(Received for publication, October 10, 1996, and in revised form, October 28, 1996)
From the Department of Medicine and
§ Department of Molecular Biology and Pharmacology,
Washington University School of Medicine, St. Louis, Missouri 63110
Lipoprotein oxidation has been implicated in the
pathogenesis of atherosclerosis. However, the physiologically relevant
pathways mediating oxidative damage have not yet been identified. Three potential mechanisms are tyrosyl radical, hydroxyl radical, and redox
active metal ions. Tyrosyl radical forms
o,o-dityrosine cross-links in proteins. The
highly reactive hydroxyl radical oxidizes phenylalanine residues to
o-tyrosine and m-tyrosine. Metal ions oxidize
low density lipoprotein (LDL) by poorly understood pathways. To explore
the involvement of tyrosyl radical, hydroxyl radical, and metal ions in
atherosclerosis, we developed a highly sensitive and quantitative
method for measuring levels of o,o
-dityrosine, o-tyrosine, and m-tyrosine in proteins,
lipoproteins, and tissue, using stable isotope dilution gas
chromatography-mass spectrometry. We showed that
o,o
-dityrosine was selectively produced in LDL oxidized with tyrosyl radical. Both o-tyrosine and
o,o
-dityrosine were major products when LDL
was oxidized with hydroxyl radical. Only o-tyrosine was
formed in LDL oxidized with copper. Similar profiles of oxidation
products were observed in bovine serum albumin oxidized with the three
different systems. Applying these findings to LDL isolated from human
atherosclerotic lesions, we detected a 100-fold increase in
o,o
-dityrosine levels compared to those in
circulating LDL. In striking contrast, levels of o-tyrosine and m-tyrosine were not elevated in LDL isolated from
atherosclerotic tissue. Analysis of fatty streaks revealed a similar
pattern of oxidation products; compared with normal aortic tissue,
there was a selective increase in
o,o
-dityrosine with no change in o-tyrosine. The detection of a selective increase of
o,o
-dityrosine in LDL isolated from vascular
lesions is consistent with the hypothesis that oxidative damage in
human atherosclerosis is mediated in part by tyrosyl radical. In
contrast, these observations do not support a role for free metal ions
as catalysts of LDL oxidation in the artery wall.
An elevated plasma level of low density lipoprotein (LDL)1 is an important risk factor for the development of atherosclerotic vascular disease (1). Many lines of evidence suggest that LDL must be oxidatively modified before it can initiate atherosclerosis (2-7). LDL is oxidized in vitro by several different mechanisms, although the physiologically relevant pathways have not yet been identified (2-10). The most widely studied model involves free metal ions. LDL oxidation by cultured arterial cells requires micromolar concentrations of either iron or copper (11) and is inhibited by metal chelators (11-13). High concentrations of iron or copper catalyze LDL oxidation in the absence of cells (11, 13).
Metal-catalyzed oxidation systems represent another potential pathway for LDL oxidation. In these systems, redox active metals bound to proteins interact with hydrogen peroxide and a reducing agent to generate a hydroxyl radical-like intermediate (9). However, it is uncertain whether the metal ions required for these pathways are present in vivo because the body has intricate mechanisms for chelating metals and rendering them redox inactive (14).
We have described a mechanism for LDL oxidation that does not require free metal ions (15). The oxidation reaction involves tyrosyl radical generated by myeloperoxidase (16), a hemeprotein secreted by activated phagocytes (17, 18). Active myeloperoxidase has been detected in human atherosclerotic tissue (19), where it co-localizes with lipid-laden foam cells, the cellular hallmark of the early atherosclerotic lesions (2, 4, 7). Immunostaining of myeloperoxidase at different stages of lesion development (19) reveals patterns that are strikingly similar to those of protein-bound oxidation products in rabbit lesions (20). Moreover, oxidation products of the enzyme (21) have been detected by immunohistochemistry in vascular lesions (22). These observations raise the possibility that myeloperoxidase promotes LDL oxidation in vivo.
