Determination of phospholipid hydroperoxides in human blood plasma by a chemiluminescence-HPLC assay

Abstract

A chemiluminescence-high performance liquid chromatography (CL-HPLC) system was developed (Miyazawa, T. et al., Anal. Lett., 20, 915-925, 1987) and applied for the hydroperoxide-specific determination of phosphatidylcholine hydroperoxide (PCOOH) in biological tissues such as human blood plasma (Miyazawa, T. et al., Anal Lett 21:1033-1044, 1988; J. Biochem. 103:744-746; 1988). This system involves separation of phosphatidylcholines from plasma total lipids with normal phase silica gel HPLC and post-column detection of hydroperoxide-dependent chemiluminescence of PCOOH. The chemiluminescence is produced by luminol oxidation during a reaction of hydroperoxide and cytochrome c-heme. The high specificity for hydroperoxide base enables a sensitive assay for a large range of PCOOH, with the detection limit of 10 picomole of hydroperoxide-O2. By use of this assay system, the presence of PCOOH in human blood plasma is confirmed quantitatively. The PCOOH concentration of healthy plasma is in the range below 10 nM to 500 nM, and much higher concentrations (500-9000 nM) of PCOOH are observed in the plasma of unhealthy donors.