Involvement of cytochrome P450 2E1 in the ( v –1)-hydroxylation of oleic acid in human and rat liver microsomes

In vitro techniques have been used to investigate the nature of microsomal cytochrome P450 involved in the metabolism of oleic acid, a physiological monounsaturated fatty acid. Like lauric acid, which is currently used as a model substrate of fatty acid metabolism, the alkyl chain of oleic acid is hydroxylated on its v and ( v –1) carbons. The identity of these hydroxylated metabolites was ascertained by GC/MS and LC/MS. The v / v –1 ratio of oleic acid metabolites (1.22 6 0.01) was found to be similar to that obtained with lauric acid in rat liver microsomes (1.10 6 0.02), while in human liver microsomes this ratio was 0.75 6 0.5 for lauric acid and 5.2 6 2.6 for oleic acid. After treatment of rats with ethanol or clofibrate, inducers of CYP2E1 and CYP4A, respectively, the hydroxylations of oleic acid were shown to be less inducible than those of lauric acid. Five in vitro approaches were used to identify the P450 isoform(s) responsible for the microsomal ( v –1)-hydroxylation of oleic acid: effect of various inducers in rats, correlation studies between specific P450 catalytic activities in a panel of 25 human liver microsomes, chemical inhibitions, immuno-inhibitions and metabolism by cDNA-expressed human P450 enzymes. From the above results, it can be ascertained that P450 2E1 is the main enzyme involved in the ( v –1)-hydroxylation of oleic acid. Furthermore, the v -hydroxylation of oleic acid was shown to be mainly catalyzed by P450 4A enzymes in human liver microsomes. The turnover number of ( v –1)-hydroxylation of lauric and oleic acids decreased from 7.8 to 1.5 min 2 1 , respectively, suggesting that the dodecane alkyl chain allows optimal binding to the active site of CYP2E1.— Adas, F., F. Berthou, D. Picart, P. Lozac’h, F. Beaugé, and Y. Amet. Involvement of cytochrome P450 2E1 in the ( v –1)-hydroxylation of oleic acid in human and rat liver microsomes. J. Lipid Res. 1998. 39: 1210–1219. Supplementary key words CYP2E1 • lauric acid • v -oxidation • monooxygenase enzymatic activities • xenobiotics • chlorzoxazone • 4-nitrophenol • mass spectrometry Cytochrome P450s (1) are the heme-thiolate proteins of the microsomal mixed function monooxygenase system. They are involved in the metabolism of xenobiotics and endogenous compounds, such as steroids and fatty acids. Many P450 enzymes are characterized by specific substrates that are regiospecifically metabolized. Accordingly, lauric acid has been described as a model substrate for hydroxylations catalyzed by P450 in human liver and kidney microsomes (2, 3). More recently, the P450 involved in these hydroxylations has been reported both in the microsomal fractions of rat (4) and in human (5, 6) livers. In both species, lauric acid was regiospecifically metabolized to form v and ( v –1)-hydroxylated metabolites, and the ratio of these two products varied significantly after starvation, diabetes (7–9), administration of clofibrate and other peroxisome proliferators (10, 11), ethanol or CYP2E1 inducers (4, 12). Fatty acid v -oxidation is a minor pathway that accounts for less than 10% of total liver fatty acid oxidation under normal physiological conditions (13). However, studies performed with mammalian systems suggest that v -hydroxylases could be involved in the first step of fatty acid catabolism (14). Moreover, it was described that starvation or intake of certain dietary fat composition could strongly enhance fatty acid hydroxylation activities (15). Oleic acid is an unsaturated physiological fatty acid present in the free fatty acid fraction and represents approximately 25% of this fraction (16). It is one of the cis unsaturated free fatty acids (with arachidonic acid) which is released from the sn -2 position of phospholipids (17). It plays an important physiological role by activating protein Abbreviations: P450, cytochrome P450 (EC 1.14.14.1) or CYP; 17OH-oleic acid, 17-hydroxyoleic acid or ( v –1)-hydroxyoleic acid; 18OH-oleic acid, 18-hydroxyoleic acid or v -hydroxyoleic acid; PKC, protein kinase C; CHZ, chlorzoxazone; 4-NP, 4-nitrophenol; 17-ODYA, 17-octadecynoic acid; BSTFA, N,O-bis-trimethylsilyl-trifluoroacetamide; TMCS, trimethylchlorosilane; HPLC, high performance liquid chromatography; APCI-LC/MS, atmospheric pressure chemical ionization liquid chromatography/mass spectrometry; GC/EIMS, gas chromatography/electron ionization mass spectrometry. 1 To whom correspondence should be addressed. by gest, on O cber 0, 2017 w w w .j.org D ow nladed fom