One strategy for determining whether free metal ions, metal-catalyzed
oxidation systems, or tyrosyl radical oxidize proteins in
vivo is to analyze normal and atherosclerotic vascular tissue for
stable end products of these three pathways identified through in
vitro studies. Hydroxyl radical generated by metal-catalyzed oxidation systems converts protein-bound phenylalanine residues to the
unnatural isomers o-tyrosine and m-tyrosine (Fig.
1; Refs. 14 and 23-25). It also cross-links tyrosine
residues into o,o-dityrosine (23, 24). It has
not been established whether LDL oxidation by free metal ions generates
a similar pattern of oxidation products. With protein oxidation by
tyrosyl radical, o,o
-dityrosine is a major
product (Fig. 1; Refs. 26 and 27). These amino acid products are stable
to acid hydrolysis, making them potentially useful markers for protein
oxidation in vivo (23, 26, 27).
We have developed a quantitative assay for measuring tissue levels of
o-tyrosine, m-tyrosine, and
o,o-dityrosine. It combines gas chromatography
with stable isotope dilution mass spectrometry (GC-MS). Using this
assay, we first investigated the relative yields of the three markers
in LDL oxidized in vitro with free copper, hydroxyl radical,
and tyrosyl radical. o-Tyrosine was a major oxidation
product when either free copper or a hydroxyl radical system was used
to oxidize LDL. In contrast, o,o
-dityrosine was
selectively produced when tyrosyl radical was the oxidizing agent. A
similar pattern of products was observed in bovine serum albumin (BSA)
oxidized with the different systems. We then used the method to
demonstrate that levels of o,o
-dityrosine, but not o-tyrosine or m-tyrosine, were elevated in
LDL isolated from human atherosclerotic tissue. Collectively, these
observations suggest that tyrosyl radical plays a role in oxidizing LDL
in the artery wall.
Unless otherwise indicated, reagents were obtained from either Sigma or Aldrich Chemical Co. All organic solvents were high performance liquid chromatography grade. Cambridge Isotope Laboratories supplied 13C-labeled amino acids. All buffers were passed over a Chelex-100 (Bio-Rad) column to remove transition metal ions.
Preparation and Isolation of Internal StandardsIsotopically labeled o-tyrosine and
m-tyrosine were synthesized using
[13C6]phenylalanine, copper, and
H2O2 (23).
o,o-[13C12]Dityrosine
was prepared with [13C6]tyrosine, horseradish
peroxidase, and H2O2 (28). Concentrations of
amino acids were determined by comparison with authentic standards using high performance liquid chromatography and monitoring of A276 (29). Compounds were analyzed at a flow
rate of 1 ml/min using an Ultrasphere ODS column (250 × 4.6 mm,
5-µm diameter particles; Beckman) equilibrated with solvent A (5%
methanol, 0.1% trifluoroacetic acid, pH 2.5) and eluted with solvent B
(90% methanol, 0.1% trifluoroacetic acid, pH 2.5). The elution
gradient was: 0-35% solvent B over 10 min; isocratic elution at 35%
solvent B for 4 min; 35-100% solvent B over 12 min; isocratic elution
at 100% solvent B for 5 min.
LDL was isolated rapidly using a two-step density gradient from plasma (EDTA, 1 mg/ml) prepared from normolipidemic, healthy subjects (30). LDL was collected by needle aspiration and subjected to size exclusion chromatography on a Bio-Rad 10 DG column equilibrated with Buffer A (50 mM sodium phosphate, pH 7.4).
