The development and clinical application of a new oxidative stress marker using glutathionylated protein

氧化应激参与动脉粥样硬化的发生和发展。活性氧是氧化应激的主要来源，它能修饰蛋白质的半胱氨酸残基，其中半胱氨基残基与谷胱甘肽之间建立二硫键，导致蛋白质的S-谷胱甘肽基化，这种修饰仍然是可逆的。因此，我们假设使用S谷胱甘肽基化蛋白的生物标志物是一个很好的反映动脉粥样硬化早期阶段的生物标志物。我们合成了针对载脂蛋白B100的S谷胱甘肽基化的抗体，载脂蛋白B100是低密度脂蛋白的一种成分。在有动脉粥样硬化危险因素的患者，如高血压、糖尿病和/或血脂异常的患者中，用该抗体进行免疫印迹分析的血清值与氧化修饰诱导的带负电荷的低密度脂蛋白亚组分相关。对颈动脉内膜切除切片的免疫组织化学分析显示，S凝集的载脂蛋白B100定位于靠近血管管腔的部位，而不是泡沫细胞聚集的部位。提示血清S谷氨酸化载脂蛋白B100可能是反映动脉粥样硬化相对早期的氧化应激标志物。

Oxidative stress contributes to the development and progression of atherosclerosis. Reactive oxygen species, major sources of oxidative stress, modifies protein cysteine residues, where a disulfide bond is established between a cysteinyl residue and glutathione, leading to the S-glutathionylation of protein, and this modification can be still reversible. Therefore, we hypothesized a biomarker using S-glutathionylated protein is a good one reflecting an early phase of atherosclerosis. We composed the antibody against S-glutathionylation of apoprotein B100, a component of low-density lipoprotein (LDL) cholesterol. The serum values by immunoblot analysis with this antibody were correlated with negatively charged LDL subfractions, which are induced by oxidative modification, in patients with atherogenetic risks, such as hypertension, diabetes mellitus, and/or dyslipidemia. Immunohistological analysis of carotid endarterectomy sections with the antibody showed S-glutationylated ApoB100 localized at the site close to the vascular lumen, but not the site of foamy cell accumulation. These suggested that serum S-glutationylated ApoB100 may be an oxidative stress marker reflecting the relatively early phase of atherosclerosis.