title: Biotransformation of coumarin derivatives. (2). Oxidative metabolism of 7-alkoxycoumarin by microsomal enzymes and a simple assay procedure for 7-alkoxycoumarin O-dealkylase.
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-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 beta-naphthoflavone-pretreated animals, similar to what was observed with the microsomal system.
mesh_terms: D015660:7-Alkoxycoumarin O-Dealkylase; D000818:Animals; D001711:Biotransformation; D003374:Coumarins / Q000378:metabolism*; D003580:Cytochromes / Q000378:metabolism; D004285:Dogs; D004355:Drug Stability; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008862:Microsomes, Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D013045:Species Specificity; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D003374:Coumarins; D003580:Cytochromes; D010105:Oxygenases; D015660:7-Alkoxycoumarin O-Dealkylase