INVOLVEMENT OF CYTOCHROME-P450 2E1 IN THE (OMEGA-1)-HYDROXYLATION OF OLEIC-ACID IN HUMAN AND RAT-LIVER MICROSOMES

In vitro techniques have been used to investigate the nature of micros omal cytochrome P450 involved in the metabolism of oleic acid, a physi ological monounsaturated fatty acid. Like lauric acid, which is curren tly used as a model substrate of fatty acid metabolism, the alkyl chai n of oleic acid is hydroxylated on its omega and (omega-1) carbons. Th e identity of these hydroxylated metabolites was ascertained by GC/MS and LC/MS, The omega/omega-1 ratio of oleic acid metabolites (1.22 +/- 0.01) was found to be similar to that obtained with lauric acid in ra t liver microsomes (1.10 +/- 0.02), while in human liver microsomes th is ratio was 0.75 +/- 0.5 for lauric acid and 5.2 +/- 2.6 for oleic ac id. After treatment of rats with ethanol or clofibrate, inducers of CY P2E1 and CYP4A, respectively, the hydroxylations of oleic acid were sh own to be less inducible than those of lauric acid. Five in vitro appr oaches were used to identify the P450 isoform(s) responsible for the m icrosomal (omega-1)-hydroxylation of oleic acid: effect of various ind ucers in rats, correlation studies between specific P450 catalytic act ivities in a panel of 25 human liver microsomes, chemical inhibitions, immune-inhibitions and metabolism by cDNA-expressed human P450 enzyme s. From the above results, it can be ascertained that P450 2E1 is the main enzyme involved in the (omega-1)-hydroxylation of oleic acid. Fur thermore, the omega-hydroxylation of oleic acid was shown to be mainly catalyzed by P450 4A enzymes in human liver microsomes, The turnover number of (omega-1)-hydroxylation of lauric and oleic acids decreased from 7.8 to 1.5 min(-1), respectively, suggesting that the dodecane al kyl chain allows optimal binding to the active site of CYP2E1.