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(The FASEB Journal. 1999;13:1113-1120.)
© 1999 FASEB

Mass spectrometric quantification of amino acid oxidation products in proteins: insights into pathways that promote LDL oxidation in the human artery wall

JAY W. HEINECKE1

Departments of Medicine and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA

1Correspondence: Division of Atherosclerosis, Nutrition and Lipid Research, Box 8046, 660 S. Euclid Ave., St. Louis, MO 63110, USA. E-mail: heinecke{at}im.wustl.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 ISOTOPE DILUTION GC/MS ANALYSIS...
 INVESTIGATING LDL OXIDATION IN...
 PROSPECTS
 REFERENCES
 
Oxidatively damaged low density lipoprotein (LDL) may play an important role in atherogenesis, but the physiologically relevant pathways have proved difficult to identify. Mass spectrometric quantification of stable compounds that result from specific oxidation reactions represents a powerful approach for investigating such mechanisms. Analysis of protein oxidation products isolated from atherosclerotic lesions implicates tyrosyl radical, reactive nitrogen species, and hypochlorous acid in LDL oxidation in the human artery wall. These observations provide chemical evidence for the reaction pathways that promote LDL oxidation and lesion formation in vivo.—Heinecke, J. W. Mass spectrometric quantification of amino acid oxidation products in proteins: insights into pathways that promote LDL oxidation in the human artery wall.


Key Words: low density lipoprotein • oxidized LDL • reactive oxygen species • myeloperoxidase • 3-chlorotyrosine • atherosclerosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
ISOTOPE DILUTION GC/MS ANALYSIS...
 INVESTIGATING LDL OXIDATION IN...
 PROSPECTS
 REFERENCES
 
OXIDATIVE DAMAGE TO low density lipoprotein (LDL),2 the major carrier of blood cholesterol (1) , is implicated in the pathogenesis of atherosclerosis. Thus oxidized LDL, but not native LDL, exhibits potentially atherogenic effects (2 3 4 5 6 7) , suggesting that oxidation may be physiologically relevant. This hypothesis is supported by many studies (reviewed in refs 8 9 10 ). For example, oxidized LDL has been isolated from human and animal atherosclerotic lesions (11 , 12) , where chemical and immunohistochemical studies have detected oxidized lipids (13 , 14) . Also, a number of structurally unrelated antioxidants retard the formation of lesions in hypercholesterolemic animals and nonhuman primates (reviewed in refs 15 , 16 ). The oxidation hypothesis raises the exciting possibility that antioxidants may prevent or retard human atherosclerosis. Indeed, the lipid-soluble antioxidant vitamin E dramatically reduces the risk of nonfatal myocardial infarction in patients with established coronary artery disease (17) .

Elucidating mechanisms of LDL oxidation may reveal other compounds that could retard artery wall damage (15 , 16 , 18) . However, pathways that are relevant in vivo have been difficult to identify because the reactive intermediates that promote oxidation are short-lived and difficult to detect. An alternative strategy is to quantify tissue and lipoprotein levels of stable products of oxidation that have been identified through in vitro studies (19) .


   ISOTOPE DILUTION GC/MS ANALYSIS OF OXIDIZED AMINO ACIDS IN PROTEINS
TOP
 ABSTRACT
 INTRODUCTION
 ISOTOPE DILUTION GC/MS ANALYSIS...
INVESTIGATING LDL OXIDATION IN...
 PROSPECTS
 REFERENCES
 
Immunohistochemical staining of tissues with antibodies that recognize protein-bound oxidation products has been widely used to study mechanisms of oxidative damage in vivo (12 13 14) .

These methods are important because they reveal which cellular locations are damaged. Disadvantages include the semiquantitative nature of immunochemistry and possible interference from structurally related molecules.

