radicals were generated by the radiolysis
of N2O-saturated aqueous solutions containing 20 mM sodium nitrite.
Under these conditions, reaction of hydroxyl radicals with nitrite ions
(NO2 + OH f NO2 + OH-) produced NO2 radicals.
-
•
•
•
Figure 1b depicts the percent inhibition of PON-induced
oxidation of dihydrorhodamine 123 to rhodamine 123 by
vanillin. The percent inhibition increased sharply with increasing
concentration of vanillin up to 100 µM and slowly thereafter.
Fifty percent inhibition was observed at ∼300 µM. It also
prevented oxidation of DHR123 effectively. DHR123 is an
efficient and sensitive dye enabling the detection of submicro-
molar concentrations of PON (36). It has been proposed that
by single electron transfer from DHR123 to PON, the dihy-
drorhodamine radical is formed, which can dismutate to form
RESULTS AND DISCUSSION
Figure 1a shows the percent inhibition of formation of
3-nitrotyrosine during the reaction of PON with tyrosine in the
presence of different concentrations of vanillin. There was a
sharp rise in the inhibition of 3-nitrotyrosine formation with
increasing concentration of vanillin, reaching ∼85% inhibition
at 100 µM. At 250 µM vanillin, the formation of 3-nitrotyrosine
was completely inhibited (Figure 1a). PON can react with both

Peroxynitrite Scavenging by Vanillin
J. Agric. Food Chem., Vol. 52, No. 1, 2004 141
Figure 2. Steady-state absorption spectra of (a) 50µM vanillin at pH 7.0
and (b) the product of the reaction of 50 µM vanillin with 500 µM PON
at pH 7.0. The absorption spectrum of blank PON is shown for comparison
(c).
Figure 3. Linear plot showing the variation of observed reaction rate for
the formation of the product at 430 nm as a function of PON concentration
after mixing with 50 µM vanillin at pH 7.0. (Inset) Formation of the product
at 430 nm with time after mixing PON with vanillin.
rhodamine 123 and dihydrorhodamine 123 (36). The fluorescent
product rhodamine 123 is stable and remains well localized
within the cells, making DHR123 a useful probe for the
detection and quantification of PON.
Figure 2a shows the absorption spectrum of vanillin at pH
7 in the wavelength region from 300 to 550 nm. The absorption
spectrum of the reaction product of PON with vanillin, with a
new peak at 430 nm, is shown in Figure 2b. For comparison,
the absorption spectrum of PON is given in Figure 2c. These
spectra demonstrated that PON reacted with vanillin to produce
a new species having an absorption that is red shifted with
respect to the parent.
The bimolecular rate constant (k1) of PON with vanillin was
also estimated independently using the fluorescence data
obtained from rhodamine 123 (R123). For this purpose, the rate
constant for the reaction of PON with DHR123 was initially
estimated. We followed the change in the rate of formation at
500 nm as a function of DHR123 concentration varying from
25 to 75 µM. Figure 4a shows a linear plot for the change in
rate of the reaction with respect to increasing concentration of
DHR123. The slope gave a bimolecular rate constant of 8200
M-1 s-1. Using this, the bimolecular rate constant of the reaction
of PON with vanillin was estimated by competition kinetics.
The reaction steps may be written as
The rate constant for the reaction of vanillin with PON was
determined using a stopped-flow spectrometer. In the absence
of vanillin the decay of PON was first-order with a rate constant
of 0.52 s-1. This decay could not be followed in the presence
of vanillin due to the strong absorption by both vanillin and its
products at 302 nm. In the presence of vanillin (50 µM), under
the same pH conditions, the absorption of vanillin at 350 nm
decreased with time with a simultaneous formation of a new
peak between 400 and 430 nm. The rate of formation of product
at 430 nm was followed as a function of PON concentration
varying from 250 µM to 2 mM by fixing the concentration of
vanillin at 50 µM. The rate constant (k1) for the reaction of
PON with vanillin was determined as follows:
k2
PON + DHR123
98 R123
(2)
PON + VAN
9
k1′8 products
(1′)
k1 and k1′ represent rate constants for the same reaction but
calculated by different methods.
