(Received for publication, September 13, 1996, and in revised form, November 12, 1996)
From the Departments of Medicine and of ** Molecular
Biology and Pharmacology, Washington University School of Medicine, St.
Louis, Missouri 63110, § Searle Research, Monsanto Company,
St. Louis, Missouri 63167, and the
Department of Medicine,
University of British Columbia, Vancouver, BC Canada V5Z 4E3
Oxidized low density lipoprotein (LDL) may be of
central importance in triggering atherosclerosis. One potential pathway
involves the production of nitric oxide (NO) by vascular wall
endothelial cells and macrophages. NO reacts with superoxide to form
peroxynitrite (ONOO), a potent agent of LDL oxidation
in vitro. ONOO
nitrates the aromatic ring of
free tyrosine to produce 3-nitrotyrosine, a stable product. To explore
the role of reactive nitrogen species such as ONOO
in the
pathogenesis of vascular disease, we developed a highly sensitive and
specific method involving gas chromatography and mass spectrometry to
quantify 3-nitrotyrosine levels in proteins. In vitro
studies demonstrated that 3-nitrotyrosine was a highly specific marker
for LDL oxidized by ONOO
. LDL isolated from the plasma of
healthy subjects had very low levels of 3-nitrotyrosine (9 ± 7 µmol/mol of tyrosine). In striking contrast, LDL isolated from aortic
atherosclerotic intima had 90-fold higher levels (840 ± 140 µmol/mol of tyrosine). These observations strongly support the
hypothesis that reactive nitrogen species such as ONOO
form in the human artery wall and provide direct evidence for a
specific reaction pathway that promotes LDL oxidation in
vivo. The detection of 3-nitrotyrosine in LDL isolated from
vascular lesions raises the possibility that NO, by virtue of its
ability to form reactive nitrogen intermediates, may promote
atherogenesis, counteracting the well-established anti-atherogenic
effects of NO.
An elevated level of low density lipoprotein (LDL)1 is a major risk factor for premature atherosclerotic vascular disease. However, a wealth of evidence suggests that LDL must be oxidatively modified to damage the artery wall (1, 2). Pathways that oxidize lipid and protein may thus be pivotal to the development of atherosclerosis. LDL oxidation has been widely studied in vitro, but the mechanisms that promote oxidation within the artery wall remain poorly understood (2).
We have described one potential pathway for LDL oxidation that involves
oxidants generated by myeloperoxidase, an enzyme secreted by phagocytes
(3). Another pathway involves nitrogen monoxide (nitric oxide; NO)
generated by vascular wall cells (4). NO is a relatively stable free
radical that fails to oxidize LDL at physiological pH (5). However, NO
reacts rapidly with superoxide to form peroxynitrite
(ONOO; Ref. 6), a reactive nitrogen species that promotes
peroxidation of the lipid moiety of LDL in vitro (7).
Cultured endothelial cells, macrophages and smooth muscle cells, all
components of the atherosclerotic lesion, generate superoxide anion
(2), suggesting that ONOO
or other reactive nitrogen
intermediates derived from NO could form in the artery wall.
In vitro studies demonstrate that ONOO
spontaneously reacts with tyrosine residues to yield the stable product
3-nitrotyrosine (Scheme 1; Ref. 8). Macrophages and
endothelial cells may play a role because antibodies for
3-nitrotyrosine detect epitopes in human atherosclerotic lesions that
are associated predominantly with these two cell types (9). However,
these studies did not quantify levels of 3-nitrotyrosine or evaluate
the extent of LDL nitration.
To explore the role of reactive nitrogen species in oxidative damage
in vivo, we developed a quantitative assay for measuring levels of 3-nitrotyrosine. The method combines gas chromatography with
stable isotope dilution mass spectrometry (GC-MS). Using this assay, we
first investigated the relative yields of protein-bound oxidation
products in bovine serum albumin (BSA) and LDL that were oxidized with
ONOO in vitro. In both cases, the major
product was 3-nitrotyrosine. Other widely studied oxidation systems
failed to generate significant levels of 3-nitrotyrosine in LDL. These
observations indicate that 3-nitrotyrosine is a specific marker for
oxidative damage by reactive nitrogen intermediates. We then
demonstrated that 3-nitrotyrosine levels are highly elevated in LDL
isolated from human atherosclerotic tissue. These observations
implicate reactive nitrogen species as one pathway for LDL oxidation in
the artery wall.
Cambridge Isotope Laboratories (Andover, MA)
supplied 13C-labeled amino acids.
