Lipid Peroxidation of a Human Hepatoma Cell Line (HepG2) after Incorporation of Linoleic Acid, Arachidonic Acid, and Docosahexaenoic Acid

Abstract

Lipid peroxidation of human heptoma cell line, HepG2, after incorporation of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) was measured with a fluorescent probe and gas chromatography–mass spectrometry (GC–MS) analysis. The analysis with a fluorescent probe showed that incorporation of each polyunsaturated fatty acid (PUFA) enhanced the cellular lipid peroxidation level, but there was little difference in the effect of LA, AA, or DHA on the enhancement of cellular lipid peroxidation. The fluorescent analysis also showed that the addition of H₂O₂ (0.5 mM) enhanced the cellular lipid peroxidation levels in LA and AA supplemented cells as compared with those without H₂O₂. However, the enhancement of lipid peroxidation by H₂O₂ was not observed in DHA-supplemented cells. The same result was obtained in the GC–MS analysis of total amounts of monohydroperoxides (MHP) formed in the cellular phospholipid oxidation. In this case, the main source for MHP was LA in LA-, AA-, and DHA-supplemented cells. A significant amount of AA–MHP and a small amount of DHA–MHP were observed in AA- and DHA-supplemented cells respectively. GC–MS analysis also indicated the specific positional distribution of DHA–MHP isomers. The isomers were formed only by hydrogen abstraction at the C-18 (16-MHP + 20-MHP; 46.5%), C-6 (4-MHP + 8-MHP; 38.5%), and C-12 (10-MHP + 14-MHP; 15.1%) positions, but not at the C-9 or C-15 positions.