Molecular action of vitamin E in lipoprotein oxidation: implications for atherosclerosis

The oxidation theory of atherosclerosis proposes that the oxidative modification of low-density lipoproteins (LDL) plays a central role in the disease. Although a direct causative role of LDL oxidation for atherogenesis has not been established, oxidized lipoproteins are detected in atherosclerotic lesions, and in vitro oxidized LDL exhibits putative pro-atherogenic activities. alpha-Tocopherol (alpha-TOH; vitamin E), the major lipid-soluble antioxidant present in lipoproteins, is thought to be antiatherogenic. However, results of vitamin E interventions on atherosclerosis in experimental animals and cardiovascular disease in humans have been inconclusive. Also, recent mechanistic studies demonstrate that the role of alpha-TOH during the early stages of lipoprotein lipid peroxidation is complex and that the vitamin does not act as a chain-breaking antioxidant. In the absence of co-antioxidants, compounds capable of reducing the alpha-TOH radical and exporting the radical from the lipoprotein particle, alpha-TOH exhibits anti- or pro-oxidant activity for lipoprotein lipids depending on the degree of radical flux and reactivity of the oxidant. The model of tocopherol-mediated peroxidation (TMP) explains the complex molecular action of alpha-TOH during lipoprotein lipid peroxidation and antioxidation. This article outlines the salient features of TMP, comments on whether TMP is relevant for in vivo lipoprotein lipid oxidation, and discusses how co-antioxidants may be required to attenuate lipoprotein lipid oxidation in vivo and perhaps atherosclerosis.     

A role for reduced coenzyme Q in atherosclerosis?

Substantial evidence implicates oxidative modification of low density lipoprotein (LDL) as an important event contributing to atherogenesis. As a result, the elucidation of the molecular mechanisms by which LDL is oxidized and how such oxidation is prevented by antioxidants has been a significant research focus. Studies on the antioxidation of LDL lipids have focused primarily on alpha-tocopherol (alpha-TOH), biologically and chemically the most active form of vitamin E and quantitatively the major lipid-soluble antioxidant in extracts prepared from human LDL. In addition to alpha-TOH, plasma LDL also contains low levels of ubiquinol-10 (CoQ10H2; the reduced form of coenzyme Q10). Recent studies have shown that in oxidizing plasma lipoproteins alpha-TOH can exhibit anti- or pro-oxidant activities for the lipoprotein's lipids exposed to a vast array of oxidants. This article reviews the molecular action of alpha-TOH in LDL undergoing "mild" radical-initiated lipid peroxidation, and discusses how small levels of CoQ10H2 can represent an efficient antioxidant defence for lipoprotein lipids. We also comment on the levels alpha-TOH, CoQ10H2 and lipid oxidation products in the intima of patients with coronary artery disease and report on preliminary studies examining the effect of coenzyme Q10 supplementation on atherogenesis in apolipoprotein E knockout mice.     

Low density lipoprotein (LDL), atherosclerosis and antioxidants

A crucial and causative role in the pathogenesis of atherosclerosis is believed to be the oxidative modification of low density lipoprotein (LDL). The oxidation of LDL involves released free radical driven lipid peroxidation. Several lines of evidence support the role of oxidized LDL in atherogenesis. Epidemiologic studies have demonstrated an association between an increased intake of dietary antioxidant vitamins, such as vitamin E and vitamin C and reduced morbidity and mortality from coronary artery diseases. It is thus hypothesized that dietary antioxidants may help prevent the development and progression of atherosclerosis. The oxidation of LDL has been shown to be reduced by antioxidants, and, in animal models, improved antioxidants may offer possibilities for the prevention of atherosclerosis. The results of several on going long randomized intervention trials will provide valuahle information on the efficacy and safety of improved antioxidants in the prevention of atherosclerosis. This review a evaluates current literature involving antioxidants and vascular disease, with a particular focus on the potential mechanisms.     

The ambivalence of vitamin E in atherogenesis.

