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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:687-696.)
© 1996 American Heart Association, Inc.

Articles

Cosupplementation With Coenzyme Q Prevents the Prooxidant Effect of -Tocopherol and Increases the Resistance of LDL to Transition Metal–Dependent Oxidation Initiation

Shane R. Thomas; Jií Neuil; Roland Stocker

From the Biochemistry Group, the Heart Research Institute, 145 Missenden Rd, Camperdown, Sydney, NSW, 2050, Australia.

Correspondence to Dr Roland Stocker, The Heart Research Institute, 145 Missenden Rd, Camperdown, Sydney, NSW 2050, Australia. E-mail r.stocker@hri.edu.au.

	Abstract

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Abstract There is considerable interest in the ability of antioxidant supplementation, in particular with vitamin E, to attenuate LDL oxidation, a process implicated in atherogenesis. Since vitamin E can also promote LDL lipid peroxidation, we investigated the effects of supplementation with vitamin E alone or in combination with coenzyme Q on the early stages of the oxidation of isolated LDL. Isolated LDL was obtained from healthy subjects before and after in vitro enrichment with vitamin E (D--tocopherol, -TOH) or dietary supplementation with D--TOH (1 g/d) and/or coenzyme Q (100 mg/d). LDL oxidation initiation was assessed by measurement of the consumption of -TOH and cholesteryl esters containing polyunsaturated fatty acids and the accumulation of cholesteryl ester hydroperoxides during incubation of LDL in the transition metal–containing Ham's F-10 medium in the absence and presence of human monocyte-derived macrophages (MDMs). Native LDL contained 8.5±2 molecules of -TOH and 0.5 to 0.8 molecules of ubiquinol-10 (CoQ₁₀H₂, the reduced form of coenzyme Q) per lipoprotein particle. Incubation of this LDL in Ham's F-10 medium resulted in a time-dependent loss of -TOH with concomitant stoichiometric conversion of the major cholesteryl esters to their respective hydroperoxides. MDMs enhanced this process. LDL lipid peroxidation occurred via a radical chain reaction in the presence of -TOH, and the rate of this oxidation decreased on -TOH depletion. In vitro enrichment of LDL with -TOH resulted in an LDL particle containing sixfold to sevenfold more -TOH, and such enriched LDL was more readily oxidized in the absence and presence of MDMs compared with native LDL. In vivo -TOH–deficient LDL, isolated from a patient with familial isolated vitamin E deficiency, was highly resistant to Ham's F-10–initiated oxidation, whereas dietary supplementation with vitamin E restored the oxidizability of the patient's LDL. Oral supplementation of healthy individuals for 5 days with either -TOH or coenzyme Q increased the LDL levels of -TOH and CoQ₁₀H₂ by two to three or three to four times, respectively. -TOH–supplemented LDL was significantly more prone to oxidation, whereas CoQ₁₀H₂-enriched LDL was more resistant to oxidation initiation by Ham's F-10 medium than native LDL. Cosupplementation with both -TOH and coenzyme Q resulted in LDL with increased levels of -TOH and CoQ₁₀H₂, and such LDL was markedly more resistant to initiation of oxidation than native or -TOH–enriched LDL. These results demonstrate that oral supplementation with -TOH alone results in LDL that is more prone to oxidation initiation, whereas cosupplementation with coenzyme Q not only prevents this prooxidant activity of vitamin E but also provides the lipoprotein with increased resistance to oxidation.

Key Words: atherosclerosis • lipid hydroperoxides • macrophage • ubiquinone • vitamin E

	Introduction

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Several lines of evidence from both in vitro and in vivo studies strongly implicate oxidative modification of LDL, the major cholesterol-carrying lipoprotein in human blood plasma, as an important early event in atherogenesis.^{1 2 3} Oxidatively modified LDL can be taken up readily by macrophages via the scavenger receptor to form lipid-laden cells or "foam cells," a hallmark of early atherosclerotic lesions.⁴ Furthermore, oxidized LDL possesses an array of additional atherogenic properties,^{1 2} and the formation of lipid hydroperoxides and products derived from them plays a central role in this. Although the precise biochemical mechanisms governing the in vivo oxidative modification of LDL remain unknown, all cell types present in the atherosclerotic lesion (ie, endothelial cells,⁵ monocyte/macrophages,⁶ and lymphocytes⁷ ) are capable of oxidizing LDL in the presence of trace amounts of the redox-active transition metals iron and copper. LDL oxidation by unactivated cells^{5 8} has an absolute requirement for transition metals. A medium commonly used to support such cell-mediated oxidation is Ham's F-10 medium.

Because of its probable pathophysiological relevance, it is important to understand the mechanism(s) of LDL lipid oxidation initiation and antioxidant defenses present in and surrounding LDL. A number of factors determine the susceptibility of isolated LDL to oxidation initiation and the rate and extent of its lipid peroxidation. These include endogenous antioxidants, the level of preformed lipid hydroperoxides, and the content and type of oxidizable substrate (ie, polyunsaturated fatty acid, particularly those of the CE_PUFAs).^{9 10} Human LDL contains a number of antioxidants that include -TOH and CoQ₁₀H₂.¹¹ -TOH, biologically and chemically the most active form of vitamin E,¹² is the most abundant lipid-soluble antioxidant in LDL extracts¹³ and as such has generated the most interest with respect to research into "antioxidation" of LDL. An increased vitamin E intake has been shown to correlate negatively with the risk of heart disease in some^{14 15} though not all¹⁶ epidemiological studies. The former are in accordance with the "oxidation hypothesis" of atherosclerosis,¹ implying that LDL antioxidants are potential antiatherogenic compounds. Consistent with this, synthetic lipid-soluble antioxidants have been shown to slow the progression of atherosclerosis in animal models.^{17 18 19 20} However, not all compounds with antioxidant activity that can inhibit Cu²⁺-initiated LDL oxidation in vitro are antiatherogenic in vivo.²¹ A necessary though not sufficient criterion for an antioxidant to have antiatherogenic activity appears to be its ability to associate with LDL at sufficiently high concentration.²² Antioxidants may also affect LDL oxidation indirectly, via modulation of cellular activities.²³

