A Simple and Sensitive Assay of 7-Ethoxycoumarin in Deethylation

Abstract

Deethylation of ethoxycoumarin as a measure of monooxygenase activity was introduced by Ullrich and Weber (Hoppe-Seyler’s Z. Physiol. Chem. 353, 1171–7, 1972). Their direct method, although convenient, was rather insensitive: the activity could not be measured in liver microsomes from control rats (Ullrich et al., ibid., 354, 514–520, 1973). A modification was proposed by Jacobson et al. (Clin. Pharmacol. Ther. 16, 701–710, 1974), with a 25 to 50 fold sensitivity. Their method made use of 4 successive heptane extractions followed by an ether extraction, and evaporation of the ether phase. The present paper describes a modified assay procedure, easier than that of Ullrich and Weber, with sensitivity comparable to that of Jacobson et al.

The incubation (0.09 mM ethoxycoumarin, NADPH regenerating system, and the tissue) is stopped by TCA. After thorough mixing, pH of the mixture is brought to 10 by adding NaOH-glycine buffer. The precipitate is spun down, and the fluorescence is measured with 390 nm as the activating and 440 nm as the emitting wavelength. The two pertinent points of the assay are 1) Measurement at an alkaline pH, where the absorption maximum of the reaction product, 7-hydroxycoumarin, shifts to 370 nm from 330 nm, and allows use of an activating wavelength long enough not to activate the substrate, ethoxycoumarin with an absorption maximum at 330 nm. 2) The use of TCA moves the absorption of NADPH from 340 nm to shorter wavelengths, and thus eliminates in addition to turbidity of the sample, also interference from fluorescence of NADPH.

The present method is capable of measuring synthesis of 5 pmoles of 7-hydroxycoumarin with a standard deviation of 4.5 per cent. Incubation of rat liver supernatant (0.7 mg wet weight) gives a fluorescence 5 fold over the background in 10 minutes, a figure about similar to that of Jacobson et al.