The Nitric Oxide Congener Nitrite Inhibits
Myeloperoxidase/H2O2/ Cl
-mediated
Modification of Low Density Lipoprotein*
Anitra C.
Carr
and
Balz
Frei
From the Linus Pauling Institute, Oregon State University,
Corvallis, Oregon 97331
Received for publication, October 4, 2000, and in revised form, October 26, 2000
 |
ABSTRACT |
Nitric oxide, a pivotal molecule in vascular
homeostasis, is converted under aerobic conditions to nitrite. Recent
studies have shown that myeloperoxidase (MPO), an abundant heme protein released by activated leukocytes, can oxidize nitrite
(NO2
) to a radical species,
most likely nitrogen dioxide. Furthermore, hypochlorous acid (HOCl),
the major strong oxidant generated by MPO in the presence of
physiological concentrations of chloride ions, can also react with
nitrite, forming the reactive intermediate nitryl chloride. Since MPO
and MPO-derived HOCl, as well as reactive nitrogen species, have been
implicated in the pathogenesis of atherosclerosis through oxidative
modification of low density lipoprotein (LDL), we investigated the
effects of physiological concentrations of nitrite (12.5-200
µM) on MPO-mediated modification of LDL in the absence
and presence of physiological chloride concentrations. Interestingly,
nitrite concentrations as low as 12.5 and 25 µM significantly decreased
MPO/H2O2/Cl
-induced modification
of apoB lysine residues, formation of N-chloramines, and
increases in the relative electrophoretic mobility of LDL. In contrast,
none of these markers of LDL atherogenic modification were affected by
the MPO/H2O2/NO2
system. Furthermore, experiments using ascorbate (12.5-200
µM) and the tyrosine analogue 4-hydroxyphenylacetic acid
(12.5-200 µM), which are both substrates of MPO,
indicated that nitrite inhibits MPO-mediated LDL modifications by
trapping the enzyme in its inactive compound II form. These data offer
a novel mechanism for a potential antiatherogenic effect of the nitric
oxide congener nitrite.
 |
INTRODUCTION |
Nitric oxide (nitrogen monoxide, NO·) is synthesized
in vivo by a family of inducible and constitutively
expressed nitric-oxide synthases
(NOS)1 (1, 2). Nitric oxide
generated by the NOS isoform present in endothelial cells (eNOS) is
critically involved in normal vascular function through regulation of
smooth muscle cell relaxation and vasodilation as well as
modulation of platelet, leukocyte, and endothelial cell
adhesion (1, 2). The inducible NOS isoform present in phagocytes (iNOS)
is thought to be involved in their antimicrobial activity, whereas
up-regulation of iNOS during chronic inflammation has been implicated
in vascular pathology (1, 3). Since nitric oxide does not readily react
with biological macromolecules, the tissue damage associated with
increased nitric oxide levels has been attributed to the generation of
peroxynitrite (3), which is formed by rapid reaction of nitric oxide
with superoxide (k2 = 1.9 × 1010 M
1
s
1) (4). Under aerobic conditions, nitric
oxide also reacts with molecular oxygen (k2 = 6 × 106 M
2
s
1) (5) to form a dinitrogen trioxide
intermediate that hydrolyzes to nitrite (Reaction 1) (5-7).
Nitrite is found in biological fluids at concentrations between 0.5 and
210 µM (8-10). In human plasma, levels of nitrite are
typically low, between 0.5 and 3.3 µM (8, 9), due to
oxidation of nitrite to nitrate by oxyhemoglobin (3). In inflammatory
conditions, however, plasma nitrite levels can significantly increase,
e.g. up to 36 µM in patients with human
immunodeficiency virus infection (11).
|
|
|
|
Leukocytes such as neutrophils, monocytes, and
macrophages, as well as endothelial cells, can synthesize both nitric
oxide and superoxide (1, 12). Thus, it is likely that peroxynitrite is
formed in vivo by these cells. 3-Nitrotyrosine, a product of the reaction of peroxynitrite with either free tyrosine or tyrosine residues in (lipo)proteins has been used as a biomarker for the generation of peroxynitrite in vivo (13). Elevated
3-nitrotyrosine levels have been detected, e.g. in
atherosclerotic lesions (14, 15), as has increased expression of the
inducible NOS isoform (16, 17). Leukocytes, however, also release the
abundant heme protein myeloperoxidase (MPO) upon activation by
inflammatory stimuli (12, 18), and recent studies show that mammalian
peroxidases can oxidize nitrite to a radical species, most likely
nitrogen dioxide (Reaction 2) (19, 20). Furthermore, hypochlorous acid (HOCl), the major strong oxidant generated by MPO in the presence of
physiological concentrations of chloride ions (Reaction 3) (12, 18),
can also react with nitrite, forming the reactive intermediate nitryl
chloride (Reaction 4) (21, 22). Both nitrogen dioxide and nitryl
chloride, like peroxynitrite, can nitrate tyrosine residues (21, 23,
24), calling into question the specificity of 3-nitrotyrosine as a
marker of peroxynitrite generation in vivo (25, 26).
Several recent studies implicate MPO and MPO-derived HOCl in the
pathogenesis of atherosclerosis (27-31). Catalytically active MPO (27)
and epitopes recognized by antibodies against HOCl-modified proteins
have been detected in human atherosclerotic lesions (28); these were
found to colocalize with monocyte/macrophages, endothelial cells, and
the extracellular matrix (29). Dityrosine and 3-chlorotyrosine, biomarkers of MPO- and HOCl-mediated protein modification, have also
been detected in atherosclerotic lesions (30, 31). Oxidative modification of low density lipoprotein (LDL) in vitro by
MPO or HOCl primarily involves chlorination of the
-amino groups of
lysine residues of apolipoprotein B-100 (apoB), the major protein component of LDL, resulting in the formation of
N-chloramines (32-34). LDL-associated
N-chloramines have been implicated in the altered
electrophoretic migration, aggregation, and subsequent uncontrolled
uptake of HOCl-modified LDL by macrophages (32, 33, 35). Since
3-nitrotyrosine has also been detected in atherosclerotic lesions (14,
15), it is possible that MPO-derived reactive nitrogen species are
involved in atherogenesis in addition to HOCl. In contrast to HOCl,
however, MPO-derived reactive nitrogen species primarily cause lipid
peroxidation in LDL (36-38). Thus, increased macrophage uptake of LDL
modified by reactive nitrogen species is most likely due to increased
levels of lipid oxidation products (36, 39).