Oxidation of BSA and LDLAll reactions were carried out at 37 °C in reaction mixture containing 1 mg of protein/ml of either BSA (fatty acid-free; Boehringer Mannheim) or LDL. Hydroxyl radical was generated by addition of 2 mM H2O2 to Buffer A supplemented with 0.1 mM CuSO4 (23). Tyrosyl radical was generated by addition of 0.1 mM H2O2 to Buffer A supplemented with 0.1 mM diethylenetriamine pentaacetic acid (DTPA, pH 7.4), 0.2 mM L-tyrosine, and 20 nM myeloperoxidase (15, 16, 26). Reactions were terminated by the addition of 300 nM catalase, 100 µM butylated hydroxytoluene, and 200 µM DTPA, and proteins were immediately precipitated at 4 °C with ice-cold trichloroacetic acid (10%, v/v). The protein pellet was washed with 0.5 ml of 10% trichloroacetic acid and delipidated with 4 ml of methanol/water-washed diethyl ether (1:3, v/v). The protein residue was hydrolyzed as described below.
Collection of Human Arterial TissueVascular tissue
resected at surgery or autopsy was immediately placed in ice-cold
antioxidant buffer (100 µM DTPA, 1 mM
butylated hydroxytoluene, 1% (v/v) ethanol, 140 mM NaCl,
10 mM sodium phosphate, pH 7.4), and then frozen at
80 °C until analysis.
LDL was isolated from vascular lesions using a modified method of Steinbrecher and Lougheed (31). Isolated thoracic aortae were thawed in Buffer B (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 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 B was added (5 ml/g tissue), and the tubes were rocked gently end-over-end overnight. Tissue powder was pelleted 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 supernatant was isolated by sequential density ultracentrifugation (d = 1.02-1.07 g/ml; Ref. 32). A metal chelator (100 µM DTPA) and myeloperoxidase inhibitor (10 mM 3-aminotriazole; Refs. 15, 16, and 26) were included in all solutions used for lipoprotein isolation. LDL was extensively dialyzed against Buffer C (50 mM phosphate, 0.1 mM DTPA, pH 7.4) and then against Buffer D (0.1 mM DTPA, pH 7.4) under N2 prior to analysis.
Preparation of TissueVascular tissue was thawed at room temperature. Regions of normal and atherosclerotic aortic tissue were defined morphologically using the criteria of the Pathobiological Determinants of Atherosclerosis in Youth Study (33). Resected arterial tissue was frozen in liquid N2 and pulverized using a stainless steel mortar and pestle. Tissue powder (~60 mg wet weight) was suspended in 1 ml of Buffer D, dialyzed (2 h) versus Buffer D at 4 °C, and then delipidated by incubation (10 min) with 13 ml of methanol/water-washed diethyl ether (3:10, v/v) on ice. The sample was centrifuged at 5000 × g for 10 min, the supernatant was removed, and the fluffy protein powder was again delipidated with 10 ml of water-washed diethyl ether.
Protein Hydrolysis and Isolation of Amino AcidsThe protein or tissue residue 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 Chemical) supplemented with 1% benzoic acid and 1% phenol. The samples were supplemented with 10% trichloroacetic acid (v/v) and passed over a reverse phase column (3 ml, LC-18 SPE Tube, Supelco Inc. Bellefonte, PA), which was previously washed with 12 ml of 50 mM NaHPO4 and 100 µM DTPA (pH 7.4) followed by 12 ml of 0.1% trifluoroacetic acid. Amino acids were eluted with 25% methanol (2 ml) and dried under vacuum for derivatization. Preliminary studies using authentic standards demonstrated that the amino acids were stable to acid hydrolysis and that >80% of tyrosine oxidation products were recovered from the C-18 column using this procedure.
Derivatization of Amino AcidsAmino acids were converted to carboxylic acid esters by the addition of 200 µl of HCl/n-propyl alcohol (1:3, v/v) and heating for 1 h at 65 °C. Excess reagent was evaporated under N2. To prepare the heptafluorobutyryl derivatives of the amino acids, 50 µl of heptafluorobutyric anhydride/ethyl acetate (1:3, v/v) was added, and the samples were heated at 65 °C for 10 min. To prepare the pentafluoropropionyl derivatives of the amino acids, 50 µl of pentafluoropropionic anhydride/ethyl acetate (1:4, v/v) was added, and the samples were heated at 65 °C for 30 min.
