Tocopherol-mediated Peroxidation Research Articles

Tunneling in tocopherol-mediated peroxidation of 7-dehydrocholesterol.

The peroxidation of 7-dehydrocholesterol (7-DHC), a biosynthetic precursor to vitamin D3 and cholesterol, has been linked to the pathophysiology of Smith-Lemli-Optiz syndrome (SLOS), a devastating human disorder. In SLOS, 7-DHC plasma and tissue levels are elevated because of defects in the enzyme that convert it to cholesterol. α-Tocopherol can mediate the peroxidation of 7-DHC under certain circumstances and this prompted us to investigate the kinetic isotope effect (KIE) during this process. Thus, 9,14-d2-7-DHC was synthesized using a photochemical cyclization of deuterium-reinforced previtamin D3 (retro to its biosynthesis). Subsequently, we carried out co-oxidation of 9,14-h2-25,26,26,26,27,27,27-d7- and 9,14-d2-7-DHC in the presence of α-tocopherol under conditions that favor TMP. By monitoring the products formed from each precursor using mass spectrometry, the KIE for the hydrogen (deuterium) atom removal at C9 was found to be 21 ± 1. This large KIE value indicates that tunneling plays a role in the hydrogen atom transfer step in the tocopherol-mediated peroxidation of 7-DHC.

Unusual Kinetic Isotope Effects of Deuterium Reinforced Polyunsaturated Fatty Acids in Tocopherol-Mediated Free Radical Chain Oxidations

Substitution of -CD2- at the reactive centers of linoleic and linolenic acids reduces the rate of abstraction of D by a tocopheryl radical by as much as 36-fold, compared to the abstraction of H from a corresponding -CH2- center. This H atom transfer reaction is the rate-determining step in the tocopherol-mediated peroxidation of lipids in human low-density lipoproteins, a process that has been linked to coronary artery disease. The unanticipated large kinetic isotope effects reported here for the tocopherol-mediated oxidation of linoleic and linolenic acids and esters suggests that tunneling makes this process favorable.

Amiodarone inhibits tocopherol-mediated human lipoprotein peroxidation

It is unknown whether lipoprotein tocopherol-mediated peroxidation (TMP) is influenced by peculiar drug physicochemical properties such as hydrophobicity. Thus, we studied the effect of the extremely hydrophobic agent amiodarone on human non-HDL TMP. The drug, albeit devoid of specific radical-scavenging effects, inhibited TMP at therapeutic concentrations and with an efficiency similar to that of the physiological co-antioxidant ascorbic acid, showing indeed an IC 50 of 5 μM. A comparable efficiency was observed with human LDL, and with a pure LDL-VLDL mixture. TMP was also inhibited by other hydrophobic cationic amphiphiles without radical-scavenging activity, namely desethylamiodarone, chlorpromazine, clomipramine, promethazine, promazine, verapamil, bromhexine, propranolol, mepivacaine, metoprolol, tramadol and ranitidine, whose anti-TMP potency was far lower than that of amiodarone and related to drug hydrophobicity degree. Further, TMP was strongly inhibited by butylhydroxytoluene, a lipophilic radical scavenger. Hydrophobic acidic (diclofenac, indomethacin, ibuprofen and ketoprofen) or neutral ( n-hexane, anthracene, o-xylene and toluene) compounds could not instead inhibit TMP, indicating a stringent requirement for drug basicity in the pharmacological inhibition of TMP. Amiodarone effectiveness was lowered by lipoprotein α-tocopherol enrichment, suggesting some drug-α-tocopherol interaction and less lipid pharmacological protection at higher α-tocopheroxyl radical generation. Drug anti-TMP activity may so be related to electrostatic and hydrophobic interactions with lipoprotein α-tocopherol and lipid moiety, resulting in decreased radical phase transfer and lipid propensity to undergo radical-driven peroxidation. In conclusions, primarily drug basicity and then hydrophobicity are solely relevant to TMP inhibition. Amiodarone, at therapeutic concentrations, has anti-TMP properties, which could occur in the clinical setting.

