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We have previously reported the detection of cholesteryl ester hydroperoxides, consisting mainly of cholesteryl linoleate hydroperoxides (Ch18:2-OOH), at nm levels in plasma from healthy humans (Y. Yamamoto and E. Niki, 1989. Biochem. Biophys. Res. Commun. 165: 988-993). To elucidate their production mechanism in vivo, we examined the distribution of Ch18:2-O(O)H regioisomers in blood plasma from nine healthy young subjects using a sequential method consisting of methanol/hexane extraction in the presence of antioxidant, reductant, and internal standard, solid phase extraction to remove unoxidized cholesteryl linoleate, purification by reversed-phase high-performance liquid chromatography (HPLC), and detection by normal phase HPLC. Furthermore, we confirm that little artifactual oxidation of cholesteryl linoleate occurred during analytical procedures indicated by the absence of oxidation products of cholesteryl 11Z,14Z-eicosadienoate (Ch20:2) when provided as an exogenous substrate to the experimental procedure. We detected nm levels of all free radical-mediated oxidation products, 13ZE-, 13EE-, 9-EZ-, and 9-EE-forms of Ch18:2-O(O)H, in blood plasma, whereas the 13ZE-isomer resulting from enzymatic 15-lipoxygenase oxidation was not evident as a major product. These results indicate that free radical chain oxidation of lipids occurs even in healthy young individuals.

Plasma glutathione peroxidase (GSHPx) has been suggested to reduce submicromolar levels of free fatty acid hydroperoxides and phosphatidylcholine hydroperoxides (PC-OOH), and therefore these hydroperoxides are undetectable in human blood plasma. The capacity for the reduction should be about 2.5 microM as the level of glutathione in human plasma is about 5 microM. However, 2 h of aerobic incubation of 58 microM PC-OOH in human plasma at 37 degrees C resulted in the formation of 36 microM phosphatidylcholine hydroxide (PC-OH). The presence of PC-OOH-reducing protein other than plasma GSHPx was suggested by the results. a) The same rates of PC-OOH decay and PC-OH formation were observed in both sera from rats with selenium-deficient and selenium-supplemented diet; b) the PC-OOH-reducing activity was observed only in the high molecular weight fraction but not in the low molecular weight fraction; and c) albumin did not work as a reducing substrate of plasma GSHPx. We have isolated two hydroperoxide-reducing protein fractions from human plasma by a sequential purification scheme, comprising an ammonium sulfate precipitation followed by sequential chromatography on anion exchange, hydrophobic interaction, and heparin columns. One of the proteins was identified as apolipoprotein A-I by N-terminal amino acid sequence analysis. Moreover, the hydroperoxide-reducing activity of one of the fractions was inhibited almost completely by the addition of anti-apolipoprotein A-I antibody. These findings demonstrate that apolipoprotein A-I in high density lipoprotein can reduce PC-OOH to PC-OH.