In vitro inhibition of linoleic acid peroxidation by primary S-Nitrosothiols

Article abstract of DOI:10.1590/S0103-50532010001000013

Nitric oxide (*NO) is an effective chain-breaking antioxidant in the inhibition of lipid peroxidation and circulates in vivo mainly as primary S-nitrosothiols (RSNOs). In this work, the in vitro peroxidation of linoleic acid-SDS comicelles (LA-SDS) catalyzed by soybean lipoxygenase (SLO) and Fe ^II ions was monitored in the presence and absence of three primary RSNOs: S-nitrosocysteine, S-nitroso-N-acetylcysteyne and S-nitrosoglutathione. Kinetic measurements based on the formation of conjugated double bonds and fluorescent oxidized LA-lysine adducts, showed that RSNOs are more potent antioxidants than their corresponding free thiols (RSHs) in equimolar conditions. These results are consistent with the blocking of LA-SDS peroxidation by RSNOs through the inactivation of peroxyl/alkoxyl (LOO*/LO*) radicals, leading to nitrogen-containing products of oxidized LA, which release free *NO. These results indicate that endogenous RSNOs may play a major role in the blocking of lipid peroxidation in vivo, through the primary inactivation of alkoxyl/peroxyl radicals and also of preformed lipid hydroperoxides.

Full text of DOI:10.1590/S0103-50532010001000013

J. Braz. Chem. Soc., Vol. 21, No. 10, 1885-1895, 2010.
Printed in Brazil - ©2010 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
In Vitro Inhibition of Linoleic Acid Peroxidation by Primary S-Nitrosothiols
Fernanda I. Simplicio, Amedea B. Seabra, Gabriela F. P. de Souza and
Marcelo G. de Oliveira*
Instituto de Química, Universidade Estadual de Campinas, CP 6154,
13083-970 Campinas-SP, Brazil
O óxido nítrico (•NO) é um antioxidante efetivo na inibição da peroxidação lipídica e circula
in vivo principalmente como S-nitrosotióis primários (RSNOs). Neste trabalho, a peroxidação in
vitro de comicelas do ácido linileico-SDS (LA-SDS) catalisada por lipoxigenase de soja (SLO) e
íons FeII foi monitorada na presença e na ausência de três RSNOs: S-nitrosocisteína, S-nitroso-N-
acetilcisteína e S-nitrosoglutationa. Medidas cinéticas baseadas na formação de duplas ligações
conjugadas e adutos fluorescentes de lisina-La oxidado mostraram que os RSNOs são antioxidantes
mais potentes que seus tióis livres (RSHs) correspondentes em condições equimolares. Esses
resultados são consistentes com o bloqueio da peroxidação de LA-SDS por RSNOs através da
inativação dos radicais peroxila/alcoxila (LOO•/LO•), levando a produtos nitrogenados do LA
•
oxidado, os quais liberam NO. Esses resultados indicam que os RSNOs endógenos podem
desempenhar um papel importante no bloqueio da peroxidação lipídica in vivo, através da inativação
primária de radicais alcoxila/peroxila bem como de hidroperóxidos lipídicos pré-formados.
Nitric oxide (•NO) is an effective chain-breaking antioxidant in the inhibition of lipid
peroxidation and circulates in vivo mainly as primary S-nitrosothiols (RSNOs). In this work, the in
vitro peroxidation of linoleic acid-SDS comicelles (LA-SDS) catalyzed by soybean lipoxygenase
(SLO) and FeII ions was monitored in the presence and absence of three primary RSNOs:
S-nitrosocysteine, S-nitroso-N-acetylcysteyne and S-nitrosoglutathione. Kinetic measurements
based on the formation of conjugated double bonds and fluorescent oxidized LA-lysine adducts,
showed that RSNOs are more potent antioxidants than their corresponding free thiols (RSHs) in
equimolar conditions. These results are consistent with the blocking of LA-SDS peroxidation
by RSNOs through the inactivation of peroxyl/alkoxyl (LOO•/LO•) radicals, leading to nitrogen-
containing products of oxidized LA, which release free •NO. These results indicate that endogenous
RSNOs may play a major role in the blocking of lipid peroxidation in vivo, through the primary
inactivation of alkoxyl/peroxyl radicals and also of preformed lipid hydroperoxides.
Keywords: nitric oxide, S-nitrosothiols, lipid peroxidation, linoleic acid, lipoxygenase
Introduction
cycles. Once formed, free radicals are also capable of
initiating other radical reactions, which may become self-
sustaining through the regeneration of propagating radicals
which are involved in the oxidation of lipids in humans
representing a key event in the atherosclerotic process.
This conclusion is reinforced by the fact that both primary
and secondary lipid oxidation products are found in human
atherosclerotic lesions.8,9
Under normal physiological conditions, endothelium-
derived nitric oxide (nitrogen monoxide, •NO) has multiple
physiological functions in humans, like the regulation of
vascular tone in both the systemic and renal circulation,10,11
the prevention of adherence and aggregation of platelets and
monocytes in the walls of blood vessels12 and the regulation
An increasing amount of evidence has demonstrated
that oxidative and nitrosative stresses, play a fundamental
role in atherosclerosis and in other diseases associated with
lipid peroxidation (LPO).1-6 In these cases, it is assumed
that free radicals which normally play an essential role in
metabolic processes, are released from the active site of
enzymes, triggering a cascade of deleterious effects on
cells.7 These effects involve the interaction of free radicals
with metal or organic redox centers and the promotion of
irreversible oxidation reactions beyond the normal catalytic
*e-mail: mgo@iqm.unicamp.br

