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

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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.