Upston J M, Neuzil J, Witting P K, Alleva R, Stocker R
Biochemistry Unit, The Heart Research Institute, 145 Missenden Road, Camperdown NSW 2050, Australia.
J Biol Chem. 1997 Nov 28;272(48):30067-74. doi: 10.1074/jbc.272.48.30067.
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.
15-脂氧合酶与体内低密度脂蛋白（LDL）的氧化有关，这一过程被认为在人类动脉粥样硬化的发生和/或发展中起重要作用。我们之前曾提出，大豆15-脂氧合酶（SLO）或人重组15-脂氧合酶（rhLO）对LDL胆固醇酯（CE）和磷脂的氧化很大程度上可归因于α-生育酚（α-TOH）介导的过氧化作用（TMP）。在本研究中，我们证明向LDL中添加未酯化的亚油酸（18:2）、其他游离脂肪酸（FFA）底物或磷脂酶A2（PLA2）可显著增强rhLO诱导的CE氢（过）氧化物（CE-O(O)H）的积累，而相应的CE和非底物FFA则无此作用。CE-O(O)H积累的增强显示出对LDL中游离18:2浓度的依赖性。相反，添加18:2对过氧自由基水溶液或Cu2+离子诱导的LDL氧化影响很小。对SLO处理的天然LDL中氧化18:2的区域异构体和立体异构体的分析表明，与脂蛋白相关的少量18:2在数分钟内被酶促氧化，而亚油酸胆固醇酯（Ch18:2）则在数小时内被非酶促且持续氧化。暴露于SLO的LDL中形成的α-生育酚自由基（α-TO.）通过添加18:2或PLA2而增加。对于rhLO和添加了18:2的LDL，18:2的氧化完全是酶促的，而Ch18:2的氧化则很大程度上（尽管不是完全）是非酶促的。酶促Ch18:2氧化的程度随酶与LDL比例的增加而增加。抗坏血酸和辅酶Q的还原形式泛醇-10都能够还原α-TO.，从而防止TMP，它们抑制了rhLO诱导的非酶促Ch18:2氧化。生育三烯酚和抗坏血酸棕榈酸酯也抑制TMP，它们改善了LDL脂质的酶促和非酶促氧化，而普罗布考是一种不能防止TMP的自由基清除剂，没有效果。这些结果表明，rhLO诱导的CE氧化在很大程度上是非酶促的，并且随着LDL中FFA底物含量的增加而增加。我们提出，增加LDL中FFA含量的条件，如脂肪酶的存在，会增加15-LO诱导的LDL脂质过氧化，并且这一过程仅需要初始的、短暂的酶促活性。