In this study we investigated the effects of physiological
concentrations of nitrite (12.5-200 µM) on MPO-mediated
modification of LDL in the absence and presence of physiological
chloride concentrations (140 mM). In addition, we examined
whether the physiological antioxidant ascorbate can inhibit LDL
modification under these conditions. Our data reveal a novel mechanism
by which nitrite inhibits MPO-mediated atherogenic modification of
LDL.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Human leukocyte MPO and anti-nitrotyrosine
monoclonal antibodies were procured from Calbiochem, HOCl was from
Aldrich, and 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide (ABD-F) was
from Molecular Probes, Eugene, OR. Alkaline phosphatase-conjugated anti-mouse IgG antibody and BCIP/TNBT color reagent were from Chemicon International, Temecula, CA. All other reagents were obtained
from Sigma. Phosphate-buffered saline (PBS) was composed of 10 mM sodium phosphate buffer, 140 mM NaCl, pH
7.4, and contained the metal chelator diethylenetriaminepentaacetic
acid (DTPA, 100 µM). Tris-buffered saline was composed of
10 mM Tris-HCl, 140 mM NaCl, pH 7.4, and
contained 0.1% Tween 20. Griess reagent was composed of 1%
sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in
2.5% H3PO4. Thionitrobenzoic acid (TNB) was prepared from 5,5'-dithiobis-(2-nitrobenzoic acid) by alkaline hydrolysis with subsequent neutralization (40).
Isolation of LDL--
LDL was isolated from fresh plasma by a
sequential centrifugation method (41) modified as described by us
previously (34). The isolated LDL (1.019 < d < 1.067 g/ml fraction) was desalted by two sequential passages through
PD-10 gel filtration columns (Amersham Pharmacia Biotech) using 10 mM sodium phosphate buffer, pH 7.4. The total protein was
estimated using the Lowry micro method kit (Sigma P5656). For
experiments the LDL was diluted to a concentration of 0.5 mg of
protein/ml (
1 µM LDL) in PBS unless indicated
otherwise. For chloride-free experiments, the LDL was diluted in
phosphate buffer containing 100 µM DTPA.
Oxidation of LDL by MPO--
MPO (50 nM) was
incubated with LDL for 30 min at 37 °C in the presence of the stated
concentrations of H2O2. The
H2O2 was freshly diluted in phosphate buffer
and standardized at 240 nm (
= 43.6 M
1 cm
1)
(42) and was added to the LDL samples in aliquots of 25-50 µM over the 30-min incubation period. Under these
conditions all of the added H2O2 was converted
into HOCl as determined by TNB oxidation using 10 mM
taurine as a trap (40).
Incubations with Alternative MPO Substrates--
LDL samples
were also incubated in the presence of the MPO substrates nitrite,
ascorbate, and 4-hydroxyphenylacetic acid (HPA) (12.5 - 200 µM each). HPA was used instead of tyrosine because it
lacks an amino group and, thus, has only minimal reactivity with HOCl.
Ascorbate was freshly diluted in phosphate buffer containing 100 µM DTPA and standardized at 265 nm (
= 15,000 M
1 cm
1)
(43). Ascorbate was measured using paired-ion, reversed-phase HPLC with
electrochemical detection (44). Nitrite was measured spectrophotometrically at 550 nm using the Griess reagent (45).
Oxidation of LDL by Reagent HOCl--
HOCl was standardized at
292 nm (
= 350 M
1
cm
1) after dilution into pH 12 buffer (46).
Standardized HOCl was freshly diluted in PBS; the
pKa of HOCl is 7.5 (46), and therefore the solution
contained both HOCl and OCl
. HOCl was added to the LDL in
the presence of nitrite (coincubation) or before the addition of
nitrite (pre-incubation), and the LDL was incubated for 30 min at
37 °C.
Quantification of Amino Acids--
Tryptophan residues were
measured directly by fluorescence (excitation = 280 nm,
emission = 335 nm) (32). In samples containing HPA, the
fluorescence of the equivalent concentration of HPA was subtracted from
the total fluorescence. Lysine residues were measured after
fluorescamine derivatization (excitation = 390 nm, emission = 475 nm) (47). Cysteine residues were measured after
7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide (ABD-F) derivatization
(excitation = 365 nm, emission = 490 nm) (48), as described
by us previously (34).
Quantification of Amino Acid
Derivatives--
N-Chloramines were determined after TNB
oxidation (
412 = 14, 100 M
1 cm
1)
(40). Carbonyls were measured after 2,4-dinitrophenylhydrazine derivatization (
450 = 22,000 M
1 cm
1)
(33). 3-Nitrotyrosine was measured by dot blot using an
anti-nitrotyrosine monoclonal antibody (49). Briefly, nitrocellulose
blots were treated for 1 h with 5% milk powder in Tris-buffered
saline, 1 h with anti-nitrotyrosine monoclonal antibody (1:200 in
Tris-buffered saline), 1 h with alkaline phosphatase-conjugated
anti-mouse IgG antibody (1:5,000 in Tris-buffered saline) and detected
with BCIP/TNBT color reagent. The change in the relative
electrophoretic mobility (REM) of LDL was determined by agarose gel
electrophoresis using a Paragon lipoprotein electrophoresis kit
(Beckman Coulter, Fullerton, CA).
Statistical Analysis--
Statistical analyses were carried out
using analysis of variance with Fisher's post hoc analysis
and regression analysis using StatView software (SAS Institute, Cary,
NC). Statistical significance was set at p < 0.05.
 |
RESULTS |
Modification of LDL by
MPO/H2O2/Cl
--
We (34) and
others (32, 33) have shown that bolus addition of reagent HOCl (25-200
µM) to human LDL results in a number of modifications to
apoB, e.g. oxidation of cysteine, tryptophan, and lysine
residues, formation of N-chloramines, and an increase in
REM. In the present study, we investigated the physiologically more
relevant system of HOCl generated by
MPO/H2O2/Cl
. The addition of
increasing amounts of H2O2 (25-200
µM) to human LDL (0.5 mg protein/ml;
1
µM) in the presence of MPO (50 nM) caused
basically identical modifications to LDL (Fig.
1) as those seen with reagent HOCl (34).
In contrast, 200 µM H2O2 alone did not cause oxidation of LDL amino acid residues or an increase in
REM (data not shown). Using the
MPO/H2O2/Cl
system, all of the
added H2O2 was converted into HOCl as
determined by TNB oxidation using taurine as a trap (40). Cysteine
residues were the most sensitive target on LDL and were completely
oxidized in the presence of 75 µM
H2O2 (Fig. 1a). Lysine and
tryptophan residues were modified at equal rates, with 39 and 43% of
the residues modified, respectively, after the addition of 200 µM H2O2 (Fig. 1a).