The early events in atherogenesis might be due to the oxidation of low- density lipoprotein. The antioxidant vitamin E, therefore, has received much attention as a potential anti-atherogenic agent. Recent mechanistic studies of the early stage of lipoprotein-lipid oxidation show that the role of vitamin E in this process is not simply that of a classical antioxidant. Unless additional compounds are present, vitamin E can have antioxidant, neutral or pro-oxidant activity. This more complex function is reflected in the results of vitamin-E-intervention studies of atherosclerosis in animals and of controlled prospective trials on the incidence of cardiovascular disease in humans, which, overall, are inconclusive.     

The role of vitamin E in atherosclerosis

Epidemiological and biochemical studies infer that oxidative processes, including the oxidation of low-density lipoprotein (LDL), are involved in atherosclerosis. Vitamin E has been the focus of several large supplemental studies of cardiovascular disease, yet its potential to attenuate or even prevent atherosclerosis has not been realised. The scientific rationale for vitamin E supplements protecting against atherosclerosis is based primarily on the oxidation theory of atherosclerosis, the assumption that vitamin E becomes depleted as disease progresses, and the expectation that vitamin E prevents the oxidation of LDL in vivo and atherogenic events linked to such oxidation. However, it is increasingly clear that the balance between vitamin E and other antioxidants may be crucial for in vivo antioxidant protection, that vitamin E is only minimally oxidised and not deficient in atherosclerotic lesions, and that vitamin E is not effective against two-electron oxidants that are increasingly implicated in both early and later stages of the disease. It also remains unclear as to whether oxidation plays a bystander or a casual role in atherosclerosis. This lack of knowledge may explain the ambivalence of vitamin E and other antioxidant supplementation in atherosclerosis.     

Human neutrophils oxidize low-density lipoprotein by a hypochlorous acid-dependent mechanism: the role of vitamin C

Oxidatively modified low-density lipoprotein (LDL) has been strongly implicated in the pathogenesis of atherosclerosis. Peripheral blood leukocytes, such as neutrophils, can oxidize LDL by processes requiring superoxide and redox-active transition metal ions; however, it is uncertain whether such catalytic metal ions are available in the artery wall. Stimulated leukocytes also produce the reactive oxidant hypochlorous acid (HOCl) via the heme enzyme myeloperoxidase. Since myeloperoxidase-derived HOCl may be a physiologically relevant oxidant in atherogenesis, we investigated the mechanisms of neutrophil-mediated LDL modification and its possible prevention by the antioxidant ascorbate (vitamin C). As a sensitive marker of LDL oxidation, we measured LDL thiol groups. Stimulated human neutrophils (5x10(6) cells/ml) incubated with human LDL (0.25 mg protein/ml) time-dependently oxidized LDL thiols (33% and 79% oxidized after 10 and 30 min, respectively). Supernatants from stimulated neutrophils also oxidized LDL thiols (33% oxidized after 30 min), implicating long-lived oxidants such as N-chloramines. Experiments using specific enzyme inhibitors and oxidant scavengers showed that HOCl, but not hydrogen peroxide nor superoxide, plays a critical role in LDL thiol oxidation by neutrophils. Ascorbate (200 microM) protected against neutrophil-mediated LDL thiol oxidation for up to 15 min of incubation, after which LDL thiols became rapidly oxidized. Although stimulated neutrophils accumulated ascorbate during oxidation of LDL, pre-loading of neutrophils with ascorbate did not attenuate oxidant production by the cells. Thus, activated neutrophils oxidize LDL thiols by HOCl- and N-chloramine-dependent mechanisms and physiological concentrations of vitamin C delay this process, most likely due to scavenging of extracellular oxidants, rather than by attenuating neutrophil oxidant production.     

