Aminopyrine metabolism by multiple forms of cytochrome P-450 from rat liver microsomes: simultaneous quantitation of four aminopyrine metabolites by high-performance liquid chromatography.
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-1-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 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 b5 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 b5. 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 Km values (0.50 and 0.62 mM, respectively) in aminopyrine N-demethylation activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Imaoka
S
S
Laboratory of Chemistry, Osaka City University Medical School, Japan.
Inoue
K
K
Funae
Y
Y
D000632:Aminopyrine / Q000031:analogs & derivatives / Q000378:metabolism*; D000675:Ampyrone / Q000378:metabolism; D000818:Animals; D000983:Antipyrine / Q000031:analogs & derivatives / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000502:physiology*; D004177:Dipyrone / Q000031:analogs & derivatives / Q000378:metabolism; D005260:Female; D007700:Kinetics; D008297:Male; D008748:Methylcholanthrene; D008797:Metyrapone / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D010634:Phenobarbital; D047069:Pyrazolones; D051381:Rats; D011919:Rats, Inbred Strains
D065607:Cytochrome P-450 Enzyme Inhibitors; D047069:Pyrazolones; D000632:Aminopyrine; D000675:Ampyrone; C011256:4-formylaminoantipyrine; D008748:Methylcholanthrene; D004177:Dipyrone; D003577:Cytochrome P-450 Enzyme System; C008667:noramidopyrine; C009077:propyphenazone; D000983:Antipyrine; D010634:Phenobarbital; D008797:Metyrapone