Since LDL contains ~3-5 free cysteine residues (50, 51), 356 lysine
residues, and 37 tryptophan residues (52), lysine residues can be
calculated to be quantitatively the major target of HOCl generated by
the MPO system. The decrease in lysine residues was mirrored by the
formation of N-chloramines, which accounted for
approximately one-third of the added H2O2 (Fig.
1b). A dose-dependent increase in REM was also
observed with increasing concentrations of H2O2
(Fig. 1b).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Modification of LDL by
MPO/H2O2/Cl . Freshly
isolated human LDL (0.5 mg of protein/ml of PBS containing 100 µM DTPA) was incubated for 30 min at 37 °C with MPO
(50 nM) and increasing concentrations of
H2O2 (25-200 µM), added in 25 or
50 µM aliquots over the incubation period. a,
modification of apoB cysteine ( ), tryptophan ( ), and lysine ( )
residues; b, formation of N-chloramines
(RNHCl, ) and increase in REM of LDL ( ) were
determined as described under "Experimental Procedures." Percent
N-chloramines was estimated using 356 lysine residues per
LDL (52). Results represent the mean ± S.D. (n = 3 - 4).
|
|
Modification of LDL by
MPO/H2O2/NO2
--
Previous
studies have investigated the modification of LDL by
MPO/H2O2/NO2
in the absence of chloride (36, 37). We found that this system caused
relatively few changes to apoB under the conditions used in the present
study (Fig. 2). No significant
modification of lysine residues occurred, and only a small
dose-dependent decrease of tryptophan residues was observed
with increasing nitrite concentrations (Fig. 2a); ~25% of
the tryptophan residues in LDL were oxidized in the presence of 200 µM nitrite. Analysis of cysteine residues was confounded
by the fact that in the absence of nitrite ~50% of the residues were
already oxidized (Fig. 2a). Since 200 µM H2O2 alone did not cause cysteine oxidation
(data not shown), this finding is most likely due to a small amount of
halide contamination in the buffers used. Nevertheless, nitrite did not
exert any dose-dependent effect on oxidation of apoB
cysteine residues (Fig. 2a). In agreement with the lack of
lysine oxidation by the
MPO/H2O2/NO2
system, there was no increase in the REM or TNB reactivity of LDL (Fig.
2c). However, dot blot analysis using an anti-nitrotyrosine monoclonal antibody showed dose-dependent formation of
3-nitrotyrosine (Fig. 2b). Interestingly, measurement of
nitrite using the Griess assay indicated that approximately twice as
much of the added nitrite was consumed in the absence of chloride than
in its presence (e.g. 124 ± 4 µM nitrite
versus 56 ± 7 µM nitrite, respectively, after the addition of 200 µM nitrite) (data not shown).
It should be noted, however, that reaction of nitrogen dioxide with
substrates that can donate hydrogens regenerates nitrite, and thus, the
Griess assay may underestimate the amount of nitrite utilized by
MPO.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 2.
Modification of LDL by
MPO/H2O2/NO2 .
LDL (0.5 mg of protein/ml of phosphate buffer containing 100 µM DTPA) was incubated for 30 min at 37 °C with MPO
(50 nM) and H2O2 (200 µM; added in 50 µM aliquots over the
incubation period) in the presence of increasing concentrations of
nitrite (12.5-200 µM). a, modification of
apoB cysteine ( ), tryptophan ( ), and lysine ( ) residues;
b, formation of 3-nitrotyrosine (3-NT);
c, formation of N-chloramines (RNHCl,
) and increase in REM of LDL ( ) were determined as described
under "Experimental Procedures." Percent N-chloramines
was estimated using 356 lysine residues per LDL (52). Results represent
the mean ± S.D. (n = 3).
|
|
Modification of LDL by
MPO/H2O2/Cl
and
NO2
--
Since nitrite is a substrate for
MPO (20), the effect of physiological concentrations of nitrite on LDL
modification in the presence of 200 µM
H2O2 and physiological chloride concentrations was investigated. The addition of increasing concentrations of nitrite
(12.5-200 µM) affected specific MPO-mediated
modifications of LDL (Fig. 3). Low
concentrations of nitrite (
12.5 µM) significantly decreased modification of apoB lysine residues induced by
MPO/H2O2/Cl
(Fig. 3a).
A concurrent, although less extensive, decrease in modification of
tryptophan residues was observed at 25-50 µM nitrite, but this effect was lost at higher nitrite concentrations (Fig. 3a). Since the cysteine residues were almost fully oxidized
with 200 µM H2O2 (Fig.
3a), LDL was treated with sufficient
H2O2 (25 µM) to oxidize
approximately one-third of the cysteine residues; the addition of
nitrite, however, did not have a significant effect on thiol oxidation
at this concentration of H2O2 (data not shown). In agreement with the decrease in lysine oxidation by low
concentrations of nitrite (Fig. 3a), formation of
N-chloramines and increase in REM were also inhibited (Fig.
3b). Interestingly, at higher concentrations of nitrite (150 and 200 µM), the REM increased again, although not to
control levels seen in the absence of nitrite (Fig. 3b).
This increase in REM could be a result of increased nitrite-dependent lipid peroxidation and subsequent
derivatization of apoB lysine residues to carbonyls by lipid
hydroperoxide breakdown products. In support of this hypothesis, we
found that nitrite dose-dependently increased apoB carbonyl
levels (e.g. 14 ± 1 µM carbonyls without
nitrite versus 60 ± 2 µM carbonyls at
200 µM nitrite) but not until the concentrations of added
nitrite reached
50 µM (data not shown).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3.
Modification of LDL by
MPO/H2O2/Cl and
NO2 . LDL (0.5 mg of protein/ml
of PBS containing 100 µM DTPA) was incubated for 30 min
at 37 °C with MPO (50 nM) and
H2O2 (200 µM; added in 50 µM aliquots over the incubation period) in the presence
of increasing concentrations of nitrite (12.5-200 µM).
a, modification of apoB cysteine ( ), tryptophan ( ),
and lysine ( ) residues; b, formation of
N-chloramines (RNHCl, ) and increase in REM of
LDL ( ) were determined as described under "Experimental
Procedures." Percent N-chloramines was estimated using 356 lysine residues per LDL (52). Results represent the mean ± S.D.
(n = 3).
|
|
Modification of LDL by Reagent HOCl in the Presence of
NO2
--
A number of mechanisms could
account for the marked decrease in lysine oxidation,
N-chloramine formation, and increase in REM by
MPO/H2O2/Cl
in the presence of
low concentrations of nitrite (Figs. 3, a and b).