The fraction of PON going to DHR123 in the presence of
vanillin (F0/F) is given by the equation
F0/F ) 1 + k1′ [VAN]/k2[DHR123]
where F0 is the fluorescence of rhodamine 123 in the absence
of vanillin and F is the fluorescence of rhodamine 123 in the
presence of vanillin. The values of F0 and F used were from
Figure 1b. Figure 4b shows a linear plot for the variation of
F0/F - 1 versus [VAN]/[DHR123].
k1
PON + VAN
98 product
(1)
The rate of change in the formation of product (kobsd) was
followed over a range of concentration of PON, under pseudo-
first-order conditions, where [PON] . [VAN]. A linear
relationship was used for the formation of the new product at
430 nm with the change in PON concentration as kobsd ) A +
k1[PON], where A is a constant and k1 the second-order rate
constant of the above reaction, which is obtained from the slope
of the linear plot of kobsd versus [PON] (Figure 3). The slope
The ratio k1′/k2 is the slope of the straight line. Using the
value of k2 (8200 M-1 s-1), the value of k1′ was obtained as
1300 M-1 s-1
.
This value of the rate constant obtained by competitive
kinetics (k1′) was comparable to the rate constant (k1) estimated
by stopped-flow measurement.
Chromatographic separation of vanillin showed a single peak
with a retention time of 7.27 min (Figure 5a). After reaction
gave a bimolecular rate constant (k1) of 1600 M-1 s-1
.

142 J. Agric. Food Chem., Vol. 52, No. 1, 2004
Kumar et al.
Figure 5. (a, top) HPLC profile of vanillin (500 µM) at pH 7.0.
(b) Separation of different products of the reaction of vanillin with
500 µM PON at pH 7.0. Column conditions: mobile phase, 0.1 M citric
acid, 15% acetonitrile, pH 2.0; flow rate, 0.5 mL/min; wavelength,
350 nm.
Figure 4. (a, top) Linear plot showing the change in rate of the reaction
at 500 nm as a function of DHR123 concentration after mixing with 5 µM
PON at pH 7.0. (b, bottom) Linear plot showing the variation of (F0/F −
1) as a function of the ratio of concentration of vanillin to that of DHR123
to estimate the rate constant by competition kinetics. Details are explained
under Results and Discussion.
comparison, reactions of vanillin with hydroxyl radicals and
nitrogen dioxide radicals generated by pulse radiolysis were
carried out (Figure 6). Reaction of hydroxyl radicals with
vanillin produced transients absorbing at 410 nm, which have
been attributed to phenoxyl radicals (42). The radicals decayed
by second-order kinetics with a 2k/ꢀl of 1.2 × 106 s-1, probably
due to radical-radical reaction (right inset of Figure 6).
In addition to hydroxyl radicals, the reaction of PON proceeds
of PON with vanillin at pH 7, five major products were obtained
as illustrated by the chromatogram shown in Figure 5b.
Retention times of these products were 2.75, 4.37, 12.93, 16.25,
and 18.80 min, respectively (Figure 5b). The peak correspond-
ing to 4.37 min was identified as a hydroxylation product of
vanillin, that is, 2,4-dihydroxy-3-methoxybenzaldehyde, using
a commercial standard (Figure 5b). The peaks at 2.37, 3.20,
and 3.61 min correspond to nitrate, nitrite, and azide, respec-
tively, which were confirmed by injecting the appropriate
standards. The other major products could be quinone and
hydroxylated, nitration, and nitrosylation products of vanillin.
HPLC analysis of the reaction of PON with phenols was
reported earlier (41), in which a number of products have been
characterized. The reaction was also monitored at 265, 285, and
425 nm, but no new products other than those observed at 350
nm were found. Only the change in the relative absorption
intensity varied.