Numerical revelation of the kinetic significance of individual steps in the reaction mechanism of methyl linoleate peroxidation inhibited by α-tocopherol

A kinetic model was constructed to describe the reactions involved in the oxidation of methyl linoleate (ML) inhibited by α-tocopherol (TH). The initial model of the reaction mechanism included 53 individual steps, which were numerically analyzed by the value method based on Hamiltonian systematization of kinetic equations. Good accord was obtained with experimental data at 40 and 50 °C. The dominant steps responsible for the antioxidant and pro-oxidant properties of TH in the process of ML peroxidation were revealed. Tocopherol-mediated peroxidation (TMP) and generation of alkoxyl radicals as a result of the reduction of hydroperoxides by TH or the decomposition tocopherol alkyl peroxides are the dominant reactions responsible for the pro-oxidant activities of α-tocopherol. The extreme behavior of reaction induction period in relation to TH initial concentration is related to the increase in the ratios of [tocopheroxyl radical]/[peroxyl radical] and the TMP rate/rate of termination by combination of tocopheroxyl and peroxyl radicals.

Vitamin E in Neurodegenerative Disorders: Alzheimer's Disease

Oxidative stress is important in the pathogenesis of Alzheimer's disease (AD). The brain contains high levels of oxidizable lipids that must be protected by antioxidants. Low concentrations of vitamin E, quantitatively the major lipophilic antioxidant in the brain, are frequently observed in cerebrospinal fluid (CSF) of AD patients, suggesting that supplementation with vitamin E might delay the development of AD. In a placebo-controlled trial, vitamin E (2000 IU/day, 2 years) slowed (-53%) functional deterioration in patients with moderate AD (Sano et al., N. Engl. J. Med. 336: 1216-1222, 1997). Recently, use of vitamin E and vitamin C supplements in combination was found to be associated with reduced prevalence (-78%) and incidence (-64%) of AD in elderly population (Zandi et al., Arch. Neurol. 61: 82-88, 2004). These results are consistent with the ability of the supplementation with vitamin E (400 IU/day, 1 month) to increase its levels in CSF (123%) and plasma (145%) of AD patients and, in combination with vitamin C (1000 g/day), to decrease the susceptibility of CSF lipoproteins (up to -32%) to in vitro oxidation (Kontush et al., Free Radic. Biol. Med. 31: 345-354, 2001). In addition, vitamin E reduced lipid peroxidation and amyloid deposition in a transgenic mice model of AD (Sung et al., FASEB J. 18: 323-325, 2004). Computer modeling of the influence of vitamin E on lipoprotein oxidation reveals that the vitamin develops antioxidative activity in CSF lipoproteins in the presence of physiologically relevant, low amounts of oxidants. By contrast, under similar conditions, vitamin E behaves as a pro-oxidant in plasma lipoproteins, consistent with the model of tocopherol-mediated peroxidation (Stocker, Curr. Opin. Lipidol. 5: 422-433, 1994). This distinction is related to major differences in the levels of vitamin E (50 nM vs. 30 microM) and oxidizable lipids (4 microM vs. 2.5 mM) between CSF and plasma, which result in major differences in oxidative conditions (per unit of vitamin E) between CSF and plasma in the presence of similar amounts of oxidants. Altogether, these data suggest that vitamin E may be effective against in vivo oxidation of CSF lipoproteins and brain lipids, and offer new perspectives in the treatment of AD and other neurodegenerative disorders.

Probabilistic kinetic model of slow oxidation of low-density lipoprotein: II. Experiments

Theoretical probabilistic kinetic model has been applied to describe the measurements of several oxidation markers as a function of time, during slow oxidation of low-density lipoprotein (LDL). It has been demonstrated that such a process could be described as tocopherol-mediated peroxidation (TMP), initiated and sustained by the action of copper ions, present in LDL in trace amounts. In that process concentration of α-tocopherol remains essentially unaltered. Tocopherol and copper ions act as catalysts, oscillating between the oxidized and reduced states. The fitting of the theoretical model to the experimental data resulted in determination of the numerical values for the kinetic parameters. It has been found that the parameter values used for the fitting of the data collected for a number of samples from various donors differ rather little. The kinetic chain length of 1.3 (in presence of co-antioxidants) and 2.9 (in the absence of co-antioxidants) is shorter than found by others. The difference probably comes from the much lower concentration of copper ions in our systems (about 0.1 ion per LDL particle).

