A new, sensitive and simple method to determine the activity of ethoxycoumarin deethylation is described.
Ethoxycoumarin
deethylation was introduced as a measure of mixedfunction oxidation by Ullrich and Weber in 1972 (1). Although the method was not very sensitive, e.g., was not able to detect monooxygenase activity in the liver of control rats (2), it gained wide use (3-7). In 1974, Jacobson and co-workers published a modification of the method, with a 25 to 50-fold sensitivity (8). Their method, however, is rather tedious: It makes use of four heptane and one ether extraction.
The present paper describes a modified assay of ethoxycoumarin deethylation, which is simpler than either of the aforementioned methods, and is comparable in sensitivity to the method of Jacobson et al. (8).
Methods
Adult male guinea pigs, Wistar strain rats, and NMRI mice were killed by decapitation (rats and guinea pigs under light ether anesthesia), and the organs were removed and cooled in ice-cold 0.25 M sucrose. A microsomal fraction was prepared with the calcium precipitation method (9) modified as described (10).
7-Ethoxycoumarin was agenerous gift from the Institute of Occupational Health, Helsinki, Finland, where it was synthesized by Eivor Elovaara. Ethoxycoumarin had a melting point of 88 to 89ยฐC and gave a single spot in paper chromatography (benzene:ethanol:acetic acid, 96.5:3.0:0.5, by volume). The incubation mixture for the ethoxycoumarin deethylation assay consisted of 200 ~1 of an NADPH-regenerating system (14.9 mg of NADP, 51.6 mg of trisodium salt of isocitric acid, 6 ml of 0.5 M Tris-HCl-0.15 M KC1 buffer, pH 7.4,2 ml of 0.1 mM MnCl,, 2 ml of 0.1 M MgCl,, 6 ml of water, and 25 ~1 of isocitrate dehydrogenase (20 mg/ml, 9 U/mg, Boehringer, Mannheim)), and 250 ~1 of 0.1 M Tris-HCl buffer, pH 7.4. These solutions contained 0.1 mrvr 7-ethoxycoumarin.
No organic