•
through the intermediacy of NO2 . Therefore, the reaction of
NO2• with vanillin was independently studied. At pH 7, vanillin
•
(200 µM) reacted with NO2 radicals, producing a transient
showing absorption in the 400-500 nm region with λmax at
400-440 nm (Figure 6), and the bimolecular rate constant for
the reaction was 4.2 × 106 M-1 s-1. The reaction of NO2• with
vanillin at the same pH gave a similar transient with reduced
absorbance at 410 nm with an additional peak at 320-330 nm,
which might be due to adduct formation. The transient produced
•
by NO2 reaction did not show any appreciable decay in 5 s
•
(left inset of Figure 6). The rate constant for NO2 reaction
•
with vanillin was much less than that of the OH radical. This
suggested that although the spectra appeared to be very similar,
they might correspond to different transients probably due to
the formation of radical adducts that might finally lead to the
Pulse radiolysis studies of vanillin with various radicals have
been reported by Mahal and co-workers (42). For the sake of

Peroxynitrite Scavenging by Vanillin
J. Agric. Food Chem., Vol. 52, No. 1, 2004 143
Figure 6. (9) Difference absorption spectrum of the transient obtained 4.5 µs after pulse radiolysis of N O-saturated aqueous solution containing 1 ×
2
10-4 M vanillin at pH 7.0. (Right inset) Absorption−time plot showing the decay of the transient at 430 nm. (b) Difference absorption spectrum of the
transient obtained 1.8 ms after pulse radiolysis of N O-saturated aqueous solution containing 200 µM vanillin and 0.02 M sodium nitrite at pH 7.0. (Left
2
inset) Transient formed during the reaction of NO • with vanillin showed no decay in 5 s time scale.
2
Scheme 1. Possible Mechanism and Products Formed during the
Reaction of PON with Vanillin
formation of nitrated products. The electron-transfer reaction
between NO2• and vanillin is a thermodynamically unfavorable
process, due to the fact that the reduction potential of the couple
(NO2 /NO2-) is 1 V versus NHE, whereas that for vanillin•+/
•
vanillin is 0.862 V versus NHE (23). Hence, the major reaction
pathway may be the radical adduct formation by the addition
•
of NO2 radical to the ortho position of phenolic OH as indicated
in Scheme 1.
PON has a pKa of 6.8 at 25 °C and, therefore, partially
undergoes protonation at physiological pH (1). The protonated
form, peroxynitrous acid (ONOOH), is very unstable and
decomposes to intermediates formed with the reactivity of •OH
•
and NO2 (2). PON can cause both one- and two-electron
oxidations of the substrates (2). The direct oxidation is mediated
by an oxygen atom transfer process. Among the various
substrates of interaction with PON, phenols are well studied
(41). The possible reaction mechanisms for the actual reaction
involving vanillin and PON are outlined in Scheme 1. The
reaction of PON with vanillin can proceed by hydroxyl-type
radical reactivity, producing hydroxylated adducts (Scheme 1).
The hydroxyl radical adducts may lose a proton to give
•
hydroxylated vanillin. PON also generates the NO2 radical,
which may add to vanillin, giving radical adducts, which may
lose a proton to yield the nitrated products (Scheme 1). The
•
•
NO2 reaction is much slower as compared to the OH-like
radical reaction and may be possible only at very high
concentrations of vanillin. Although thermodynamically not a
favorable process, NO2• may also oxidize vanillin to give same
phenoxyl radicals.
Because vanillin is used for direct human consumption and
PON is generated inside cells, these observations, if extended
to in vivo systems, may have functional significance. However,
dietary compounds may undergo structural changes or degrada-
tion during metabolism. These processes are important in
determining their role in disease conditions and also for
understanding their mechanism of action. The “bioavailability”
of exogenous antioxidants depends on their absorption, biodis-
tribution, and bioactivity after reaching the site of action. Some
of these dietary antioxidants may be modified by xenobiotic
metabolizing enzymes such as cytochrome P450 dependent

144 J. Agric. Food Chem., Vol. 52, No. 1, 2004
Kumar et al.
(9) Ischiropoulos, H. Biological tyrosine nitration: A pathophysi-
ological function of nitric oxide and reactive oxygen species.
Arch. Biochem. Biophys. 1998, 356, 1-11.
(10) Gatto, E. M.; Riobo, N. A.; Carreras, M. C.; Chernavsky, A.;
Rubio, A.; Satz, M. L.; Poderoso, J. J. Overexpression of
neutrophil neuronal nitric oxide synthase in Parkinson’s disease.
Nitric Oxide: Biol. Chem. 2000, 4, 534-539.
(11) Muijsers, R. B. R.; Folkerts, G.; Henricks, P. A. J.; Sadeghi-
Hashjin, G.; Nijkamp, P. Peroxynitrite: a two faced metabolite
of nitric oxide. Life Sci. 1997, 60, 1833-1845.