It is commonly assumed that the antioxidant action of -TOH in LDL is the reason for its putative antiatherogenic effect. Studies have shown that enriching LDL with -TOH by dietary supplementation increases the protection of the lipoprotein lipids to in vitro copper-initiated^{24 25 26} or cell-facilitated²⁷ LDL oxidation. However, many studies have also documented a lack of significant correlation between -TOH content and oxidizability in native, unsupplemented LDL,^{9 10 24 27} raising doubts as to the efficacy of -TOH as an important antioxidant in isolated, intact LDL. Also, a beneficial effect of vitamin E supplementation on the development of atherosclerosis in animal models, other than its hypocholesterolemic activity, remains equivocal.²⁸

Most of the in vitro studies showing an antioxidant protective effect of -TOH supplementation^{24 25 26 27} on LDL oxidation used high concentrations of copper, whereby the lipoprotein was exposed to a high flux of radicals. The relevance of such strong oxidizing conditions and relatively "late" oxidation parameters measured in these studies (such as LDL oxidation after the complete consumption of -TOH, maximal accumulation of conjugated dienes, or relative electrophoretic mobility) to the in vivo extent of LDL oxidation is not clear. For example, LDL isolated from rabbit or human atherosclerotic lesions exhibits only modest signs of oxidative change,^{29 30} so that it may not be recognized by the scavenger receptor. Also, a recent survey showed that advanced human atherosclerotic lesions and normal arteries contained comparable amounts of -TOH, even though lesions but not normal arteries contained large concentrations of oxidized cholesteryl esters.³¹ Since "early" events are likely to precede "late" events in LDL oxidation, it appears important to understand the mechanism(s) of LDL lipid oxidation initiation.

Recent studies have shown that in the absence of reducing agents, such as CoQ₁₀H₂,^{11 32} ascorbate,³³ and bilirubin,³⁴ capable of reducing the -tocopheroxyl radical (-TO), -TOH can promote the initial stages of lipid peroxidation in isolated LDL.^{33 35 36} This process, referred to as TMP, is most pronounced under conditions of low radical flux, ie, when the -TOH of LDL is consumed slowly. Here, we examined the effect of enriching LDL with -TOH either alone or in combination with CoQ₁₀H₂ on the initial stages of LDL lipid oxidation initiated by Ham's F-10 medium in the absence and presence of human MDMs. This medium represents a transition metal–dependent oxidative stress substantially milder than that of the common Cu²⁺/LDL oxidation test and therefore more appropriate for the study of LDL oxidation initiation. We observed that under these oxidizing conditions, enrichment of LDL with -TOH alone afforded LDL with greater susceptibility to oxidation initiation, whereas coenrichment of the LDL with CoQ₁₀H₂ and -TOH not only prevented the prooxidant effect of vitamin E supplementation but also afforded LDL that was markedly more resistant to oxidation than native LDL.

	Methods

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Materials
EDTA, RPMI 1640 (powder), Hanks' balanced salt solution (Ca²⁺- and Mg²⁺-free), and PBS tablets were obtained from Sigma Chemical Co. D--TOH (335 mg) and coenzyme Q (50 mg) capsules for dietary supplementation were a generous gift from Blackmores Ltd. Potassium bromide was from British Drug House, and Ham's F-10 medium was from Gibco. Nanopure water (MODULAB) was used for all aqueous buffers, which were subsequently treated with Chelex-100 (Bio-Rad Laboratories) to chelate and hence remove contaminating amounts of redox-active transition metals. All organic solvents (HPLC quality) were from Mallinckrodt, Inc. Buffers and media used for cell isolation and culture (except for F-10 media) were sterile-filtered through Zetapor membranes (CUNO) and stored in heat-treated (250°C for 3 hours) glassware to minimize contamination with endotoxin (LPS), tested for regularly by use of a chromogenic Limulus amebocyte lysate test (Associates of Cape Cod/American Diagnostica); LPS found to be <50 pg/mL.

Isolation of Human Monocytes and Culture of MDMs
Isolation of human peripheral blood mononuclear cells from white blood cell concentrates (kindly provided by the Red Cross Blood Bank, NSW, Australia) and subsequent isolation of monocytes by centrifugal elutriation were carried out as described in detail previously.³⁷ Monocytes (1x10⁶ cells/well) were cultured in 12-well plates (Falcon) containing 1.5 mL/well RPMI 1640 medium supplemented with 10% human serum for 6 days so that they matured into MDMs.³⁷

Preparation of LDL
Human LDL (d1.06 g/mL) was isolated from anticoagulated (lithium heparin vacutainers, Becton Dickinson) fresh plasma obtained from nonfasted, healthy, and normolipidemic donors by 2 hours of ultracentrifugation (15°C) with the TL-100.4 rotor in a TL-100 benchtop centrifuge (Beckman).³⁸

In Vitro Enrichment of LDL With -TOH
For in vitro enrichment of LDL, freshly prepared human plasma was incubated at 37°C for 3 to 5 hours in the presence of added D--TOH (500 µmol/L final concentration; Eastman Kodak) dissolved in DMSO.³⁹ The final concentration of DMSO in the plasma was <3% (vol/vol). Control plasma received an equal amount of DMSO and was incubated in the same manner. The LDL was subsequently isolated from the plasma as described above. To remove potassium bromide and aqueous antioxidants (ie, ascorbic acid and uric acid), the enriched LDL was dialyzed against four changes of deoxygenated and chelated PBS buffer (10 mmol/L, pH 7.0). Chelation treatment of the buffer was omitted in the last buffer change. Alternatively, LDL was passed through two consecutive PD-10 gel-filtration columns (Pharmacia). These two procedures resulted in LDL, when isolated from a single donor, of similar oxidizability (data not shown). LDL was always freshly prepared and sterile-filtered (Acrodisk 0.2 µm, Gelman) before addition to the culture medium.