1886
In Vitro Inhibition of Linoleic Acid Peroxidation by Primary S-Nitrosothiols
J. Braz. Chem. Soc.
of the proliferation and migration of smooth muscle cells.13
In addition to the actions related to the mediation of signal
transduction, via stimulation of guanylate cyclase-mediated
cGMP synthesis, NO has been shown to exert several
antiatherogenic properties assigned to its ability to react
directly with free radicals, blocking the propagation of
radical reactions. This protective effect has already been
observed in model lipid systems,14,15 low-density lipoproteins
(LDL)16-18 and cells19,20 and is supported by several in vitro
studies which have demonstrated the formation of nitrogen-
containing products of polyunsaturated fatty acids (PUFA),
including alkylnitrites (RONO), alkylnitrates (ROONO and
RONO2), alkylepoxynitrite (R(O)NO2), alkylnitrohydroxy
(RNO2OH), and nitrolipids (RNO2), when PUFAs are
oxidized in the presence of •NO. Such products have already
been characterized in other works by mass spectrometry and
can be taken as markers of the in vivo pro-oxidant and/or
“tertiary” RSNO. Evaluating the antioxidant properties of
primary RSNOs may have an additional relevance, once
•
the lability of NO in primary and tertiary RSNOs can be
different.37-39 In any case, one of the important characteristics
•
of having NO carried as RSNOs is its preservation from
inactivationcausedbyreactionwithoxygen, leadingtonitrite
(NO2–) and further to nitrate (NO3–),40 two of the main end
products of •NO metabolism. Although several exogenous
•NO sources (which are not found endogenously) have been
used as antioxidants in LPO studies like organic nitrites and
NONOates,41 only one work has reported the protective role
of primary RSNOs in blocking LPO reactions.42
In this work, the in vitro peroxidation of linoleic acid SDS
comicelles (LA-SDS) catalyzed by soybean lipoxygenase
(SLO) and by FeII ions was monitored in the presence and
absenceofthreeprimaryRSNOs:CySNO, SNACandGSNO
andoftheircorrespondingfreethiols(RSHs),at37ºC.Kinetic
data showed that RSNOs can block LA peroxidation much
more efficiently than RSHs, by inactivating alkoxyl/peroxyl
(LO•/LOO•) radicals and LA hydroperoxides, (LOOH =
13-hydroperoxy-octadecadienoic acid, 13-HPODE) through
nitration and transnitrosation reactions. These results suggest
that endogenous primary RSNOs may play a major role in
blocking lipid peroxidation in vivo.
•
antioxidant actions of NO.14,15,21-28 These results stimulate
new therapeutic approaches for treating lipid peroxidation-
related diseases by enhancing •NO synthesis and/or activity
by administration of L-arginine and antioxidants.29 As an
alternative strategy, compounds that act as NO donors could
be administrated as exogenous NO sources, as already
demonstrated for the treatment of hepatic steatosis via oral
administration of the S-nitrosothiol (RSNO) S-nitroso-N-
acetylcysteine (SNAC).3-6 RSNOs are peptides or proteins
carrying the S-NO moiety and were shown to occur in the
plasma and cells of mammals where they have the same
Experimental
Reagents
•
physiological functions of free NO like vasodilation,30,31
inhibition of platelet activation and aggregation32,33 and
post-translational modification of protein function.34,35
S-nitrosoglutathione (GSNO), S-nitrosoalbumin and
S-nitrosohemoglobin, for example, have been considered
to be •NO carriers and donors in humans and are the focus
of several studies both in vivo and in vitro.36 Other RSNOs,
like S-nitrosocysteine (CySNO) have also been described.37
What classifies a RSNO as primary is the fact that the
sulfur atom of its SNO moiety is bound to a primary carbon
atom (Figure 1), while in S-nitroso-N-acetylpenicillamine
(SNAP), which is widely used in experimental studies, the
sulfur atom is bound to a tertiary carbon atom, making it a
Ascorbic acid, cysteine (CySH), ferrous sulfate
(FeSO4), copper sulfate (CuSO4), glutathione (g-Glu-Cys-
Glu, GSH), linoleic acid (LA), L-lysine monohydrochloride
(Lys), malonaldehyde bis(dimethyl acetal) (MDA),
N-acetyl-L-cysteine (NAC), phosphate buffer saline (PBS,
pH 7.4), sodium dodecil sulfate (SDS), sodium nitrite
(NaNO2), soybean lipoxygenase (SLO), hydrochoric acid
(HCl), mercury chloride (HgCl2), tert-butyl hydroperoxide
(tBOOH, C4 H10 O2) (Sigma/Aldrich, St. Louis, MO) and
sulfanilamide (Merck, Germay), were used as received.All
the experiments were carried out using analytical grade
water from a Millipore Milli-Q gradient filtration system.
Figure 1. Molecular structures of S-nitroso-L-cysteine (CySNO), S-nitroso-N-acetylcysteine (SNAC) and S-nitrosoglutathione (GSNO).

Vol. 21, No. 10, 2010
Simplicio et al.
1887
Synthesis of GSNO, CySNO and SNAC in aqueous solution
in PBS solution at room temperature and the emission
spectrum was obtained in the range 375-600 nm, with
excitation wavelength of 360 nm.
Aqueous GSNO solution was prepared by the reaction
of GSH with sodium nitrite in acidic medium as described
elsewhere.37,43 GSNO was obtained as stable reddish
crystals in the pure form and was dried by freeze-drying.
Freshly prepared GSNO solutions in PBS were used in
the experiments. S-nitroso-N-acetylcysteine (SNAC) and
S-nitrosocysteine (CySNO) cannot be precipitated from
solution and stored as dry solids because of their high
solubility in water. Therefore, aqueous SNAC or CySNO
solutions were synthesized through the equimolar reaction of
NAC or CyS, respectively, with NaNO2 in acidified aqueous
solution. Stock acidic SNAC and CySNO solutions freshly
prepared, were diluted in PBS and used immediately.
Spectrofluorimetric characterization and monitoring of
oxidized LA-lysine adduct formation
LA peroxidation was induced through the addition of
aqueous FeSO4 solution (final concentration 5.0 µmol L-1) to
aqueous LA (final concentration 1.2 mmol L-1) dispersions
in SDS solution (final concentration 0.01 mol L-1) in the
absence or presence of GSNO (final concentrations 5 and
500 mmol L-1) for 2 h in PBS (pH 7.4).After LA oxidation,
lysine solution (final concentration 1.0 mmol L-1) was
transferred to the dispersions followed by incubation for
48 h. The kinetics of formation of fluorescent oxidized
LA-lysine adduct (oxLA-Lys) in the reaction between
oxLA and Lys during the incubation time was characterized
based on the spectral changes in the range 375 to 600 nm
and on the emission intensity at 430 nm, under excitation
at 360 nm. All the spectrofluorimetric measurements were
performed using a Perkin-Elmer LS55 spectrofluorimeter
with a temperature-controlled cuvette holder at 37 ºC.
Characterization of linoleic acid peroxidation
Linoleic acid (LA) peroxidation was induced through
the addition of SLO to aqueous LA dispersions (final
concentration 19 mmol L-1) in SDS solution (0.01 mol L-1)
(LA-SDS comicelles). Each LA dispersion was transferred
to a quartz cuvette, blowed with O2 for 2 min and SLO (final
concentration 56 nmol L-1) was added to the cuvette with
a syringe to start the peroxidation reaction. Peroxidation
reactionsweremonitoredintheabsenceorpresenceofCySH,
NAC and GSH (final concentrations 560 mmol L-1) and of
their corresponding RSNOs, CySNO, SNAC and GSNO,
respectively (final concentrations 56 mmol L-1), through the
increase in absorbance at 234 nm, due to conjugated diene
formation. A Hewlett Packard spectrophotometer, model
8453 (Palo Alto, CA, USA) with a temperature-controlled
cuvette holder was used to monitor the spectral changes in
the range 200-600 nm in the dark at 37 °C, in time intervals
of 2 s. Spectra of the solutions were obtained in 1 cm quartz
cuvette, under stirring (1,000 r min-1). Each point in the
kinetic curves of absorbance vs. time is the average of two
experiments with error bars expressed by their standard
errors of the mean (SEM). Statistical differences among the
initial rates (Ir) of LA peroxidation, catalyzed by SLO, in
the absence and presence of RSHs and their corresponding
RSNOs were evaluated using ANOVA followed by Tukey-
Kramer multiple test.
