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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.

After investigation of the contents and redox status of antioxidants and lipids in homogenates of both normal artery and atherosclerotic plaque, we now investigated them in the density fractions (very low, low, high, and protein fractions) of atherosclerotic plaque freshly obtained from carotid endarterectomy. By using the optimum extraction method (homogenization in carbonate buffer) and after density gradient ultracentrifugation, we isolated and characterized density fractions of plaque for apolipoproteins, size and contents of alpha-tocopherol (alpha-TOH), unesterified cholesterol, cholesteryl linoleate (Ch18:2), and hydroxides and hydroperoxides of Ch18:2, ie, Ch18:2-O(O)H. The distribution of apolipoproteins was more heterogeneous than that in the corresponding lipoproteins isolated from blood, and the majority of material in all plaque density fractions was present in large particles eluting in the void volume of gel-filtration columns. The content of unesterified cholesterol per unit of protein in low- and high-density fractions was 10-fold that in corresponding plasma lipoproteins. Low- and very-low-density fractions contained most of the lesion lipids and alpha-TOH. Two to five percent of lesion Ch18:2 was present as Ch18:2-O(O)H and distributed more or less equally among all density fractions, yet the content of alpha-TOH per unit of Ch18:2 was higher than that in corresponding plasma lipoproteins. These results demonstrate that alpha-TOH and oxidized lipids coexist in all lesion density fractions, further supporting the notion that large proportions of lipids in lipoproteins of advanced stages of atherosclerosis are oxidized. However, although not ruling it out, our results do not support the suggestion that advanced stages of atherosclerosis are associated with gross deficiencies in the lipoproteins' vitamin E content.

The 'oxidation theory' of atherosclerosis proposes that oxidation of low density lipoprotein (LDL) contributes to atherogenesis. Although little direct evidence for a causative role of 'oxidized LDL' in atherogenesis exists, several studies show that, in vitro, oxidized LDL exhibits potentially proatherogenic activities and lipoproteins isolated from atherosclerotic lesions are oxidized. As a consequence, the molecular mechanisms of LDL oxidation and the actions of alpha-tocopherol (alpha-TOH, vitamin E), the major lipid-soluble lipoprotein antioxidant, have been studied in detail. Based on the known antioxidant action of alpha-TOH and epidemiological evidence, vitamin E is generally considered to be beneficial in coronary artery disease. However, intervention studies overall show a null effect of vitamin E on atherosclerosis. This confounding outcome can be rationalized by the recently discovered diverse role for alpha-TOH in lipoprotein oxidation; that is, alpha-TOH displays neutral, anti-, or, indeed, pro-oxidant activity under various conditions. This review describes the latter, novel action of alpha-TOH, termed tocopherol-mediated peroxidation, and discusses the benefits of vitamin E supplementation alone or together with other antioxidants that work in concert with alpha-TOH in ameliorating lipoprotein lipid peroxidation in the artery wall and, hence, atherosclerosis.

15-Lipoxygenase has been implicated in the in vivo oxidation of low density lipoprotein (LDL) a process thought to be important in the origin and/or progression of human atherogenesis. We have suggested previously that oxidation of LDL's cholesteryl esters (CE) and phospholipids by soybean (SLO) or human recombinant 15-lipoxygenase (rhLO) can be ascribed largely to alpha-tocopherol (alpha-TOH)-mediated peroxidation (TMP). In this study we demonstrate that addition to LDL of unesterified linoleate (18:2), other free fatty acid (FFA) substrates, or phospholipase A2 (PLA2) significantly enhanced the accumulation of CE hydro(pero)xides (CE-O(O)H) induced by rhLO, whereas the corresponding CE and nonsubstrate FFA were without effect. The enhanced CE-O(O)H accumulation showed a dependence on the concentration of free 18:2 in LDL. In contrast, addition of 18:2 had little effect on LDL oxidation induced by aqueous peroxyl radicals or Cu2+ ions. Analyses of the regio- and stereoisomers of oxidized 18:2 in SLO-treated native LDL demonstrated that the small amounts of 18:2 associated with the lipoprotein were oxidized enzymically and within minutes, whereas cholesteryl linoleate (Ch18:2) was oxidized nonenzymically and continuously over hours. alpha-Tocopheroxyl radical (alpha-TO.) formed in LDL exposed to SLO was enhanced by addition of 18:2 or PLA2. With rhLO and 18:2-supplemented LDL, oxidation of 18:2 was entirely enzymic, whereas that of Ch18:2 was largely, though not completely, nonenzymic. The small extent of enzymic Ch18:2 oxidation increased with increasing enzyme to LDL ratios. Ascorbate and the reduced form of coenzyme Q, ubiquinol-10, which are both capable of reducing alpha-TO. and thereby preventing TMP, inhibited nonenzymic Ch18:2 oxidation induced by rhLO. Trolox and ascorbyl palmitate, which also inhibit TMP, ameliorated both enzymic and nonenzymic oxidation of LDL's lipids, whereas probucol, a radical scavenger not capable of preventing TMP, was ineffective. These results demonstrate that rhLO-induced oxidation of CE is largely nonenzymic and increases with LDL's content of FFA substrates. We propose that conditions which increase LDL's FFA content, such as the presence of lipases, increase 15-LO-induced LDL lipid peroxidation and that this process requires only an initial, transient enzymic activity.

