Determination of phospholipid hydroperoxides using luminol chemiluminescence—high-performance liquid chromatography

Phospholipid peroxidation in biomembranes has received much attention in connection with its pathological effects and possible contributions to diseases, such as atherosclerosis, cancer, and infection and also to aging. To determine that peroxidation occurs in membrane lipids, the direct measurement of the primary products, phospholipid hydroperoxides, is important and necessary, rather than the detection of their secondary breakdown products or metabolites. It is possible to measure phosphatidylcholine hydroperoxide (PC–OOH) and phosphatidylethanolamine hydroperoxide (PE–OOH) present in biological tissues using the method of chemiluminescence–high-performance liquid chromatography (CL–HPLC) in which a mixture of luminol and cytochrome c is employed as a hydroperoxide-specific postcolumn chemiluminescence reagent. This chapter presents the recommended conditions for the CL–HPLC assay for analyzing phospholipid hydroperoxides in human blood plasma and rat liver and brain. It illustrates the extraction procedure for total lipids of rat liver and brain. The extraction procedure is almost the same as that for human plasma except for the homogenizing procedure for tissues.