Infrared characterization of linoleic acid peroxidation
Linoleic acid peroxidation was induced by heating
a sample of pure LA at 80 ºC for 4 h under stirring in a
glass flask with O2 atmosphere, obtained by continuously
blowing O2 from a cylinder into the headspace of the flask.
Aliquots of LA were removed from the reaction flask 2
and 4 h after the beginning of the peroxidation reaction.
Capillary films of non-oxidized and peroxidized LA were
obtained between two calcium fluoride (CaF2) windows,
which were mounted in special sample holder. IR spectra
were obtained in the range 4000-1000 cm-1 using an FTIR
Bomem MB-series, model B-100. An IR spectrum of non-
oxidized LA was obtained as a control.
•
Detection of NO released from nitrogen-containing
products of oxidized LA
Nitric oxide released from nitrogen-containing
products of oxLA formed in the peroxidation of LA
in the presence of GSNO, was detected using a gas-
phase chemiluminescence-based nitric oxide analyzer
(NOA, Sievers, Bolder Co, USA). Peroxidation of
LA was induced by two different procedures. In the
first, the LA-SDS dispersion in the presence of CuII
(900 mmol L-1) ions was previously blowed with O2 for
2 min, followed by incubation with GSNO (900 mmol L-1)
Characterization of fluorescent MDA-lysine adduct
To characterize the emission spectrum of the fluorescent
adduct formed in the reaction of oxidized linoleic acid
(oxLA) and lysine, an MDA-lysine adduct was prepared
as a model adduct by reacting MDA with L-lysine in
equimolar condition (final concentration 0.25 mol L-1)

1888
In Vitro Inhibition of Linoleic Acid Peroxidation by Primary S-Nitrosothiols
J. Braz. Chem. Soc.
for 30 min at room temperature. In the second,
peroxidation of LA was induced by heating a sample of
pure LA at 80 ºC for 1 h under stirring in a glass flask with
O2 atmosphere, obtained by continuously blowing O2 from
a cylinder into the headspace of the flask. After oxidation,
oxLA was dispersed in SDS solution (0.01 mol L-1) and
incubated with GSNO, for 30 min at room temperature.
In both cases, after incubation, non-reacted excess
GSNO was removed from the solution by adding HgCl2
(final concentration 29.4 mmol L-1) and allowing GSNO
the spectral changes of RSNOs solutions in the range
220-1100 nm in the dark, in a 1 cm quartz cuvette
referenced against air. Kinetic curves of GSNO, SNAC and
CySNO decomposition were obtained from the absorption
changes at 336 nm in time intervals of 15 s, at 37 ºC, for
8 min. The control experiment was performed by incubating
RSNOs with pure water at pH 12, adjusted with NaOH
solution. Each point in the kinetic curves of concentration
vs. time is the average of two experiments with error bars
expressed by their standard errors of the mean (SEM).
•
decomposition to GS-SG and free NO to proceed for
•
15 min. In this condition, NO is quantitatively released
Results and Discussion
from excess GSNO by mercuric catalysis and is rapidly
and quantitatively converted to its stable end product,
nitrite (NO2 ). Nitrite formed was removed by adding a
Kinetic characterization of linoleic acid peroxidation
–
10% v/v solution of sulfanilamide (6.0 mmol L-1 in HCl
2 mol L-1), followed by incubation for 15 min. A volume
of 5 mL of aqueous saturated ascorbic acid solution, used
as a reducing agent, was added in the glass purge vessel
of the NOA. Antifoaming agent was used to prevent
foaming caused by injection of the samples. Volumes
of 100 mL of the final nitrogen-containing products
of oxLA suspension were injected in the glass purge
vessel containing ascorbic acid, through an impermeable
septum. Nitrogen gas (Air Liquid, Brazil) was bubbled
Figure 2 shows the kinetic curves corresponding to
the spectral changes monitored at 234 nm in the first
80 s of LA oxidation by SLO in the presence of RSHs
and RSNOs. The band at 234 nm is associated with the
formation of conjugated double bonds in LA, as a result
of LA peroxidation.35 In this time range, the curves reach
an apparent plateau after ca. 20 s in all cases. The initial
rates of reaction (Ir), as well as the height of the plateaus,
are decrease significantly in the presence of RSHs and
RSNOs, compared to the peroxidation of LA alone. As
the height of the plateaus can be taken as a measurement
of the extent of the peroxidation reaction in its initial
phase, this result indicates that RSNOs in concentrations
ten times lower than their corresponding RSHs (56 versus
560 mmol L-1) exert a much more extensive blockage of
the peroxidation reaction than the corresponding RSHs
in this time range.
The initial rates of LA peroxidation (Ir), extracted
from the curves of Figure 2 are shown in the bar graphs of
Figure 3 for comparison. It can be seen that the Ir of LA
(19 mmol L-1) peroxidation is decreased to about ½ of the
control value in the presence of RSHs (560 mmol L-1),
i.e., at a molar ratio RSH/LA = 29.5, with no significant
differences among the antioxidant actions of CySH,
NAC and GSH. In contrast, the presence of RSNOs at a
concentration ten times lower (56 mmol L-1), i.e., at a molar
ratio RSH/LA = 2.9 reduced Ir values to ca. 1/5 of the
control value, also with no significant differences among
the action of CysNO, SNAC and GSNO.
•
through the solution and free NO formed due to the
reduction of nitrogen-containing products of oxLA by
ascorbic acid in the glass purge vessel was carried into
the reaction chamber for detection. Control curves are
for the measurements of samples of water incubated with
GSNO 900 mmol L-1 without LA-SDS and CuII for 30 min
at room temperature (control 1), and of water incubated
with GSNO 900 mmol L-1 and CuII without LA-SDS for
30 min at room temperature (control 2). In both cases,
the incubations were followed by the addition of HgCl2
(final concentration 29.4 mmol L-1) for promoting GSNO
•
decomposition to GS-SG and free NO for 15 min and
by the addition of a 10% v/v solution of sulfanilamide
(6.0 mmol L-1 in HCl 2 mol L-1), for 15 min for eliminating
nitritre formed in the reaction between NO and O2.