Oxidant Stress as a Marker for Cardiovascular Events

During the past decade, numerous experimental and clinical studies have demonstrated that many common conditions predisposing to atherosclerosis, such as hypercholesterolemia, hypertension, diabetes, and smoking, are associated with a reduced vascular availability of nitric oxide (NO•). Nitric oxide not only produces vasodilation but also has potent antiatherogenic properties. These properties include inhibition of platelet aggregation, prevention of smooth muscle cell proliferation, reduction of lipid peroxidation, and inhibition of adhesion molecule expression. Thus, the loss of NO• observed in these various conditions not only alters vascular tone but also may explain in part why these conditions are risk factors for atherosclerosis. See p 2673 Given this apparent link between loss of nitric oxide and atherosclerosis, several groups have been interested in the concept that endothelium-dependent vasodilation, a surrogate for NO• bioavailability, may predict cardiovascular events. Indeed, Suwaidi et al1 followed 157 patients with mildly diseased coronary arteries for an average of 28 months and observed cardiac events only in the patients with the lowest tertile of coronary vasodilation to acetylcholine. Similarly, in a study of 147 patients, Schachinger et al2 used 3 different stimuli for endothelial release of NO: acetylcholine, cold pressor testing, and increased blood flow. The authors showed that responses to each of these stimuli were independent predictors of cardiovascular events during a follow-up period of ≈8 years. Perticone et al3 also demonstrated that endothelial dysfunction in the forearm circulation predicts cardiovascular events in hypertensive subjects. There have been several explanations for why the various risk factors impair endothelial function. One that has received substantial attention is increased production of reactive oxygen species within the vessel.4 In particular, superoxide (O2•-) reacts rapidly with NO•, resulting in the formation of the peroxynitrite anion and loss of NO• …

Quantitative studies on the peroxidation of human low-density lipoprotein initiated by superoxide and by charged and neutral alkylperoxyl radicals.

Rates of peroxidation of human LDL and rates of consumption of the LDL's alpha-tocopherol (TocH) have been measured at 37 degrees C. Peroxidation was initiated by radicals generated in the aerated aqueous phase at known rates by thermal decomposition of appropriate precursors: superoxide (O2(*-)/HOO(*)) from a hyponitrite and alkylperoxyls (ROO(*), two positively charged, one negatively charged and one neutral) from azo compounds. The efficiencies of escape from the solvent cage of the geminate pair of neutral carbon-centered radicals was found to be 0.1, but it was 0.5 for the three charged radicals, a result attributed to radical/radical Coulombic repulsion within the cage. All four alkylperoxyls initiated and terminated tocopherol-mediated peroxidation (TMP) with about equal efficiency and essentially all of these radicals that were generated were consumed in these two reactions. TMP is a radical chain process, and when initiated by the alkylperoxyls, the rate of LDL peroxidation was faster in the early stages while TocH was present than later, after all of this "antioxidant" had been consumed. In contrast, only about 3-4% of the generated superoxide radicals reacted in any measurable fashion with TocH-containing LDL at pH's from 7.6 to 6.5 and peroxidation was much slower than with a similar rate of generation of alkylperoxyls. After all the TocH had been consumed, LDL peroxidation was negligible at pH 7.6 and 7.4, but at pH 6.8 and 6.5, the peroxidation rates showed a large increase over the rates while the TocH had been present. That is, endogenous TocH behaves as an antioxidant in LDL subjected to attack by the physiologically relevant superoxide radical, whereas TocH behaves as a prooxidant in LDL subjected to attack by the probably far less physiologically important alkylperoxyls. Rates of LDL peroxidation initiated by superoxide increased as the pH was decreased, and the results are consistent with the initiation of peroxidation of fresh LDL occurring via H-atom abstraction from TocH by HOO(*) to form the Toc(*) radical and termination by reaction of O2(*-) with Toc(*), a process that occurs partly by addition leading to TocH consumption and partly by electron plus proton transfer leading to the regeneration of TocH.

Kinetic analysis of copper-induced peroxidation of HDL, autoaccelerated and tocopherol-mediated peroxidation