For example, nitrite could "scavenge" MPO-derived HOCl (21, 22) or
react with preformed N-chloramines. To investigate the
former mechanism, LDL was exposed to reagent HOCl (200 µM) in the presence of increasing concentrations of
nitrite. As shown in Table I, nitrite had
little effect on the oxidation of LDL by HOCl, with only a small
dose-dependent decrease in lysine oxidation and
N-chloramine formation. Thus, nitrite does not appear to
protect LDL to a significant extent by scavenging HOCl. Similarly, when LDL was pretreated with HOCl and subsequently incubated with nitrite, no reversal of N-chloramine formation or lysine modification
was observed (data not shown).
View this table:
[in this window]
[in a new window]
|
Table I
Modification of LDL by 200 µM HOCl in the presence of
increasing concentrations of nitrite
LDL (0.5 mg of protein/ml of PBS containing 100 µM DTPA)
was incubated for 30 min at 37 °C with HOCl (200 µM)
in the presence of increasing concentrations of nitrite (12.5-200
µM). Modification of apoB lysine (Lys), tryptophan (Trp),
and cysteine (Cys) residues, formation of N-chloramines
(RNHCl), and increase in REM of LDL were determined as described under
"Experimental Procedures." Percent N-chloramines was
estimated using 356 lysine residues per LDL (52). Results for Lys, Trp,
and Cys are expressed as percent control and for RNHCl and REM as
percent increase and represent the mean ± S.D. (n = 3). NS, not significant.
|
|
Effects of Ascorbate on LDL Modification by
MPO/H2O2/Cl
and
NO2
--
Another potential mechanism for
low concentrations of nitrite decreasing
MPO/H2O2/Cl
-mediated modification
of LDL is inhibition of enzyme activity. This is conceivable because
nitrite has a relatively high reaction rate with compound I and a low
reaction rate with compound II of MPO (20), thus effectively trapping
the enzyme in a form incapable of generating HOCl (Scheme
1). To investigate this possibility, the
complete system
(MPO/H2O2/Cl
/NO2
)
was incubated with ascorbate, a "cosubstrate" of MPO that has a
relatively high reaction rate with compound II (53) (54) and, thus, can
cycle the enzyme (Scheme 1). Indeed, the addition of ascorbate to LDL
in the presence of the complete MPO system strongly affected LDL
modification (Fig. 4). Low concentrations of ascorbate (12.5-50 µM) abrogated the inhibitory
effect of the equivalent concentrations of nitrite on lysine oxidation
by MPO (Fig. 4a). In contrast to the complete system (Fig.
3a), the addition of ascorbate provided almost
stoichiometric protection against lysine and tryptophan oxidation (Fig.
4a). Another noticeable difference between the complete
system in the absence (Fig. 3a) and the presence of
ascorbate (Fig. 4a) was the significant inhibition of
cysteine oxidation at high concentrations of ascorbate (150 and 200 µM). In agreement with the dose-dependent
protection against lysine modification in the presence of ascorbate,
dose-dependent inhibition of N-chloramine
formation and an increase in REM was also observed under these
conditions (Fig. 4b), with complete protection by 150 and
200 µM ascorbate.

View larger version (15K):
[in this window]
[in a new window]
|
Scheme 1.
Catalytic cycle of MPO. The addition
of H2O2 to MPO forms the redox intermediate
compound I, which oxidizes either Cl in a two-electron
step to form HOCl and regenerate the native enzyme or
NO2 in a one-electron step to form
NO2· and compound II. Compound II subsequently
carries out another one-electron oxidation step to regenerate the
native enzyme. The second order rate constants
(k2) for the reactions of Cl (62),
NO2 (20), and ascorbate
(AH ) (64) with compound I and/or II are shown. The
rate constants for the reaction of ascorbate with compound I is
1.1 × 106 M 1
s 1 (64) and tyrosine with compounds I and II
are 7.7 × 105
M 1 s 1
and 1.6 × 104
M 1 s 1,
respectively (68).
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of ascorbate on LDL modification by
MPO/H2O2/Cl and
NO2 . LDL (0.5 mg of protein/ml
of PBS containing 100 µM DTPA) was incubated for 30 min
at 37 °C with MPO (50 nM) and
H2O2 (200 µM; added in 50 µM aliquots over the incubation period) in the presence
of increasing concentrations of ascorbate (12.5-200 µM)
and nitrite (12.5-200 µM). a, modification of
apoB cysteine ( ), tryptophan ( ), and lysine ( ) residues;
b, formation of N-chloramines (RNHCl,
) and increase in REM of LDL ( ) were determined as described
under "Experimental Procedures." Percent N-chloramines
was estimated using 356 lysine residues per LDL (52). Results represent
the mean ± S.D. (n = 3).
|
|
Effects of Ascorbate on LDL Modification by
MPO/H2O2/Cl
without
NO2
--
In the absence of nitrite,
ascorbate provided identical protection as in the presence of nitrite
against MPO-mediated oxidation of lysine, tryptophan, and cysteine
residues, formation of N-chloramines, and increases in REM
(compare Figs. 5, a and
b with Figs. 4, a and b,
respectively). Since low µM concentrations of ascorbate have been shown to stimulate HOCl production by MPO (53), LDL was
treated with sufficient H2O2 (25 µM) to oxidize approximately one-third of the cysteine
residues, and low concentrations (0.5-5 µM) of ascorbate
were added. These concentrations of ascorbate, however, failed to
enhance thiol oxidation, whereas slightly higher concentrations (5-50
µM) dose-dependently inhibited thiol
oxidation (data not shown). The above findings (Figs. 5, a
and b) agree with our previous data using bolus addition of
HOCl to LDL in the presence of increasing concentrations of ascorbate
(34). The one exception, however, is protection against modification of
cysteine residues; 200 µM ascorbate gave approximately
2-fold greater protection in the
MPO/H2O2/Cl
system compared with
oxidation by 200 µM reagent HOCl (80 ± 15% versus 39 ± 6% protection, respectively).
Interestingly, HPLC analysis showed that all of the added ascorbate
(200 µM) was consumed in the
MPO/H2O2/Cl
system, whereas
36 ± 12 µM (n = 3) ascorbate
remained in the presence of HOCl (data not shown). This difference is
likely due to more efficient scavenging by ascorbate of HOCl
continuously generated by MPO rather than added as a bolus.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of ascorbate on LDL modification by
MPO/H2O2/Cl without
NO2 . LDL (0.5 mg of protein/ml
of PBS containing 100 µM DTPA) was incubated for 30 min
at 37 °C with MPO (50 nM) and
H2O2 (200 µM; added in 50 µM aliquots over the incubation period) in the presence
of increasing concentrations of ascorbate (12.5-200 µM).
a, modification of apoB cysteine ( ), tryptophan ( ),
and lysine ( ) residues; b, formation of
N-chloramines (RNHCl, ) and increase in REM of
LDL ( ) were determined as described under "Experimental
Procedures." Percent N-chloramines was estimated using 356 lysine residues per LDL (52). Results represent the mean ± S.D.