Anti-atherogenic effect of coenzyme Q10 in apolipoprotein E gene knockout mice

Oxidation of low-density lipoprotein (LDL) lipid is implicated in atherogenesis and certain antioxidants inhibit atherosclerosis. Ubiquinol-10 (CoQ10H2) inhibits LDL lipid peroxidation in vitro although it is not known whether such activity occurs in vivo, and, if so, whether this is anti-atherogenic. We therefore tested the effect of ubiquinone-10 (CoQ10) supplemented at 1% (w/w) on aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E-deficient (apoE-/-) mice fed a high-fat diet. Hydroperoxides of cholesteryl esters and triacylglycerols (together referred to as LOOH) and their corresponding alcohols were used as the marker for lipoprotein lipid oxidation. Atherosclerosis was assessed by morphometry at the aortic root, proximal and distal arch, and the descending thoracic and abdominal aorta. Compared to controls, CoQ10-treatment increased plasma coenzyme Q, ascorbate, and the CoQ10H2:CoQ10 + CoQ10H2 ratio, decreased plasma alpha-tocopherol (alpha-TOH), and had no effect on cholesterol and cholesterylester alcohols (CE-OH). Plasma from CoQ10-supplemented mice was more resistant to ex vivo lipid peroxidation. CoQ10 treatment increased aortic coenzyme Q and alpha-TOH and decreased the absolute concentration of LOOH, whereas tissue cholesterol, cholesteryl esters, CE-OH, and LOOH expressed per bisallylic hydrogen-containing lipids were not significantly different. CoQ10-treatment significantly decreased lesion size in the aortic root and the ascending and the descending aorta. Together these data show that CoQ10 decreases the absolute concentration of aortic LOOH and atherosclerosis in apoE-/- mice.     

Oxidized amino acids: culprits in human atherosclerosis and indicators of oxidative stress

Oxidized low-density lipoprotein (LDL) is implicated in atherogenesis, but the mechanisms that oxidize LDL in the human artery wall have proven difficult to identify. A powerful investigative approach is mass spectrometric quantification of the oxidized amino acids that are left in proteins by specific oxidation reactions. Comparison of these molecular fingerprints in biological samples with those produced in proteins by various in vitro oxidation systems can indicate which biochemical pathway has created damage in vivo. For example, the pattern of oxidized amino acids in proteins isolated from atherosclerotic lesions implicates reactive intermediates generated by myeloperoxidase, a major phagocyte enzyme. These intermediates include hypochlorous acid, tyrosyl radical, and reactive nitrogen species, each of which generates a different pattern of stable end products. Despite this strong evidence that myeloperoxidase promotes LDL oxidation in vivo, the antioxidant that has been tested most extensively in clinical trials, vitamin E, fails to inhibit myeloperoxidase pathways in vitro. Because the utility of an antioxidant depends critically on the nature of the pathway that inflicts tissue damage, interventions that specifically inhibit myeloperoxidase or other physiologically relevant pathways would be more logical candidates for the prevention of cardiovascular disease. Moreover, levels of oxidized amino acids in urine and plasma might reflect those in tissues and therefore identify individuals with high levels of oxidative stress. Trials with such subjects would seem more likely to uncover effective antioxidant therapies than trials involving the general population.     

Effect of vitamin E on aortic lipid oxidation and intimal proliferation after arterial injury in cholesterol-fed rabbits.

Oxidized low-density lipoproteins (LDL) are implicated in atherosclerosis. However, large-scale intervention studies designed to test whether antioxidants, such as vitamin E, can ameliorate cardiovascular disease have generated ambivalent results. This may relate to the fact that the mechanism whereby lipid oxidation is initiated in vivo is unknown and the lack of direct evidence for a deficiency of antioxidants in atherosclerotic lesions. Further, there is little evidence to suggest that vitamin E acts as an antioxidant for lipid peroxidation in vivo. Here we tested the antioxidant effect of dietary vitamin E (alpha-tocopherol) supplementation on intimal proliferation and lipid oxidation in balloon-injured, hypercholesterolemic rabbits. alpha-Tocopherol supplementation increased vascular content of alpha-tocopherol over 30-fold compared to nonsupplemented and alpha-tocopherol-deficient chows. Balloon injury resulted in oxidized lipid deposition in the aorta. Maximum levels of primary lipid oxidation products, measured as hydroperoxides of esterified lipid (LOOH) and oxidized linoleate (HODE), were 0.22 and 1.10 nmol/mg, representing 0.21 and 0.39% of the precursor molecule, respectively. Secondary lipid oxidation products, measured as oxysterols, were maximal at 5.60 nmol/mg or 1.48% of the precursor compound. Vascular HODE and oxysterols were significantly reduced by vitamin E supplementation. However, the intima/media ratio of aortic vessels increased with vitamin E supplementation, suggesting that the antioxidant promoted intimal proliferation. Thus, the study demonstrates a dissociation of aortic lipid oxidation and lesion development, and suggests that vitamin E does not prevent lesion development in this animal model.     