Comparison of the kinetic profiles of copper-induced peroxidation of HDL and LDL at different copper concentrations reveals that under all the studied experimental conditions HDL is more susceptible to oxidation than LDL. The mechanism responsible for HDL oxidation is a complex function of the copper/HDL ratio and of the tocopherol content of the HDL. At high copper concentrations, the kinetic profiles were similar to those observed for LDL oxidation, namely, relatively rapid accumulation of oxidation products, via an autoaccelerated, noninhibited mechanism, was preceded by an initial “lag phase.” Under these conditions, the maximal peroxidation rate (V max) of HDL and LDL depended similarly on the molar ratio of bound copper/lipoprotein. Analysis of this dependency in terms of the binding characteristics of copper to lipoprotein, yielded similar dissociation constant (K = 10 −6 M) but different maximal binding capacities for the two lipoproteins (8 Cu +2/HDL as compared to 17 Cu +2/LDL). Given the size difference between HDL and LDL, these results imply that the maximal surface density of bound copper is at least 2-fold higher for HDL than for LDL. This difference may be responsible for the higher susceptibility of HDL to copper-induced oxidation in the presence of high copper concentrations. At relatively low copper concentrations, the kinetic profile of HDL oxidation was biphasic, similar to but more pronounced than the biphasic kinetics observed for the oxidation of LDL lipids at the same concentration of copper. Our results are consistent with the hypothesis that the first phase of rapid oxidation occurs via a tocopherol-mediated-peroxidation (TMP) mechanism. Accordingly, enrichment of HDL with tocopherol resulted in enhanced accumulation of hydroperoxides during the first phase of copper-induced oxidation. Notably, the maximal accumulation during the first phase decreased upon increasing the ratio of bound copper/HDL. This behavior can be predicted theoretically for peroxidation via a TMP mechanism, in opposition to autoaccelerated peroxidation. The possible pathophysiological significance of these findings is discussed.

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. α-Tocopherol (α-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 α-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 α-TOH radical and exporting the radical from the lipoprotein particle, α-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 α-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.

Lipid Oxidation in Human Low-Density Lipoprotein Induced by Metmyoglobin/H2O2: Involvement of α-Tocopheroxyl and Phosphatidylcholine Alkoxyl Radicals

Metmyoglobin (metMb) and H(2)O(2) can oxidize low-density lipoprotein (LDL) in vitro, and oxidized LDL may be atherogenic. The role of alpha-tocopherol (alpha-TOH) in LDL oxidation by peroxidases such as metMb is unclear. Herein, we show that during metMb/H(2)O(2)-induced oxidation of native LDL, alpha-tocopheroxyl radical (alpha-TO(*)) and hydroperoxides and alcohols of cholesteryl esters [CE-O(O)H] and phosphatidylcholine [PC-O(O)H] accumulate concomitantly with alpha-TOH consumption. The ratio of accumulating CE-O(O)H to PC-O(O)H remains constant as long as alpha-TOH is present. Accumulation of CE-O(O)H is dependent on, and correlates with, LDL's alpha-TOH content, yet does not require preformed lipid hydroperoxides or H(2)O(2). This indicates that in native LDL alpha-TOH can act as a phase-transfer agent and alpha-TO(*) as a chain-transfer agent propagating LDL lipid peroxidation via tocopherol-mediated peroxidation (TMP). After alpha-TOH depletion, CE-O(O)H continues to accumulate, albeit at a slower rate than in the presence of alpha-TOH. This second phase of LDL oxidation is accompanied by depletion of PC-OOH, a rapid increase in the CE-O(O)H/PC-O(O)H ratio, formation of lipid-derived alkoxyl radicals and phosphatidylcholine hydroxides (PC-OH), and accumulation of a second organic radical, characterized by a broad singlet EPR signal. The latter persists for several hours at 37 degrees C. We conclude that metMb/H(2)O(2)-induced peroxidation of LDL lipids occurs initially via TMP. After alpha-TOH depletion, cholesteryl esters peroxidize at higher fractional rates than surface phospholipids, and this appears to be mediated at least in part via reactions involving alkoxyl radicals derived from the peroxidatic activity of metMb on PC-OOH.

Tocopherol-mediated peroxidation of lipoproteins: implications for vitamin E as a potential antiatherogenic supplement.

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.

Secondary radicals derived from chloramines of apolipoprotein B-100 contribute to HOCl-induced lipid peroxidation of low-density lipoproteins

