Aminopyrine metabolism by multiple forms of cytochrome P-450 from rat liver microsomes: Simultaneous quantitation of four aminopyrine metabolites by high-performance liquid chromatography

Abstract

Four aminopyrine metabolites generated by hepatic microsomes were simultaneously assayed by high-performance liquid chromatography. The metabolites were 4-monomethylaminoantipyrine (MAA), 4-aminoantipyrine (AA), 3-hydroxymethyl-2-methyl-4-dimethylamino-l-phenyl-3-pyrazoline-5-one (AM-OH), and one unidentified metabolite. MAA was the major metabolite generated by the microsomes; its formation was induced by phenobarbital but not by 3-methylcholanthrene. Female rats had lower N-demethylation activity of aminopyrine than male rats. The production of AA by microsomes was low. The formation of AM-OH was strongly induced by phenobarbital, but treatment with 3-methylcholanthrene reduced its formation. These differences in the microsomal aminopyrine monooxygenase activity are dependent on the relative amounts of the individual cytochrome P-450 isozymes. Therefore, we examined aminopyrine metabolism in a reconstituted system with purified cytochrome P-450s. P-450 UT-2 ( P-450h) had high aminopyrine N-demethylation and hydroxylation activities, but P-450 F-2 ( P-450i) had low N-demethylation activity and no hydroxylation activity. P-450 PB-4 ( P-450b) and P-450 PB-5 ( P-450e) had high aminopyrine hydroxylation activity and their N-demethylation activity also was high. The 3-methylcholanthrene-inducible forms P-450 MC-1 ( P-450d) and MC-5 ( P-450c) had aminopyrine N-demethylation activity but no hydroxylation activity. P-450 UT-4 (RLM2) is a unique form that produced a large amount of the unknown metabolite. P-450 UT-7 had the highest N-demethylation activity. Addition of cytochrome b 5 to the reconstituted system enhanced the aminopyrine hydroxylation activities of P-450s UT-1, UT-2, PB-2, and PB-5. Also, the N-demethylation activities of P-450s UT-1, PB-1, PB-2, and MC-1 were increased by cytochrome b 5. Metyrapone inhibited the catalytic activities of P-450s PB-4, PB-5, MC-1, and MC-5, and especially those of P-450s UT-4, and UT-7. The kinetics of the four major cytochrome P-450s ( P-450 UT-2, UT-4, PB-4, and MC-5) for aminopyrine N-demethylation and hydroxylation activities were studied. P-450s PB-4 and UT-2 had similar K m values (0.50 and 0.62 m m, respectively) in aminopyrine N-demethylation activity. These K m values were similar to the K m of aminopyrine N-demethylase in microsomes from phenobarbital-treated rats. P-450 UT-4 had a higher K m (3.23 m m) than P-450 UT-2 by an order of magnitude. P-450 MC-5 gave the highest K m value for aminopyrine N-demethylation (8.55 m m).