3-Nitro[13C6]tyrosine was synthesized using
tetranitromethane (10), and its concentration was determined by reverse
phase HPLC analysis by comparison with authentic material (11). All
organic solvents were HPLC grade. Autopsy material was supplied by the
Division of Surgical Pathology, Washington University School of
Medicine. Vascular tissue resected within 10 h of death was
immediately placed in ice-cold antioxidant buffer (100 µM
diethylenetriamine pentaacetic acid (DTPA), 1 mM butylated
hydroxytoluene, 1% (v/v) ethanol, 10 mM 3-aminotriazole,
50 mM NaHPO4, pH 7.4), and then frozen at
80 °C until analysis.
Reactions were carried out at
37 °C in Buffer A (50 mM phosphate, 0.1 mM
DTPA, pH 7.4) supplemented with 1 mg protein/ml LDL or BSA (fatty
acid-free; Boehringer Mannheim). ONOO was synthesized
from 2-ethoxyethyl nitrite and H2O2 (12) and stored at
80 °C. ONOO
was thawed just prior to the
experiment, and its concentration was determined spectrophotometrically
(
302 = 1670 M
1
cm
1; Refs. 6 and 8). Reactions were initiated by the
addition of ONOO
. Samples were incubated for 5 min at
37 °C.
LDL (d = 1.02-1.07 g/ml) was isolated by sequential density ultracentrifugation from plasma (1 mg/ml EDTA) prepared from normolipidemic, healthy volunteers (13). LDL was extensively dialyzed against Buffer A (50 mM phosphate, 0.1 mM DTPA, pH 7.4) at 4 °C prior to experiments.
Lesion LDL was isolated from thoracic aortae that were thawed in Buffer C (0.15 M NaCl, 10 mM sodium phosphate (pH 7.4), 0.3 mM EDTA, 100 µM DTPA, 50 µg/ml soybean trypsin inhibitor, 100 µM butylated hydroxytoluene, and 10 mM 3-aminotriazole). Fatty streaks and intermediate lesions from a single individual were resected from aortic tissue (~9 g wet weight per aorta), frozen in liquid N2, and pulverized under liquid N2 with a stainless steel mortar and pestle. All subsequent procedures were carried out at 4 °C. Tissue powder was collected into 50-ml sterile conical tubes, Buffer C was added (5 ml/g tissue), and the tubes were rocked gently overnight. Tissue was removed by centrifugation at 5000 × g for 15 min, the supernatant was subjected to centrifugation at 100,000 × g for 30 min, and the pellet and uppermost lipemic layer were discarded. LDL in the remaining supernatant was isolated by sequential density ultracentrifugation (d = 1.02-1.07 g/ml; Ref. 13). A metal chelator (100 µM DTPA) and NO synthase inhibitor (10 mM 3-aminotriazole; Ref. 14) were included in all solutions used for lipoprotein isolation. Lesion LDL was extensively dialyzed at 4 °C under N2 against Buffer A (50 mM phosphate, 0.1 mM DTPA, pH 7.4) and then against 0.1 mM DTPA (pH 7.4) prior to analysis.
Mass Spectrometric AnalysisBSA and LDL were precipitated at 4 °C with ice-cold trichloroacetic acid (10% v/v). LDL lipids were extracted with methanol/water-washed diethyl ether and water-washed diethyl ether.2 Protein residue (~0.5 mg) was dried under N2, 13C-labeled internal standards were added, and the sample was then hydrolyzed at 110 °C for 24 h in 0.5 ml of 6 N HCl (Sequenal grade, Pierce) supplemented with 1% benzoic acid and 1% phenol. Amino acids were isolated from the acid hydrolysate using a C18 column.2 Authentic 3-nitrotyrosine was stable to acid hydrolysis under these conditions, and recovery of the amino acid from the C18 column was >80%. The N-propylheptafluorobutyryl derivatives of the amino acids were prepared,2 dried under N2, and redissolved in 50 µl of ethyl acetate, and 1-µl aliquots were analyzed on a Hewlett Packard 5890 Gas Chromatograph equipped with a 12-m DB-1 capillary column (0.20 mm inside diameter, 0.33-µ film thickness, J & W Scientific) interfaced with a Hewlett Packard 5988A Mass Spectrometer equipped with extended mass range. Both the injection and detector temperature of the gas chromatograph were set at 250 °C. Full scan mass spectra and selected ion monitoring were obtained in the negative ion chemical ionization mode with methane as the reagent gas. The limit of sensitivity for all of the amino acids was <1 nmol (signal to noise > 10).