Mass Spectrometric AnalysisAmino acids were quantified by stable isotope dilution GC-MS. Derivatized samples were dried under N2 and redissolved in 50 µl of ethyl acetate, and 1 µl aliquots were then 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 with extended mass range (26, 34). The injector and ion source temperature were set at 250 °C and 150 °C, respectively. Full scan mass spectra and selected ion monitoring were obtained with both the n-propyl heptafluorobutyryl and the n-propyl pentafluoropropionyl derivatives of both authentic and isotopically labeled amino acids in the negative-ion chemical ionization mode with methane as the reagent gas.
For phenylalanine and p-tyrosine, an aliquot of derivatized
amino acid was diluted 1:100 (v/v) with ethyl acetate. A 1-µl sample
was injected into the gas chromatograph with a 1:100 split prior to
mass analysis. The initial column temperature of 120 °C was
maintained for 1 min and then increased to 220 °C at 10 °C/min. The mass spectrum of the n-propyl heptafluorobutyryl
derivative of phenylalanine included a small molecular ion
(M) at mass-to-charge (m/z) 403 and
a prominent ion at m/z 383 (M
HF). Phenylalanine was quantified using the m/z
383 ion. The mass spectrum of the n-propyl
heptafluorobutyryl derivative of p-tyrosine revealed
prominent ions at m/z 595 (M
HF)
and 417 (M
CF3
(CF2)2CHO). The m/z 417 ion was used to quantify tyrosine.
Tyrosine and phenylalanine oxidation products were analyzed without
dilution and injected in the splitless mode. The initial column
temperature was 150 °C: it was increased to 300 °C at
40 °C/min. The mass spectrum of the n-propyl
heptafluorobutyryl derivative of o,o-dityrosine
included a small molecular ion at m/z 1228 (M
) and prominent ions at m/z 1208 (M
HF) and 1030 (M
CF3
(CF2)2CHO). The m/z 1208 ion was used to quantify o,o
-dityrosine levels.
The mass spectrum of the n-propyl heptafluorobutyryl
derivative of o-tyrosine exhibited prominent ions at
m/z 595 (M
HF) and 417 (M
CF3 (CF2)2CHO).
The m/z 595 ion was used for quantification. The
mass spectrum of the n-propyl heptafluorobutyryl derivative of m-tyrosine exhibited prominent ions at
m/z 595 (M
HF) and 417 (M
CF3 (CF2)2CHO).
The m/z 417 ion was used for quantification.
The generation of oxidized amino acids by the analytical procedure was
investigated in preliminary experiments. The increase in the level of
o-tyrosine in phenylalanine subjected to acid hydrolysis was
less than 10 µmol of oxidation product/mol of precursor amino acid,
which is less than 5% of the value found in tissue. Under these
conditions, there was no detectable conversion of tyrosine to
o,o-dityrosine. The level of oxidation products
in model proteins (BSA, RNase, IgG) subjected to acid hydrolysis, derivatization, and GC-MS analysis was variable. For
o-tyrosine it ranged from 100 to 1,100 µmol/mol, and for
o,o
-dityrosine it ranged from undetectable (less
than 5 µmol/mol) to 60 µmol/mol. The level of oxidation products in
different model proteins remained consistent during repeated analyses,
strongly suggesting that the oxidized amino acids were present
endogenously. These observations indicate that the analytical procedure
itself was not a source of protein oxidation.
To determine whether post-mortem changes were likely to be contributing
to protein oxidation, human aortic tissue obtained at either surgery or
autopsy was subjected to acid hydrolysis, derivatization, and
negative-ion chemical ionization GC-MS analysis. There were no
significant differences in the levels of o-tyrosine and
o,o-dityrosine in the surgical and autopsy
tissue, suggesting that post-mortem changes are unlikely to result in
significant alterations in the levels of o-tyrosine,
m-tyrosine, and o,o
-dityrosine in
human vascular tissue.
The base peak ion of each amino acid was used for quantification. To ensure that interfering ions were not co-eluting with the analyte, the ratio of ion currents of the two most abundant ions of each amino acid were monitored in all analyses. Under these chromatographic conditions, authentic compounds and isotopically labeled standards were baseline separated and exhibited retention times identical to those of analytes derived from tissue samples.