beta-Carotene inhibits the oxidative modification of low-density lipoprotein

Several lines of evidence indicate that oxidized LDL (Ox-LDL) may promote atherogenesis. Hence, the role of antioxidants in the prevention of LDL oxidation needs to be determined. beta-Carotene, in addition to being an efficient quencher of singlet oxygen, can also function as a radical-trapping antioxidant. Since previous studies have failed to show that beta-carotene inhibits LDL oxidation, we re-examined its effect on the oxidative modification of LDL. For these studies, LDL was oxidized in both a cell-free (2.5 microM Cu2+ in PBS) and a cellular system (human monocyte macrophages in Ham's F-10 medium). beta-Carotene inhibited the oxidative modification of LDL in both systems as evidenced by a decrease in the lipid peroxide content (thiobarbituric-acid-reacting substances activity), the negative charge of LDL (electrophoretic mobility) and the formation of conjugated dienes. By inhibiting LDL oxidation, beta-carotene substantially decreased its degradation by macrophages. beta-Carotene (2 microM) was more potent than alpha-tocopherol (40 microM) in inhibiting LDL oxidation. Thus, beta-carotene, like ascorbate and alpha-tocopherol, inhibits LDL oxidation and might have an important role in the prevention of atherosclerosis.     

Copper supplementation in humans does not affect the susceptibility of low density lipoprotein to in vitro induced oxidation (FOODCUE project)

The oxidative modification of low-density lipoprotein cholesterol (LDL) has been implicated in the pathogenesis of atherosclerosis. Copper (Cu) is essential for antioxidant enzymes in vivo and animal studies show that Cu deficiency is accompanied by increased atherogenesis and LDL susceptibility to oxidation. Nevertheless, Cu has been proposed as a pro-oxidant in vivo and is routinely used to induce lipid peroxidation in vitro. Given the dual role of Cu as an in vivo antioxidant and an in vitro pro-oxidant, a multicenter European study (FOODCUE) was instigated to provide data on the biological effects of increased dietary Cu. Four centers, Northern Ireland (coordinator), England, Denmark, and France, using different experimental protocols, examined the effect of Cu supplementation (3 or 6 mg/d) on top of normal Cu dietary intakes or Cu-controlled diets (0.7/1.6/6.0 mg/d), on Cu-mediated and peroxynitrite-initiated LDL oxidation in apparently healthy volunteers. Each center coordinated its own supplementation regimen and all samples were subsequently transported to Northern Ireland where lipid peroxidation analysis was completed. The results from all centers showed that dietary Cu supplementation had no effect on Cu- or peroxynitrite-induced LDL susceptibility to oxidation. These data show that high intakes (up to 6 mg Cu) for extended periods do not promote LDL susceptibility to in vitro-induced oxidation.     

