Biotransformation of coumarin derivatives (2). Oxidative metabolism of 7-alkoxycoumarin by microsomal enzymes and a simple assay procedure for 7-alkoxycoumarin O-dealkylase.

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Abstract
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-hydroyxcoumarin produced during the incubation. TLC 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 mixtures 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-dealkylated reaction and in the responsiveness of rats, mice, guinea pigs, rabbits and dogs to the inducer were observed even with the use of the liver homogenate obtained from untreated and phenobarbital- or .beta.-naphthoflavone-pretreated animals, similar to what was observed with the microsomal system.