Reaction between RSNOs and tert-butyl hydroperoxide
The formation of tert-butyl peroxynitrites (tBOONOs)
in the reactions between tert-butyl hydroperoxide
(tBOOH) and RSNOs was characterized by following
the decomposition of GSNO, SNAC and CySNO (initial
concentrations 1 mmol L-1) upon the addiction of tBOOH
(initial concentration 25 mmol L-1) in basic medium
(pH 12). The decomposition of RSNOs in absence or
presence of tBOOH was characterized by following
Fluorimetric characterization and monitoring of oxidized
LA-lysine adduct formation
Figure 4 shows the emission spectra obtained after
LA oxidation catalyzed by FeII ions for 2 h, followed
by incubation of the solution with lysine for 48 h at

Vol. 21, No. 10, 2010
Simplicio et al.
1889
37 ºC. The peroxidation reactions were performed in
the absence (a) and presence of GSNO 5.0 mmol L-1 (b)
and 500.0 mmol L-1 (c). The inset in Figure 4 shows the
emission spectrum obtained after the incubation of MDA
with lysine in equimolar concentrations of 0.25 mol L-1,
as a control experiment. Figure 5 shows the kinetic curves
of the fluorescent oxLA-lysine adduct formation in the
reaction between oxidized LA and lysine in conditions
(a), (b) and (c) of Figure 4. The curves were based on the
spectral changes monitored at 430 nm, during 48 h after
lysine addition. It can be observed that the formation of
the oxLA-Lys adduct follows a sigmoid pattern with an
apparent induction or “lag” phase, which is evident in
curves (a) and (b). In curve (c) the reaction is still in an
apparent lag phase until 48 h of monitoring, although a
small rate of fluorophore formation can be detected since
the beginning of the reaction.
Figure 2. Kinetic curves of LA (19 mmol L-1) peroxidation catalyzed by
(SLO) (56 nmol L-1), in the absence of RSH or RSNO (Control, A) and
in the presence of CySH 560 mmol L-1 and CySNO 56 mmol L-1 (A);
NAC 560 mmol L-1 and SNAC 56 mmol L-1 (B) and GSH 560 mmol L-1
and GSNO 56 mmol L-1 (C). Absorbance changes were monitored at
234 nm at 37 ºC.
Figure 4. Emission spectra obtained after linoleic acid (LA) oxidation
(final concentration 1.2 mmol L-1) catalyzed by FeII ions (FeSO4, final
concentration 5.0 mmol L-1) for 2 h, followed by incubation of the solution
with lysine (Lys) (final concentration 1.0 mmol L-1) for 48 h at 37 ºC in the
absence (a) and presence of GSNO 5.0 mmol L-1 (b) and 500.0 mmol L-1
(c). Excitation/emission wavelengths 360/430 nm. Inset: Emission spectra
of MDA incubated with Lys in equimolar concentrations of 0.25 mol L-1,
used as a control.
Figure 3. Initial rates (Ir) achieved after ca. 20 s of LA peroxidation,
catalyzed by SLO, in the absence and presence of RSHs and
their corresponding RSNOs at concentrations ten times lower.
[LA] = 19 mmol L-1; [SLO] = 56 nmol L-1; [RSHs] = 560 mmol L-1
and [RSNOs] = 56 mmol L-1. Data extracted from the kinetic curves of
Figure 2. * p < 0.001; ** p < 0.05.
Figure 5. Kinetic curves of fluorescent oxidized LA-Lys adduct formation
during the reaction between oxidized LA and Lys in the conditions (a), (b)
and (c) of Figure 4, based on the spectral changes monitored at 430 nm
during 48 h, after Lys addition.

1890
In Vitro Inhibition of Linoleic Acid Peroxidation by Primary S-Nitrosothiols
J. Braz. Chem. Soc.
Detection of LA peroxidation by infrared spectroscopy
by NO2– trapping by sulfanilamide, before injection in the
ascorbic acid soluti
Figure 6 shows the spectral change in the normal
infrared region, obtained after 2 and 4 h of heating of pure
LA at 80 ºC under O2 atmosphere. The new absorption
band with maximum at ca. 1180 cm-1 can be assigned to
the C–O–O vibration of hydroperoxides (LOOH).45
Figure 7. Light emission peaks obtained in the chemiluminescence
reaction of ozone with free NO, released in the reduction of nitrogen-
containing products of oxidized linoleic acid (oxLA) formed in the
peroxidation reaction of LA in the presence of GSNO (900 µmol L-1).
For details see experimental part. Peak (a) was obtained for the product
formed after the incubation of LA with CuII ions (900 µmol L-1) in the
presence of GSNO. Peak (b) was obtained for the product formed after the
incubation of LA previously oxidized by heating under O2 with GSNO.
Control curves are for the measurements of samples of water incubated
with GSNO without LA-SDS and CuII (control 1), and of water incubated
with GSNO and CuII without LA-SDS (control 2).
Figure 6. Infrared spectral changes associated with the formation of
hydroperoxides in the oxidation of linoleic acid at 80 ºC under O2
atmosphere for 2 and 4 h.
•
Detection of NO released from nitrogen-containing
products of oxidized LA
Reaction between RSNOs and tert-butyl hydroperoxide
Figure 7 shows the peaks of light emission obtained
•
in the chemiluminescence reaction of free NO, released
Figure 8 shows the kinetic curves corresponding to
the spectral changes due the disappearance of the RSNOs,
CySNO, SNAC and GSNO, during their reaction with tert-
butyl hydroperoxide (tBOOH) with formation of tert-butyl
peroxynitrites (tBOONOs). Control curves correspond to
the monitoring of RSNOs solutions at the same temperature
from nitrogen-containing products of oxLA, formed in
the reaction between oxLA and GSNO. The two peaks
shown were obtained after reduction of nitrogen-containing
products of oxLA by ascorbate, according to the procedures
described above. Peak (a) was obtained in the reduction of
a sample of LA-SDS dispersion oxidized in the presence
of CuII ions and GSNO. The same result was observed
when SNAC or CySNO were used in the place of GSNO
(data not shown). Peak (b) was obtained in the reduction
of a sample of LA-SDS dispersion incubated with GSNO,
where pure LA had been previously oxidized by heating
under O2 at 80 °C. The detection of free •NO in these cases
shows that nitrogen-containing products of oxLA are
formed either when LA is oxidized in aqueous dispersion
in the presence of GSNO or when a dispersion of oxLA-
SDS is subsequently incubated with GSNO. Control curves
are for the measurements of samples of water incubated
with GSNO without LA-SDS and CuII (control 1), and of
water incubated with GSNO and CuII without LA-SDS
(control 2), which show that GSNO was completely
eliminated through decomposition by HgII ions, followed
t
and pH conditions, but in the absence of BOOH. It can
be seen that the RSNOs solutions are quite stable in the
absence of tBOOH and that their thermal decompositions
are negligible in this time range. On the other hand, the
t
presence of BOOH leads to the fast disappearance of
the absorption bands of the three RSNOs, indicating that
t
they react with BOOH. The rates of reaction of SNAC
and GSNO are very similar and follow pseudo-first order
kinetics. However, CySNO shows a different kinetic
pattern, with an apparent bimodal behaviour. In this case,
the rate of reaction is lower and approximately constant up
to ca. 3 min and increases after this time, becoming similar
to the rates observed in the last 4 min for SNAC and GSNO.