Quantification was based on an external calibration curve using each
amino acid as a standard and the corresponding isotopically labeled
amino acid as internal standard. The ratio of ion currents for each
amino acid divided by that of the internal standard was a linear
function of unlabeled amino acid for all ranges over which the amino
acids were measured. The limit of detection (signal/noise > 10)
was 1 pmol for all of the amino acids. The coefficients of variation
for the analyses of tyrosine and phenylalanine in different LDL
preparations were 0.8% and 4.8%, respectively.
To investigate the relative yields of the amino acid markers after
protein oxidation by hydroxyl radical, metal ions, and tyrosyl radical,
BSA and LDL were exposed to either a
copper-H2O2 oxidation system (hydroxyl
radical), free copper (metal ion), or a
myeloperoxidase-H2O2-tyrosine system (tyrosyl
radical). We selected BSA for our initial studies because albumin is
abundant in plasma and has been widely investigated as a target for
oxidative damage in vitro. To determine whether lipid
components might affect the pattern of oxidation products, we performed
similar experiments with LDL. LDL was rapidly isolated using a
procedure that generates very low levels of endogenous lipid
hydroperoxides (30). The presence of o-tyrosine,
m-tyrosine, and o,o-dityrosine was
determined using stable isotope dilution GC-MS analysis.
Hydroxyl radical was generated
using a system containing 0.1 mM copper and 2 mM H2O2. The accumulation of
o-tyrosine in BSA oxidized by hydroxyl radical was initially
linear and then reached a plateau after 2 h (data not shown).
o-Tyrosine was the major product when BSA was oxidized by
hydroxyl radical, and smaller amounts of m-tyrosine and
o,o-dityrosine were also formed (Fig. 2A). When LDL was oxidized by hydroxyl
radical under the same experimental conditions, the major products were
o-tyrosine and o,o
-dityrosine, with
smaller amounts of m-tyrosine (Fig. 2B). These
results indicate that o-tyrosine is a major product of
protein oxidation by hydroxyl radical.
Detection of o-Tyrosine, m-Tyrosine, and o,o
Copper is a potent catalyst for LDL
oxidation in vitro (11, 13), but the yield of aromatic
oxidation products in the modified lipoprotein has not been reported.
We therefore monitored the formation of o-tyrosine,
m-tyrosine, and o,o-dityrosine in LDL exposed to copper. To explore the effect of the lipid environment of
LDL on its susceptibility to oxidation, we also determined the product
yield of the oxidation products in BSA. The levels of
o-tyrosine, m-tyrosine, and
o,o
-dityrosine were unaffected by exposing BSA
to high concentrations of copper alone (Fig.
3A). The oxidation of tyrosine and
phenylalanine residues in BSA by the hydroxyl radical generating system
thus requires both H2O2 and copper under
these experimental conditions (compare Figs. 2A and
3A).
In contrast to BSA, both o-tyrosine and
m-tyrosine increased in LDL exposed to copper (Fig.
3B). However, there was little change in the level of
o,o-dityrosine in LDL incubated with copper alone (Fig. 3B). When LDL was exposed to copper, the
kinetics of protein oxidation (monitored as o-tyrosine
formation) and lipid oxidation (monitored as thiobarbituric acid
reacting substances; Ref. 32) were similar. These results suggest that
alkoxyl and/or peroxyl radicals formed during metal-catalyzed lipid
peroxidation play a role in the hydroxylation of protein-bound
phenylalanine. In contrast, reactive intermediates generated during
copper-catalyzed LDL oxidation apparently fail to promote
o,o
-dityrosine formation.
