Abstract

Many cholesterol-laden foam cells in atherosclerotic lesions are macrophages and much of their cholesterol is present in their lysosomes and derived from low density lipoprotein (LDL). LDL oxidation has been proposed to be involved in the pathogenesis of atherosclerosis. We have shown previously that LDL can be oxidised in the lysosomes of macrophages. α-Tocopherol has been shown to inhibit LDL oxidation in vitro, but did not protect against cardiovascular disease in large clinical trials. We have therefore investigated the effect of α-tocopherol on LDL oxidation at lysosomal pH (about pH 4.5). LDL was enriched with α-tocopherol by incubating human plasma with α-tocopherol followed by LDL isolation by ultracentrifugation. The α-tocopherol content of LDL was increased from 14.4 ± 0.2 to 24.3 ± 0.3 nmol/mg protein. LDL oxidation was assessed by measuring the formation of conjugated dienes at 234 nm and oxidised lipids (cholesteryl linoleate hydroperoxide and 7-ketocholesterol) by HPLC. As expected, LDL enriched with α-tocopherol was oxidised more slowly than control LDL by Cu²⁺ at pH 7.4, but was not protected against oxidation by Cu²⁺ or Fe³⁺ or a low concentration of Fe²⁺ at pH 4.5 (it was sometimes oxidised faster by α-tocopherol with Cu²⁺ or Fe³⁺ at pH 4.5). α-Tocopherol-enriched LDL reduced Cu²⁺ and Fe³⁺ into the more pro-oxidant Cu⁺ and Fe²⁺ faster than did control LDL at pH 4.5. These findings might help to explain why the large clinical trials of α-tocopherol did not protect against cardiovascular disease.

摘要

动脉粥样硬化病变中的许多载有胆固醇的泡沫细胞是巨噬细胞，它们的大部分胆固醇存在于溶酶体中并来源于低密度脂蛋白 (LDL)。已提出低密度脂蛋白氧化与动脉粥样硬化的发病机制有关。我们之前已经表明 LDL 可以在巨噬细胞的溶酶体中被氧化。α-生育酚已被证明可在体外抑制 LDL 氧化，但在大型临床试验中并不能预防心血管疾病。因此，我们研究了 α-生育酚在溶酶体 pH 值（约 pH 值 4.5）下对 LDL 氧化的影响。通过将人血浆与 α-生育酚一起孵育，然后通过超速离心分离 LDL，从而使 LDL 富含 α-生育酚。低密度脂蛋白的 α-生育酚含量从 14.4 ± 0.2 增加到 24.3 ± 0.3 nmol/mg 蛋白质。通过在 234 nm 处测量共轭二烯和氧化脂质（胆固醇亚油酸酯氢过氧化物和 7-酮胆固醇）的形成来评估 LDL 氧化。正如预期的那样，富含 α-生育酚的 LDL在 pH 7.4 时被 Cu ²⁺氧化的速度比对照 LDL 慢，但不能防止 Cu ²⁺或 Fe ³⁺氧化或在 pH 4.5 时使用低浓度的 Fe ²⁺（有时在 pH 4.5 时被 α-生育酚与 Cu ²⁺或 Fe ³⁺氧化得更快）。在 pH 4.5 时，富含 α-生育酚的 LDL比对照 LDL 更快地将 Cu ²⁺和 Fe ³⁺还原为更促氧化的 Cu ⁺和 Fe ²⁺。这些发现可能有助于解释为什么 α-生育酚的大型临床试验不能预防心血管疾病。