Purification of lipid hydroperoxide reducing proteins from blood plasma, and analysis of its clinical roles

脂质过氧化损伤内皮细胞，是动脉粥样硬化形成的危险因素。我们发现，人血浆中含有脂质氢过氧化物还原蛋白，而不是血浆谷胱甘肽过氧化物酶，一种主要的脂质氢过氧化物还原酶。在这项研究中，我们初步纯化的脂质过氧化氢减少蛋白从人血浆中使用硫酸铵分级，QAE琼脂糖凝胶，肝素琼脂糖凝胶和凝胶过滤柱层析的组合。氨基酸序列分析表明，载脂蛋白A1和B100具有脂质过氧化氢还原活性。利用合成的寡肽研究了这些载脂蛋白还原脂质过氧化物的分子机制，发现蛋氨酸残基被氧化为蛋氨酸亚砜，同时脂质过氧化氢被还原为相应的醇。为了阐明该活性的病理生理作用，我们开发了一种使用合成的1-棕榈酰-2-亚麻油-磷脂酰胆碱过氧化氢（PLPC-OOH）作为底物来测量血浆脂质过氧化氢还原活性的测定方法。应用此方法测定了52例孕妇、11例先兆子痫患者、18例男性不育患者、24例慢性肾功能衰竭患者和34例糖尿病患者的PLPC-OOH还原活性。子痫前期患者PLPC-OOH还原活性较正常妊娠降低90%，慢性肾功能衰竭和糖尿病患者PLPC-OOH还原活性较正常妊娠升高1.5倍。提示PLPC-OOH还原活性在清除血浆脂质过氧化氢中可能起重要作用，并可能参与这些疾病的发生发展。

Plasma lipid hydroperoxide has been known as a risk factor for atherogenesis, because lipid peroxide can injury the endothelial cells. We found that human blood plasma contains lipid hydroperoxide reducing proteins other than plasma glutathione peroxidase, a major lipid hydroperoxide reducing enzyme. In this study, we initially purified this lipid hydroperoxide reducing protein from human blood plasma by using the combinations of ammonium sulfate fractionation, QAE sepharose, Heparin sepharose and gel filtrations column chromatography. Amino acids sequence analysis indicated that apolipoprotein A1 and B100 possess the lipid hydroperoxide reducing activity. The molecular mechanism of reduction of lipid peroxide by those apolipoproteins were investigated by using the synthetic oligopeptides, and revealed that methionine residues are oxidized to methionine-sulfoxide with concomitant reduction of lipid hydroperoxide to corresponding alcohols. To clarify the patho-physiological roles of the activity, we have developed an assay method to measure plasma lipid hydroperoxide reducing activity using synthetic 1-palmitoyl-2-linoleoil-Phosphatidylcholine hydroperoxide (PLPC-OOH) as the substrate. By using this method, PLPC-OOH reducing activities were determined in the 52 pregnant women, 11 pre-eclamptic, 18 male infertile, 24 chronic renal failure, and 34 diabetes mellitus patients. PLPC-OOH reducing activity was decreased in the pre-eclamptic patients to 90% than that of normal pregnancy, and increased in the patients with chronic renal failure and diabetes mellitus to about 1.5 fold than that of healthy controls. These data indicated that PLPC-OOH reducing activity may have important roles in the elimination of the plasma lipid hydroperoxide, and also suggested the activity may involved in the development of those diseases.