The mass spectrum of the N-propylheptafluorobutyryl
derivative of 3-nitrotyrosine included prominent ions at
m/z 464 (M) and
m/z 444 (M
-HF). The base ion
(m/z 464) and its 13C-labeled
internal standard ion (m/z 470) were used for
quantification. A 1:10 split injection was used for analysis of
3-nitrotyrosine; the initial column temperature of 70 °C was
increased to 180 °C at 60 °C/min and then raised to 205 °C at
4 °C/min. p-Tyrosine o,o
-dityrosine, phenylalanine, and
o-tyrosine were subjected to GC-MS analysis as
described.2
All buffers were passed over a Chelex 100 column (Bio-Rad) to remove transition metal ions. Myeloperoxidase was isolated as described (11). Protein was measured using the Markwell-modified Lowry procedure (16) with BSA as the standard. Results are presented as means ± S.E.
After exposing BSA to ONOO, we used stable
isotope dilution GC-MS analysis to quantify the level of protein-bound
3-nitrotyrosine. Native BSA contained very low levels of
3-nitrotyrosine, but there was a dramatic,
concentration-dependent increase after ONOO
exposure (Fig. 1A). When BSA was added to the
reaction mixture 2 min after ONOO
, however, its
3-nitrotyrosine content barely increased. These results indicate that
ONOO
or short-lived intermediates derived from
ONOO
mediated nitration of protein tyrosyl residues.
ONOO also behaves like a hydroxyl radical in that it
oxidizes the amino acids phenylalanine and tyrosine to the unnatural compounds o-tyrosine and
o,o
-dityrosine (6, 17, 18). To determine whether
these products were formed in proteins oxidized by ONOO
,
we quantified o-tyrosine and
o,o
-dityrosine levels in BSA incubated with 0.3 mM ONOO
. The levels of o-tyrosine
and o,o
-dityrosine increased in oxidized BSA;
however, the yield of the modified amino acids was <5% of the level
of 3-nitrotyrosine. Nitration and hydroxylation rates of free amino
acids are strongly influenced by H+ (8, 17, 18). However,
the pH of the reaction mixture measured immediately after addition of
ONOO
was not significantly altered. These results
indicate that 3-nitrotyrosine is the major product when
ONOO
oxidizes BSA tyrosyl residues at physiological
pH.
To explore the possibility that a lipid environment affects the
susceptibility of tyrosine to nitration, we isolated LDL from plasma
and exposed it to the concentrations of ONOO described
above. Native LDL contained very low levels of 3-nitrotyrosine, but
3-nitrotyrosine increased dramatically when LDL was exposed to
ONOO
(Fig. 1B). The product yield of
o-tyrosine and o,o
-dityrosine in LDL
oxidized by ONOO
was <1% of the yield of
3-nitrotyrosine. These results confirm that 3-nitrotyrosine is a major
product of protein oxidation by ONOO
. They also suggest
that nitration of protein tyrosine residues is favored over
hydroxylation and hydrogen atom abstraction by this reactive nitrogen
intermediate.
The 3-nitrotyrosine content of LDL depended on ONOO
concentration (Fig. 1B), and adding LDL 2 min after
ONOO
drastically reduced the extent of protein nitration.
At equal concentrations of ONOO
, the yield of
3-nitrotyrosine (expressed as millimoles of nitrotyrosine per mol of
tyrosine) in LDL was only half that in BSA, perhaps because its lipids
(or lipid-soluble antioxidants) compete with the apolipoprotein for
ONOO
(19) or because its tyrosine residues are less
accessible to the short-lived nitrating intermediate.
To evaluate the specificity of 3-nitrotyrosine as a marker for protein damage by reactive nitrogen species, we examined a variety of in vitro oxidation systems for their ability to generate 3-nitrotyrosine in apolipoprotein B100, the major protein of LDL.
Significant levels of the nitrated amino acid were present in LDL
exposed to ONOO (Fig. 2). In contrast,
there was little change in the 3-nitrotyrosine content of LDL oxidized
by copper, iron, a hydroxyl radical generating system
(H2O2 plus copper), myeloperoxidase,
lactoperoxidase, horseradish peroxidase, glucose, or lipoxygenase (Fig.
2). All of the systems oxidized LDL as monitored by the appearance of
lipid oxidation products (thiobarbituric reacting substances assay and
lipid hydroperoxides; Refs. 13 and 20). Collectively, these results
demonstrate that 3-nitrotyrosine is a highly specific marker for LDL
oxidation by reactive nitrogen species.