In Vivo Enrichment of LDL With -TOH and/or CoQ₁₀H₂
Nonfasted, healthy, normolipidemic, and nonsmoking human subjects (n=14; age, 21 to 39 years) were used for the in vivo supplementation studies. Some of these subjects (n=4) underwent multiple supplementation regimens. However, only after the subjects' LDL levels of -TOH and/or CoQ₁₀H₂ had returned to baseline did they undertake a new supplementation regimen. -TOH returned to baseline levels 4 weeks after 5 days of supplementation with -TOH, whereas CoQ₁₀H₂ levels returned to baseline after 1 week after 5 days of coenzyme Q supplementation (data not shown).

Before supplementation, a blood sample was withdrawn from each subject, the blood cells were removed immediately, and the resulting control plasma was stored under an atmosphere of argon at 4°C in two aliquots, one for 6 hours and the other for up to 6 days, after which time LDL was isolated as described above. Preliminary studies showed that there was no discernible difference in the oxidizability of control LDL isolated from freshly obtained plasma and tested immediately compared with LDL isolated and tested subsequent to storage of plasma under argon and at 4°C for up to 6 days (data shown in part in Figs 3 and 4).

View larger version (18K): [in this window] [in a new window]   Figure 3. Resistance of vitamin E–deficient LDL, isolated from a FIVE patient, to oxidation initiated by Ham's F-10 medium. Isolated LDL (final concentration, 0.1 mg/mL) prepared from the plasma of the FIVE patient either before (-TOH–deficient LDL; open symbols) or after (-TOH–supplemented LDL; closed symbols) dietary supplementation with -TOH was incubated in Ham's F-10 medium. At the time points indicated, aliquots were extracted and analyzed for -TOH (circles) and CE-OOH (squares).

View larger version (16K): [in this window] [in a new window]   Figure 4. Effect of dietary enrichment with either -TOH or coenzyme Q on LDL oxidation initiated by Ham's F-10 medium. LDL was incubated in Ham's F-10 medium at a final concentration of 0.1 to 0.2 mg/mL. Enriched LDL samples were obtained from subjects supplemented with either -TOH (A) (n=4) or coenzyme Q (B) for 6 hours (n=3) () or 5 days (n=8) (), and their oxidation was compared with the corresponding native LDL isolated from nonsupplemented plasma after 6 hours () or 5 days () of storage (see "Methods"). The results shown represent the mean±SEM of three to eight separate experiments, each carried out in duplicate. *Significant difference between lines of CE-OOH values of supplemented vs the corresponding control LDL.

After collection of a control blood sample, the subjects received supplements of the appropriate antioxidant(s). For the single-antioxidant supplementation regimen, 1 g -TOH (3 capsules of 500 IU or 335 mg D--tocopherol) or 100 mg coenzyme Q (2 capsules of 50 mg) was administered orally to subjects as a single daily dose for up to 5 days. For the cosupplementation experiments, 1 g -TOH was administered initially to subjects for the first day of supplementation, followed by a daily cosupplement of 1 g -TOH plus 100 mg coenzyme Q for the ensuing 5 days. During each type of supplementation, a blood sample was taken at 6 hours and at 5 days after the initial supplement administration, and antioxidant-enriched LDL was isolated from the plasma as described above. Plasma CoQ₁₀H₂ concentrations increase maximally 6 to 8 hours after a single oral dose of coenzyme Q.³² Coenzyme Q was used for the in vivo enrichment of LDL with CoQ₁₀H₂ because the latter is neither stable nor commercially available, and coenzyme Q is efficiently reduced to CoQ₁₀H₂ during its intestinal uptake (D. Mohr, Y. Umeda, T.G. Redgrave, R. Stocker, unpublished data, 1993) and subsequently becomes incorporated into LDL.³² No side effects or alterations in the LDL lipid profiles were noted in any of the human subjects undergoing -TOH and/or coenzyme Q supplementation. We are aware of the limitations of this supplementation protocol with respect to clinical intervention studies that require randomization of the subjects and the use of placebo controls.

In Vivo -TOH–Deficient LDL
Plasma was obtained from a patient (male, 28 years old) with FIVE syndrome from the University of Hamburg, Germany. This deficiency is characterized by a very low level of plasma -TOH unless the patient receives daily supplements of vitamin E.^{40 41} Initially, the patient was without vitamin E supplements for 5 consecutive days, after which time a first blood sample was taken, and this provided the -TOH–deficient sample. After this, the patient received supplementation with 400, 1200, and 1800 mg vitamin E for the ensuing 3 days, after which a second blood sample was taken that provided the -TOH–supplemented sample. Plasma from both samples was prepared immediately, supplemented with the lipoprotein cryopreservative sucrose (0.6% wt/vol), frozen, and shipped on dry ice to Sydney. The plasma was kept at -80°C until it was used. LDL was prepared from both the in vivo -TOH–deficient and -TOH–supplemented plasma samples as described above and used immediately. Such LDL did not contain detectable CE-OOH (detection limit, 1 to 5 pmol).

Assessment of the Oxidizability of LDL
LDL (0.5 to 0.8 mg protein/mL) (1 vol) was incubated in Ham's F-10 medium (5 vol) at 37°C in a humidified atmosphere (5% CO₂ in air) in the absence or presence of human MDMs. According to the manufacturer's information, Ham's F-10 medium contains 0.01 and 3 µmol/L copper and iron ions, respectively. Aliquots (100 to 300 µL) of cell-free medium or cell supernatants were withdrawn at various times up to 24 hours. For MDM experiments, 100 µL of LDL was added per well (12-well plate) containing 500 µL Ham's F-10 medium and 1x10⁶ cells. A separate well was used for each time point, since otherwise the ratio of cell number to LDL particles would increase with increasing numbers of aliquots removed.