Oxidation of BSA by
myeloperoxidase-generated tyrosyl radical was initially rapid and then
leveled off after 0.5 h as monitored by
o,o-dityrosine formation (data not shown). The
progress curve for protein dityrosine cross-linking by myeloperoxidase
was similar to that previously reported for the synthesis of free
o,o
-dityrosine (16) and protein tyrosylation
(26). o,o
-Dityrosine was the major product when
BSA was exposed to tyrosyl radical, with little increase in the levels
of either o-tyrosine or m-tyrosine (Fig. 4A). o,o
-Dityrosine
also was the only product formed in LDL exposed to tyrosyl radical,
with no significant change in the levels of either
o-tyrosine or m-tyrosine (Fig. 4B).
These results indicate that o,o
-dityrosine
is a major product of protein oxidation by tyrosyl radical generated by
myeloperoxidase.
The concentrations of oxidant and redox catalyst were very different in
the hydroxyl radical and tyrosyl radical systems. There was 20 times as
much H2O2 in the hydroxyl radical system as in
the tyrosyl radical system (2 mM versus 0.1 mM), and there was 5,000 times as much copper as
myeloperoxidase (100 µM versus 20 nM). Despite the marked differences in concentrations of
redox active components in the reaction mixtures, the relative yields of o-tyrosine (per mol of phenylalanine) and
o,o-dityrosine (per mol of tyrosine) were
similar when BSA was exposed either to hydroxyl radical or to tyrosyl
radical. These observations imply that under our experimental
conditions myeloperoxidase-generated tyrosyl radical is much more
efficient than metal-catalyzed hydroxyl radical at oxidizing
protein-bound aromatic amino acids.
To determine whether free metal ions, hydroxyl radical, or tyrosyl radical damage lipoproteins in vivo, we looked for the amino acid markers in LDL isolated from human artery wall. LDL (d = 1.02 to 1.07 g/ml) was prepared by sequential ultracentrifugation from aortic atherosclerotic tissue obtained at autopsy (31, 32). The buffers used for tissue processing contained high concentrations of DTPA (a metal chelator), butylated hydroxytoluene (a lipid soluble antioxidant), and 3-aminotriazole (a peroxidase inhibitor) to prevent artifactual oxidation of lipoproteins. Western blotting with a rabbit antibody monospecific for human apolipoprotein B100 (35), the major protein of circulating LDL, confirmed that intact apolipoprotein B100 was present in lesion LDL. As previously noted by other investigators (5, 31), aggregated and a range of lower molecular weight forms of immunoreactive proteins also were present in LDL isolated from lesions.
Compared with circulating LDL isolated from plasma, there was a
striking 100-fold increase in o,o-dityrosine
levels in LDL isolated from atherosclerotic lesions (Fig.
5). In contrast, the levels of o-tyrosine and
m-tyrosine in circulating LDL and lesion LDL were similar.
The distribution of oxidation products in LDL isolated from aortic
tissue was thus similar to that found in LDL oxidized by tyrosyl
radical.
Detection of o-Tyrosine, m-Tyrosine, and o,o
To determine whether metal ions, hydroxyl
radical, or tyrosyl radical damage proteins in vivo, we
looked for the amino acid markers in human atherosclerotic tissue.
Amino acids isolated from acid hydrolysates of advanced lesions were
treated with n-propyl alcohol and pentafluoropropionyl
anhydride and then analyzed by GC-MS in the negative-ion chemical
ionization mode. We detected compounds that exhibited major ions and
retention times identical to those of o-tyrosine,
m-tyrosine, and o,o-dityrosine.
Selected ion monitoring showed that the ions derived from the amino
acids co-eluted with the ions derived from 13C-labeled
internal standards, as shown in Fig. 6 for
o,o
-dityrosine. The identity of each compound
was confirmed by comparison with authentic standards using both
heptafluorobutyryl and pentafluoropriopionyl derivatives of each
oxidized amino acid. These results indicate that
o,o
-dityrosine, o-tyrosine, and
m-tyrosine are present in acid hydrolysates of proteins from
human atherosclerotic lesions.