(12) Pryor, W. A.; Xia-Jin; Squadrito, G. L. One and two electron
oxidations of methionine by peroxynitrite. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 11173-11177.
monooxygenases, conjugating enzymes of phase II biotrans-
formation (UDP glucuronosyl transferases or phenol sulfotrans-
ferases), esterases, â-glucosidases, or some very specific
enzymes. The metabolic products may retain or lose their
antioxidant activity in the body. Carotenoids are cleaved to
apocarotenoids (43), whereas R-tocopherol is oxidized to
R-tocopherol hydroquinones (44) and polyphenols are conju-
gated to glucuronic acid and/or sulfate in the liver (45). The
blocking of hydroxyl groups on the parent molecule due to
conjugation results in loss of antioxidant activity of polyphenols,
whereas R-tocopherol hydroquinones retain their antioxidant
activity in the body (46). Antioxidants such as glutathione and
vitamin C are regenerated enzymatically from their metabolites
(47).
In conclusion, our studies show that vanillin inhibits PON-
induced nitration and oxidation reactions. With the help of a
stopped-flow technique, pulse radiolysis, and HPLC analysis,
the kinetics of the reaction was studied and some of the products
have been identified. Some of the important mechanistic steps
involved during the progress of the reaction are proposed.
(13) Szabo, C.; Ohshima, H. DNA damage induced by peroxyni-
trite: subsequent biological effects. Nitric Oxide: Biol. Chem.
1997, 1, 373-385.
(14) Salgo, M. G.; Stone, K.; Squadrito, G. L.; Battista, J. R.; Pryor,
W. A. Peroxynitrite causes DNA nicks in plasmid pBR322.
Biochem. Biophys. Res. Commun. 1995, 210, 1025-1030.
(15) Rubbo, H.; Radi, R.; Trujillo, M.; Telleri, R.; Kalyanaraman,
B.; Barnes, S.; Kirk, M.; Freeman, B. A. Nitric oxide regulation
of superoxide and peroxynitrite-dependent lipid peroxidation.
Formation of novel nitrogen-containing lipid derivatives. J. Biol.
Chem. 1994, 269, 26066-26075.
ABBREVIATIONS USED
(16) Darley-Usmar, V. M.; Hogg, N.; O’Leary, V. J.; Wilson, M. T.;
Moncada, S. The simultaneous generation of superoxide and
nitric oxide can initiate lipid peroxidation in human low-density
lipoprotein. Free Radical Res. Commun. 1992, 17, 9-20.
(17) Pannala, A. S.; Rice-Evans, C.; Sampson, J.; Singh, S. Interaction
of peroxynitrite with carotenoids and tocopherols within low-
density lipoprotein. FEBS Lett. 1998, 423, 297-301.
(18) Bartlett, D.; Church, D. F.; Bounds, P. L.; Koppenol, W. H. The
kinetics of the oxidation of 1-ascorbic acid by peroxynitrite. Free
Radical Biol. Med. 1995, 18, 85-92.
PON, peroxynitrite; ROS, reactive oxygen species; HPLC,
•
high-performance liquid chromatography; NO2 , nitrogen diox-
ide; •OH, hydroxyl radical; NO•, nitric oxide; N3 , azide radical;
•
H2O2, hydrogen peroxide; DPPH, diphenylpicrylhydrazyl radi-
cal.
ACKNOWLEDGMENT
We acknowledge the encouragement and support of Dr. T.
Mukherjee and Dr. Hari Mohan of the Radiation Chemistry and
Chemical Dynamics Division, BARC.
(19) Hogg, N.; Joseph, J.; Kalyanaraman, B. The oxidation of
R-tocopherol and trolox by peroxynitrite. Arch. Biochem. Bio-
phys. 1994, 314, 153-158.
(20) Radi, R.; Beckman, J. S.; Bush, K. M.; Freeman, B. A.
Peroxynitrite oxidation of sulfhydryls. J. Biol. Chem. 1991, 266,
4244-4250.
LITERATURE CITED
(1) Patel, R. P.; McAndrew, J.; Sellak, H.; White, C. R.; Jo, H.;
Freeman, B. A.; Darley-Usmar, V. M. Biological aspects of
reactive nitrogen species. Biochim. Biophys. Acta 1999, 1411,
385-400.
(2) Radi, R.; Peluffo, G.; Alvarez, M. N.; Naviliat, M.; Cayota, A.
Unraveling peroxynitrite formation in biological systems. Free
Radical Biol. Med. 2001, 30, 463-488.