The oxidative modification hypothesis of atherogenesis: an overview

The literature relating lipid and lipoprotein oxidation to atherosclerosis has expanded enormously in recent years. Papers on the “oxidative modification hypothesis” of atherogenesis have ranged from the most basic studies of the chemistry and enzymology of LDL oxidation, through studies of the biological effects of oxidized LDL on cultured cells, and on to in vivo studies of the effects of antioxidants on atherosclerosis in animals and humans. The data in support of this theory are mounting but many key questions remain unanswered. For example, while it is generally agreed that LDL undergoes oxidation and that oxidized LDL is present in arterial lesions, it is still not known how and where LDL gets oxidized in vivo nor which of its many biological effects demonstrable in vitro are relevant to atherogenesis in vivo. This brief review is not intended to be comprehensive but rather to offer a perspective and a context for this Forum. We discuss the strengths and weaknesses of each line of evidence, try to identify areas in which further research is needed, assess the relevance of the hypothesis to the human disease, and point to some of the potential targets for therapy.     

The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL

For more than two decades, there has been continuing evidence of lipid oxidation playing a central role in atherogenesis. The oxidation hypothesis of atherogenesis has evolved to focus on specific proinflammatory oxidized phospholipids that result from the oxidation of LDL phospholipids containing arachidonic acid and that are recognized by the innate immune system in animals and humans. These oxidized phospholipids are largely generated by potent oxidants produced by the lipoxygenase and myeloperoxidase pathways. The failure of antioxidant vitamins to influence clinical outcomes may have many explanations, including the inability of vitamin E to prevent the formation of these oxidized phospholipids and other lipid oxidation products of the myeloperoxidase pathway. Preliminary data suggest that the oxidation hypothesis of atherogenesis and the reverse cholesterol transport hypothesis of atherogenesis may have a common biological basis. The levels of specific oxidized lipids in plasma and lipoproteins, the levels of antibodies to these lipids, and the inflammatory/anti-inflammatory properties of HDL may be useful markers of susceptibility to atherogenesis. Apolipoprotein A-I (apoA-I) and apoA-I mimetic peptides may both promote a reduction in oxidized lipids and enhance reverse cholesterol transport and therefore may have therapeutic potential.     

Comparison of LDL trap assay to other tests of antioxidant capacity; effect of vitamin E and lovastatin treatment

Oxidized low density lipoprotein (LDL) has a major impact in the development of atherosclerosis. Risk for oxidative modification of LDL is usually determined indirectly by measuring the capability of LDL to resist radical insult. We compared three different methods quantifying the antioxidative capacity of LDL ex vivo in dyslipidemic patients with coronary heart disease. Plasma samples were obtained from two double-blinded cross-over trials. The duration of all interventions (placebo, lovastatin 60 mg/day, RRR-alpha-tocopherol 300 mg/day and lovastatin + RRR-alpha-tocopherol combined) was 6 weeks. The total radical capturing capacity of LDL (TRAP) in plasma was determined using 2,2-azo-bis(2,4-dimethyl-valeronitrile) (AMVN) -induced oxidation, and measuring the extinction time of chemiluminescence. TRAP was compared to the variables characterizing formation of conjugated dienes in copper-induced oxidation. Also the initial concentrations and consumption times of reduced alpha-tocopherol (alpha-TOH) and ubiquinol in AMVN-induced oxidation were determined. Repeatability of TRAP was comparable to that of the lag time in conjugated diene formation. Coefficient of variation within TRAP assay was 4.4% and between TRAP assays 5.9%. Tocopherol supplementation produced statistically significant changes in all antioxidant variables except those related to LDL ubiquinol. TRAP increased by 57%, the lag time in conjugated diene formation by 34% and consumption time of alpha-TOH by 88%. When data of all interventions were included in the analyses, TRAP correlated with the lag time (r = 0.75, p < 10(-6)), with LDL alpha-TOH (r = 0.50, p < 0.001) and with the consumption time of alpha-TOH (r = 0.58, p < 0.0001). In the baseline data, the associations between different antioxidant variables were weaker. TRAP correlated with the lag time (r = 0.55, p < 0.001) and alpha-TOH consumption time (r = 0.48, p < 0.05), and inversely with apolipoprotein Al (r = -0.51, p < 0.05). Lag time at the baseline did not correlate with ubiquinol or tocopherol parameters, or with any plasma lipid or lipoprotein levels analyzed. Lovastatin treatment did not significantly affect the antioxidant capacity of LDL. In conclusion, TRAP reflects slightly different properties of LDL compared to the lag time. Thus, LDL TRAP assay may complement the other methods used to quantify the antioxidant capacity of LDL. However, TRAP and the lag time react similarly to vitamin E supplementation.     