Gaschromatography/mass spectrometry (GC/MS) is a highly sensitive and specific analytical method (20 , 21) . It uses a stable, isotopically labeled internal standard that, apart from its heavy isotope, is structurally identical to the target analyte and therefore behaves nearly identically during extraction, processing, and chromatographic analyses. Including such a standard corrects for analyte loss during processing and increases the precision of quantitative measurements. Using isotope dilution GC/MS, it is possible to unambiguously quantify trace amounts of analyte in a complex biological mixture.

In vitro oxidation of arachidonic acid yields a family of prostaglandin F2-like compounds, the F2-isoprostanes (reviewed in refs 22 , 23 ). Isotope dilution GC/MS measurement of plasma and urine levels of these compounds has been extensively investigated both in animal models and humans. These pioneering studies provide strong evidence that F2-isoprostanes are reliable markers of lipid peroxidation in vivo. Indeed, quantification of F2-isoprostanes has implicated oxidative stress in the pathogenesis of human disease.

Lipid peroxidation involves hydrogen atom abstraction and the subsequent formation of oxidizing intermediates that promote the formation of additional oxidation products (24 , 25) . The initial products of lipid oxidation may not be unique, because many different oxidants promote hydrogen atom abstraction. In addition, oxidation products generated during the propagation phase of lipid peroxidation may mask the primary oxidative event. In contrast, electrophilic addition and substitution reactions, radical addition and substitution reactions, and radical-radical recombination reactions yield compounds that retain the initial oxidant as a covalent adduct (25) . Lipids, proteins and nucleic acids modified by such reactions may therefore represent more specific markers of oxidative damage than oxidation products generated in chain-propagating reactions.

Oxidized amino acids in proteins that are formed by electrophilic and radical reactions are emerging as attractive candidates markers of oxidative damage (19 , 21) . Recent studies suggest that isotope dilution GC/MS analysis of oxidized amino acids in tissue proteins provides evidence for the pathways that promote oxidative stress in vivo. In a typical analysis, tissue or lipoproteins are delipidated, isotope-labeled internal standards are added, and protein is hydrolyzed with acid. Amino acids are isolated from the hydrolysate by chromatography on a solid-phase extraction column and derivatized to increase their volatility. We typically prepare perfluoroacyl derivatives of amino acids to obtain detectable negative-ion electron capture GC/MS signals at subpicomole levels (21) . The derivatized amino acids are then subjected to gas chromatography and quantification by isotope dilution MS analysis.


   INVESTIGATING LDL OXIDATION IN THE HUMAN ARTERY WALL
TOP
 ABSTRACT
 INTRODUCTION
 ISOTOPE DILUTION GC/MS ANALYSIS...
 INVESTIGATING LDL OXIDATION IN...
PROSPECTS
 REFERENCES
 
Known agents that can oxidize LDL include extracellular metal ions and products of cells in the artery wall. The latter include reactive nitrogen species and oxidants generated by myeloperoxidase, a major phagocytic enzyme. Because each pathway generates a different pattern of oxidation products (19 , 21) , GC/MS provides a way to assess the relative importance of various oxidative agents in vivo.

Extracellular metal ions
One important pathway for LDL oxidation in vitro involves redox-active transition metal ions. Increasing concentrations of iron or copper modify LDL incubated with smooth muscle cells (5) or even in the absence of cells if the ions are present at sufficiently high concentrations (5 , 7 , 26) . Metal chelators also inhibit LDL oxidation by cultured cells of the artery wall (5 6 7) . Moreover, protein-bound metal ions in ceruloplasmin (27) and hemin (28) promote LDL oxidation, though the mechanisms may differ from those involving free metal ions.

Despite extensive study, the mechanisms by which metal ions stimulate LDL oxidation are poorly understood. Reduced metal ions (Mn+) catalyze the decomposition of lipid peroxides (LOOH) into alkoxyl radical (RO•), a potent oxidizing species (9 , 10 , 24 , 29) .

The hydroperoxide-derived radicals then are scavenged by antioxidants or attack polyunsaturated fatty acids (LH) to form carbon-centered radicals (L·) that initiate the radical chain reaction of lipid peroxidation (9 , 10 , 24 , 29) . The reaction cycle continues until antioxidants or radical-radical cross-linking reactions terminate lipid peroxidation.

The relevance of this mechanism is uncertain because it is unclear whether free metal ions are available in vivo and because LDL isolated from plasma contains extremely low levels of hydroperoxides (30) . A small fraction of LDL in plasma, termed LDL-, contains elevated levels of lipid oxidation products (31) , suggesting that the conversion of preformed hydroperoxides into further lipid oxidation products might be physiologically relevant. The origin of LDL- is uncertain, but it may be generated in blood or come from LDL that has been oxidized in peripheral tissue.

Alternatively, metal ions may be reduced by cells (26 , 32 , 33) or compounds present in lipoproteins (34 , 35) yielding intermediates capable of peroxidizing LDL lipid. One possible mechanism is the cellular conversion of cystine into thiols (32) that subsequently generate superoxide and sulfur-centered radicals. Another is the conversion of {alpha}-tocopherol to {alpha}-tocopherol radical (34 , 35) . The radical then attacks a polyunsaturated fatty acid to initiate lipid peroxidation.


Studies with a widely used model, in vitro oxidation of LDL by free copper ions, support this idea. Reduction of bound Cu2+ to Cu1+ by endogenous vitamin E, for example, appears to be a key step in initiating LDL lipid peroxidation (34 , 35) . This has led to the counterintuitive proposal that vitamin E, normally considered to be an antioxidant, can promote the peroxidation of LDL lipid under certain conditions (34) . The biological relevance of tocopherol-mediated lipid peroxidation is not established.

It has not yet been determined whether extracellular free metal ions (or low molecular weight chelates of metal ions) exist extracellularly in vivo. Redox-active metal ions have been found in tissue homogenates of atherosclerotic tissue, but results from normal aortic tissue subjected to the same homogenization procedure were not reported (36 37 38) . Transferrin is the major carrier of iron in plasma, but its high-affinity binding sites for iron and copper are only partly saturated in normal individuals (39) . Moreover, low concentrations of albumin, the most abundant protein in plasma, inhibit metal ion-dependent LDL oxidation (40) . This protein also avidly binds free copper (41) , suggesting that extracellular free metal ions are unlikely to be present in normal arterial tissue.

One way to explore this question involves quantifying protein oxidation products generated by hydroxyl radical (HO ·), an extremely reactive agent generated by metal ion-dependent reactions (42) . The radical is formed, for example, when a reduced metal ion, such as Fe2+ or Cu1+, reacts with hydrogen peroxide (H2O2).

LDL and model proteins exposed to hydroxyl radical generated by metal ions exhibit a dramatic increase in ortho-tyrosine and meta-tyrosine, isomers of the common amino acid tyrosine (Fig. 1 ; refs 43 , 44 ).



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  		Figure 1. Generation of ortho-tyrosine, and meta-tyrosine by hydroxyl radical.

LDL oxidized by copper also demonstrates large increases in ortho-tyrosine and meta-tyrosine (44) . These observations suggest that these unusual tyrosine isomers might be useful markers of metal ion-mediated damage in vivo.

We used isotope dilution GC/MS to quantify levels of ortho-tyrosine and meta-tyrosine in plasma LDL and LDL isolated from human atherosclerotic lesions (44) . Neither oxidation product was more abundant in lesion LDL than in plasma LDL. Moreover, levels of the two markers were similar in normal aortic tissue and fatty streaks, the earliest lesion of atherosclerosis. These observations suggest that metal ions are unlikely to play a role in oxidizing LDL early in atherogenesis. In contrast, we observed a two- to threefold increase in levels of ortho-tyrosine and meta-tyrosine in advanced atherosclerotic lesions, though it was not statistically significant (44) . This raises the possibility that redox-active metal ions, perhaps released from necrotic or dystrophic cells, promote LDL oxidation late in the atherosclerotic process.

Epidemiological studies examining the relationship between iron stores and risk for atherosclerosis have provided inconsistent results, which supports the notion that free iron is unlikely to be a major risk factor for atherosclerosis (reviewed in ref 45 ). Moreover, premature atherosclerosis is not a prominent feature of hemochromatosis (46) , a common genetic disorder that causes iron to accumulate in plasma and liver. Quantitative angiographic analysis of postmortem hearts revealed less atherosclerosis in subjects suffering from classic hemochromatosis (47) . Animal studies also have yielded inconsistent results regarding the effect of iron on atherosclerosis (48 , 49) . Whether excess copper promotes LDL oxidation in vivo is unclear. Individuals with Wilson's disease appear not to be at increased risk for atherosclerosis, even though this disorder raises copper levels in liver, plasma, and brain (50) . However, it is important to note that levels of ceruloplasmin, a proposed catalyst of LDL oxidation, are low in this disorder.

Oxidants from cells of the artery wall
Pioneering studies suggested that cultured arterial cells convert LDL into a form that binds to scavenger receptors (4) , promoting the conversion of macrophages into foam cells, the hallmark of the early atherosclerotic lesion. Investigations of the major cell types in lesions—endothelial cells, smooth muscle cells, and monocyte/macrophages—have shown that oxidative reactions are involved, and a number of possible mechanisms have been proposed.

Reactive nitrogen species
An oxidation system of broad interest involves reactive nitrogen species, which can be formed through a variety of mechanisms (reviewed in ref 51 ). Nitric oxide, for example, is generated by endothelial cells as a major regulator of vascular tone in muscular arteries, and it mediates numerous biological effects that are potentially antiatherogenic (reviewed in ref 52 ). However, it is also a relatively stable radical that may indirectly oxidize lipoproteins (10) and promote atherosclerosis. This may be especially true for the much larger quantities generated by inducible nitric oxide synthase as part of the inflammatory response.

Nitric oxide also can be converted into more reactive species, as when it interacts with superoxide (O2 · -), a product of activated phagocytes. The reaction generates the powerful oxidizing intermediate peroxynitrite (ONOO-) (53) .

Peroxynitrite peroxidizes LDL lipids and converts the lipoprotein into a form that promotes the formation of macrophage foam cells, suggesting that nitric oxide is proatherogenic (54) . In contrast, other studies suggest that nitric oxide is protective because it inhibits LDL oxidation by both murine macrophages and copper (55 56 57 58 59) . It might suppress LDL oxidation by inhibiting heme-containing enzymes, scavenging superoxide, reacting with lipid radicals, or nitrosylating important cellular proteins.

Peroxynitrite is a potent nitrating reagent that converts tyrosine to 3-nitrotyrosine in vitro (Fig. 2 ; ref 51 ). Peroxynitrite also hydroxylates phenylalanine and converts tyrosine to dityrosine, though these reactions occur less readily than tyrosine nitration. Studies of LDL oxidized by a variety of oxidation systems indicate that 3-nitrotyrosine is a specific marker of damage by reactive nitrogen species (60) . Immunohistochemical studies have detected 3-nitrotyrosine in human atherosclerotic lesions, suggesting that reactive nitrogen species may promote LDL oxidation in vivo (61) .



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  		Figure 2. Tyrosine nitration by reactive nitrogen species produces 3-nitrotyrosine.

To explore the possibility that reactive nitrogen species mediate LDL oxidation in the human artery wall, we used isotope dilution GC/MS to quantify 3-nitrotyrosine levels in LDL isolated from atherosclerotic lesions (60) . The level of 3-nitrotyrosine in lesion LDL was 80-fold higher than in circulating LDL. This observation raises the possibility that nitric oxide can render LDL atherogenic, counteracting in part its well-established antiatherogenic effects. Alternatively, the detection of elevated levels of 3-nitrotyrosine in lesion LDL may indicate that nitric oxide provides one mechanism for inhibiting lipid peroxidation in the artery wall. Studies in hypercholesterolemic rabbits and mice show that nitric oxide inhibits fatty streak formation (62 , 63) , suggesting that it is antiatherogenic in these animal models.

Myeloperoxidase from phagocytes
We are particularly interested in oxidative damage mediated by neutrophils, monocytes and macrophages because activated phagocytic white blood cells produce a variety of potent oxidants while defending their host against invading microorganisms (64 , 65) .

Macrophages and other phagocytic white blood cells generate superoxide (O2 ·-) through the action of a membrane-associated NADPH oxidase that directly reduces molecular oxygen.

Because superoxide dismutates, the cells also generate H2O2 (42) .

Phagocytes also secrete the heme protein myeloperoxidase, which interacts with hydrogen peroxide to generate antimicrobial toxins (64 , 65) . In contrast to many in vitro oxidation reactions, this enzyme does not require free metal ions. Its major action is to convert chloride ion to hypochlorous acid (HOCl; ref 66 ).

Hypochlorous acid is a potent bactericidal oxidant that may inadvertently damage host proteins at sites of inflammation in vivo. We have shown that it chlorinates tyrosine to 3-chlorotyrosine, a highly specific marker for myeloperoxidase-initiated protein oxidation (67 , 68) .

Recent studies have shown that active myeloperoxidase is present in human atherosclerotic tissue (69) . The enzyme colocalizes with macrophages in intermediate lesions and is closely associated with cholesterol clefts in extracellular lipid deposits of advanced atherosclerotic lesions. A similar pattern of immunostaining for protein-bound lipid oxidation products has been reported for rabbit atherosclerotic lesions (14) . LDL exposed to hypochlorous acid undergoes aggregation and promotes macrophage foam cell formation (70) . The enzyme's ability to oxidize LDL in vitro by reactions that do not require free metal ions, together with its presence in atherosclerotic lesions, suggests that this heme protein may be one important agent for lipoprotein oxidation in vivo.

Biochemical studies and electron paramagnetic resonance spectroscopy demonstrate that myeloperoxidase can convert the phenolic amino acid L-tyrosine to tyrosyl radical (Fig. 3 ; refs 71 , 72 ), which then initiates the peroxidation of LDL lipid (73) . This process bears remarkable biochemical similarities to the enzymatic peroxidation of polyunsaturated fatty acids by cyclooxygenase, a reaction also thought to involve tyrosyl radical (74) . Protein tyrosyl residues are damaged when tyrosyl radical converts protein-bound tyrosines to o,o'-dityrosine (Fig. 3 ; refs 75 , 76 ).



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  		Figure 3. Radical-radical recombination of tyrosyl radical yields o,o'-dityrosine.

Both dityrosine formation and the initiation of LDL oxidation by myeloperoxidase are greatly enhanced by plasma concentrations of tyrosine (73 , 75) . Dityrosine, which is intensely fluorescent and stable to acid hydrolysis, may therefore serve as a marker for proteins that activated phagocytes have oxidatively damaged.

LDL and model proteins exposed to free tyrosyl radical generated by myeloperoxidase become markedly enriched in dityrosine but not in ortho-tyrosine, one of the markers of protein oxidation by metal ions (44) . The mechanism involves a radical-radical recombination reaction between protein-bound tyrosyl radical and free or protein-bound tyrosyl radical (44 , 72 , 75 , 76) .

To determine whether tyrosyl radical might play a role in oxidizing LDL in vivo, we quantified dityrosine levels using isotope dilution GC/MS (44) . LDL isolated from atherosclerotic lesions exhibited a dramatic 100-fold increase in dityrosine levels compared with circulating LDL. In striking contrast, there was no evidence of ortho-tyrosine enrichment. Tissue dityrosine levels also increased markedly in fatty streaks (the earliest lesion of atherosclerosis) and advanced atherosclerotic lesions. These results suggest that tyrosyl radical, perhaps generated in part by myeloperoxidase, contributes to LDL oxidation both early and late in the disease process.