(3) Radi, R.; Denicola, A.; Alvarez, B.; Ferrer-Sueta, G.; Rubbo,
H. The biological chemistry of peroxynitrite. In Nitric Oxide;
Ignarro, L., Ed.; Academic Press: San Diego, CA, 2000; pp 57-
82.
(4) Haddad, I. Y.; Pataki, G.; Hu, P.; Beckman, J. S.; Matalon, S.
Quantification of nitrotyrosine levels in lung sections of patients
and animals with acute lung injury. J. Clin. InVest. 1994, 94,
2407-2413.
(5) Kaur, H.; Halliwell, B. Evidence for nitric oxide mediated
oxidative damage in chronic inflammation. Nitrotyrosine in
serum and synovial fluid from rheumatoid patients. FEBS Lett.
1994, 350, 9-12.
(6) Carreras, M. C.; Paragament, G. A.; Catz, S. D.; Poderosso, J.
J.; Boveris, A. Kinetics of nitric oxide and hydrogen peroxide
production and formation of peroxynitrite during respiratory burst
of human neutrophils. FEBS Lett. 1994, 341, 65-68.
(7) Smith, M. A.; Richey, H. P.; Sayre, L. M.; Beckman, J. S.; Perry,
G. Widespread peroxynitrite mediated damage in Alzheimer’s
disease. J. Neurosci. 1997, 17, 2653-2657.
(21) Vanillin RHOVANIL; Monograph RP 10/95; Rhone-Poulenc
Chimie: France, 1995.
(22) Spillman, P. J.; Pollnitz, A. P.; Liacopoulos, D.; Skouroumounis,
G. K.; Sefton, M. A. Accumulation of vanillin during barrel-
aging of white, red, and model wines. J. Agric. Food Chem.
1997, 45, 2584-2589.
(23) Santosh Kumar, S.; Priyadarsini, K. I.; Sainis, K. B. Free radical
scavenging activity of vanillin and o-vanillin using 1,1-diphenyl-
2-picrylhydrazyl (DPPH) radical. Redox Rep. 2002, 7, 35-40.
(24) Prince, R. C.; Gunson, D. E. Just plain vanilla? Trends Biochem.
Sci. 1994, 19, 521.
(25) Santosh Kumar, S.; Gosh, A.; Devasagayam, T. P. A.; Chauhan,
P. S. Effect of vanillin on methylene blue plus light-induced
single-strand breaks in plasmid pBR322 DNA. Mutat. Res. 2000,
469, 207-214.
(26) Kamat, J. P.; Gosh, A.; Devasagayam, T. P. A. Vanillin as an
antioxidant in rat liver mitochondria: Inhibition of protein
oxidation and lipid peroxidation induced by photosensitization.
Mol. Cell. Biochem. 2000, 209, 47-53.
(27) Ohta, T. Modification of genotoxicity by naturally occurring
flavorings and their derivatives. Crit. ReV. Toxicol. 1993, 23,
127-146.
(28) Tamai, K.; Tezuka, H.; Kuroda, Y. Different modifications by
vanillin in cytotoxicity and genetic changes induced by EMS
and H2O2 in cultured Chinese hamster cells. Mutat. Res. 1992,
268, 231-237.
(29) Keshava, C.; Keshava, N.; Ong, T.; Nath, J. Protective effect of
vanillin on radiation-induced micronuclei and chromosomal
aberrations in V79 cells. Mutat. Res. 1997, 397, 149-159.
(8) White, C. R.; Brock, T. A.; Chang, L. Y.; Crapo, J.; Briscoe,
P.; Ku, D.; Bradley, W. A.; Gianturco, S. H.; Gore, J.; Freeman,
B. A. Superoxide and peroxynitrite in atherosclerosis. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 1044-1048.

Peroxynitrite Scavenging by Vanillin
J. Agric. Food Chem., Vol. 52, No. 1, 2004 145
(30) Fahrig, R. Anti-mutagenic agents are also co-recombinogenic
and can be converted into co-mutagens. Mutat. Res. 1996, 350,
59-67.
(31) Tsuda, H.; Uehara, N.; Iwahori, Y.; Asamoto, M.; Ligo, M.;
Nagao, M.; Matsumoto, K.; Ito, M.; Hirono, I. Chemopreventive
effects of â-carotene, R-tocopherol and five naturally occurring
antioxidants on initiation of hepatocarcinogenesis by 2-amino-
3-methylimidazo sbd (4,5-f)-quinoline in the rat. Jpn. J. Cancer
Res. 1994, 85, 1214-1219.