Oxidation of low-density lipoproteins: effect of antioxidant content,fatty acid composition and intrinsic phospholipase activity on susceptibility to metal ion-induced oxidation

The oxidative modification of low-density lipoprotein (LDL) may play an important role in atherogenesis. Our understanding of the mechanism of LDL oxidation and the factors that determine its susceptibility to oxidation is still incomplete. We have isolated LDL from 45 healthy individuals and studied the relationship between LDL fatty acid, vitamin E and β-carotene composition, intrinsic phospholipase A₂-like activity and parameters of LDL oxidation. LDL was exposed to a copper ion-dependent oxidising system and the kinetics of oxidation studied by monitoring formation of fatty acid conjugated dienes. The length of the lag phase of inhibited lipid peroxidation was measured as well as the rate of lipid peroxidation during the propagation phase. There was no significant correlation between LDL antioxidant vitamin or fatty acid composition and lag time to LDL oxidation. Oleic acid was negatively correlated with the rate of LDL oxidation (r = −0.41, P < 0.01) whilst linoleic acid was significantly correlated with the extent of LDL oxidation measured by the production of total dienes (r = 0.34, P < 0.05). Interestingly, LDL vitamin E content was positively correlated with both the rate (r = 0.28, P < 0.05) and extent of LDL oxidation (r = 0.43, P < 0.01). LDL isolated from this group of subjects showed significant intrinsic phospholipase-like activity. The phospholipase activity, whilst not correlated with lag time, was significantly correlated with both rate (r = 0.43, P < 0.01) and total diene production (r = 0.44, P < 0.01) of LDL oxidation. We conclude that antioxidant content, fatty acid composition and intrinsic phospholipase activity have little influence on the lag time of Cu-induced LDL oxidation. These components do however, significantly influence both the rate and extent of LDL oxidation, with increased vitamin E, linoleic acid content and phospholipase activity associated with faster and more extensive oxidation. The possible pro-oxidant effect of vitamin E has interesting implications for the postulated ‘protective’ effects of vitamin E on atherogenesis.     

Oxidation of low-density lipoprotein in atherosclerosis from basic biochemistry to clinical studies

Although it has been known for long time that atherosclerosis is associated with lipid deposition, only recently it has been accepted that the plasmatic concentration of cholesterol, especially LDL cholesterol, is a risk factor for atherosclerosis. However, chemically modified LDL, but not native LDL, is able to induce the formation of foam cells, the hallmark of atherosclerosis. LDL oxidation is likely to be the most important form of LDL modification in humans. In biochemical terms, LDL oxidation is a free radical driven chain reaction where polyunsaturated fatty acids are converted to lipid peroxides, which easily decompose to many products, including biologically active aldehydes. The assay of LDL oxidation in biological fluids is problematic; direct assays detect a product of LDL oxidation whereas indirect assays give an indicator of LDL oxidation susceptibility. In general, epidemiological studies support the concept that the level of plasmatic lipophilic antioxidants, tocopherols and carotenoids, is low in populations at increased risk for atherosclerosis. However, clinical trials based on vitamin E as antioxidant showed inconclusive results, suggesting that supplementation with vitamin E is not generically recommended for atherosclerotic patients. These results, however, do not contradict that oxidation of lipoprotein is involved in atherosclerosis; rather, this negative outcome raises a number of considerations such as the need for a reliable marker of lipoprotein oxidation in plasma and a more complete information about the physiological triggers of lipoprotein oxidation.     