Phenylalanine and Tyrosine Oxidation Products at Different Stages of Atherosclerosis in Human Aortic Tissue
To determine the levels of oxidative damage to proteins at different stages of atherosclerosis, we analyzed autopsy samples of human aortic tissue. The abundance of this material allowed us to identify areas of normal tissue and areas at different stages of atherosclerotic disease from each individual donor. Tissue was prepared and analyzed by stable isotope dilution negative-ion chemical ionization GC-MS as described above. Four independent analyses of tissue obtained from 16 different individuals were performed, and each donor provided all four types of aorta, for a total of 64 analyses.
There was a striking 11-fold elevation in the level of protein-bound
o,o-dityrosine in fatty streaks compared with
normal aorta (Fig. 7). The content of protein-bound
o,o
-dityrosine also was elevated in advanced
lesions, being 6 times greater than in normal tissue. In contrast,
levels of o-tyrosine and m-tyrosine were not
significantly different at any stage of the atherosclerotic process,
although there was a trend toward higher levels of both markers in
advanced atherosclerotic lesions (Fig. 7). The pattern of elevation of
oxidation products was identical in each analysis, with marked
increases in fatty streaks and advanced lesions observed in all four
independent experiments. These results indicate that aortic tissue
levels of protein-bound o,o
-dityrosine, but not o-tyrosine or m-tyrosine, increase in fatty
streaks and advanced lesions obtained through autopsy from humans.
The long-term goal of our research is to identify the molecular
mechanisms of oxidative damage in human vascular disease. In the
current studies we have focused on three pathways, free metal ions,
hydroxyl radical, and tyrosyl radical, that potentially play a role in
LDL oxidation and the pathogenesis of atherosclerosis. We selected
o-tyrosine, m-tyrosine, and
o,o-dityrosine as markers for oxidation because
they are unnatural amino acids and therefore should represent
post-translational modifications of proteins.
We first performed in vitro studies to establish the product
yields of o-tyrosine, m-tyrosine, and
o,o-dityrosine in LDL and BSA exposed to the
different oxidation systems. LDL oxidized by copper exhibited large
increases in o-tyrosine and m-tyrosine. In
contrast, there was little change in the level of
o,o
-dityrosine in copper-oxidized LDL. There was
no evidence of protein oxidation, as monitored by the amino acid
oxidation products, in BSA exposed to high concentrations of copper
alone. For both LDL and BSA, o-tyrosine was a major end
product when the oxidant was hydroxyl radical-generated by a
metal-catalyzed oxidation system. The level of
o,o
-dityrosine in proteins exposed to hydroxyl
radical was variable but was always accompanied by a striking increase
in o-tyrosine. Similar results were reported by Huggins
et al. (23) for RNase and lysozyme damaged with hydroxyl
radical generated by metal ions and ionizing radiation. In contrast to
copper and hydroxyl radical, there was a selective increase in the
level of o,o
-dityrosine in BSA and LDL oxidized
by tyrosyl radical generated by myeloperoxidase, with no significant
increase in the amount of either o-tyrosine or
m-tyrosine. Collectively, these results indicate that
proteins damaged in vitro by free metal ions, hydroxyl
radical, and tyrosyl radical exhibit distinct patterns of
phenylalanine and tyrosine oxidation products (Table
I).
|
We next determined the levels of the oxidation products in LDL isolated
from human vascular tissue resected at autopsy. There was a remarkable
100-fold increase in o,o-dityrosine levels
compared with circulating LDL. In striking contrast, the levels of
o-tyrosine and m-tyrosine in lesion LDL and
circulating LDL were similar. The pattern of a selective increase in
o,o
-dityrosine levels without significant change
in o-tyrosine levels was remarkably similar to that observed
in both LDL and BSA oxidized by tyrosyl radical (Table I). Furthermore,
the selective increase in o,o
-dityrosine levels
with little change in either o-tyrosine or
m-tyrosine was different from that observed with LDL
oxidized with either copper or the hydroxyl radical system. LDL exposed
to either of the metal ion-dependent systems exhibited
large increases in o-tyrosine with a variable increase in
o,o
-dityrosine. These observations suggest that
tyrosyl radical, but not free copper, may play a role in oxidizing LDL
in vivo.