Heme-containing (per)oxidases including horse radish peroxidase (HRP)/H2O2 have been shown to oxidatively modify isolated low-density lipoprotein (LDL) in vitro and oxidized LDL is implicated in the early events leading to atherosclerosis. The role of alpha-tocopherol (alpha-TOH) in the oxidation of LDL by HRP/H2O2 is unclear, although alpha-tocopheroxyl radical (alpha-TO.), which is formed during this process, can act as a chain transfer agent of lipid peroxidation in LDL. By combining HPLC and EPR spectroscopy, we hereby show that during HRP/H2O2-induced oxidation of human LDL: (i) the accumulation of cholesteryl linoleate hydroperoxides and hydroxides (CE-O(O)H) occurs concomitantly with the formation of alpha-TO. and consumption of alpha-TOH in the absence of other detectable organic (g approximately 2) radicals; (ii) the rates of alpha-TO. formation and subsequent decay reflect the rates of both alpha-TOH consumption and CE-O(O)H accumulation; (iii) CE-O(O)H accumulation is directly dependent on the level of endogenous alpha-TOH, and vitamin E supplementation results in increased lipid oxidizability; (iv) the inhibition of HRP activity by catalase plus urate results in a persistent alpha-TO. signal, the decay (t1/2 approximately 20 min) of which is accompanied by continued accumulation of CE-O(O)H, with complete cessation of lipid peroxidation upon loss of the chromanoxyl signal. These results demonstrate a direct correlation between alpha-TOH/alpha-TO. and the extent of HRP/H2O2-induced LDL lipid peroxidation, and that this type of oxidative modification can occur in the absence of g approximately 2 radicals other than alpha-TO.. Together, the results support a role for tocopherol-mediated peroxidation but not the involvement of a protein radical in the initiation of LDL lipid peroxidation induced by HRP/H2O2.

Various lipoxygenases (LO) oxidize low density lipoprotein (LDL) in vitro and 15-LO has been implicated in the development of atherosclerosis in vivo. Direct oxidation of phospholipids (PL) and cholesteryl esters (CE) by LO has been proposed as a mechanism whereby these enzymes cause or contribute to LDL lipid peroxidation. Herein we show that the extent to which recombinant human 15-LO (rhLO) caused peroxidation of LDL's esterified core and surface lipids depended on, and directly related to, the alpha-tocopherol (alpha-TOH) content of the lipoprotein. Thus, CE and PL of in vivo alpha-TOH-depleted LDL, isolated from a patient with familial isolated vitamin E deficiency, were resistant to oxidation by rhLO, whereas those in alpha-TOH-containing LDL from the same patient receiving vitamin E supplements readily oxidized. The extent to which rhLO caused oxidation of CE and PL directly and linearly correlated with LDL's content of vitamin E, as demonstrated by studies with in vitro alpha-TOH-depleted lipoproteins. The geometric isomers of oxidized cholesteryl linoleate formed in LDL during oxidation initiated by rhLO, matched those obtained during non-enzymic, peroxyl radical-initiated oxidation of LDL whilst alpha-TOH was present. Ascorbate, an efficient co-antioxidant for alpha-TOH, completely prevented rhLO-initiated oxidation of LDL's CE, but did not inhibit rhLO-mediated oxidation of unesterified linoleate. These results are inconsistent with direct oxidation of LDL esterified lipids by rhLO. Isolated LDL contained free fatty acids (FFA), and its exposure to rhLO caused a rapid formation of linoleate hydroperoxide. To reconcile these data, we propose that during rhLO-initiated oxidation of LDL, enzymic oxidation of FFA preceeds the oxidation of CE and PL, which occurs largely via a tocopherol-dependent process.