3-Nitrotyrosine Is Present at Greatly Elevated Levels in LDL Isolated from Human Vascular Lesions
To assess the possible role
of reactive nitrogen intermediates such as ONOO in
oxidizing lipoproteins in vivo, we isolated LDL
(d = 1.02-1.07 g/ml) from human atherosclerotic tissue
recovered at autopsy and then determined its 3-nitrotyrosine content.
3-Aminotriazole, an inhibitor of NO synthase (14) and myeloperoxidase
(11), was included during tissue processing and lipoprotein isolation. Lesion LDL subjected to SDS-polyacrylamide gel electrophoresis and
immunoblotting analysis with a rabbit polyclonal antibody monospecific
for human apolipoprotein B100 (21) demonstrated a protein with the
predicted molecular mass of apolipoprotein B100. Forms of
immunoreactive apolipoprotein B100 with lower molecular mass were also
present, as previously reported by other investigators (1, 2).
LDL isolated from human atherosclerotic lesions was delipidated,
hydrolyzed, and derivatized, and the derivatized amino acids were
subjected to GC-MS analysis in the negative ion chemical ionization
mode. We detected a compound in the amino acid hydrolysate that
exhibited major ions and retention times identical to those of
authentic 3-nitrotyrosine. Selected ion monitoring showed that the ions
derived from the amino acid co-eluted with those derived from
3-nitro[13C6]tyrosine (Fig.
3). The identity of 3-nitrotyrosine was confirmed further by comparison with an authentic standard using both
heptafluorobutyryl and pentafluoropropionyl derivatives. These results
indicate that 3-nitrotyrosine is present in LDL isolated from human
atherosclerotic lesions.
To determine whether protein nitration may contribute to the oxidation
of artery wall lipoproteins, we isolated LDL from plasma and from human
atherosclerotic aortic tissue. We then delipidated and hydrolyzed the
proteins and subjected the derivatized amino acid hydrolysates to
stable isotope dilution GC-MS analysis (Fig. 4). There
was a striking 90-fold increase in the level of protein-bound 3-nitrotyrosine in lesion LDL (840 ± 140 µmol/mol of tyrosine; n = 10) compared with circulating LDL (9 ± 7 µmol/mol of tyrosine; n = 6).
In this study, we examined the potential role of reactive nitrogen
species in LDL oxidation. In vitro studies demonstrated that
3-nitrotyrosine was a highly specific marker for LDL oxidized by
ONOO, a reactive nitrogen species. Analysis of LDL
isolated from human atherosclerotic lesions revealed a remarkable
90-fold elevation of the level of 3-nitrotyrosine compared with that in
circulating LDL. These observations strongly support the hypothesis
that reactive nitrogen intermediates derived from NO contribute to LDL
oxidation in the artery wall.
A key question is whether NO generation by cells of the artery wall
promotes or inhibits the development of atherosclerotic plaque.
Cultured endothelial cells, macrophages, and smooth muscle cells
generate superoxide (2), which may rapidly react with NO to form
ONOO (6). Moreover, ONOO
peroxidizes the
lipid moieties of LDL in vitro, converting the lipoprotein
to a form that is recognized by the macrophage scavenger receptor (7).
Unregulated uptake of such modified lipoprotein may play a role in
cholesterol accumulation by macrophages, a critical early step in
atherogenesis (1, 2). In keeping with this hypothesis, our detection of
elevated levels of 3-nitrotyrosine in LDL isolated from atherosclerotic
lesions suggests that reactive nitrogen intermediates derived from NO
may indeed promote atherosclerotic vascular disease.
In contrast, other studies have shown that NO inhibits LDL oxidation by cultured macrophages (2, 5) and that NO may act as a chain-breaking antioxidant during lipid peroxidation (19). NO also may exert other anti-atherogenic effects in vivo, including inhibition of platelet aggregation and suppression of smooth muscle cell proliferation (4). Studies in hypercholesterolemic rabbits suggest that NO inhibits fatty streak formation, the cellular hallmark of the early atherosclerotic lesion (22). These results suggest that NO is anti-atherogenic in this animal model.
These apparently conflicting findings could be explained if the
relatively stable free radical NO exerts potent anti-atherogenic effects while reactive intermediates derived from NO damage artery wall
targets. The availability of superoxide may be critical in controlling
this balance. Superoxide would both remove anti-atherogenic NO and
produce pro-atherogenic ONOO. Thus, an imbalance in the
pro- and anti-atherogenic effects of NO may be one important factor in
the pathogenesis of atherosclerotic vascular disease.