BIOTRANSFORMATION OF COUMARIN DERIVATIVES (2) OXIDATIVE METABOLISM OF 7-ALKOXYCOUMARIN BY MICROSOMAL ENZYMES AND A SIMPLE ASSAY PROCEDURE FOR 7-ALKOXYCOUMARIN O-DEALKYLASE

• Published in: Japanese journal of pharmacology Vol. 33; no. 1; pp. 41 - 56
• Main Authors: MATSUBARA, Takashi, OTSUBO, Shigemi, YOSHIHARA, Emiko, TOUCHI, Akira
• Format: Journal Article
• Language: English
• Published: The Japanese Pharmacological Society 1983
• ISSN: 0021-5198; 1347-3506
• DOI: 10.1254/jjp.33.41

The in vitro biotransformation of 7-alkoxycoumarin by rat liver microsomes was studied to develop a simple and accurate assay procedure for 7-alkoxycoumarin O-dealkylase. 7-Alkoxycoumarin was converted to the O-dealkylated metabolite, 7-hydroxycoumarin, by aerobic incubation of the parent compound with microsomes and NADPH, but the decreased amount of 7-alkoxycoumarin in the reaction mixture was several times higher than that of the 7-hydroxycoumarin produced during the incubation. The thin-layer chromatogram of the ether extractable metabolites in the reaction mixture showed the existence of several fluorescent metabolites including 7-hydroxycoumarin. Fluorescent properties of the parent compound, 7-alkoxycoumarin, and most of the metabolites differed from that of 7-hydroxycoumarin, but the reaction cofactor, NADPH, showed similar properties. Treatment of the reaction mixture with perchloric acid resulted in conversion of NADPH to the non-fluorescent form without any effect upon the fluorescent properties of 7-hydroxycoumarin and its related compounds. Based on these properties, an improved and simple in vitro fluorometric assay of the O-dealkylation of 7-alkoxycoumarin was developed. The method is applicable to routine determination of O-dealkylase activity in both isolated microsomes and whole homogenate. Species differences in the substrate specificity of the O-dealkylation reaction and in the responsiveness of animals to the inducer were observed even with use of the liver homogenate obtained from untreated and phenobarbital- or β-naphthoflavone-pretreated animals, similar to what was observed with the microsomal system.