Dynamics of Vitamin E Action against LDL Oxidation

Vitamin E acts as an important antioxidant against oxidative modification of low density lipoprotein (LDL) which is accepted as an initial event in the pathogenesis of atherosclerosis. In spite of the numerous studies and reports, the action and role of vitamin E have not been fully elucidated yet. In this brief overview, the dynamics of action of vitamin E as an antioxidant have been discussed and it is emphasized that the total antioxidant potency is determined by the relative importance of many competing reactions which is determined by the reactivities and concentrations of substrates, radicals and antioxidant and by physical factors of the environment.     

Free radical-mediated chain oxidation of low density lipoprotein and its synergistic inhibition by vitamin E and vitamin C

The oxidation of human low density lipoprotein (LDL) initiated by free radical initiator and its inhibition by vitamin E and water-soluble antioxidants have been studied. It was found that the kinetic chain length was considerably larger than 1, suggesting that LDL was oxidized by a free radical chain mechanism. Vitamin E acted as a lipophilic chain-breaking antioxidant. Water-soluble chain-breaking antioxidants such as ascorbic acid and uric acid suppressed the oxidation of LDL initiated by aqueous radicals but they could not scavenge lipophilic radicals within LDL to break the chain propagation. Ascorbic acid acted as a synergistic antioxidant in conjunction with vitamin E.     

Inhibition by a coantioxidant of aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E and low density lipoprotein receptor gene double knockout mice.

Antioxidants can inhibit atherosclerosis in animals, though it is not clear whether this is due to the inhibition of aortic lipoprotein lipid (per)oxidation. Coantioxidants inhibit radical-induced, tocopherol-mediated peroxidation of lipids in lipoproteins through elimination of tocopheroxyl radical. Here we tested the effect of the bisphenolic probucol metabolite and coantioxidant H 212/43 on atherogenesis in apolipoprotein E and low density lipoprotein (LDL) receptor gene double knockout (apoE-/-;LDLr-/-) mice, and how this related to aortic lipid (per)oxidation measured by specific HPLC analyses. Dietary supplementation with H 212/43 resulted in circulating drug levels of approximately 200 microM, increased plasma total cholesterol slightly and decreased plasma and aortic alpha-tocopherol significantly relative to age-matched control mice. Treatment with H 212/43 increased the antioxidant capacity of plasma, as indicated by prolonged inhibition of peroxyl radical-induced, ex vivo lipid peroxidation. Aortic tissue from control apoE-/-;LDLr-/- mice contained lipid hydro(pero)xides and substantial atherosclerotic lesions, both of which were decreased strongly by supplementation of the animals with H 212/43. The results show that a coantioxidant effectively inhibits in vivo lipid peroxidation and atherosclerosis in apoE-/-;LDLr-/- mice, consistent with though not proving a causal relationship between aortic lipoprotein lipid oxidation and atherosclerosis in this model of the disease.     

On the role of vitamin C and other antioxidants in atherogenesis and vascular dysfunction

Oxidative stress has been implicated as an important etiologic factor in atherosclerosis and vascular dysfunction. Antioxidants may inhibit atherogenesis and improve vascular function by two different mechanisms. First, lipid-soluble antioxidants present in low-density lipoprotein (LDL), including alpha-tocopherol, and water-soluble antioxidants present in the extracellular fluid of the arterial wall, including ascorbic acid (vitamin C), inhibit LDL oxidation through an LDL-specific antioxidant action. Second, antioxidants present in the cells of the vascular wall decrease cellular production and release of reactive oxygen species (ROS), inhibit endothelial activation (i.e., expression of adhesion molecules and monocyte chemoattractants), and improve the biologic activity of endothelium-derived nitric oxide (EDNO) through a cell- or tissue-specific antioxidant action. alpha-Tocopherol and a number of thiol antioxidants have been shown to decrease adhesion molecule expression and monocyte-endothelial interactions. Vitamin C has been demonstrated to potentiate EDNO activity and normalize vascular function in patients with coronary artery disease and associated risk factors, including hypercholesterolemia, hyperhomocysteinemia, hypertension, diabetes, and smoking.