In the lipid pool of plasma and cells, polyunsaturated
fatty acids (PUFAs) have higher propensity to oxidation due
to the fact that bis-allylic methylene hydrogens are more

Vol. 21, No. 10, 2010
Simplicio et al.
1891
degree of coronary disease.52 Linoleic acid has a double
bond configuration with bis-allylic methylene hydrogens
(Scheme 1). Due to this characteristic, and due to the
reasons mentioned above, LA is an appropriate model
compound for LPO studies. The kinetic monitoring of
conjugated double bond formation in LA-SDS comicelles
catalyzed by SLO (Figure 2) shows that LA is effectively
oxidized in aqueous dispersion by dissolved O2. It must be
considered that, in this particular condition, SLO is also
an appropriate catalyzer as a member of a well known
group of enzymes able to induce enzymatic peroxidation
of polyunsaturated fatty acids in biological membranes
and lipoproteins.53,54 In general, such enzymes contain
an essential iron atom, which is present as FeII in the
inactive enzyme form; enzymatic activation occurs through
hydroperoxide-driven oxidation of FeII to FeIII.
Figure 8. Kinetic curves corresponding to the spectral changes of
CySNO, SNAC and GSNO (initial concentration 1.0 mmolL-1) solutions
in the presence and absence of tBOOH (final concentration 25 mmol L-1),
monitored at 336 nm for 8 min, at 37 ºC. Control curves correspond to
the thermal decomposition of RSNOs solutions in the same temperature
and pH conditions but in the absence of tBOOH.
Although the kinetics of lipoperoxidation induced by
SLO, CuII and FeII ions may be different, the formation
of alkoxyl and peroxyl radicals is a common result in
all cases, since hydrogen abstraction by any initiator in
the presence of O2 will lead to the formation of these
propagating species. The results of all experiments herein
presented are in accordance with the inactivation of alkoxyl
and peroxyl species by the RSNOs. The expected products
in all cases are LOONO and LONO species. In fact,
nitrated lipid formation have been shown to occur in vivo
as potential “footprint” of the critical role that •NO and/or
•NO-derived reactive species play during lipid oxidation
processes.8 The RSNOs used here cannot be considered
classical antioxidants like a-tocopherol (a-TOH) or
ascorbic acid. Although a-TOH reacts with LOO• forming
a tocopheroxyl radical (a-TO•) which can scavenge another
LOO• species, allowing two LOO• radicals to be scavenged
by one a-TOH molecule, the primary product of this
reaction is LOOH and its accumulation may expose lipids
to subsequent oxidation mediated by metal ions.48 A similar
process can be described for other classical antioxidants
found in cells, like ascorbic acid and glutathione (GSH).
susceptible to hydrogen abstraction by oxidizing radicals
than the methylene hydrogens from fully saturated lipids.44
After such initiation process, the rapid reaction between the
formed carbon-centered radical and dioxygen (O2) forms
a lipid peroxyl radical (LOO•). Propagation occurs via the
reaction between LOO• and intact fatty acid (LH) molecules
forming lipid hydroperoxides (LOOH), which leads to the
formation of more LOO• species through the decomposition
of LOOH catalyzed by CuII or FeIII ions either free or in the
form of heme proteins.46-48 In addition, LOOH can lead to
the formation of alkoxyl radicals mediated by FeII. These
processes are represented in Scheme 1.
Linoleic acid (LA), one of the components of LDL
particles, is a major unsatured fatty acid in the american
diet and is considered to be atherogenic because of its
pro-oxidative and pro-inflammatory response through the
activation of endothelial cells.49,50 An increase in LA levels
has been reported in the phospholipid fractions of human
coronary arteries in cases of sudden cardiac death due to
ischemic heart disease.51 Additionally, concentrations of
LA in adipose tissue were positively correlated with the
Scheme 1. Formation of lipid fluorescent oxidized LA-lysine adducts.

1892
In Vitro Inhibition of Linoleic Acid Peroxidation by Primary S-Nitrosothiols
J. Braz. Chem. Soc.
Both are highly susceptible to hydrogen abstraction,
generating ascorbyl and thyil radicals and leading to LOOH
formation in their primary reactions. In the case of GSH, the
fate of the glutathionyl radical (GS•) formed is dimerization,
forming oxidized glutathione (GS-SG), the ration
GSH/GS-SG being a well-known marker of oxidative
stress.55 The protective actions of RSNOs are primarily
process once it neutralizes the cluster of positive charges
on the surface of LDL particles, conferring to them a
higher anionic electrophoretic mobility and a reduced
recognition by the LDL receptor on fibroblasts, while
increasing their recognition by macrophages.60 As Schiff
bases have fluorescence properties,61,62 it was assumed in
this work that such adducts could be used to characterize
the formation of aldehydes from hydroperoxides in the
peroxidation of LA. It was found here that the fluorescence
emission spectra obtained after LA oxidation catalyzed by
FeII ions followed by incubation of the solution with Lys,
has exactly the same shape and position of the spectrum
obtained in the incubation of MDA with Lys (Figure 4).
This result shows that products of LA peroxidation are
also reactive toward lysine, forming the same fluorescent
Schiff base adduct formed in oxidized LDL (like the MDA-
lysine adduct). The fluorescent adduct identified in this
work was assigned to the reaction between the aldehydes
formed from the reduction and b-scission of LOOH, with
lysine (Scheme 2). More specifically, the reaction involves
the nucleophilic addition between the amino group of
lysine and the carbonyl group of the aldehydes, forming
hemiaminals, followed by dehydration to generate stable
imines. In Scheme 2, these reactions are represented for
the two possible aldehydic fragments of LOOH b-scission:
12-oxododecanoic acid and hexanal. Of course, in different
oxidative situations several other aldehydic products may
form after LA peroxidation, which may also generate
fluorescent adducts with lysine. In addition to the aldehyde-
type lysine adducts, amide-type-lysine adducts have also
been described by Kawai and co-workers63,64 as a new class
of protein adducts derived from lipid peroxidation.
