NONRANDOM PEROXIDATION OF DIFFERENT CLASSES OF MEMBRANE PHOSPHOLIPIDSIN LIVE CELLS DETECTED BY METABOLICALLY INTEGRATED CIS-PARINARIC ACID

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 mole cule 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 techniq ue to metabolically integrate PnA into all major classes of membrane p hospholipids, i.e., phosphatidylcholine, phosphatidylethanolamine, pho sphatidylserine, phosphatidylinositol and cardiolipin, that can be qua ntified by HPLC with fluorescence detection. Integrated PnA constitute d less than 1% of lipid fatty acid residues, suggesting that membrane structure and characteristics were not significantly altered. Low conc entrations (20-40 mu M) of tert-butyl hydroperoxide (t-BuOOH) caused s elective oxidation of PnA residues in phosphatidylserine and phosphati dylethanolamine of K562 cells and K/VP.5 cells while cell viability wa s unaffected. At higher t-BuOOH concentrations (exceeding 100 mu M), h owever, a progressive, random oxidation of all major phospholipid clas ses occurred and was accompanied by significant cell death. In HL-60 c ells, phosphatidylethanolamine, phosphatidylserine and cardiolipin wer e sensitive to low concentrations of t-BuOOH, while phosphatidylcholin e and phosphatidylinositol were not affected. Phosphatidylinositol was the only phospholipid that responded to the low concentrations of t-B uOOH in CHO cells, At high t-BuOOH concentrations, again, all phosphol ipid classes underwent extensive oxidation, All phospholipids were nea rly equally affected by peroxidation induced by a initiator of peroxyl radicals, 2,2'-azobis-(2,4-dimethylvaleronitrile) (AMVN), in K562 cel ls. In gamma-irradiated (4-128 Gy) CHO cells, phosphatidylserine was t he most affected phospholipid class (34% peroxidation) followed by pho sphatidylinositol (24% peroxidation) while the other three phospholipi d classes were apparently unaffected, Since loss of PnA fluorescence i s a direct result of irreparable oxidative loss of its conjugated doub le bond system, the method described allows for selective and sensitiv e monitoring of oxidative stress in live cells without interference fr om cell repair mechanisms.