LDL lipid oxidation initiation was assessed by the measurement of three different parameters: the loss of -TOH; the loss of cholesteryl linoleate and cholesteryl arachidonate, the major CE_PUFAs and hence lipid substrates for peroxidation in LDL; and the accumulation of the corresponding hydroperoxides (CE-OOH), the major lipid oxidation product formed initially in oxidizing LDL. Special care was taken to minimize inadvertent loss of the labile CoQ₁₀H₂ during the time between LDL isolation and onset of oxidation. Thus, gel filtration of the isolated LDL through two consecutive PD-10 columns was carried out under an argon atmosphere and using cold chelated and argon-flushed PBS as the eluant. LDL prepared in this way contained 50% to 80% of its total coenzyme Q, as CoQ₁₀H₂, as determined from an aliquot before addition to Ham's F-10 medium. This is in accordance with previous work,³² indicating that <30% loss of CoQ₁₀H₂ occurred during LDL isolation and preparation. To directly compare the oxidizability of antioxidant-enriched versus nonenriched (native) LDL from the same subject and under identical oxidation conditions, the native LDL was isolated from aliquots of control plasma stored for the appropriate length of time (ie, 6 hours or 5 days), and its susceptibility to oxidation was compared directly with that of -TOH and/or CoQ₁₀H₂-enriched LDL prepared from freshly obtained plasma after supplementation.

Determination of -TOH, Neutral Lipids, and CE-OOH by HPLC
Lipid-soluble antioxidants, neutral lipids, and CE-OOH were quantitatively extracted from aliquots of the LDL-containing supernatants as described.^{37 38} Lipid extracts were stored for up to 48 hours at -80°C before analysis. The hexane extracts of the LDL-containing supernatants were dried under vacuum and redissolved in isopropanol (200 µL) for analysis by various HPLC methods. The levels of -TOH, neutral lipids (mainly free cholesterol and CE_PUFA), and CE-OOH were determined by reverse-phase HPLC with electrochemical, UV, and postcolumn chemiluminescence detection, respectively, as described originally in Reference 42⁴² , with the modifications detailed in Reference 38³⁸ . Chemiluminescence detection is a very sensitive and selective method for analysis of CE-OOH, with a detection limit of 1 to 5 pmol, and as such provides an ideal method for analysis of the early events of LDL lipid oxidation. Lipid-soluble components were standardized internally against free cholesterol.

The method described for the use of -TOH determination³⁸ is principally suitable for the measurement of CoQ₁₀H₂ as well. We observed, however, that the method was not readily applicable to the transition metal–containing samples of this study as a result of carryover of transition metals onto the HPLC system, which caused inadvertent oxidation of CoQ₁₀H₂ on the column to an increasing extent with increasing numbers of samples injected. Therefore, accurate determination of CoQ₁₀H₂ on a routine basis (ie, other than that in LDL before its addition to Ham's F-10 medium) was not possible, because this would have required the use of a new HPLC column for each set of experiments. For both the in vivo CoQ₁₀H₂ enrichment and the CoQ₁₀H₂ plus -TOH enrichment experiments, the LDL samples of two (of the eight) subjects were analyzed with new HPLC columns, which allowed estimation of the time-dependent loss of CoQ₁₀H₂ during LDL oxidation.

Statistical Data Analysis
Repeated-measures ANOVA comparing lines of CE-OOH values of supplemented LDL samples and those of the corresponding native LDL samples was used to evaluate significant differences. Data were analyzed by SPSS Windows 6.0, with significance accepted at the P.01 level.

	Results

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Incubation in Ham's F-10 medium resulted in the time-dependent oxidation of LDL lipids indicated by alteration of all three oxidation parameters measured (Fig 1). That is, -TOH and CE_PUFAs were consumed simultaneously with the concomitant formation of CE-OOH. While -TOH was present, stoichiometric conversion of CE_PUFAs to CE-OOH was observed. This indicated that formation of CE-OOH can be used as a reliable measurement for LDL oxidation in F-10 medium and that no significant breakdown of CE-OOH occurred during this initial period of LDL oxidation. Significant lipid peroxidation occurred in the presence of -TOH, with 50 molecules of CE-OOH being formed per molecule of -TOH consumed, indicating that LDL peroxidation proceeded via a radical chain reaction despite the presence of -TOH. With -TOH depletion, the rates of CE_PUFA consumption and CE-OOH formation decreased. At the time point coinciding with complete -TOH consumption (at 20 hours of oxidation), 40% of endogenous CE_PUFAs were oxidized (Fig 1), even though the surface charge of the LDL was unaltered, as judged by the electrophoretic mobility of the lipoprotein in an agarose gel (data not shown). In the post–-TOH oxidation period examined, consumption of CE_PUFAs slightly exceeded the formation of CE-OOH (Fig 1), suggesting some degradation of CE-OOH and/or formation of oxidation products other than hydroperoxides.

View larger version (21K): [in this window] [in a new window]   Figure 1. Oxidation of human LDL in Ham's F-10 medium. LDL (final concentration, 0.1 mg protein/mL) was incubated in Ham's F-10 medium, and aliquots were extracted and analyzed for -TOH (), CE-OOH (), and CEPUFAs (). CEPUFA data represent the amount of cholesteryl linoleate plus cholesteryl arachidonate consumed. The results shown represent mean±SEM of four separate experiments, each using LDL from a single donor.