The dose-dependent reduction in the amounts of
aldehyde-type lysine adducts formed in the presence
of GSNO 5.0 µmol L-1 and 500.0 µmol L-1 in Figure 4,
compared to the peroxidation reaction performed in the
absence of GSNO, reflect the protective effect of GSNO in
this particular peroxidation condition. This protective action
is also reflected in the reduction of the rates and increase in
the apparent lag phase of the aldehyde-type lysine adducts
formation (Figure 5).
•
linked to the well-known antioxidant action of NO as
a radical-chain terminator, which arises from the fact
•
that NO is itself a free radical. Like its reaction with
superoxide (O2•–) generating peroxynitrite (ONOO–,
•
k = 6.7 × 109 L mol-1 s-1)56 free NO may react extremely
fast with LOO• (k = 2 × 109 L mol-1 s-1)48 removing this
chain carrying radical from the reaction scene. Although
formation of ONOO– is usually associated with a pro-
oxidant response reflected also in the nitration of tyrosine
residues,48,57 the deleterious actions of ONOO– have been
shown to depend strongly on the balance between •NO and
O2•–.48 More generally, the balance between oxidant species
and •NO seems to be fundamental in allowing a protective
action of •NO against LPO. Hummel et al.20 for example,
have shown that quite low levels of •NO (ca. 50 nmol L-1)
are enough to suppress FeII-O2 lipid oxidation in vitro in
cell models.
Although aqueous RSNOs solutions may spontaneous
•
release free NO in vitro through the homolytic cleavage
of the S–N bond, the chemical stability of RSNOs
solutions at low concentrations is high enough to allow
these compounds to react directly with other substrates
in a bimolecular mechanism. The decomposition of
RSNOs with NO release may occur in several hours and
days, depending on the experimental condition.19 In the
present work, as shown in Figure 2, the kinetic curves
of LA peroxidation were monitored for only 80 s. In this
time frame RSNOs are stable and the inhibition of lipid
peroxidation can be assigned to the primary reactions
between intact RSNOs molecules and oxyl/peroxyl species.
In the case of LA peroxidation, the primary reaction
of RSNOs with LO• or LOO• species will lead to the
formation of LOONO or LONO species and not to LOOH
or LOH species, as in the case of hydrogen abstraction from
classical antioxidants.
As a complementary analysis of the protective action
of RSNOs in LPO, one may also consider that linoleate
hydroperoxide (LOOH) formed as a primary oxidation
product of LA is expected to undergo b-scission generating
free aldehydes. It is generally assumed that adducts formed
in the conjugation of free aldehydes generated during
peroxidation of PUFAs with amino groups on LDL particles
are proteins with Schiff bases (containing the -C=N- group).
Formation of such adducts is central in the atherosclerotic
The formation of nitrogen-containing products of oxLA,
induced by CuII ions and heat, during the peroxidation of
LA in the presence of RSNOs was demonstrated in the
present work by reducing these products to free •NO and
hydroperoxides with ascorbic acid, according to procedures
already described in other works.14 The reaction involved
can be written as:
2 LOONO/LONO + AscH– + H+ →
2LOOH/LOH + DHA + 2 •NO (1)

Vol. 21, No. 10, 2010
Simplicio et al.
1893
Scheme 2. Key sites of primary S-nitrosothiol action on pathways of linoleic acid peroxidation.
where AscH– is the ascorbate anion and DHA is
dehydroascorbic acid formed in the oxidation (hydrogen
products of oxLA, detected by their reduction to •NO with
ascorbate. This result points to the ability of RSNOs to
inactivate preformed LA hydroperoxides (LOOH). In this
case, the bimolecular reaction is expected to proceed via a
transnitrosation mechanism and can be written as:
•
abstraction) of AscH–. Free NO released in this reaction
was unequivocally detected by chemiluminescence, after
its removal from the solution by bubbling with N2 and its
reaction with ozone (O3). It must be emphasized here that
•
to avoid any possible interference of NO released from
LOOH + RSNO → LOONO + RSH
(2)
excess GSNO, instead of •NO released from nitrated LA,
excess GSNO (which did not reacted with LOO•) was
completely eliminated from the solution by addition of HgII
ions and sulfanilamide. It is know that •NO is quantitatively
liberated from GSNO• by mercuric catalysis, and it is
rapidly and quantitatively converted to its stable solution
end-product, nitrite (NO2–) in aerated medium. By adding
sulfanilamide to the solution, NO2– formed is completely
It must be noted that the formation of LOOH in the
LA oxidized by heating under O2 was proven by observing
the appearance of an IR absorption band with maximum
around 1180 cm-1, assigned to the C-O-O vibration of
hydroperoxides (LOOH).45 The reactivity of RSNOs against
tert-butyl hydroperoxide (tBOOH) is an additional evidence
for reaction 2. Although the different kinetic behavior of
CysNO compared to SNAC and GSNO in Figure 8 is not
reflected in the initial rates of LA peroxidation in Figure 3, it
must be noted that reaction rates in Figure 3 were calculated
over the first 20 s of reaction, while differences in the
kinetic behavior among the three RSNOs in Figure 8 begin
to appear after 60 s. In addition, reaction rates in Figure 3
reflect not only the inactivation of LOOH by RSNOs but
mainly of LO• and LOO• species. Therefore, the evidences
of the ability of primary RSNOs to block the propagation of
•
removed, and therefore the NO signal detected in this
•
analysis can be attributed solely to NO released from
nitrogen-containing products of oxLA. This conclusion is
supported by the control experiments, which confirmed that
GSNO is completely eliminated by the mercuric catalysis/
sulfanilamide procedure. In addition, it was observed
that RSNOs react with LA previously oxidized through
heating, once incubation of oxLA-SDC comicelles with
GSNOs, led also to the formation of nitrogen-containing

1894
In Vitro Inhibition of Linoleic Acid Peroxidation by Primary S-Nitrosothiols
J. Braz. Chem. Soc.
lipid peroxidation reaction not only by inactivating LO• or
LOO• radicals, but also by inactivating preformed LOOH,
reinforces the potential role of endogenous RSNOs as
modulators of peroxidation reactions in vivo. The relevance
of this evidence can be better appreciated by considering
that the reduction of LOOH to LO• radical by metal ions as
FeII or CuII represents an important via for the propagation of
radical reactions in the lipid peroxidation process.Although
SLO, FeII and CuII have chemical peculiarities, it might be
consider that the three compounds lead to the formation of
nitrogen containing products of oxLA,8 which can release
NO upon reduction with ascorbic acid. For example, it has
been reported that lipid peroxidation of LA by lipoxygenase
yielded nitrogen-containing lipid derivatives.8
Thus, if the role of endogenous •NO as an antioxidant
species is linked in vivo to its presence in primary RSNOs,
this role must be extended beyond its classical radical
chain termination action, to encompass the inactivation
of deleterious hydroperoxides also present in the cellular
milieu. These multiple protective actions of RSNOs are
summarized in Scheme 1 for the case of LA peroxidation
and can be generalized for other lipids. These results
raise the possibility that primary RSNOs are involved in
the formation of nitrogen-containing lipids (and perhaps
of nitroalkanes), which all may be natural membrane
components and may have biological activities intimately
linked with the biological activities of RSNOs.
