Analysis of membrane phospholipid peroxidation by isocratic high-performance liquid chromatography with ultraviolet...

A new high-performance liquid chromatography (HPLC) for separation of phospholipid classes with ultraviolet (UV) detection at 210 nm was applied to study of peroxidation of the human erythrocyte membranes induced by soybean lipoxygenase. Phospholipid hydroperoxide production of each phospholipid was monitored at the same time by UV at 234 nm. Each phospholipid class was collected directly from the HPLC of phospholipids and was subjected to fatty acid analysis. All phospholipid classes except sphingomyelin were significantly decreased by lipoxygenase. Production of each phospholipid hydroperoxide was according to the decrease of its corresponding phospholipid class. Polyunsaturated fatty acids of each phospholipid were preferentially decreased with lipoxygenase, and degrees of the changes of the phospholipid classes corresponded to the amount of polyunsaturated fatty acids of each phospholipid. alpha-Tocopherol suppressed the decrease of the membrane phospholipids by peroxidation and suppressed also the production of malondialdehyde. However, production of phospholipid hydroperoxides appeared to be not suppressed by alpha-tocopherol. The present HPLC method proved to be sensitive to peroxidation of phospholipids and could detect the changes of each phospholipid class including phosphatidylserine and phosphatidylinositol at a single chromatographic elution. Production of hydroperoxide of each phospholipid could be detected simultaneously.