Incubation of LDL in the presence of MDMs accelerated lipid oxidation, as indicated by increased rates of -TOH consumption and accumulation of CE-OOH in LDL (Fig 2). In vitro enrichment of LDL with -TOH resulted in a lipoprotein that contained sixfold to sevenfold more -TOH. Compared with native LDL, such -TOH–enriched LDL was more susceptible to oxidation initiation by F-10 medium in both the absence and presence of MDMs as indicated by the approximately twofold increased rates of CE-OOH formation (Fig 2A). Similar to the situation in cell-free Ham's F-10 medium, CE_PUFAs were converted stoichiometrically into the corresponding CE-OOH in the presence of cells, as long as -TOH was detected in LDL (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 2. In vitro enrichment of LDL with -TOH increases the susceptibility of LDL to oxidation initiation by human MDMs cultured in Ham's F-10 medium. LDL (final concentration, 0.1 mg protein/mL), either -TOH–enriched (squares) or native (circles), was incubated in Ham's F-10 medium in the absence (open symbols) or presence (closed symbols) of human MDMs (1x106 cells/well). Aliquots were extracted and analyzed for CE-OOH (A) and -TOH (B). Note that the -TOH level is expressed as percent of the vitamin content present in each LDL sample at the beginning of the oxidation. The 100% values of -TOH were 15.7±4.9 µmol/L and 2.3±0.3 µmol/L for enriched and native LDLs, respectively. The results shown represent mean±SEM of four separate experiments carried out in duplicate using LDL from a single donor. *Significant difference between lines of CE-OOH values of supplemented vs corresponding control LDL.

In the absence of MDMs, the rates of -TOH consumption in native and -TOH–enriched LDL were 85 and 194 pmol·L^-1·s^-1 in Ham's F-10 medium, respectively. In the presence of MDMs, these rates increased to 166 and 334 pmol·L^-1·s^-1, respectively (Fig 2B). The rate of lipid peroxidation initiation (R_i) in the presence of a phenolic antioxidant is commonly determined by the length of strongly inhibited peroxidation, ie, the "lag phase." However, since there was no well-defined lag phase under the oxidizing conditions used here, the rate of -TOH consumption was used instead to determine R_i (R_i=-2d[-TOH]/dt), assuming that one -TOH molecule scavenges two radicals⁴³ and that the decay of -TOH is due to interaction with the initiating radicals only. R_i determined in this way was approximately twofold greater in -TOH–enriched than in native LDL, whether cells were present or not, in direct support of the notion that enrichment of the lipoprotein with the vitamin made it more susceptible to oxidation initiation.

For both enriched and native LDL in the absence and presence of cells, 20 to 30 molecules of CE-OOH were formed per molecule of -TOH consumed. From these and previous observations,³⁷ we conclude that LDL lipid peroxidation proceeded via a radical chain reaction in the presence of -TOH and that MDMs facilitated rather than caused the early stages of LDL oxidation in Ham's F-10. Others have shown that this oxidation is initiated by and dependent on the transition metals present in the medium^{5 8} and that the peroxidation-enhancing activity of cells may be due to the cellular release of reductants, such as thiols, that aid the redox cycling of the metal ions.⁴⁴ These reductants may facilitate formation of Cu⁺ and thereby enhance the efficacy of LDL lipid peroxidation initiation, because Cu⁺ is a stronger oxidant than Cu²⁺. Since MDMs appeared simply to enhance LDL oxidation initiated by components in Ham's F-10 medium, we carried out all subsequent experiments in Ham's F-10 medium in the absence of cells.

Having shown that -TOH enrichment increased the susceptibility of LDL to Ham's F-10–initiated LDL oxidation, we next examined the effect of -TOH deficiency on the oxidation of the lipoprotein. For this we isolated LDL from a FIVE patient. Before vitamin E supplementation, such LDL contained on average only 0.8 molecules of -TOH per LDL particle and was highly resistant to initiation of oxidation in Ham's F-10 medium (Fig 3). After dietary supplementation, the -TOH levels of the LDL increased to 12.5 -TOH molecules per LDL particle, and this afforded a readily oxidizable lipoprotein, indicating that -TOH is required for efficient Ham's F-10–initiated oxidation of peroxide-free LDL (Fig 3).

We next examined the effect of dietary supplementation of healthy subjects with either -TOH or coenzyme Q on Ham's F-10–initiated LDL oxidation. A potential problem with antioxidant supplementation studies is that in addition to the antioxidant(s), other parameters affecting the oxidizability of LDL (eg, its CE_PUFA content) may also be altered. To overcome this potential problem, we decided to compare the oxidizability of LDL from each individual before and 6 hours after a single oral dose of either -TOH or coenzyme Q as well as after 5 days of daily oral antioxidant supplementation. At the dosages used, supplementation for 5 days is known to result in maximal increases in LDL levels of -TOH⁴⁵ or CoQ₁₀H₂.³² To eliminate potential variations between different sets of oxidation experiments, we also simultaneously tested the oxidizability of native and supplemented LDLs. For this purpose, we stored plasma prepared from each subject immediately before the onset of antioxidant supplementation and isolated LDL from these stored plasma samples on the day of the oxidation experiments (see "Methods"). Importantly, there was no discernible difference in the oxidizability of native LDL isolated from freshly obtained plasma and tested immediately compared with that of native LDL isolated and tested subsequent to storage of plasma under argon and at 4°C for up to 6 days (data shown in part in Figs 4 and 5).

View larger version (16K): [in this window] [in a new window]   Figure 5. Cosupplementation with coenzyme Q efficiently protects LDL against the prooxidant effect of -TOH supplementation alone. LDL was incubated in Ham's F-10 medium at a final concentration of 0.1 to 0.2 mg protein/mL. The oxidation of enriched LDL samples from 8 subjects supplemented initially with -TOH alone for 6 hours () and then cosupplemented with -TOH and coenzyme Q for 5 days () was compared with that of native LDL isolated from plasma taken before supplementation and stored for 6 hours () or 5 days (). Note that the -TOH level is expressed as percentage of the vitamin content present in each LDL sample at the beginning of the oxidation. The 100% values for -TOH were 1.8±0.6 µmol/L for control LDL, 3.25±0.7 µmol/L for -TOH–enriched LDL, and 4.7±0.8 µmol/L for coenriched LDL. The results shown represent mean±SEM of (A) (n=8) and (B) (n=4) separate experiments carried out in duplicate. *Significant difference between lines of CE-OOH values of supplemented vs the corresponding control LDL.