6. de Oliveira, C. P. M. S.; Alves, V. A. F.; de Lima, V. M. R.;
Stefano, J. T.; Debbas, V.; de Sá, S. V.; Wakamatsu, A.; Corrêa-
Giannella, M. L.; de Mello, E. S.; Havaki, S.; Tiniakos, D. G.;
Marinos, E.; de Oliveira, M. G.; Giannella-Neto, D.; Laurindo,
F. R.; Caldwell, S.; Carrilho, F. J.; Biochem. Pharmacol. 2007,
74, 290.
7. Girotti, A. W.; J. Lipid Res. 1998, 39, 1529.
8. Rubbo, H.; O`Donnell, V.; Toxicology 2005, 208, 305.
9. Letters, J. M.; Witting, P. K.; Christison, J. K.; Eriksson, A. W.;
Pettersson, K.; Stocker, R.; J. Lipid Res. 1999, 40, 1104.
10. McMackin, C. J.;Vita, J.A.; Methods Enzymol. 2005, 396, 541.
11. Walkowska, A.; Kompanowska-Jezierska, E.; Sadowski, J.;
Kidney Int. 2004, 66, 705.
12. Napoli, C.; Ignarro, L. J.; Nitric Oxide 2001, 5, 88.
13. Cooke, J. P.; Atheroscler. Suppl. 2003, 4, 53.
14. Lima, E.S.; Bonini, M. G.;Augusto, O.; Barbeiro, H.V.; Souza,
H. P.; Abdalla, D. S. P.; Free Radic. Biol. Med. 2005, 39, 532.
15. Schopfer, F. J.; Baker, P. R. S.; Giles, G.; Chumley, P.; Batthyany,
C.; Crawford, J.; Patel, R. P.; Hogg, N.; Branchaud, B. P.;
Lancaster, J. R.; Freeman, B.A.; J. Biol. Chem. 2005, 280, 19289.
16. Hogg, N.; Kalyanaraman, B.; Joseph, J.; Struck, A.;
Parthasarathy, S.; FEBS Lett. 1993, 334, 170.
17. Yamanaka, N.; Oda, O.; Nagao, S.; FEBS Lett. 1996, 398, 53.
18. Rubbo, H.; Trostchansky, A.; Botti, H.; Batthyány, C.; Biol.
Chem. 2002, 283, 547.
19. Kelley, E. E.;Wagner, B.A.; Buettner, G. R.; Burns, C. P.; Arch.
Biochem. Biophys. 1999, 370, 97.
20. Hummel, S. G.; Fischer, A. J.; Martin, S. M.; Schafer, F. Q.;
Buettner, G. R.; Free Radic. Biol. Med. 2006, 40, 501.
21. Lima, E. S.; Di Mascio, P.; Rubbo, H.; Abdalla, D. S. P.;
Biochemistry 2002, 41, 10717.
Acknowledgments
F. I. S. held a studentship from CNPq, project
140702/2003-2. G. F. P. S. holds a studentship from
FAPESP, project 07/55877-3. We thank FAPESP and CNPq
for financial support.
22. Freeman, B.A.; Baker, P. R. S.; Schopfer, F. J.;Woodcock, S. R.;
Napolitano, A.; d’Ischia, M.; J. Biol. Chem. 2008, 283, 15515.
23. Coler, B.; Bloodsworth, A.; Clark, S. R.; Lewis, M. J.; Cross, A.
R.; Freeman, B. A.; O’Donnell, V. B.; Circ. Res. 2002, 91, 375.
24. Schopfer, F. J.; Lin, Y.; Baker, P. R. S.; Cui, T.; Garcia-Barrio,
M.; Zhang, J.; Chen, K.; Chen,Y. E.; Freeman, B.A; Proc. Natl.
Acad. Sci. U.S.A. 2005, 102, 2340.
References
1. Whitlock, D. R.; Nitric Oxide-Biol. Chem. 2006, 14, 68.
2. Kunsch, C.; Medford R. M.; Circ. Res. 1999, 85, 753.
3. de Oliveira, C. P. M. S.; Simplicio, F. I.;. de Lima, V. M. R.;
Yuahasi, K.; Lopasso, F. P.; Alves, V. A. F.; Abdalla, D. S. P.;
Carrilho, F. J.; Laurindo, F. R. M.; de Oliveira, M. G.; World J.
Gastroenterol. 2006, 12, 1905.
25. Wright, M. M.; Schopfer, F. J.; Baker, P. R. S.; Vidyasagar, V.;
Powell, P.; Chumley, P.; Iles, K. E.;. Freeman, B. A.; Agarwal,
A.; Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 4299.
26. Cui, T.; Schopfer, F. J.; Zhang, J.; Chen, K.; Ichikawa, T.;.
Baker, P. R. S.; Batthyany, C.; Chacko, B. K.; Feng, X.; Patel,
R. P.; Agarwal, A.; Freeman, B. A.; Chen,Y. E.; J. Biol. Chem.
2006, 281, 35686.
4. de Oliveira, C. P. M. S.; Stefano, J. T.;. de Lima, V. M. R.;
Simplicio, F. I.; de Mello, E. S.; de Sá,V. M.; Corrêa-Giannella,
M. L.; Alves, V. A. F.; Laurindo, F. R. M.; de Oliveira, M. G.;
Giannela-Neto, D.; Carrilho, F. J.; J. Hepatol. 2006, 15, 725.
5. de Oliveira, C. P. M. S.; de Lima, V. M. R.; Simplicio, F. I.;
Soriano, F. G.; de Mello, E. S.; de Souza, H. P.; Alves, V. A.;
Laurindo, F. R.; Carrilho, F. J.; de Oliveira, M. G.; J. Am. Coll.
Nutr. 2008, 27, 299.
27. Villacorta, L.;Zhang, J.;Garcia-Barrio, M.T.;Chen, X.;Freeman,
B. A.; Chen, Y. E.; Cui, T.; Am. J. Physiol. 2007, 293, H770.
28. Baker, P. R. S.; Lin, Y.; Schopfer, F. J.; Woodcock, S. R.;
Groeger, A. L.; Batthyany, C.; Sweeney, S.; Long, M. H.; Iles,
K. E.; Baker, L. M. S.; Branchaud, B. P.; Chen,Y. E.; Freeman,
B. A.; J. Biol. Chem. 2005, 280, 42464.

Vol. 21, No. 10, 2010
Simplicio et al.