(n = 3).
|
|
Effects of HPA on LDL Modification by
MPO/H2O2/Cl
and
NO2
--
Ascorbate not only cycles MPO by
reducing compound II to the native enzyme (54) but also scavenges HOCl
in a stoichiometric manner (43) and regenerates amines from
N-chloramines (34). To avoid these "confounding"
reactions, the tyrosine analogue HPA was used as an alternative
substrate to cycle MPO since it has only minimal reactivity with HOCl
and N-chloramines. Like ascorbate, the addition of low
concentrations of HPA (12.5 and 25 µM) to the complete
MPO/H2O2/Cl
/NO2
system abrogated the inhibitory effect of nitrite on lysine oxidation (compare Figs. 3a and
6a). However, unlike
ascorbate, higher concentrations of HPA (50-200 µM) did
not dose-dependently protect against modification of
lysine, tryptophan, and cysteine residues (compare Figs. 4a and 6a). Modification of tryptophan residues was actually
increased in the presence of HPA (Fig. 6a), most likely due
to enhanced turnover of the enzyme. The small decrease in
N-chloramines and change in REM (Fig. 6b) and
modification of lysine residues (Fig. 6a) could be due to
increased competition for reaction of chloride with compound I of MPO
by the additional substrates nitrite and HPA (see Scheme 1). In the
absence of nitrite, HPA had little effect on the modification of apoB
by the MPO/H2O2/Cl
system (data
not shown). The lack of an effect of HPA on tryptophan oxidation in the
latter system (data not shown) suggests that it is nitrogen dioxide
radicals rather than HPA-derived phenoxyl radicals that are causing
tryptophan oxidation in the complete system (Fig. 6a; see
also Fig. 2a).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of HPA on LDL modification by
MPO/H2O2/Cl and
NO2 . LDL (0.5 mg of protein/ml
of PBS containing 100 µM DTPA) was incubated for 30 min
at 37 °C with MPO (50 nM) and
H2O2 (200 µM; added in 50 µM aliquots over the incubation period) in the presence
of increasing concentrations of HPA (12.5-200 µM) and
nitrite (12.5-200 µM). a, modification of
apoB cysteine ( ), tryptophan ( ), and lysine ( ) residues;
b, formation of N-chloramines (RNHCl,
) and increase in REM of LDL ( ) were determined as described
under "Experimental Procedures." Percent N-chloramines
was estimated using 356 lysine residues per LDL (52). Results represent
the mean ± S.D. (n = 3).
|
|
 |
DISCUSSION |
The present study shows for the first time that the nitric oxide
congener nitrite inhibits MPO-mediated modification of LDL. Modification of LDL by MPO in the presence of physiologically relevant
concentrations of H2O2 (25-200
µM) and chloride ions (140 mM) results in a
number of potentially atherogenic alterations to LDL. These include
modification of lysine residues to form N-chloramines and an
increase in REM of LDL, which reflects an increase in its net negative
charge. We have shown that modification of LDL with 200 µM HOCl (34) or with MPO, 200 µM
H2O2, and chloride ions (this study) causes
modification of 26 and 39% of LDL lysine residues, respectively.
Previous studies using chemically modified LDL show that modification
of as little as 16% of the apoB lysine residues results in recognition
of LDL by the scavenger receptor(s) of macrophages (55, 56). LDL
modified with HOCl also exhibits increased uptake by macrophages (32,
35), likely due to modification of the apoB lysine residues by HOCl
(32). In the presence of physiologically relevant concentrations of
nitrite (12.5-50 µM) (8, 11), a major oxidation product
of nitric oxide, a dose-dependent decrease in the
MPO/H2O2/Cl
-mediated oxidation of
LDL lysine residues was observed. A similar decrease in
N-chloramines and change in REM was also observed.
There are a number of mechanisms that could account for the marked
decrease in MPO/H2O2/Cl
-induced
lysine oxidation, N-chloramine formation, and increase in
REM by low concentrations of nitrite. HOCl reacts with nitrite in a
stoichiometric manner to form intermediates such as nitryl chloride
(21, 22), which may have decreased reactivity with amines. However, we
found that exposure of LDL to HOCl (200 µM) in the
presence of increasing concentrations of nitrite (25-200 µM) only minimally decreased oxidation of lysine residues
and formation of N-chloramines. Others have shown that the
ratio of nitrite to HOCl needs to be greater than unity to afford
protection against HOCl-mediated cytotoxicity (57-59). The protective
effects of nitrite would also be highly dependent on other available
targets. HOCl reacts with nitrite with a second order rate constant of 7.4 × 103 M
1
s
1 (22); in contrast, the reaction of HOCl
with amines is almost 2 orders of magnitude faster
(k2 = 4.8 × 105
M
1 s
1),
and with thiols, approximately 4 orders of magnitude faster (k2 > 107
M
1 s
1)
(60). Thus, 20 mM nitrite would be required to completely protect LDL amines from oxidation by 200 µM HOCl.
Furthermore, since preformed LDL-associated N-chloramines
did not react with nitrite, as has been observed previously for taurine
chloramine (59, 61), this mechanism is unlikely to account for the
decreased levels of N-chloramines and lysine modification
observed in the present study.
An alternative inhibitory mechanism of nitrite could involve
competition between nitrite and chloride ions for reaction with compound I of MPO (see Scheme 1). In the absence of chloride ions, the
MPO/H2O2/NO2
system failed to significantly modify lysine residues or increase REM
of LDL, implying that the MPO-derived products of nitrite are unable to
cause significant oxidation of amines. The rate constant for the
reaction of nitrite with compound I (k2 = 2.0 × 106 M
1
s
1) (20) is 2 orders of magnitude greater
than that for chloride with compound I (k2 = 2.5 × 104 M
1
s
1) (62). However, chloride was present in
our experiments at a concentration approximately 4 orders of magnitude
higher than that of nitrite (140 mM and 12.5 µM, respectively). Furthermore, the activity of MPO
(compound I) is not saturated at plasma concentrations of chloride
(63). Therefore, it is unlikely that competition between these
physiological concentrations of nitrite and chloride for compound I are
the major mechanism.