In the case of -TOH, the dietary supplementation regimen resulted in LDL that contained 1.8 (15.3±3.5 molecules/LDL particle) and 2.6 (22.1±4.0 molecules/LDL particle) times more -TOH than native LDL (8.5±2 molecules/LDL particle) after 6 hours and 5 days, respectively. Increasing the content of -TOH afforded LDL that was significantly and increasingly more susceptible to peroxidation initiation in F-10 medium (Fig 4A). Thus, LDL isolated from subjects who received supplements for 5 days oxidized earlier and exhibited greater rates of CE-OOH formation than LDL obtained after a single oral dose of vitamin E.

Dietary supplementation with coenzyme Q resulted in LDL that contained 1.5 to 2.0 (or, on average, 1.0 to 1.5 molecules/LDL particle) and 3 to 4 (2.0 to 2.5 molecules/LDL particle) times more CoQ₁₀H₂ after 6 hours and 5 days of supplementation, respectively, than native LDL (0.5 to 0.8 molecules/LDL particle). The extent of this enrichment is consistent with published values.³² Wherever it was measured (see "Methods"), CoQ₁₀H₂ in control LDL was depleted within the initial 30 to 60 minutes of incubation in Ham's F-10 medium (data not shown). In LDL isolated from subjects who received supplements of coenzyme Q or coenzyme Q plus -TOH for 5 days, 2 to 4 hours was required for complete oxidation of CoQ₁₀H₂, and only minor oxidation ([CE-OOH] <0.5 µmol/L) was noted as long as CoQ₁₀H₂ was present (data not shown). CoQ₁₀H₂-enriched LDL was significantly better protected against Ham's F-10 oxidation initiation than native LDL, and the degree of oxidation resistance increased with the CoQ₁₀H₂ content of lipoprotein (Fig 4B). Interestingly, although at present this is not understood, LDL from subjects supplemented for 5 days with coenzyme Q alone contained 1.2 to 1.5 times more -TOH than native LDL, consistent with a previous report.³²

Having shown that enrichment with -TOH alone increased the susceptibility of LDL to Ham's F-10–initiated oxidation while enrichment with CoQ₁₀H₂ alone protected LDL from such oxidation, we examined the effect of cosupplementation with both antioxidants on the oxidizability of LDL in Ham's F-10 medium (see "Methods"). In agreement with the results shown in Fig 4, supplementation with -TOH alone for 6 hours again increased the -TOH content of LDL 1.8-fold and with this, its oxidizability in Ham's F-10 medium (Fig 5A). Cosupplementation resulted in LDL with 2.6 and 3 to 4 times more -TOH and CoQ₁₀H₂, respectively, than native LDL. Such coenriched LDL was highly resistant to oxidation, even though its -TOH content was greater than that of LDL enriched with -TOH alone (Fig 5A). Wherever examined, CE-OOHs were not detected as long as CoQ₁₀H₂ was present, indicating that the latter prevents the prooxidant activity of -TOH (data not shown). There was no discernible difference in the oxidizability of LDL enriched with CoQ₁₀H₂ alone or CoQ₁₀H₂ plus -TOH after 5 days, at least for the time period examined (compare Fig 4B with Fig 5A). The strongly diminished oxidizability of the cosupplemented LDL was also reflected in a decreased rate of -TOH consumption (an index of the initiation of peroxidation of LDL) (Fig 5B). Hence, LDL coenriched in both -TOH and CoQ₁₀H₂ was very resistant to the initiation of oxidation mediated by transition metals in Ham's F-10 medium.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study demonstrates for the first time that -TOH can promote the initial stages of LDL oxidation mediated by the transition metal–containing Ham's F-10 medium in the absence and presence of human MDMs. This prooxidant activity of -TOH is effectively prevented in the presence of CoQ₁₀H₂, thereby providing the lipoprotein with increased resistance to oxidative modification.

The kinetics of the early stages of LDL lipid peroxidation in Ham's F-10 medium and the effects of increasing -TOH or CoQ₁₀H₂ content(s) on it are reminiscent of the situation with peroxyl radical–initiated LDL oxidation.^{11 32 33 35 46} In particular, lipid peroxidation proceeded via a radical chain reaction in the presence of -TOH; the rate of LDL lipid oxidation was faster in the presence of -TOH than immediately after its consumption and increased with increasing concentrations of -TOH in LDL. Furthermore, enrichment with relatively small amounts of CoQ₁₀H₂ efficiently inhibited LDL oxidation. Together, these results strongly suggest that the initial stages of LDL oxidation in Ham's F-10 medium, in both the absence and the presence of human MDMs, proceed largely via TMP, the proposed novel model of molecular action of -TOH in isolated lipoproteins (Fig 6).³³ According to TMP, -TOH in the absence of suitable reductants that eliminate its one electron oxidation product, -TO, can promote LDL lipid peroxidation by acting as both a phase-transfer and a chain-transfer agent.

View larger version (23K): [in this window] [in a new window]   Figure 6. Model of TMP for LDL lipid oxidation and antioxidation. As the most reactive component of LDL, -TOH is oxidized first when LDL encounters an aqueous radical (eg, ROO or Cu2+). On reaction with the aqueous radical, -TOH is converted to -TO , which itself can undergo at least three different reactions resulting in regeneration of -TOH. Reaction pathways 1 and 2 represent antioxidant activity of -TOH (which requires coantioxidants), whereas pathway 3 represents prooxidant activity of -TOH. In pathways 1 and 2, -TO reacts with either LDL-associated CoQ10H2 or the aqueous ascorbate (AH-) or albumin-bound bilirubin (HSA-BR). The resulting ubisemiquinone radical (CoQ10-), formed due to the interaction of -TO with CoQ10H2, autooxidizes to CoQ10 inside LDL, and the resulting charged O2- escapes to the aqueous phase, where it decays to nonradical products (NRP). In the case of the aqueous coantioxidants, the putative bilirubin (BR) and ascorbyl (A-) radicals decay to aqueous albumin-bound biliverdin (HSA-BV) and NRP, respectively. In the absence of coantioxidants (AH-, HSA-BR, and CoQ10H2) and under conditions of low radical flux, the prooxidant activity of -TOH is apparent (pathway 3), where -TO is forced to react with a polyunsaturated lipid (LH), producing a carbon-centered lipid radical (L), with the estimated rate constant for this TMP rate-limiting reaction being 0.1 mol·L-1·s-1.33 L will add molecular oxygen (O2), forming a lipid peroxyl radical (LOO) that propagates lipid peroxidation by reacting with another -TOH, thereby regenerating -TO. Under conditions of high radical flux, pathway 4 becomes prevalent, which is characterized by increased levels of radical-radical termination reactions involving -TO. In pathway 4, -TO is eliminated by reaction with a second -TO (not shown) or oxidation-initiating aqueous radical, resulting in net -TOH consumption and LDL lipid antioxidation. It is proposed that this pathway predominates in the action of vitamin E in the commonly used high radical flux Cu2+/LDL oxidation system.

