Determination of metabolites of cytochrome P-450 model systems using high-performance liquid chromatography.
High-performance liquid chromatographic techniques were developed for the simultaneous detection of metabolites in a cytochrome P-450 model system composed of NADH, haemoglobin and methylene blue. Monohydroxylated metabolites were determined following aniline, acetanilide and phenol hydroxylations. 4-Aminoantipyrine, 7-hydroxycoumarin and p-nitrophenol were determined after dealkylation of 4-N,N-dimethylamino-antipyrine, 7-ethoxycoumarin and p-nitroanisole. These substrates are commonly used for measuring cytochrome P-450 activities. Treatment of the samples was minimal, consisting of a simple deproteinization, and did not involve any organic extraction. Separations were carried out on reversed-phase columns and the products were detected by UV adsorption. Separations were completed in less than 15 min and the detection limits were between 0.5 and 4 microM.
D015660:7-Alkoxycoumarin O-Dealkylase; D000083:Acetanilides / Q000378:metabolism; D000815:Aniline Hydroxylase / Q000378:metabolism; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003640:Dealkylation; D004579:Electron Transport; D006454:Hemoglobins / Q000378:metabolism; D006900:Hydroxylation; D066298:In Vitro Techniques; D008751:Methylene Blue / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology; D008954:Models, Biological; D009575:Nitroanisole O-Demethylase / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism; D010636:Phenols / Q000378:metabolism; D051381:Rats; D014238:Trichloroacetic Acid
D000083:Acetanilides; D006454:Hemoglobins; D010636:Phenols; D014238:Trichloroacetic Acid; D003577:Cytochrome P-450 Enzyme System; D009575:Nitroanisole O-Demethylase; D010105:Oxygenases; D015660:7-Alkoxycoumarin O-Dealkylase; D000815:Aniline Hydroxylase; D010089:Oxidoreductases, N-Demethylating; C023680:dimethylaminoantipyrine N-demethylase; D008751:Methylene Blue