Abstract
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.
Highlights
TWOmajor metabolites, accountingfor 78%of the total 1989;McGiff, 1991;Bonventre and Nemenoff, 1991;Capdevmetabolism, were produced with arachidonicacid
18%of the total metabolite produced of the cynomolgus monkey (Oliw, 1989), causes contraction from arachidonic acid coeluted with epoxyeicosatriof guinea pig lung strips andrelaxation of guinea pig arteries
NADPH generating system, purifiedP450 2E1 metabolized arachidonic acid and several otherfatty acids to multiple products (Fig. 1).Maximal metabolism of all six fatty acids examined was dependent on the presence of cytochrome b5
Summary
Reactions were stopped by acidification to pH 4.5 with 10 p1 of6.7% (v/v) formic acid, transferred to new tubes, and extracted three times with 2 volumes of ethyl acetate.The organic phases were pooled and dried under reduced pressure and metabolites were sepadonic acid This alteration of P450 by ethanol could affect rated as described below. This mixture was incubated for 15 PFB esters of standards and metabolites M1 and M2 were separately min on ice. When the reconstituted system was used, 15 pgof purified by reverse-phase HPLC (isocratic solvent, methanol/water, sonicated 1,2-dilauroyl-sn-3-phosphatidylcholinweas added, and the 95:5; column: Ultrasphere Cl8,flow 1ml/min) using UV absorbance mixture was incubated for an additional 15min on ice. The remainder detection at 220 nm.
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