Formation of 19(S)-, 19(R)-, and 18(R)-hydroxyeicosatetraenoic acids by alcohol-inducible cytochrome P450 2E1.
When reconstituted with cytochrome b5 and NADPH cytochrome P450 oxidoreductase, cytochrome P450 2E1 metabolized lauric, stearic, oleic, linoleic, linolenic, and arachidonic acid to multiple metabolites. Two major metabolites, accounting for 78% of the total metabolism, were produced with arachidonic acid. The Vmax for total metabolite formation from arachidonic acid was 5 nmol/min/nmol P450 with an apparent Km of 62 microM. Gas chromatography-mass spectrometry analysis identified the two major metabolites as monohydroxylated eicosatetraenoic acids (HETEs). The major HETE was 19-hydroxyeicosatetraenoic acid (19-HETE) and comprised 46% of the total metabolite produced. The second metabolite was the omega-2 hydroxylated metabolite (18-HETE) and comprised 32% of the total product formed. Chiral analysis demonstrated that 19-HETE was 70% 19(S)-HETE and 30% 19(R)-HETE. In contrast, 18-HETE was essentially 100% R isomer. Approximately 18% of the total metabolite produced from arachidonic acid coeluted with epoxyeicosatrienoic acid (EET) standards. The EET metabolites were 56.4% 14,15-EET and 43.6% as a mixture of 11,12-EET and 8,9-EET. 5,6-EET was not detected. Anti-P450 2E1 IgG inhibited arachidonic acid metabolism by renal and hepatic microsomes prepared from acetone-treated rabbits. With renal cortex microsomes, the formation of 18-HETE and 19-HETE was inhibited 67 and 25%, respectively, by the antibody. Liver microsomal formation of 18-HETE was inhibited by 87% and 19-HETE by 70%. Thus, under conditions where cytochrome P450 2E1 is induced, the enzyme could contribute significantly to the formation of the omega-1 and omega-2 hydroxylated metabolites of arachidonic acid.
Department of Pharmacology, Oregon Health Sciences University, Portland 97201.
R M
RM
Laethem
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M
Balazy
J R
JR
Falck
C L
CL
Laethem
D R
DR
Koop
D000096:Acetone / Q000494:pharmacology*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000378:metabolism*; D004790:Enzyme Induction; D000431:Ethanol / Q000494:pharmacology*; D005227:Fatty Acids / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006893:Hydroxyeicosatetraenoic Acids / Q000302:isolation & purification / Q000378:metabolism*; D007672:Kidney Cortex / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D013058:Mass Spectrometry; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology*; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D009928:Organ Specificity; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis / Q000378:metabolism*; D011817:Rabbits; D013237:Stereoisomerism; D013379:Substrate Specificity
D005227:Fatty Acids; D006893:Hydroxyeicosatetraenoic Acids; D000096:Acetone; C061444:18-hydroxy-5,8,11,14-eicosatetraenoic acid; D000431:Ethanol; C055986:19-hydroxy-5,8,11,14-eicosatetraenoic acid; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating