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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1716-1723

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1716.)
© 2000 American Heart Association, Inc.

Brief Reviews

Oxidation of LDL by Myeloperoxidase and Reactive Nitrogen Species

Reaction Pathways and Antioxidant Protection

Anitra C. Carr; Mark R. McCall; Balz Frei

From the Linus Pauling Institute, Oregon State University, Corvallis.

Correspondence to Balz Frei, PhD, Linus Pauling Institute, Oregon State University, 571 Weniger Hall, Corvallis, OR 97331-6512. E-mail balz.frei{at}orst.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMPO-Dependent Oxidation of LDL
 down arrowAntioxidant Protection Against...
 down arrowOxidation of LDL by...
 down arrowMPO and RNS
 down arrowAntioxidant Protection of LDL...
 down arrowConclusions and Perspectives
 down arrowReferences
 
Abstract—Oxidative modification of low density lipoprotein (LDL) appears to play an important role in atherogenesis. Although the precise mechanisms of LDL oxidation in vivo are unknown, several lines of evidence implicate myeloperoxidase and reactive nitrogen species, in addition to ceruloplasmin and 15-lipoxygenase. Myeloperoxidase generates a number of reactive species, including hypochlorous acid, chloramines, tyrosyl radicals, and nitrogen dioxide. These reactive species oxidize the protein, lipid, and antioxidant components of LDL. Modification of apolipoprotein B results in enhanced uptake of LDL by macrophages with subsequent formation of lipid-laden foam cells. Nitric oxide synthases produce nitric oxide and, under certain conditions, superoxide radicals. Numerous other sources of superoxide radicals have been identified in the arterial wall, including NAD(P)H oxidases and xanthine oxidase. Nitric oxide and superoxide readily combine to form peroxynitrite, a reactive nitrogen species capable of modifying LDL. In this review, we examine the reaction pathways involved in LDL oxidation by myeloperoxidase and reactive nitrogen species and the potential protective effects of the antioxidant vitamins C and E.


Key Words: low density lipoproteins • myeloperoxidase • reactive nitrogen species • vitamin C • vitamin E


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMPO-Dependent Oxidation of LDL
 down arrowAntioxidant Protection Against...
 down arrowOxidation of LDL by...
 down arrowMPO and RNS
 down arrowAntioxidant Protection of LDL...
 down arrowConclusions and Perspectives
 down arrowReferences
 
The hypothesis that oxidative stress plays an important role in the pathogenesis of atherosclerosis has gained considerable support. Although there are many determinants in the development of an atherosclerotic lesion, substantial in vitro evidence links LDL oxidation to potentially atherogenic processes at the molecular and cellular level.1 2 3 However, the in vivo mechanism(s) of the initiation and progression of LDL oxidation is presently unclear and is a topic of much research. The most relevant information concerning this mechanism has come from immunohistochemical and biochemical analyses of animal and human atherosclerotic lesions and lipoproteins extracted from these lesions. Ceruloplasmin, 15-lipoxygenase, myeloperoxidase (MPO), and inducible (in addition to endothelial) nitric oxide synthase (NOS) have been found in animal and human lesions and can cause or contribute to LDL oxidation in vitro.2 4 5 6 7 8 9 10

With respect to a role for ceruloplasmin in in vivo LDL oxidation, specific markers of metal ion–catalyzed protein damage are not elevated in early or intermediate lesions, in contrast to advanced lesions.9 11 Immunohistochemical detection of aldehyde-modified LDL in human and animal lesions12 13 also has been used as evidence for metal ion–mediated oxidation; however, the specificity of antibodies raised against aldehyde-modified or copper-oxidized LDL has been questioned, because these can cross-react with LDL modified by hypochlorous acid (HOCl).14 Therefore, it appears unlikely that ceruloplasmin and other sources of redox-active metal ions contribute significantly to LDL oxidation in vivo during the early stages of atherosclerotic lesion development.

The role of 15-lipoxygenase in LDL oxidation and atherogenesis is controversial, because the mechanism by which the intracellular enzyme "seeds" extracellular LDL with hydroperoxides is unclear.15 16 Furthermore, there is limited evidence for the presence of 15-lipoxygenase–modified lipids in lesions, because only small increases in the S/R enantiomeric ratio of lipid hydro(pero)xides were detected.17 18 Nevertheless, immunohistochemical studies have demonstrated the presence of 15-lipoxygenase in macrophage-rich regions of human and rabbit atherosclerotic lesions,19 20 and epitopes of oxidized LDL colocalize with 15-lipoxygenase.20 Interestingly, cholesterol-fed transgenic rabbits overexpressing the human 15-lipoxygenase gene in macrophages develop significantly less atherosclerosis than do their nontransgenic littermates.21 In contrast, 12/15-lipoxygenase–knockout mice crossbred with atherosclerosis-prone apoE-deficient mice exhibit significantly reduced lesion development.22 The lack of agreement between these studies is intriguing and indicates that more studies are needed to determine the role of 15-lipoxygenase in LDL oxidation and atherogenesis.

MPO and MPO-derived HOCl have been implicated in in vivo LDL modification and atherogenesis (Figures 1Down and 2Down).23 Immunoreactive and catalytically active MPO has been detected in human atherosclerotic lesions and colocalizes with macrophages.10 In addition, specific markers of MPO- and HOCl-mediated protein modification, ie, dityrosine and 3-chlorotyrosine, have been isolated from human atherosclerotic lesions.9 24 Similarly, epitopes recognized by antibodies against HOCl-modified proteins have been detected in early and advanced human lesions.25 Interestingly, the extent of atherosclerosis was increased in LDL-receptor–deficient MPO-knockout mice fed a high fat diet compared with MPO wild-type mice.26 However, it is not known whether MPO and MPO- or HOCl-modified proteins are present in atherosclerotic lesions of mice and contribute to atherogenesis. In vitro studies have shown that HOCl-modified LDL stimulates foam cell formation,27 28 enhances leukocyte oxidant and cytokine production, degranulation, migration, and adherence to endothelial cells, and increases vascular cell permeability.29 30 31 These pathophysiological properties of HOCl-modified LDL toward leukocytes and vascular cells could contribute to the development of atherosclerosis. In addition, we have found that, like mildly oxidized LDL,32 HOCl-modified LDL impairs the activity of lecithin:cholesterol acyltransferase, a key component of the antiatherogenic reverse cholesterol transport (McCall, Carr, and Frei, unpublished data, 1999).



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  		Figure 1. MPO-mediated modification of LDL and inhibition by vitamin C. MPO uses H2O2, generated by dismutation of superoxide (O2·-), to oxidize chloride ions (Cl-) to HOCl. HOCl oxidizes LDL either directly or via conversion of amine-containing compounds (RNH2) to chloramines (RNHCl). Chloramines retain sufficient oxidizing capacity to directly modify LDL; in addition, chloramines can break down to form reactive aldehydes (RCHO) or nitrogen-centered radicals. MPO also oxidizes phenolic compounds, such as tyrosine (Tyr-OH), to phenoxyl radical intermediates (Tyr-O·) that can modify LDL. MPO-derived oxidants modify the protein (apoB) and lipid components of LDL as well as deplete LDL-associated antioxidants. Vitamin C can inhibit MPO-dependent LDL modifications at several junctures (X), including regeneration of amines from chloramines (+).



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  		Figure 2. RNS-mediated modification of LDL and inhibition by vitamin C. Constitutive endothelial and inducible phagocytic NOS (eNOS and iNOS, respectively) generate nitric oxide (NO·) from arginine. NO· diffuses out of the endothelial and phagocytic cells and rapidly reacts with superoxide (O2·-) to form peroxynitrite (ONOO-). ONOO- either isomerizes or reacts with carbon dioxide (CO2) to form reactive intermediates, which can modify LDL. Aerobic degradation of NO· forms nitrite (NO2-), which can act as a substrate for MPO; this reaction generates nitrogen dioxide (NO2·), which can directly modify LDL. RNS modify the protein (apoB) and lipid components of LDL as well as deplete LDL-associated antioxidants. Vitamin C can inhibit modification of LDL by RNS at several junctures (X).

Although nitric oxide (NO·) has been implicated as an antiatherogenic molecule,33 34 the reactive nitrogen species (RNS) formed by the reaction of NO· with oxygen and superoxide are potentially proatherogenic (Figure 2Up). Inducible NOS has been detected in lesions,7 8 as has the reaction product of RNS with protein tyrosine residues, 3-nitrotyrosine.7 8 35 36 3-Nitrotyrosine, detected immunocytochemically, was localized to macrophages, foam cells, and smooth muscle cells.7 8 35 Interestingly, 3-nitrotyrosine was detected even in the presence of extracellular superoxide dismutase,7 suggesting that the enzymatic dismutation of superoxide cannot effectively compete with the formation of peroxynitrite in vivo.37 One study reported that the levels of 3-nitrotyrosine are significantly higher in LDL derived from atherosclerotic intima than in LDL isolated from plasma,36 although another study found no difference in 3-nitrotyrosine levels between atherosclerotic lesions and normal intima.38 LDL modified in vitro by RNS is recognized by the macrophage scavenger receptor39 and thus may contribute to foam cell formation.

Thus, several lines of evidence indicate that MPO and RNS contribute to atherogenic modification of LDL in vivo.7 8 9 10 24 25 In the present review, we examine the reaction pathways involved in LDL oxidation by these 2 mechanisms and the potential protective effects of the antioxidant vitamins C and E.


*    MPO-Dependent Oxidation of LDL
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *MPO-Dependent Oxidation of LDL
down arrowAntioxidant Protection Against...
 down arrowOxidation of LDL by...
 down arrowMPO and RNS
 down arrowAntioxidant Protection of LDL...
 down arrowConclusions and Perspectives
 down arrowReferences
 
MPO is a heme-containing enzyme secreted by human phagocytes after activation by respiratory burst stimulants.40 MPO has 2 major activities: halogenation and peroxidation (reactions 1 through 4). During the halogenation activity of MPO, hydrogen peroxide (H2O2) reacts with native MPO to form the redox intermediate compound I, which oxidizes halides to hypohalous acids via a 2-electron oxidation step (reactions 1 and 2). HOCl is the major strong oxidant generated by the MPO system of stimulated phagocytes at physiological concentrations of halide ions.40 MPO also oxidizes a number of organic substrates (RH) to free radical intermediates by a classical peroxidase cycle involving compounds I and II (reactions 3 and 4). Reactions 1 through 4 are as follows:

(1)

(2)

(3)

(4)

In vitro reaction of reagent or MPO-derived HOCl with LDL results primarily in modifications of apoB, with little oxidation of the lipids (Table 1Down).27 41 HOCl reacts readily with the {epsilon}-amino groups of apoB lysine residues, resulting in the formation of N-chloramines (reactions 5 and 6), which are quantitatively the major products in HOCl-modified LDL.27 LDL-associated chloramines have been shown to alter LDL charge characteristics, leading to the uncontrolled uptake of HOCl-modified LDL by macrophages in culture.27 28 41 A small proportion of the LDL-associated chloramines break down to form aldehydes,41 42 which may be involved in the cross-linking and aggregation of HOCl-exposed LDL particles via Schiff base formation, as well as the formation of advanced glycation end products.41 Other amino acid residues of apoB, such as cysteine, methionine, tryptophan, and tyrosine, are also susceptible to oxidation by reagent or MPO-derived HOCl.27 Among the latter amino acid residues and lysine, cysteine and methionine exhibit the highest reactivity with HOCl (Table 1Down).41 43 Although tyrosine is only a minor target, one of its products, namely, 3-chlorotyrosine, is a useful specific biomarker of HOCl-mediated protein modification.24 Reactions 5 and 6 are as follows:

(5)

(6)


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  		Table 1. Modification of Specific LDL Targets by MPO-Derived Oxidants

Several investigators have reported lipid peroxidation in isolated LDL exposed to reagent or MPO-derived HOCl, but the amounts of lipid oxidation products formed were small.41 44 45 One mechanism proposed by Panasenko46 involves homolytic cleavage of preformed lipid hydroperoxides by HOCl to generate alkoxyl radicals. More recently, it has been reported that LDL-associated chloramines break down to produce radicals that can initiate lipid peroxidation.47 Formation of chlorinated lipids in LDL exposed to MPO has also been reported and has been proposed to be due to the formation of molecular chlorine.48 However, chlorinated lipids are only minor products of MPO-mediated reactions with LDL.

MPO can also modify LDL via reactive amino acid intermediates. MPO-derived tyrosyl radicals, produced by 1-electron oxidation of free tyrosine (see reactions 3 and 4),49 can initiate LDL lipid peroxidation and dityrosine formation (Table 1Up)50 as well as catalyze oxidative cross-linking of proteins.51 However, because tyrosine is present in plasma at levels several orders of magnitude lower than chloride ions (20 to 80 µmol/L versus 100 to 140 mmol/L, respectively),52 it is uncertain whether significant amounts of tyrosyl radicals are formed by MPO in the presence of physiological concentrations of chloride ions. Reaction of MPO-derived HOCl with free amino acids produces N-chloramine derivatives, which subsequently can break down to form reactive aldehydes (Figure 1Up).53 Reactive aldehydes derived from tyrosine and serine have been shown to modify protein lysine residues.54 55 Although N-chloramines are less reactive than HOCl, we have found that stable N-chloramine derivatives of N{alpha}-acetyl-lysine and taurine, as well as ammonia, still retain sufficient oxidizing capacity to oxidize apoB cysteine residues.56


*    Antioxidant Protection Against MPO-Dependent LDL Modification
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMPO-Dependent Oxidation of LDL
 *Antioxidant Protection Against...
down arrowOxidation of LDL by...
 down arrowMPO and RNS
 down arrowAntioxidant Protection of LDL...
 down arrowConclusions and Perspectives
 down arrowReferences
 
Vitamin E ({alpha}-tocopherol), the major lipid-soluble antioxidant in LDL, and the carotenoids ß-carotene and lycopene are susceptible to HOCl-mediated LDL oxidation, but only at relatively high concentrations of HOCl.27 42 LDL-associated ubiquinol, in contrast, is fully oxidized with physiological (200 µmol/L) concentrations of HOCl.27 In vitro supplementation of LDL with vitamin E does not protect against HOCl-mediated modification of the lipoprotein57 and may actually enhance lipid oxidation via {alpha}-tocopheroxyl radical–mediated abstraction of bis-allylic hydrogens from unsaturated lipids.47 Similarly, vitamin E is unable to protect LDL lipids from oxidation by tyrosyl radicals generated by the MPO/H2O2/tyrosine system.50 As such, vitamin E does not appear to provide effective antioxidant protection against MPO-mediated modification of LDL, most likely a result of apoB being the major target of HOCl and the ability of vitamin E to promote in vitro lipid oxidation in the absence of suitable co-antioxidants.58

One likely defense against MPO-mediated LDL oxidation is vitamin C (ascorbate), an important water-soluble antioxidant in biological fluids (Figure 1Up).59 Vitamin C scavenges HOCl in a stoichiometric manner (reaction 7)60 and can regenerate amines from HOCl-derived N-chloramines (reaction 8).27 56 Moreover, as a co-antioxidant, vitamin C can prevent the pro-oxidant properties of the {alpha}-tocopheroxyl radical (reaction 9).58 We found that physiological concentrations of vitamin C can protect LDL lysine and tryptophan residues from oxidation by HOCl and partially protect LDL cysteine residues.56 We also observed that vitamin C protects against LDL cysteine loss mediated by N{alpha}-acetyl-lysine-, taurine-, and mono-chloramines; protection occurred to different extents depending on the type and hydrophobic tendencies of the chloramine used.56 Reactions 7 through 9 are as follows:

(7)

(8)

(9)

Although generation of HOCl by the MPO/H2O2/Cl- system (reactions 1 and 2) produces similar modifications to LDL as reagent HOCl,41 the interaction of vitamin C in the enzymatic system may be complex in view of the fact that vitamin C not only scavenges HOCl and N-chloramines (reactions 7 and 8) but can also paradoxically enhance the chlorinating activity of MPO.61 62 The latter activity of vitamin C involves regeneration of the native enzyme from the compound II intermediate (reaction 4), which is catalytically inactive in the chlorination reaction. The predominant effect of vitamin C most likely depends on its concentration, with catalytic (low micromolar) concentrations stimulating enzymatic activity and higher concentrations scavenging HOCl and chloramines. Although vitamin C reacts with compound I of MPO at a rate similar to that of chloride ions [reaction 3, k2=1.1x106 (mol/L)-1 · s-1, versus reaction 2, k2=4.7x106 (mol/L)-1 · s-1, respectively],63 64 vitamin C is unlikely to act as a competitive inhibitor of HOCl formation by MPO because of its relatively low physiological plasma concentration compared with that of chloride ions (25 to 150 µmol/L versus 100 to 140 mmol/L, respectively).65

Vitamin C also protects against LDL lipid peroxidation initiated by MPO-derived tyrosyl radicals.50 66 This is likely due to vitamin C’s scavenging of these radicals (reaction 10) or possibly preventing their formation by MPO, because the rate constants of the reaction of vitamin C and tyrosine with compounds I and II are comparable63 64 and because their extracellular concentrations are similar (20 to 80 µmol/L versus 25 to 150 µmol/L, respectively).52 65 In addition, because tyrosyl radical–mediated lipid peroxidation is presumably mediated via an {alpha}-tocopheroxyl radical intermediate,58 vitamin C could also be acting as a co-antioxidant in this system (see reaction 9). Thus, vitamin C appears to provide efficient antioxidant protection from HOCl- and MPO-mediated damage to LDL in vitro by a number of mechanisms (reactions 7 through 10). Reaction 10 is as follows:

(10)


*    Oxidation of LDL by RNS
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMPO-Dependent Oxidation of LDL
 up arrowAntioxidant Protection Against...
 *Oxidation of LDL by...
down arrowMPO and RNS
 down arrowAntioxidant Protection of LDL...
 down arrowConclusions and Perspectives
 down arrowReferences
 
Nitric oxide (nitrogen monoxide, NO·) is an important regulatory molecule in vascular homeostasis.67 NO· is synthesized from L-arginine by the NADPH-dependent enzyme NOS (reaction 11), which is present as constitutive or inducible isoforms in several cell types, including endothelial cells and phagocytes, respectively.67 NO· itself is a relatively unreactive radical, but it readily interacts with molecular oxygen and superoxide radicals to produce a multitude of RNS.68 Potential sources of superoxide in the arterial wall include xanthine oxidase, cyclooxygenases, NAD(P)H oxidases, leakage from the mitochondrial respiratory chain,69 and, under certain conditions, NOS itself.70 Reaction 11 is as follows:

(11)

The reaction of NO· with oxygen [k3=6x106 (mol/L)-2 · s-1]71 is thought to lead to the formation of the nitrosating intermediate dinitrogen trioxide (N2O3, reaction 12), which can convert amines and thiols into, respectively, nitrosamines (reaction 13) and S-nitrosothiols (reaction 14), an important "storage" form of NO·. 68 Reducing agents, such as ascorbate, have been implicated in the rapid release of NO· from S-nitrosothiols (reaction 15).72 73 74 Reactions 12 through 15 are as follows:

(12)

(13)

(14)

(15)

The reaction of NO· with superoxide, a diffusion-limited radical-radical reaction [k2=1.9x1010 (mol/L)-1 · s-1],37 produces peroxynitrite and peroxynitrous acid (pKa 6.5, reaction 16).37 Peroxynitrite mediates oxidation and nitration reactions, although the intermediate(s) involved is as yet unidentified; this is possibly an activated isomer (*ONOOH) rather than the products of homolytic cleavage, ie, hydroxyl radical (·OH) and nitrogen dioxide (NO2·), as originally suggested.75 Peroxynitrite also reacts with carbon dioxide present in biological fluids [k2=5.8x104 (mol/L)-1 · s-1, reaction 17] to form reactive intermediates that can oxidize thiols and nitrate phenolic compounds, such as tyrosine.68 Reactions 16 and 17 are as follows:


In vitro experiments have shown that NO· produces very little, if any, modification of LDL particles, as determined by antioxidant consumption, lipid peroxidation, and electrophoretic properties.76 77 78 79 To the contrary, numerous studies have shown that NO· inhibits oxidation of LDL by copper ions, aqueous peroxyl radicals, lipoxygenases, and macrophages.76 80 81 82 83 84 85 86 One mechanism proposed for this antioxidant activity is the rapid reaction of NO· with lipid peroxyl radicals [k2=1.3x109 (mol/L)-1 · s-1) to form nonradical adducts, thus terminating the lipid peroxidation chain reaction.82

In the presence of oxygen or superoxide, NO· forms RNS (reactions 12 and 16) that can modify LDL in vitro (Table 2Down).76 77 78 The sydnonimine compound SIN-1, which breaks down to simultaneously generate NO· and superoxide, is often used as an in vitro source of peroxynitrite.87 Exposure of LDL to reagent peroxynitrite or SIN-1 results in depletion of vitamin E and ß-carotene88 89 and oxidation of lipids to lipid hydroperoxides, F2-isoprostanes, and oxysterols.76 77 90 91 Specific amino acid residues of apoB are also modified, including lysine, tryptophan, and cysteine92 93 ; modification of the lysine and cysteine residues is probably due to secondary reactions with lipid-derived aldehydes and radicals, respectively.93 Interestingly, bicarbonate was found to increase LDL oxidation by peroxynitrite (Table 2Down),88 92 most likely acting via reaction 17 above. These data suggest that bicarbonate-containing biological fluids could enhance peroxynitrite-mediated damage in vivo.


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  		Table 2. Modification of Specific LDL Targets by RNS


*    MPO and RNS
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMPO-Dependent Oxidation of LDL
 up arrowAntioxidant Protection Against...
 up arrowOxidation of LDL by...
 *MPO and RNS
down arrowAntioxidant Protection of LDL...
 down arrowConclusions and Perspectives
 down arrowReferences
 
Although 3-nitrotyrosine was initially used as a specific biomarker for peroxynitrite formation, this notion has been questioned recently, because MPO can also generate 3-nitrotyrosine via RNS (see below).94 95 NO· itself may modulate the activity of MPO because NO· reacts rapidly with the iron center of a number of heme-containing proteins and enzymes, resulting in either activation (eg, guanylate cyclase) or inactivation (eg, cytochrome P-450).68 Peroxynitrite has also been shown to interact with MPO, converting the enzyme to its compound II form.96

Recent work has indicated that peroxidases can convert nitrite, a physiological oxidation product of NO· (reaction 12),68 into RNS, most likely nitrogen dioxide (NO2·, reaction 18).97 The MPO/H2O2/nitrite system, presumably via NO2· production, modifies apoB and lipids in LDL (Table 2Up),98 99 resulting in increased uptake of the modified LDL by macrophages in culture.98 HOCl can also react with nitrite to form nitryl chloride (NO2Cl, reaction 19), which can chlorinate and nitrate tyrosine residues100 as well as oxidize LDL lipids and antioxidants (Table 2Up).101 However, nitryl chloride is unlikely to be formed from nitrite in vivo because of the presence of higher concentrations of alternative targets that are more reactive with HOCl, particularly thiols.102 Because RNS, such as NO2·, can be formed by MPO at physiological concentrations of nitrite and chloride ions,98 99 these could contribute to the formation of nitrated (lipo)proteins in vivo.94 95 It should be noted, however, that HOCl may cause the loss of 3-nitrotyrosine103 and thus result in an underestimation of the levels of this biomarker at sites where HOCl and RNS are being produced. Reactions 18 and 19 are as follows:


(19)


*    Antioxidant Protection of LDL Against RNS
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMPO-Dependent Oxidation of LDL
 up arrowAntioxidant Protection Against...
 up arrowOxidation of LDL by...
 up arrowMPO and RNS
 *Antioxidant Protection of LDL...
down arrowConclusions and Perspectives
 down arrowReferences
 