The most likely mechanism by which low concentrations of nitrite
decrease MPO/H2O2/Cl
-mediated
modifications of LDL is inhibition of MPO enzyme activity. This could
occur if nitrite has a low reaction rate with compound II of MPO (20),
thus effectively trapping the enzyme in a form incapable of generating
HOCl (see Scheme 1). To determine whether nitrite is inhibiting MPO by
this mechanism, a cosubstrate with a relatively high reaction rate with
compound II can be used to cycle the enzyme. One such substrate is
ascorbate, which has been shown to increase the chlorinating activity
of MPO (53) by reducing compound II back to the native enzyme (54). The
second order rate constant for the reaction of ascorbate with compound
II of MPO is 1.1 × 104
M
1 s
1
(64), which is approximately 2 orders of magnitude higher than the
reaction of nitrite with compound II (k2 = 5.5 × 102 M
1
s
1) (20). As such, ascorbate should be able
to effectively cycle the enzyme back to its native ferric form. We
found that the addition of low concentrations of ascorbate (12.5-50
µM) significantly attenuated the inhibition of lysine
oxidation, N-chloramine formation, and change in REM by
equivalent concentrations of nitrite, whereas higher concentrations of
ascorbate dose-dependently inhibited MPO-mediated
modifications of apoB. Thus, although ascorbate protects against
modification of LDL by HOCl (34) and
MPO/H2O2/Cl
(this study), nitrite
appears to be a better "antioxidant" than ascorbate at low
µM concentrations due to inhibition of the chlorinating activity of MPO. However, at higher concentrations, ascorbate was a
better antioxidant than nitrite, as indicated by
dose-dependent protection against tryptophan and cysteine
oxidation and an increase in REM, most likely due to direct scavenging
of HOCl.
Since ascorbate not only cycles MPO by reducing compound II to the
native enzyme (54) but also scavenges HOCl in a stoichiometric manner
(43) and regenerates amines from N-chloramines (34), we
sought to compare the effects of ascorbate with an alternative substrate. Others have shown that MPO-mediated nitration of tyrosine residues can be enhanced by addition of free tyrosine (65). Since the
rate constant for the reaction of tyrosine with compound II of MPO is
even greater than that of ascorbate (i.e.
k2 = 1.6 × 106
M
1 s
1)
(66), tyrosine also effectively cycles the enzyme. We used HPA instead
of tyrosine to avoid interfering reactions of MPO-derived oxidants with
the amino group of tyrosine and found a significant decrease in the
nitrite-dependent inhibition of LDL lysine oxidation, N-chloramine formation, and change in REM. These results
agree with those observed in the presence of ascorbate (see above) and support our hypothesis that nitrite inhibits MPO-mediated modification of LDL by trapping the enzyme in its "inactive" compound II form.
Several groups have investigated the reaction of HOCl-, MPO-, and
leukocyte-derived-nitrating species with LDL (22, 36-39). The reactive
nitrogen species generated by
MPO/H2O2/NO2
and activated leukocytes is most likely the one-electron oxidation product nitrogen dioxide (19, 20), although generation of the
two-electron oxidant peroxynitrite by MPO has not been ruled out (24).
Peroxynitrite is likely formed by leukocytes at high fluxes of NO due
to rapid reaction of NO with leukocyte-derived superoxide (38, 61). The
above studies (36-39) primarily focused on
nitrite-dependent LDL lipid peroxidation in chloride-free
systems, although the addition of physiological concentrations of
chloride decreased lipid peroxidation by only a small extent (36, 37). Oxidative modification of LDL by reactive nitrogen species results in
enhanced macrophage cholesterol accumulation (22, 36, 39), likely due
to increased levels of lipid oxidation products (36, 39). MPO-derived
HOCl, in contrast, causes very little LDL lipid peroxidation (32, 33)
but still converts LDL into a high uptake form for macrophages (32,
35), likely due to direct modification of lysine residues.
A number of the above studies also measured formation of
3-nitrotyrosine (22, 36, 38, 39). Chloride does not appear to inhibit
nitrite-dependent 3-nitrotyrosine formation in LDL (36,
38), likely due to incomplete saturation of the activity of MPO
(compound I) at physiological concentrations of chloride (63). Although
a potentially useful biomarker of nitration reactions, 3-nitrotyrosine
is only a minor product, e.g. ~0.3 mmol/mol of tyrosine
formed in the presence of 50 to 500 µM nitrite (36, 38),
and the (patho)physiological significance of this type of modification
for atherosclerosis is unknown (39).
In this study we observed a protective effect of low concentrations of
nitrite against MPO-dependent modification of LDL lysine residues, most likely due to inhibition of the chlorinating activity of
MPO. The nitrite-dependent decrease in oxidative
modification of LDL may translate into decreased LDL uptake by
macrophages, thus attenuating the formation of lipid-laden foam cells,
the hallmark of atherosclerotic lesions. Since NO itself has been shown
to inhibit leukocyte-dependent modification of LDL by
reacting with lipid radicals (67), this study provides a novel
mechanism by which metabolites of NO may exert an antiatherogenic effect.
 |
ACKNOWLEDGEMENT |
We thank Dr. Anthony Kettle for critically
reviewing the manuscript.
 |
FOOTNOTES |
*
This work was supported by American Heart Association
Northwest Affiliate Grant 9920420Z (to A. C.) and National Institutes of Health Grant HL-56170 (to B. F.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Linus Pauling
Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR
97331-6512. Tel.: 541-737-5085; Fax: 541-737-5077; E-mail: anitra.carr@orst.edu.
Published, JBC Papers in Press, October 27, 2000, DOI 10.1074/jbc.M009082200
 |
ABBREVIATIONS |
The abbreviations used are:
NOS, nitric-oxide
synthase;
apoB, apolipoprotein B-100;
DTPA, diethylenetriaminepentaacetic acid;
HPA, 4-hydroxyphenylacetic acid;
LDL, low density lipoprotein;
MPO, myeloperoxidase;
PBS, phosphate-buffered saline (10 mM sodium phosphate, 140 mM NaCl, pH 7.4);
REM, relative electrophoretic mobility;
TNB, thionitrobenzoic acid: HPLC, high performance liquid
chromatography.
 |
REFERENCES |
1.
|
Moncada, S.,
and Higgs, A.
(1993)
N. Engl. J. Med.
329,
2002-2012
|
2.
|
Ignarro, L. J.
(1989)
FASEB J
3,
31-36[Abstract/Free Full Text]
|
3.
|
Beckman, J. S.,
and Koppenol, W. H.
(1996)
Am. J. Physiol.
271,
C1424-C1437[Abstract/Free Full Text]
|
4.
|
Koppenol, W. H.
(1998)
Free Radic. Biol. Med.
25,
385-391[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Wink, D. A.,
Darbyshire, R. W.,
Nims, J. E.,
Sasvedra, J. E.,
and Ford, P. C.
(1993)
Chem. Res. Toxicol.
6,
23-27[Medline]
[Order article via Infotrieve]
|
6.
|
Ignarro, L. J.,
Fukuto, J. M.,
Griscavage, J. M.,
Rogers, N. E.,
and Byrns, R. E.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8103-8107
|
7.
|
Kharitonov, V. G.,
Sundquist, A. R.,
and Sharma, V. S.