Oxidation of low-density lipoproteins (LDL) is thought to contribute to atherogenesis. Although there is increasing evidence for a role of myeloperoxidase-derived oxidants such as hypochlorite (HOCl), the mechanism by which HOCl modifies LDL remains controversial. Some studies report the protein component to be the major site of attack, whereas others describe extensive lipid peroxidation. The present study addresses this controversy. The results obtained are consistent with the hypothesis that radical-induced oxidation of LDL's lipids by HOCl is a secondary reaction, with most HOCl consumed via rapid, non-radical reaction with apolipoprotein B-100. Subsequent incubation of HOCl-treated LDL gives rise to lipid peroxidation and antioxidant consumption in a time-dependent manner. Similarly, with myeloperoxidase/H2O2/Cl- (the source of HOCl in vivo), protein oxidation is rapid and followed by an extended period of lipid peroxidation during which further protein oxidation does not occur. The secondary lipid peroxidation process involves EPR-detectable radicals, is attenuated by a radical trap or treatment of HOCl-oxidized LDL with methionine, and occurs less rapidly when the lipoprotein was depleted of alpha-tocopherol. The initial reaction of low concentrations of HOCl (400-fold or 800-fold molar excess) with LDL therefore seems to occur primarily by two-electron reactions with side-chain sites on apolipoprotein B-100. Some of the initial reaction products, identified as lysine-residue-derived chloramines, subsequently undergo homolytic (one-electron) reactions to give radicals that initiate antioxidant consumption and lipid oxidation via tocopherol-mediated peroxidation. The identification of these chloramines, and the radicals derived from them, as initiating agents in LDL lipid peroxidation offers potential new targets for antioxidative therapy in atherogenesis.

Secondary radicals derived from chloramines of apolipoprotein B-100 contribute to HOCl-induced lipid peroxidation of low-density lipoproteins

Oxidation of low-density lipoproteins (LDL) is thought to contribute to atherogenesis. Although there is increasing evidence for a role of myeloperoxidase-derived oxidants such as hypochlorite (HOCl), the mechanism by which HOCl modifies LDL remains controversial. Some studies report the protein component to be the major site of attack, whereas others describe extensive lipid peroxidation. The present study addresses this controversy. The results obtained are consistent with the hypothesis that radical-induced oxidation of LDL's lipids by HOCl is a secondary reaction, with most HOCl consumed via rapid, non-radical reaction with apolipoprotein B-100. Subsequent incubation of HOCl-treated LDL gives rise to lipid peroxidation and antioxidant consumption in a time-dependent manner. Similarly, with myeloperoxidase/H2O2/Cl- (the source of HOCl in vivo), protein oxidation is rapid and followed by an extended period of lipid peroxidation during which further protein oxidation does not occur. The secondary lipid peroxidation process involves EPR-detectable radicals, is attenuated by a radical trap or treatment of HOCl-oxidized LDL with methionine, and occurs less rapidly when the lipoprotein was depleted of α-tocopherol. The initial reaction of low concentrations of HOCl (400-fold or 800-fold molar excess) with LDL therefore seems to occur primarily by two-electron reactions with side-chain sites on apolipoprotein B-100. Some of the initial reaction products, identified as lysine-residue-derived chloramines, subsequently undergo homolytic (one-electron) reactions to give radicals that initiate antioxidant consumption and lipid oxidation via tocopherol-mediated peroxidation. The identification of these chloramines, and the radicals derived from them, as initiating agents in LDL lipid peroxidation offers potential new targets for antioxidative therapy in atherogenesis.

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.

33] Assessment of prooxidant activity of vitamin E in human low-density lipoprotein and plasma

This chapter discusses the assessment of pro-oxidant activity of vitamin E in human low-density lipoprotein and plasma. The chapter discusses a series of simple tests that manipulate both the phase- and the chain-transfer activity of α-tocopherol (α-TOH) in low-density lipoprotein (LDL). While these tests can provide useful information on the mechanism of action of α -TOH in vitro , they do not give information on the action of vitamin E in vivo , nor propose that α -TOH acts as a prooxidant in vivo . The chapter presents various procedures for testing the pro-oxidant activity of α-TOH in lipoproteins and lipid emulsions. The methods described in the chapter allow investigations on the mechanism of in vitro lipoprotein lipid peroxidation and the way this process is affected by various (co-)antioxidants surrounding or associated with lipoproteins. The methods discussed also allow investigation of a possible role of tocopherol-mediated peroxidation (TMP) in cellular lipid peroxidation and antioxidation, particularly once analytical tools comparable to those for lipoproteins are available for the assessment of cellular lipid peroxidation.