Vitamins C and E may protect LDL from atherogenic modifications mediated by RNS (Figure 2Up). A recent study showed that in vitro enrichment of LDL with vitamin E decreased or increased peroxynitrite-mediated lipid peroxidation, depending on the peroxynitrite to LDL ratio; at ratios <100:1, vitamin E acted as a pro-oxidant, whereas at ratios >200:1, vitamin E acted as an antioxidant.92 Vitamin E enrichment did not protect against peroxynitrite-induced lysine or tryptophan loss. In contrast, vitamin C inhibited protein and lipid oxidation and prevented consumption of vitamin E.92

Vitamin E is able to partially protect LDL lipids from oxidation by RNS generated by the MPO/H2O2/nitrite system,50 suggesting that {alpha}-tocopherol is acting as a chain-breaking antioxidant. In contrast, vitamin C scavenges peroxidase-generated nitrating species97 and effectively inhibits LDL oxidation by the MPO/H2O2/nitrite system.99 It should be noted, however, that the reaction of NO2· with vitamin C regenerates nitrite (reaction 20),97 suggesting that vitamin C is acting as a competitive inhibitor rather than a radical scavenger. Thus, based on the in vitro findings discussed above, vitamin C may be able to prevent the oxidation of LDL by reactive oxygen, chlorine, and nitrogen species generated in vivo; in contrast, vitamin E would be expected to be less effective and sometimes may act as a pro-oxidant in LDL. Reaction 20 is as follows:

(20)


*    Conclusions and Perspectives
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMPO-Dependent Oxidation of LDL
 up arrowAntioxidant Protection Against...
 up arrowOxidation of LDL by...
 up arrowMPO and RNS
 up arrowAntioxidant Protection of LDL...
 *Conclusions and Perspectives
down arrowReferences
 
As discussed in the present brief review, in vitro and in vivo studies have linked MPO and RNS to the oxidative, and thus atherogenic, modification of LDL. In light of the efficacy of plasma and interstitial fluid antioxidants, in particular, vitamin C, to quench reactive oxygen, chlorine, and nitrogen species,59 104 it appears that localized microenvironments in which antioxidant defenses have been overwhelmed may, in part, underlie the focal nature of atherosclerosis. Defining this intimal microenvironment, however, has proven difficult, as illustrated by the recent observations that relatively large amounts of vitamins C and E coexist with oxidized lipids in human atherosclerotic lesions.105 106

Future progress in defining the oxidative pathways involved in atherogenesis and evaluating effective strategies to limit oxidative damage are dependent on the resolution of conceptual and methodological problems. The limited number of stable and mechanism-specific biomarkers of oxidative damage has hindered the characterization of the precise pathway(s) involved in LDL oxidation in vivo. Additional impediments to research in the area include the ubiquitous nature of in vitro transition metal contamination, the continued use of relatively insensitive and nonspecific indices of LDL oxidation, eg, thiobarbituric reactive substances, and artifactual ex vivo formation of lipid hydroperoxides during LDL isolation and storage. In addition, the various forms of oxidized LDL generated in vitro need to be carefully characterized.

Understanding the specific pathway(s) of LDL oxidation in vivo is essential for evaluating whether a causal link exists between LDL oxidation and atherosclerotic lesion development, as the "oxidative modification hypothesis" of atherosclerosis postulates. Moreover, if such a link exists, understanding these pathways is requisite for the design of specific strategies to inhibit LDL oxidation and its pathobiological and clinical sequelae. If, as current evidence suggests, MPO and RNS are involved in atherogenesis through the oxidative modification of LDL, then dietary or supplemental vitamin C should prove useful as an antiatherogenic agent.


*    Acknowledgments
 
The authors are supported by grants from the US National Institutes of Health (HL-56170 to B.F.) and the American Heart Association (No. 9920420Z to A.C.C.). We thank Jace Carson for graphic design.

Received December 7, 1999; accepted April 14, 2000.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMPO-Dependent Oxidation of LDL
 up arrowAntioxidant Protection Against...
 up arrowOxidation of LDL by...
 up arrowMPO and RNS
 up arrowAntioxidant Protection of LDL...
 up arrowConclusions and Perspectives
 *References
 

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