The phase-transfer activity refers to the ability of -TOH, the most reactive molecule present on the surface of LDL, to "pull" radical reactions from the aqueous phase into the lipoprotein particle. The phase-transfer activity of -TOH is best demonstrated by the fact that in vivo -TOH–deficient LDL (obtained from the FIVE patient) was highly resistant to initiation of oxidation, suggesting that -TOH is required for transition metal–mediated LDL oxidation in the absence of preformed lipid hydroperoxides. Also, R_i in LDL enriched with the vitamin was increased compared with that in native LDL (Fig 2). This means that the more -TOH LDL contains, the greater the phase-transfer activity of -TOH, and hence the more likely that aqueous radicals are drawn into the lipoprotein particle. We proposed earlier³³ that in peroxide-free LDL the peroxidation-initiating reaction by transition metals is that between Cu²⁺ and -TOH, producing Cu⁺ and -TO. Indeed, -TOH in LDL has been shown to react with Cu²⁺, resulting in the formation of Cu⁺ and -TO.^{47 48} Whether Cu⁺ or -TO mediates lipid peroxidation in LDL remains to be clarified. However, the fact that core and surface lipids of LDL exposed to Cu²⁺ peroxidize at comparable relative rates (J. Neuzil, S.R. Thomas, R. Stocker, unpublished data, 1995) favors -TO as the active compound, because the hydrophilic Cu⁺ would be expected to initially oxidize surface lipids to a greater extent than core lipids. Even if Cu⁺ rather than -TO were to oxidize lipids directly, -TO would still be expected to be generated subsequently (ie, after reaction of -TOH with the lipid peroxyl radical formed initially) and to represent the predominant radical, since it is the most stable of all possible radicals in an oxidizing LDL particle.^{33 36}

A reason for -TO being the lipid peroxidation–mediating species is that once produced, the -TO cannot readily escape from LDL because of its hydrophobicity. In the absence of suitable reducing agents (ie, coantioxidants) and under conditions of mild radical flux, the -TO is forced to react with LDL CE_PUFA, thereby initiating and propagating a radical chain reaction (pathway 3 in Fig 6) and thus exhibiting chain-transfer activity.^{33 35 36} The length of this chain reaction can be substantial in Ham's F-10 medium, since 20 to 50 molecules of CE-OOH were formed per molecule of -TOH consumed and hydroperoxides of phospholipid (which we did not measure in this study) are formed at relative rates comparable to those of cholesteryl esters.³⁵ Such long lipid peroxidation chains mean that substantial amounts (eg, 40% in Fig 1 and 50% in Fig 8 of Reference 33³³ ) of LDL CE_PUFAs can become oxidized in F-10 medium before total -TOH consumption. Despite oxidation of such large proportions of LDL lipids, the relative electrophoretic mobility of the lipoprotein particle does not change as long as -TOH is present. This is because in the presence of -TOH, lipid hydroperoxides are stable, so that significant amounts of degradation products that could modify apoprotein B-100 are not formed.

The radical chain reaction is inhibited strongly in the presence of even small amounts of CoQ₁₀H₂. In fact, wherever examined, CE-OOHs were not detected in samples that still contained CoQ₁₀H₂, and enrichment of the LDL with this antioxidant provided significantly increased protection, similar to the situation with peroxyl radical–initiated LDL oxidation.³² It has been proposed³⁶ that rather than direct radical scavenging, the strong antioxidant activity of CoQ₁₀H₂ is based on its ability to reduce and hence eliminate -TO (pathway 2 in Fig 6); the resulting superoxide anion radical (O₂ -) may react with another molecule of -TO or decay to nonradical products. As -TO propagates LDL lipid peroxidation (see above), each molecule of CoQ₁₀H₂ can terminate one to two free-radical chains (each with a length of 20 to 50 molecules of CE-OOH formed per -TOH molecule consumed), which may explain how even small amounts of this compound can have a large antioxidant effect. In accordance with this, recent studies have shown that the CoQ₁₀H₂ content of LDL correlated negatively with the susceptibility of the lipoprotein to the initial stages of copper-induced oxidation.^{10 49}

The proposed interaction between LDL -TOH and CoQ₁₀H₂ (pathway 2 in Fig 6) can also explain why cosupplementation with the latter inhibits the prooxidant activity of vitamin E. In fact, despite containing more -TOH, coenriched LDL was better protected against transition metal–initiated oxidation than native LDL. The conventional view of -TOH action predicts that up to one molecule of lipid hydroperoxide is formed for each molecule of vitamin E consumed.⁴³ In contrast, the TMP model predicts that the formation of lipid hydroperoxides is strongly suppressed as long as -TOH and a reductant capable of eliminating -TO are present. In addition to CoQ₁₀H₂, vitamin C and albumin-bound bilirubin are such reductants (pathway 1 in Fig 6), which have been called coantioxidants.⁵⁰ In other words, coantioxidants can make -TOH a more effective antioxidant for LDL.