(1994)
J. Biol. Chem.
269,
5881-5883[Abstract/Free Full Text]
|
8.
|
Leone, A. M.,
Francis, P. L.,
Rhodes, P.,
and Moncada, S.
(1994)
Biochem. Biophys. Res. Commun.
200,
951-957[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Ueda, T.,
Maekawa, T.,
Sadamitsu, D.,
Oshita, S.,
Ogino, K.,
and Nakamura, K.
(1995)
Electrophoresis
16,
1002-1004[Medline]
[Order article via Infotrieve]
|
10.
|
Green, L. C.,
Wagner, D. A.,
Glogowski, J.,
Skipper, P. L.,
Wishnok, J. S.,
and Tannenbaum, S. R.
(1982)
Anal. Biochem.
126,
131-138[Medline]
[Order article via Infotrieve]
|
11.
|
Torre, D.,
Ferrario, G.,
Speranza, F.,
Orani, A.,
Fiori, G. P.,
and Zeroli, C.
(1996)
J. Clin. Pathol.
49,
574-576[Abstract]
|
12.
|
Weiss, S. J.
(1989)
N. Engl. J. Med.
320,
365-376
|
13.
|
Beckman, J. S.
(1996)
Chem. Res. Toxicol.
9,
836-844[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Beckman, J. S.,
Ye, Y. Z.,
Anderson, P. G.,
Chen, J.,
Accavitti, M. A.,
Tarpey, M. M.,
and White, C. R.
(1994)
Biol. Chem. Hoppe-Seyler
375,
81-88[Medline]
[Order article via Infotrieve]
|
15.
|
Leeuwenburgh, C.,
Hardy, M. M.,
Hazen, S. L.,
Wagner, P.,
Oh-ishi, S.,
Steinbrecher, U. P.,
and Heinecke, J. W.
(1997)
J. Biol. Chem.
272,
1433-1436[Abstract/Free Full Text]
|
16.
|
Buttery, L. D.,
Springall, D. R.,
Chester, A. H.,
Evans, T. J.,
Standfield, E. N.,
Parums, D. V.,
Yacoub, M. H.,
and Polak, J. M.
(1996)
Lab. Invest.
75,
77-85[Medline]
[Order article via Infotrieve]
|
17.
|
Luoma, J. S.,
Stralin, P.,
Marklund, S. L.,
Hiltunen, T. P.,
Sarkioja, T.,
and Yla-Herttuala, S.
(1998)
Arterioscler. Thromb. Vasc. Biol.
18,
157-167[Abstract/Free Full Text]
|
18.
|
Kettle, A. J.,
and Winterbourn, C. C.
(1997)
Redox Rep.
3,
3-15
|
19.
|
Reszka, K. J.,
Matuszak, Z.,
Chignell, C. F.,
and Dillon, J.
(1999)
Free. Radic. Biol. Med.
26,
669-678[CrossRef][Medline]
[Order article via Infotrieve]
|
20.
|
Burner, U.,
Furtmuller, P. G.,
Kettle, A. J.,
Koppenol, W. H.,
and Obinger, C.
(2000)
J. Biol. Chem.
275,
20597-20601[Abstract/Free Full Text]
|
21.
|
Eiserich, J. P.,
Cross, C. E.,
Jones, A. D.,
Halliwell, B.,.,
and van der Vliet, A.
(1996)
J. Biol. Chem.
271,
19199-19208[Abstract/Free Full Text]
|
22.
|
Panasenko, O. M.,
Briviba, K.,
Klotz, L.,
and Sies, H.
(1997)
Arch. Biochem. Biophys.
343,
254-259[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
van der Vliet, A.,
Eiserich, J. P.,
Halliwell, B.,
and Cross, C. E.
(1997)
J. Biol. Chem.
272,
7617-7625[Abstract/Free Full Text]
|
24.
|
Sampson, J. B.,
Ye, Y.,
Rosen, H.,
and Beckman, J. S.
(1998)
Arch. Biochem. Biophys.
356,
207-213[CrossRef][Medline]
[Order article via Infotrieve]
|
25.
|
Halliwell, B.
(1997)
FEBS Lett.
411,
157-160[CrossRef][Medline]
[Order article via Infotrieve]
|
26.
|
Kettle, A. J.,
Van Dalen, C. J.,
and Winterbourn, C. C.
(1997)
Redox Rep.
3,
257-258[Medline]
[Order article via Infotrieve]
|
27.
|
Daugherty, A.,
Dunn, J. L.,
Rateri, D. L.,
and Heinecke, J. W.
(1994)
J. Clin. Invest.
94,
437-444
|
28.
|
Hazell, L. J.,
Arnold, L.,
Flowers, D.,
Waeg, G.,
Malle, E.,
and Stocker, R.
(1996)
J. Clin. Invest.
97,
1535-1544[Abstract/Free Full Text]
|
29.
|
Malle, E.,
Waeg, G.,
Schreiber, R.,
Grone, E. F.,
Sattler, W.,
and Grone, H. J.
(2000)
Eur. J. Biochem.
267,
4495-4503[Abstract/Free Full Text]
|
30.
|
Leeuwenburgh, C.,
Rasmussen, J. E.,
Hsu, F. F.,
Mueller, D. M.,
Pennathur, S.,
and Heinecke, J. W.
(1997)
J. Biol. Chem.
272,
3520-3526[Abstract/Free Full Text]
|
31.
|
Hazen, S. L.,
and Heinecke, J. W.
(1997)
J. Clin. Invest.
99,
2075-2081[Abstract/Free Full Text]
|
32.
|
Hazell, L. J.,
and Stocker, R.
(1993)
Biochem. J.
290,
165-172[Medline]
[Order article via Infotrieve]
|
33.
|
Hazell, L. J.,
van den Berg, J. J.,
and Stocker, R.
(1994)
Biochem. J.
302,
297-304[Medline]
[Order article via Infotrieve]
|
34.
|
Carr, A. C.,
Tijerina, T.,
and Frei, B.
(2000)
Biochem. J.
346,
491-499[CrossRef][Medline]
[Order article via Infotrieve]
|
35.
|
Ryu, B. H.,
Mao, F. W.,
Lou, P.,
Gutman, R. L.,
and Greenspan, P.
(1995)
Biosci. Biotechnol. Biochem.
59,
1619-1622[Medline]
[Order article via Infotrieve]
|
36.
|
Podrez, E. A.,
Schmitt, D.,
Hoff, H. F.,
and Hazen, S. L.
(1999)
J. Clin. Invest.
103,
1547-1560[Abstract/Free Full Text]
|
37.
|
Byun, J.,
Mueller, D. M.,
Fabjan, J. S.,
and Heinecke, J. W.
