Development of quantification method for lipid hydroperoxides inbiological tissues

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A chemiluminescence-high performance liquid chromatography (CL-HPLC) system was developed and applied for the hydroperoxide-specific determination of phosphatidylcholine hydroperoxide (PCOOH), phosphatidylethanolamine hydroperoxide (PEOOH), triacylglycerol hydroperoxide (TGOOH), squalene hydroperoxide (SQOOH)and 8a-hydroperoxy tocopherone (TocOOH) in biological tissues such as human blood plasma, plasma lipoproteins, erythrocytes, cultured diploid cells, and liver, brain, heart, lung of the rodents.The method involves separation of lipid hydroperoxide in each lipid class from total lipids of the biological tissues with HPLC column and post-column detection of hydroperoxide-dependent chemiluminescence of lipid hydroperoxides. The chemiluminescence is produced by luminol oxidation during a reaction of hydroperoxide and cytochrome c-heme. The high specificity for hydroperoxide group enables a sensitive assay for a wide range of hydroperoxides, with the detection limit of 1 picomole of hydroperoxide-O_2. By use of this method, the presence of phospholipid hydroperoxides in human blood plasma and erythrocytes is confirmed quantitatively.