a-Tocopheryl hydroquinone is an efficient multifunctional inhibitor of radical-initiated oxidation of low density lipoprotein lipids (vitamin Eytocopherol-mediated peroxidationyco-antioxidationyatherogenesis)

As the oxidation of low density lipoprotein (LDL) lipids may be a key event in atherogenesis, there is interest in antioxidants as potential anti-atherogenic compounds. Here we report that a-tocopheryl hydroquinone (a-TQH2) strongly inhibited or completely prevented the (per)oxidation of ubiquinol-10 (CoQ10H2), a-tocopherol (aTOH), and both surface and core lipids in LDL exposed to either aqueous or lipophilic peroxyl radicals, Cu21, soybean lipoxygenase, or the transition metal-containing Ham’s F-10 medium in the absence or presence of human monocytederived macrophages. The antioxidant activity of a-TQH2 was superior to that of several other lipophilic hydroquinones, including endogenous CoQ10H2, which is regarded as LDL’s first line of antioxidant defence. At least three independent activities contributed to the antioxidant action of a-TQH2. First, a-TQH2 readily associated with LDL and instantaneously reduced the lipoprotein’s ubiquinone-10 to CoQ10H2, thereby maintaining this antioxidant in its active form. Second, a-TQH2 directly intercepted aqueous peroxyl radicals, as indicated by the increased rate of its consumption with increasing rates of radical production, independent of LDL’s content of CoQ10H2 and a-TOH. Third, a-TQH2 rapidly quenched a-tocopheroxyl radical in oxidizing LDL, as demonstrated directly by electron paramagnetic resonance spectroscopy. Similar antioxidant activities were also seen when a-TQH2 was added to high-density lipoprotein or the proteinfree Intralipid, indicating that the potent antioxidant activity of a-TQH2 was neither lipoprotein specific nor dependent on proteins. These results suggest that a-TQH2 is a candidate for a therapeutic lipid-soluble antioxidant. As a-tocopherylquinone is formed in vivo at sites of oxidative stress, including human atherosclerotic plaque, and biological systems exist that reduce the quinone to the hydroquinone, our results also suggest that a-TQH2 could be a previously unrecognized natural antioxidant. The oxidation of low density lipoprotein (LDL) is regarded as one of the early and key events in atherogenesis (1). As a result of the breakdown of oxidized lipids, LDL’s apolipoprotein B-100 (apoB) may become modified, and this can result in the uncontrolled cellular uptake of the lipoprotein, leading to the formation of lipid-laden ‘‘foam’’ cells (1, 2). Oxidized lipid component(s) may also be responsible for recognition of modified LDL, e.g., by the thrombospondin CD 36 receptor (3). In addition, oxidized LDL has many additional proatherogenic activities, so that inhibition of LDL lipid (per)oxidation might be beneficial and retard atherogenesis (4). Recently, we proposed a novel molecular mechanism of radical-initiated lipid peroxidation in isolated LDL (5–7) and extended it to other isolated lipoproteins and to lipoproteins in human plasma (8–10). This model, referred to as tocopherol-mediated peroxidation (TMP), predicts that a-tocopherol (a-TOH, biologically the most active form of vitamin E) present in lipoproteins will aid the ‘‘entry’’ of radical oxidants into the particle by acting as a phase-transfer agent. Once inside, the radical will be present predominantly as a-tocopheroxyl radical (a-TOz) that, under relatively mild oxidizing conditions, will initiate and propagate the formation of lipid hydroperoxides by acting as the lipid peroxidation chaincarrying species. This chain transfer activity of a-TOH is inhibited by either high rates of radical entry into the lipoprotein particle (resulting in radical-radical termination reactions) (6, 10) or the presence of suitable reductants capable of ‘‘exporting’’ the radical from the lipoprotein back into the aqueous compartment (11). Human blood plasma (12) and interstitial f luids (13) contain several such reductants, referred to as co-antioxidants (11). Of these, ubiquinol-10 (CoQ10H2) (14) and ascorbate (15) form the first line of nonproteinaceous antioxidant defence; in their presence, a-TOH efficiently protects the lipids in isolated LDL and plasma against in vitro oxidation (15–17). It is not known how and where LDL becomes oxidized during atherogenesis. However, oxidation most likely takes place in the subendothelial space where, at least at the late stages of the disease, the levels of oxidized lipids are approximately 105-fold higher (17) than in plasma of severely diseased subjects (18). Despite such high levels of oxidized lipids, human atherosclerotic plaque contains large amounts of ascorbate and a-TOH when expressed per protein and oxidizable lipid, respectively (17). This could suggest that lipid peroxidation in the intima proceeds via TMP, perhaps within microenvironments from which aqueous co-antioxidants such as ascorbate are excluded. In such a case, lipid-soluble coantioxidants that associate with LDL could conceivably be of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y947885-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: AAPH, 2,29-azobis(2-amidinopropane) dihydrochloride; AMVN, 2,29-azobis(2, 4-dimethylvaleronitrile); apoB, apolipoprotein B-100; CE-O(O)H, cholesteryl ester hydroperoxides plus cholesteryl ester hydroxides; CoQ10, ubiquinone-10; CoQ10H2, ubiquinol-10; 2,5-DTBHQ, 2,5-di-tert-butylhydroquinone; 3,5-DTBHQ, 2,6di-tert-butylhydroquinone; EPR, electron paramagnetic resonance; FIVE, vitamin E-deficient patient; HDL, high density lipoproteins; LDL, low density lipoprotein; MDM, human monocyte-derived macrophage; 2OCl, hypochlorite; ONOO2, peroxynitrite; PC-OOH, phosphatidyl choline hydroperoxides; a-PQH2, 1,4-di-hydroxy-2-(3hydroxy-3-methylbutyl)-3,5,6-trimethylbenzene; ROOz, peroxyl radical; SLO, soybean 15-lipoxygenase; TMP, tocopherol-mediated peroxidation; a-TOH, a-tocopherol; a-TOz, a-tocopheroxyl radical; a-TQH2, a-tocopheryl hydroquinone; a-TQ, a-tocopheryl quinone. *To whom reprint requests should be addressed: The Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, NSW, 2050, Australia; e-mail: [email protected].