Metabolism of Leukotriene B 4 in Cultured Hepatoma Cells

Abstract

Incubation of leukotriene B 4 (LTB 4) with Hep G2 cells (a human-derived hepatoma cell line) resulted in the production of several metabolites indicative of alternative pathways of LTB 4 metabolism not previously observed in normal hepatocytes. The major extracellular LTB 4-derived metabolites were structurally identified using mass spectrometry and ancillary techniques including electrospray ionization. The major metabolite was 10 hydroxy-4,6,8,12-octadecatetraenoic acid (10-HOTE), an unexpected metabolite which lost the hydroxy group at carbon 5 from the parent LTB 4. Two other major metabolites were 3(R)-hydroxy-LTB 4 and 3(S)-hydroxy-LTB 4. The formation of these three metabolites revealed that β-oxidation from the carboxyl terminus can be a significant metabolic pathway for degradation of this hydroxy unsaturated fatty acid. The normal hepatocyte LTB 4-derived metabolite, 20-carboxy-LTB 4, was observed as only a minor product. The metabolic profile for Hep G2 cells suggests that the efficient cytochrome P-450 pathway involved in ω-oxidation in typical hepatocytes is absent in these cells. Several minor metabolites were also identified which included dihydro products resulting from metabolism by a 12-hydroxydehydrogenase/Δ 10-reductase pathway. The formation of the major metabolite reveals the operation of steps in β-oxidation of hydroxy, unsaturated fatty acids not anticipated by previously identified steps of fatty acid β-oxidation.