Finally, we determined the levels of the oxidation products in human
vascular tissue resected at autopsy. Compared with normal aortic
tissue, there was an 11-fold elevation of
o,o-dityrosine in fatty streaks, the earliest
lesion of atherosclerosis. Protein-bound o,o
-dityrosine was elevated 6-fold in advanced
atherosclerotic lesions. There was no significant increase in
o-tyrosine or m-tyrosine in aortic tissue at any
stage of the atherosclerotic process. The selective increase in
o,o
-dityrosine levels without significant change
in o-tyrosine levels in atherosclerotic aortic tissue is consistent with the hypothesis that tyrosyl radical represents one
pathway for protein oxidation in the artery wall (Table I).
Because o,o-dityrosine levels in aortic tissue
were highest in fatty streaks, and also were elevated in advanced
lesions, tyrosyl radical may contribute both to the initiation and
progression of vascular disease. The trend toward higher levels of
protein-bound o,o
-dityrosine in intermediate
aortic lesions did not achieve statistical significance, perhaps
reflecting large biological variations or a real decrease in tissue
o,o
-dityrosine levels. The decline in
o,o
-dityrosine levels seen in intermediate
lesions may result in part from degradation of oxidized proteins during vascular remodeling. Indeed, o,o
-dityrosine has
been proposed to target oxidized proteins for proteolytic breakdown
(36). The variations in o,o
-dityrosine levels at
different stages of the atherosclerotic process may also reflect
variations in the degree or mechanism of oxidative stress, as well as
differences in the protein composition of normal and atherosclerotic
tissue.
Previous studies have shown that tissue homogenates prepared from
atherosclerotic lesions contain detectable levels of catalytically active metal ions (37, 38), suggesting that free metal ions or low
molecular weight chelates of metal ions may promote LDL oxidation
in vivo. However, it is difficult to exclude the possibility that homogenization of the tissue artifactually generated the redox
active components observed in these experiments. Neither study reported
the level of catalytically active metal ions in normal aortic tissue
subjected to the same procedure. Because levels of
o-tyrosine were similar in circulating LDL and lesion LDL,
and because LDL oxidation by copper yielded large amounts of
o-tyrosine with no change in
o,o-dityrosine, our results suggest that free
metal ions are unlikely to promote LDL oxidation in the artery wall
(Table I). Indeed, premature atherosclerosis is not a prominent feature
of genetic disorders that cause iron and copper to accumulate in tissue
and plasma (39, 40). It is possible, however, that endogenous
o-tyrosine masked a metal ion-dependent increase
in protein oxidation. There was a trend toward higher levels of
o-tyrosine and m-tyrosine in advanced atherosclerotic lesions, suggesting that metal ion-catalyzed oxidation reactions may be important late in the disease process when cellular dissolution might promote the release of redox active metal ions.
The detection of a selective increase in
o,o-dityrosine in lesion LDL and atherosclerotic
tissue, together with the distinct patterns of phenylalanine and
tyrosine oxidation products formed in BSA and LDL oxidized in
vitro, suggests that tyrosyl radical may play a role in
lipoprotein oxidation in vivo. A potential pathway involves
myeloperoxidase, a well-characterized source of tyrosyl radical (15,
16, 26). Active myeloperoxidase is present in human atherosclerotic
tissue (19), and oxidation products of the enzyme have been detected by
immunohistochemistry in vascular lesions (21, 22), suggesting that
tyrosyl radical generated by myeloperoxidase may represent one pathway
for LDL oxidation in vivo. In contrast, the failure to
detect an increase of o-tyrosine in lesion LDL and
atherosclerotic tissue suggests that free redox active metal ions are
unlikely to serve as a catalyst for LDL oxidation in the artery
wall.
We thank Drs. L. Sage, S. Hazen, and J. Turk for critical reading of the manuscript. Gas chromatography-mass spectrometry experiments were performed at the Washington University School of Medicine Mass Spectrometry Resource.