Non-random peroxidation of different classes of membrane phospholipids in live cells detected by metabolically integrated cis-parinaric acid

Abstract

Quantitative assays of lipid peroxidation in intact, living cells are essential for evaluating oxidative damage from various sources and for testing the efficacy of antioxidant interventions. We report a novel method based on the use of cis-parinaric acid (PnA) as a reporter molecule for membrane lipid peroxidation in intact mammalian cells. Using four different cell lines (human leukemia HL-60, K562 and K/VP.5 cells, and Chinese hamster ovary (CHO) fibroblasts), we developed a technique to metabolically integrate PnA into all major classes of membrane phospholipids, i.e., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and cardiolipin, that can be quantified by HPLC with fluorescence detection. Integrated PnA constituted less than 1 % of lipid fatty acid residues, suggesting that membrane structure and characteristics were not significantly altered. Low concentrations (20–40 μM) of tert-butyl hydroperoxide (t-BuOOH) caused selective oxidation of PnA residues in phosphatidylserine and phosphatidylethanolamine of K562 cells and K/VP.5 cells while cell viability was unaffected. At higher t-BuOOH concentrations (exceeding 100 μM), however, a progressive, random oxidation of all major phospholipid classes occurred and was accompanied by significant cell death. In HL-60 cells, phosphatidylethanolamine, phosphatidylserine and cardiolipin were sensitive to low concentrations of t-BuOOH, while phosphatidylcholine and phosphatidylinositol were not affected. Phosphatidylinositol was the only phospholipid that responded to the low concentrations of t-BuOOH in CHO cells. At high t-BuOOH concentrations, again, all phospholipid classes undewent extensive oxidation. All phospholipids were nearly equally affected by peroxidation induced by a initiator of peroxyl radicals, 2,2′-azobis-(2,4-di-methylvaleronitrile) (AMVN), in K562 cells. In gamma-irradiated (4–128 Gy) CHO cells, phosphatidylserine was the most affected phospholipid class (34% peroxidation) followed by phosphatidylinositol (24% peroxidation) while the other three phospholipid classes were apparently unaffected. Since loss of PnA fluorescence is a direct result of irreparable oxidative loss of its conjugated double bond system, the method described allows for selective and sensitive monitoring of oxidative stress in live cells without interference from cell repair mechanisms.