(32) Whiteman, M.; Halliwell, B. protection against peroxynitrite
dependent tyrosine nitration and R 1-antiproteinase inactivation
by ascorbic acid. A comparison with other biological antioxi-
dants. Free Radical Res. 1996, 25, 275-283.
(33) Pannala, A.; Rice-Evans, C. A.; Halliwell, B.; Singh, S. Inhibition
of peroxynitrite-mediated tyrosine nitration by catechin polyphe-
nols. Biochem. Biophys. Res. Commun. 1997, 232, 164-168.
(34) Arteel, G. E.; Sies, H. Protection against peroxynitrite by cocoa
polyphenol oligomers. FEBS Lett. 1999, 462, 167-170.
(35) Pryor, W. A.; Cueto, R.; Jin, X.; Ngu-Schwemlein, M.; Squadrito,
G. L.; Uppu, P. L.; Uppu, R. M. A practical method for preparing
peroxynitrite solutions of low ionic strength and free of hydrogen
peroxide. Free Radical Biol. Med. 1995, 18, 75-83.
(36) Kooy, N. W.; Royall, J. A.; Ischiropoulos, H.; Beckman, J. S.
Peroxynitrite mediated oxidation of dihydrorhodamine 123. Free
Radical Biol. Med. 1994, 16, 149-156.
(40) Yu, S. M.; Hung, L. M.; Lin, C. C. cGMP-elevating agents
suppress proliferation of vascular smooth muscle cells by
inhibiting the activation of epidermal growth factor signaling
pathway. Circulation 1997, 95, 1269-1277.
(41) Diaber, A.; Mehl, M.; Ullrich, V. New aspects in the reaction
mechanism of phenol with peroxynitrite: the role of phenoxyl
radicals. Nitric Oxide: Biol. Chem. 1998, 2, 259-269.
(42) Mahal, H. S.; Badheka, L. P.; Mukherjee, T. Radical scavenging
properties of flavoring agent-vanillin. Res. Chem. Intermed. 2001,
27, 595-604.
(43) Wang, X. D.; Tang, G. W.; Fox, J. G.; Krinsky, N. I.; Russel,
R. M. Enzymatic conversion of â-carotene into â-apo-carotenals
and retinoids by human, monkey, ferret and rat tissues. Arch.
Biochem. Biophys. 1991, 285, 8-16.
(44) Pope, S. A.; Clayton, P. T.; Muller, D. P. A new method for
analysis of urinary vitamin E metabolites and the tentative
identification of novel group of compounds. Arch. Biochem.
Biophys. 2000, 381, 8-15.
(45) Griffiths, L. A. In The FlaVonoids: AdVances in Research;
Harborne, J., Mabry, T., Eds.; Chapman and Hall: London, U.K.,
1982; pp 681-718.
(46) Neuzil, J.; Witting, P. K.; Stocker, R. R-tocopheryl hydroquinone
is an efficient multifunctional inhibitor of radical initiated
oxidation of low-density lipoprotein lipids. Proc. Natl. Acad.
Sci. U.S.A. 1997, 94, 7885-7890.
(37) Fielden, E. M. In The Study of Fast Processes and Transient
Species by Electron Pulse Radiolysis; Baxendale, J. H., Busi,
F., Eds.; Reidel: Boston, MA, 1984; p 54.
(47) Stahl, W.; van den Berg, H.; Arthur, J.; et al. Bioavailability
(38) Buxton, G. V.; Greenstock, C. L.; Helman, W. P.; Ross, A. B.
Critical review of rate constants for reactions of hydrated
electrons, hydrogen atoms and hydroxyl radicals (•OH/•O-) in
aqueous solution. J. Phys. Chem. Ref. Data 1988, 17, 513-523.
(39) Herce-Pagliai, C.; Kotecha, S.; Shuker, D. E. G. Analytical
methods for 3-nitrotyrosine as a marker of exposure to reactive
nitrogen species: A review. Nitric Oxide: Biol. Chem. 1998, 2,
324-336.
and metabolism. Mol. Aspects Med. 2002, 23, 39-100.
Received for review April 28, 2003. Revised manuscript received
October 23, 2003. Accepted October 24, 2003.
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