Chlorinated biomolecules should be specific markers of oxidative damage by activated phagocytes, because at plasma concentrations of halide, myeloperoxidase is the only human enzyme know to generate hypochlorous acid (66 , 77) . 3-Chlorotyrosine is formed in the protein component of LDL oxidized by the myeloperoxidase-peroxide-chloride system (Fig. 4 ; ref 68 ).



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  		Figure 4. Hypochlorous acid converts tyrosine into 3-chlorotyrosine.

In contrast, 3-chlorotyrosine was undetectable in LDL oxidized by hydroxyl radical, copper, iron, horseradish peroxidase, hemin, glucose, or peroxynitrite, indicating that the chlorinated amino acid is a specific marker of oxidation by myeloperoxidase. We have recently used isotope dilution GC/MS to show that 3-chlorotyrosine is dramatically elevated in LDL isolated from human atherosclerotic tissue and in atherosclerotic lesions harvested at surgery (68) . These results provide strong evidence that oxidative damage to LDL—and perhaps to other macromolecules involved in plaque formation—can result from the action of myeloperoxidase in the human artery wall (Fig. 5 ).



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  		Figure 5. Potential roles of reactive intermediates generated by myeloperoxidase in LDL oxidation. In the intima of the artery wall, where mononuclear cells are abundant and local antioxidants that scavenge oxidants might be depleted, stimulated phagocytes generate superoxide that dismutates to hydrogen peroxide. Myeloperoxidase secreted by phagocytes uses the peroxide to generate an array of oxidizing intermediates. One important product is hypochlorous acid, which chlorinates tyrosine residues. Myeloperoxidase generated tyrosyl radical may promote LDL oxidation by forming o,o'-dityrosine cross-links in proteins and initiating lipid peroxidation. Myeloperoxidase also generates other intermediates (such as amino acid-derived aldehydes and reactive nitrogen species) that may play a role in converting LDL into an atherogenic particle.

Immunohistochemical studies of atherosclerotic lesions suggest that oxidation-specific epitopes are localized to phagolysosomal-like structures in macrophages (14) . Phagocytosis is a potent stimulus for the secretion of both peroxide and myeloperoxidase into the phagolysosome, a cellular microenvironment where high concentrations of oxidants might overwhelm antioxidant defense mechanisms (64 , 65) . These observations suggest that the phagolysosome might represent an ideal location for promoting LDL oxidation.

Recent studies indicate that myeloperoxidase also uses nitrite, a decomposition product of nitric oxide, to generate potent chlorinating and nitrating intermediates (78) . Moreover, human neutrophils use the myeloperoxidase system to both chlorinate and nitrate tyrosine residues (67 , 79) . These observations, together with our demonstration that levels of 3-nitrotyrosine and 3-chlorotyrosine are elevated in human atherosclerotic tissue (60 , 68) , raise the possibility that the myeloperoxidase pathway both nitrates and chlorinates host tissues in vivo.


   PROSPECTS
TOP
 ABSTRACT
 INTRODUCTION
 ISOTOPE DILUTION GC/MS ANALYSIS...
 INVESTIGATING LDL OXIDATION IN...
 PROSPECTS
REFERENCES
 
The ability to quantify distinct amino acid oxidation products in proteins and tissues is invaluable for exploring the roles of various oxidation pathways in the pathogenesis of atherosclerosis. The specificity, sensitivity, and precision of isotope dilution GC/MS make this a remarkably powerful technique for this purpose. This approach cannot establish the relevance of oxidation chemistry to the pathogenesis of disease, however. Using animal models that overexpress or lack enzymes that generate specific oxidants or oxidation products, in concert with the use of mass spectrometry to detect specific chemical markers in vivo, should provide further insight into the contributions of oxidative damage to the onset and progression of atherosclerosis.


   FOOTNOTES
 
2 Abbreviations: GC, gas chromatography; LDL, low density lipoprotein; MS, mass spectrometry.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 ISOTOPE DILUTION GC/MS ANALYSIS...
 INVESTIGATING LDL OXIDATION IN...
 PROSPECTS
 REFERENCES
 

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