Our results appear to be in contrast to a number of previous studies showing that supplementation of LDL with -TOH alone results in a lipoprotein that is more resistant to oxidation.^{24 25 27} However, most of these studies used the oxidation susceptibility test, in which isolated LDL is exposed to high concentrations of Cu²⁺ (ie, 12 to 16 Cu²⁺ molecules per LDL particle), and an inhibitory action of the vitamin is noted during the post–-TOH period, suggesting that under these strongly oxidizing conditions, later stages of LDL oxidation are inhibited, perhaps by oxidation products derived from -TOH. Under these conditions, LDL is exposed to a high radical flux (indicated by the rapid consumption of -TOH), in which radical termination reactions (pathway 4 in Fig 6) occur more frequently and therefore can effectively compete with the peroxidation chain-transfer activity (pathway 3 in Fig 6).³³ This is seen as an antioxidant activity of -TOH for LDL and could explain the increase in duration of the lag phase observed under these oxidizing conditions. However, pathway 3 predominates under conditions of low radical flux,³³ resulting in a net prooxidant activity of -TOH in LDL. In a separate study, we showed that for Cu²⁺-initiated LDL oxidation, -TOH switches from an antioxidant to a prooxidant, a copper-to-lipoprotein ratio of less than 3.⁵¹

The extent to which our results are physiologically relevant depends on the importance in vivo of the early stages of LDL oxidation, the radical flux to which LDL is exposed in the subendothelial space (where the in vivo oxidation of LDL is thought to take place), and the contents of -TOH and CoQ₁₀H₂ in lesion LDL. The same is true, of course, for the oxidizability parameters determined by the Cu²⁺/LDL test. The early stages in LDL oxidation studied here may be important for atherogenesis, since they most likely precede the processes leading to more severely oxidized forms of LDL with known proatherogenic properties. Although little is known about the in vivo radical fluxes in the subendothelial space, cosupplementation with both vitamin E and coenzyme Q would be advantageous under both high and low radical flux conditions, whereas lack of coantioxidants is clearly a disadvantage at low radical fluxes. Supplementation with coenzyme Q alone or in combination with -TOH also protects LDL against oxidation induced by high concentrations of copper, ie, Cu²⁺:LDL 10 (S.R. Thomas, J. Neuzil, R. Stocker, unpublished data, 1995).

If low radical fluxes were apparent in vivo, TMP and its prevention by coantioxidants like CoQ₁₀H₂ would become more relevant. This is consistent with the suggestion that "mild" as well as extensive LDL oxidation contributes to atherogenesis,⁵² that advanced human atherosclerotic lesions contain substantial amounts of both -TOH and lipid oxidation products,³¹ and that a low ratio of coenzyme Q to LDL may be a coronary risk factor.⁵³ For prevention of the initiation of LDL oxidation in the subendothelial space, it may be necessary for LDL -TOH to have access to CoQ₁₀H₂. Aqueous coantioxidants present in lesions, such as ascorbate,³¹ may be restricted in their ability to interact with LDL vitamin E (eg, as a result of formation of large complexes of the lipoprotein with extracellular matrix). Consistent with this, previous animal studies suggest that antioxidants need to associate with LDL to a substantial level to have antiatherogenic activity.²² We therefore suggest that coenzyme Q is a good candidate for a potential antiatherogenic compound, particularly since its supplementation can readily increase the CoQ₁₀H₂ content of LDL from <1 to >1 molecule/particle and hence provide every lipoprotein particle with a molecule of this efficient coantioxidant.

Some retrospective studies suggest that a high vitamin E intake is associated with lower risk of cardiovascular events.^{14 15} However, these studies may be confounded by a "self-selection" bias and unmeasured indexes of healthy lifestyle. In apparent contrast, a large prospective, randomized study¹⁶ has failed to demonstrate any benefit of antioxidants, including vitamin E, on cardiovascular outcome, although the dose and form of the vitamin administered in that study may not have been optimal. No studies to date have examined the effect of supplementation of coenzyme Q on atherosclerosis in humans and animal models. Our results demonstrate that during the initial phase of LDL oxidation initiated by low amounts of transition metals in both the absence and the presence of human MDMs, -TOH supplementation increases the oxidizability of LDL, whereas cosupplementation with coenzyme Q inhibits the prooxidant activity of -TOH. It may also be important to maximize the antioxidant efficacy of vitamin E for lipoprotein protection against in vivo oxidation. Therefore, we propose that future animal studies and/or clinical trials should consider supplementation with coenzyme Q, either alone or in combination with vitamin E, rather than supplementation with -TOH alone, as has been proposed.⁵⁴ Since LDL oxidation is thought to occur and be of greater importance during the initial stages of atherosclerosis, we feel it is important to test such a supplementation regimen in people without advanced clinical indexes of atherosclerosis.

	Selected Abbreviations and Acronyms

 

CE-OOH = cholesteryl ester hydroperoxides CEPUFA = major cholesteryl ester containing polyunsaturated fatty acids CoQ10H2 = ubiquinol-10 FIVE = familial isolated vitamin E deficiency HPLC = high-performance liquid chromatography MDM = human monocyte–derived macrophage Ri = rate of lipid peroxidation initiation TMP = tocopherol-mediated peroxidation -TOH = -tocopherol -TO = -tocopheroxyl radical

	Acknowledgments

 
This work received support from the National Health and Medical Research Council of Australia, grant number 940915 to Dr Stocker. We thank Drs A. Kohlschütter, B. Finckh, and A. Kontush of the Department of Pediatrics, University of Hamburg, Germany, for providing us with the FIVE patient plasma. We also thank Prof Roger Dean and Dr David Celermajer for critically reading the manuscript and partaking in many helpful discussions, Dr Mark Adams for his help with the statistical analyses, and Blackmores Ltd (Sydney, Australia) for their financial support and generous donation of -tocopherol and coenzyme Q capsules.

Received April 4, 1995; accepted January 4, 1996.

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