1895
29. Adams, M. R.; McCredie, R.; Jessup,W.; Robinson, J.; Sullivan,
D.; Celermajer, D. S.; Atherosclerosis 1997, 129, 261.
30. Gladwin, M. T.; Adv. Exp. Med. Biol. 2006, 588, 189.
31. Seabra, A. B.; Fitzpatrick, A.; Paul, J.; de Oliveira, M. G.;
Weller, R.; Brit. J. Dermatol. 2004, 151, 977.
49. Hennig, B.; Lei, W.; Arzuaga, X.; Ghosh, D. D.; Saraswathi,
V.; Toborek, M.; J. Nutr. Biochem. 2006, 17, 766.
50. Young, V. M.; Toborek, M.; Yang, F.; McClain, C. J.; Hennig,
B.; Metabolism 1998, 47, 566.
51. Luostarinen, R.; Boberg, M.; Saldeen, T.; Atherosclerosis 1993,
99, 187.
32. Pignatelli, P.; Di Santo, S.; Buchetti, B.; Sanguigni,V.; Brunelli,
A.; Violi, F.; FASEB J. 2006, 20, 1082.
52. Hodgson, J. M.; Wahlqvist, M. L.; Boxall, J. A.; Balazs, N. D.;
Am. J. Clin. Nutr. 1993, 58, 228.
33. Marcondes, S.; Cardoso, M. H. M.; Morganti, R. P.; Thomazzi,
S. M.; Lilla, S.; Murad, F.; De Nucci, G.; Antunes, E.; Proc.
Natl. Acad. Sci. U. S. A. 2006, 103, 3434.
53. Lapenna, D.; Ciofani, G.; Pierdomenico, S. D.; Giamberardino,
M. A.; Cuccurullo, F.; Free Radical Biol. Med. 2003, 35, 1203.
54. Brash, A. R.; J. Biol. Chem. 1999, 274, 23679.
55. Rahman, I.; Biswas, S. K.; Jimenez, L. A.; Torres, M.; Forman,
H. J.; Antioxid. Redox Signal. 2005, 7, 42.
34. Chen, C.; Huang, B.; Han, P. W.; Duan, S. J.; Prog. Biochem.
Biophys. 2006, 33, 609.
35. Batthyany, C.; Schopfer, F. J.; Baker, P. R. S.; Duran, R.;
Baker, L. M. S.; Huang, Y.; Cervenansky, C.; Branchaud, B.
P.; Freeman, B. A.; J. Biol. Chem. 2006, 281, 20450.
36. Giustarini, D.; Milzani,A.; Colombo, R.; Dalle-Donne, I.; Rossi,
R.; Clin. Chim. Acta 2003, 330, 85.
56. O’Donnell, V. B.; Eiserich, J. P.; Chumley, P. H.; Jablonsky, M.
J.; Krishna, N. R.; Kirk, M.; Barnes, S.; Darley-Usmar, V. M.;
Chem. Res. Toxicol. 1999, 12, 83.
57. O`Donnell, V. B.; Freeman, B. A.; Cir. Res. 2001, 88, 12.
58. Nakano, E.; Williamson, M. P.; Williams, N. H.; Powers, H. J.;
Biochim. Biophys. Acta 2004, 1688, 33.
37. de Oliveira, M. G.; Shishido, S. M.; Seabra, A. B.; Morgon, N.
H.; J. Phys. Chem. A 2002, 106, 8963.
38. Roy, B.; Dhardemare, A. D.; Fontecave, M.; J. Org. Chem.
1994, 59, 7019.
59. Zhang, Y. H.; Hogg, N.; Proc. Natl. Acad. Sci. U. S. A. 2004,
21, 7891.
39. Bainbrigge, N.; Butler, A. R.; Gorbitz, C. H.; J. Chem. Soc.,
Perkin Trans. 2 1997, 351.
60. Cominacini, L.; Garbin, U.; Davoli, A.; Micciolo, R.; Bosello,
O.; Gaviraghi, G.; Scuro, L. A.; Pastorino, A. M.; J. Lipid Res.
1991, 32, 349.
40. Stamler, J. S.; Singel, D. J.; Loscalzo, J.; Science 1992, 258, 1898.
41. Nicolescu ,A. C.; Reynolds, J. N.; Barclay, L. R. C.; Thatcher,
G. R. J.; Chem. Res. Toxicol. 2004, 17, 185.
61. Fruebis, J.; Parthasarathy, S.; Steinberg, D.; Proc. Natl. Acad.
Sci. U. S. A. 1992, 89, 10588.
42. Krieger, M.H.; Santos, K.F.R.; Shishido, S.M.; de Oliveira,
M.G.; Franchini, K.G.; Spadari-Bratfish, R.C.; Wanschel, A.
A. C. B.; Estrela, H.F.G.; Santos, L.; Laurindo, F. R. M.; Nitric
Oxide 2006, 14, 12.
62. de Oliveira, F. G.; Rossi, C. L.; de Oliveira, M. G.; Saad, M. J.
A.; Velloso, L. A.; Cardiovasc. Res. 2000, 47, 567.
63. Kawai, Y.; Kato, Y.; Fujii, H.; Makino, Y.; Mori, Y.; Naito, M.;
Osawa, T.; J. Lipid Res. 2003, 44, 1124.
43. Hart, T. W.; Tetrahedron Lett. 1985, 26, 2013.
44. Hogg, N.; Kalyanaraman, B.; Biophys. Acta 1999, 1411, 378.
45. Silvertein, R. M.; Franciz, X.; Spectrometric Identification of
Organic Compounds, JohnWiley & Sons Inc.: NewYork, 1998.
46. Ohyashiki, T.; Kadoya, A.; Kushida, K.; Chem. Pharm. Bull.
2002, 50, 203.
64. Kawai, Y.; Fujii, H.; Kato, Y.; Kodama, M.; Naito, M.; Uchida,
K.; Osawa, T.; Biochem. Biophys. Res. Commun. 2004, 313, 271.
Submitted: November 16, 2009
Published online: June 23, 2010
47. Pinchuk, I.; Lichtenberg, D.; Prog. Lipid Res. 2002, 41, 279.
48. Patel, R. P.; Moellering, D.; Murphy-Ullrich, J.; Jo, H.;
Beckman, J. S.; Darley-Usmar, V. M.; Free Radical Biol. Med.
2000, 28, 1780.
FAPESP has sponsored the publication of this article.