Very slow autoxidation of low-density lipoprotein spares α-tocopherol

Low-density lipoprotein (LDL) in solution, supplemented with EDTA, is spontaneously oxidized at physiological temperature. In closed systems, three distinct phases of oxidation are present. In the lag period no change of any of the usually measured ‘markers’ of oxidation have been detected. In the second phase oxygen is consumed and subsequently the level of lipid hydroperoxides is increased. In the third phase, after consumption of oxygen, hydroperoxides decrease in concentration and the apoprotein-associated free radicals are formed. In the entire process α-tocopherol is conserved. The participation and preservation of α-tocopherol in the process is interpreted in terms of the tocopherol-mediated peroxidation. In the latter two phases α-tocopherol oscillates between the oxidized and reduced states.

Radical-induced lipoprotein and plasma lipid oxidation in normal and apolipoprotein E gene knockout (apoE–/–) mice: apoE–/– mouse as a model for testing the role of tocopherol-mediated peroxidation in atherogenesis

Exposure of plasma from apolipoprotein E gene knockout (apoE–/–) and control (CBA or C57BL/6J) mice plasma to a constant rate of aqueous peroxyl radicals (ROO.) resulted in the depletion of ascorbate, urate and α-tocopherol (α-TOH), with substantial and little lipid peroxidation, respectively. α-TOH levels were 3-times higher in plasma from apoE–/– than control mice and its addition enhanced the oxidizability of control mouse plasma. In apoE–/– mouse plasma, α-TOH was associated primarily with very low density lipoprotein (VLDL), whereas in plasma from control mice, the vitamin was located largely in high density lipoproteins. Oxidation of isolated lipoproteins by ROO. resulted in the accumulation of lipid hydroperoxides to an extent that reflected the plasma concentration and α-TOH content of the different lipoprotein fractions. Oxidation of 'plasma’ reconstituted from components of apoE–/– mice and/or human plasma showed that human and apoE–/– mouse lipoproteins peroxidized with similar kinetics, although the initiation of lipid peroxidation was greater in the presence of mouse than human lipoprotein-deficient plasma. Also, the chain length of lipid peroxidation in apoE–/– mouse plasma after ascorbate depletion appeared to be independent of the rate of ROO. generation. Together, these results show that the ROO. induced peroxidation of plasma lipoproteins in atherogenesis-susceptible apoE–/– mice exhibits some, though not all, features of tocopherol-mediated peroxidation (TMP). Therefore, apoE–/– mice may represent a suitable animal model to test a role for TMP in atherogenesis and the prevention of this disease by anti-TMP agents.—Neuzil, J., J. K. Christison, E. Iheanacho, J-C. Fragonas, V. Zammit, N. H. Hunt, and R. Stocker. Radical-induced lipoprotein and plasma lipid oxidation in normal and apolipoprotein E gene knockout (apoE–/–) mice: apoE–/– mouse as a model for testing the role of tocopherol-mediated peroxidation in atherogenesis.

Role of alpha-tocopheroxyl radical in the initiation of lipid peroxidation in human low-density lipoprotein exposed to horse radish peroxidase.

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.

α-Tocopherol Enrichment of High-Density Lipoproteins: Stabilization of Hydroperoxides Produced During Copper Oxidation

In the aim to study the effect of an in vitro enrichment of high-density lipoprotein (HDL) with α-tocopherol in alcoholic solution on a copper-induced peroxidation, we monitored several markers of lipid peroxidation ( α-tocopherol consumption, formation of conjugated dienes and of fatty acid hydroperoxides, production of thiobarbituric acid-reactive substances) and the integrity of apolipoprotein A-I. High-density lipoproteins (1.063 < d < 1.21) with a mean of 0.58 α-tocopherol molecules per HDL particle were enriched whith α-tocopherol in alcoholic solution to obtain an average of 3.7 and 21 α-tocopherol molecules per HDL particle. HDL oxidation with 5 μM CuSO 4 at 37°C resulted in the total disappearance of endogenous α-tocopherol after 2 h, but after 24 h about 19% of α-tocopherol remained in the most enriched HDL. In agreement with the tocopherol-mediated peroxidation, the formation of conjugated dienes and of fatty acid hydroperoxides was very fast and increased with α-tocopherol concentration, whereas TBARS production decreased. These results showed that α-tocopherol enrichment stabilized the production of hydroperoxides in HDL and decreased the formation of secondary oxidation products. These latter products are known for deleterious effects towards apolipoproteins. This could explain why we observed that the apolipoprotein A-I of the most enriched HDL was only slightly altered after incubation with CuSO 4. Copyright © 1996 Elsevier Science Inc.