(1999)
FEBS Lett.
455,
243-246[CrossRef][Medline]
[Order article via Infotrieve]
|
38.
|
Hazen, S. L.,
Zhang, R.,
Shen, Z.,
Wu, W.,
Podrez, E. A.,
MacPherson, J. C.,
Schmitt, D.,
Mitra, S. N.,
Mukhopadhyay, C.,
Chen, Y.,
Cohen, P. A.,
Hoff, H. F.,
and Abu-Soud, H. M.
(1999)
Circ. Res.
85,
950-958[Abstract/Free Full Text]
|
39.
|
Podrez, E. A.,
Febbraio, M.,
Sheibani, N.,
Schmitt, D.,
Silverstein, R. L.,
Hajjar, D. P.,
Cohen, P. A.,
Frazier, W. A.,
Hoff, H. F.,
and Hazen, S. L.
(2000)
J. Clin. Invest.
105,
1095-1108[Abstract/Free Full Text]
|
40.
|
Kettle, A. J.,
and Winterbourn, C. C.
(1994)
Methods Enzymol.
233,
502-512[Medline]
[Order article via Infotrieve]
|
41.
|
Sattler, W.,
Mohr, D.,
and Stocker, R.
(1994)
Methods Enzymol.
233,
469-489[Medline]
[Order article via Infotrieve]
|
42.
|
Kettle, A. J.,
and Winterbourn, C. C.
(1988)
Biochem. J.
252,
529-536[Medline]
[Order article via Infotrieve]
|
43.
|
Chesney, J. A.,
Mahoney, J. R.,
and Eaton, J. W.
(1991)
Anal. Biochem.
196,
262-266[Medline]
[Order article via Infotrieve]
|
44.
|
Frei, B.,
England, L.,
and Ames, B. N.
(1989)
Proc. Natl. Acad. Sci. U. S. A.
86,
6377-6381
|
45.
|
Vodovotz, Y.
(1996)
Biotechniques
20,
390-394[Medline]
[Order article via Infotrieve]
|
46.
|
Morris, J. C.
(1966)
J. Phys. Chem.
70,
3798-3805
|
47.
|
Bohlen, P.,
Stein, S.,
Dairman, W.,
and Udenfriend, S.
(1973)
Arch. Biochem. Biophys.
155,
213-220[Medline]
[Order article via Infotrieve]
|
48.
|
Toyo'oka, T.,
and Imai, K.
(1984)
Anal. Chem.
56,
2461-2464
|
49.
|
Ye, Y. Z.,
Strong, M.,
Huang, Z. Q.,
and Beckman, J. S.
(1996)
Methods Enzymol.
269,
201-209[Medline]
[Order article via Infotrieve]
|
50.
|
Sommer, A.,
Gorges, R.,
Kostner, G. M.,
Paltauf, F.,
and Hermetter, A.
(1991)
Biochemistry
30,
11245-11249[Medline]
[Order article via Infotrieve]
|
51.
|
Yang, C. Y.,
Kim, T. W.,
Weng, S. A.,
Lee, B. R.,
Yang, M. L.,
and Gotto, A. M. J.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
5523-5527[Abstract]
|
52.
|
Esterbauer, H.,
Gebicki, J.,
Puhl, H.,
and Jurgens, G.
(1992)
Free Radic. Biol. Med.
13,
341-390[CrossRef][Medline]
[Order article via Infotrieve]
|
53.
|
Bolscher, B. G. J. M.,
Zoutberg, G. R.,
Cuperus, R. A.,
and Wever, R.
(1984)
Biochim. Biophys. Acta
784,
189-191[Medline]
[Order article via Infotrieve]
|
54.
|
Marquez, L. A.,
Dunford, H. B.,
and Van Wart, H.
(1990)
J. Biol. Chem.
265,
5666-5670[Abstract/Free Full Text]
|
55.
|
Haberland, M. E.,
Fogelman, A. M.,
and Edwards, P. A.
(1982)
Proc. Natl. Acad. Sci. U. S. A.
79,
1712-1716
|
56.
|
Haberland, M. E.,
Olch, C. L.,
and Fogelman, A. M.
(1984)
J. Biol. Chem.
259,
11305-11311[Abstract/Free Full Text]
|
57.
|
Kono, Y.
(1995)
Biochem. Mol. Biol. Int.
36,
275-283[Medline]
[Order article via Infotrieve]
|
58.
|
Klebanoff, S. J.
(1993)
Free Radic. Biol. Med.
14,
351-360[CrossRef][Medline]
[Order article via Infotrieve]
|
59.
|
Marcinkiewicz, J.,
Chain, B.,
Nowak, B.,
Grabowska, A.,
Bryniarski, K.,
and Baran, J.
(2000)
Inflamm. Res.
49,
280-289[CrossRef][Medline]
[Order article via Infotrieve]
|
60.
|
Folkes, L. K.,
Candeias, L. P.,
and Wardman, P.
(1995)
Arch. Biochem. Biophys.
323,
120-126[CrossRef][Medline]
[Order article via Infotrieve]
|
61.
|
Eiserich, J. P.,
Hristova, M.,
Cross, C. E.,
Jones, A. D.,
Freeman, B. A.,
Halliwell, B.,
and van der Vliet, A.
(1998)
Nature
391,
393-397[CrossRef][Medline]
[Order article via Infotrieve]
|
62.
|
Furtmuller, P. G.,
Burner, U.,
and Obinger, C.
(1998)
Biochemistry
37,
17923-17930[CrossRef][Medline]
[Order article via Infotrieve]
|
63.
|
Van Dalen, C. J.,
Whitehouse, M.,
Winterbourn, C. C.,
and Kettle, A. J.
(1997)
Biochem. J.
327,
487-492[Medline]
[Order article via Infotrieve]
|
64.
|
Hsuanyu, Y.,
and Dunford, H. B.
(1999)
Arch. Biochem. Biophys.
368,
413-420[CrossRef][Medline]
[Order article via Infotrieve]
|
65.
|
Van Dalen, C. J.,
Winterbourn, C. C.,
Senthilmohan, R.,
and Kettle, A. J.
(2000)
J. Biol. Chem.
275,
11638-11644[Abstract/Free Full Text]
|
66.
|
Marquez, L. A.,
and Dunford, H. B.
(1995)
J. Biol. Chem.
270,
30434-30440[Abstract/Free Full Text]
|
67.
|
Hogg, N.,
and Kalyanaraman, B.
(1998)
Free Radic. Res.
28,
593-600[Medline]
[Order article via Infotrieve]
|
68.
|
Kuhn, H.,
and Chan, L.
(1997)
Curr. Opin. Lipidol.
8,
111-117[Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.