Critical evaluation of 7-ethoxycoumarin O-deethylase activity measurement in intact isolated rat hepatocytes

Abstract

1. In the determination of 7-ethoxycoumarin O-deethylase activity in intact isolated rat hepatocytes various factors influence the assay, including: (i) the decay of 7-ethoxycoumarin fluorescence which is temperature and pH dependent; (ii) the measured fluorescence which has to be adjusted for the inner filter effect; (iii) glucose addition to the medium which influences the activity, (iv) all organic solvents which inhibit the enzymic activity, with dimethylformamide provoking the smallest effect (partial competitive inhibition); (v) the enzymic reaction which is inhibited by the product of reaction; and (vi) the presence of bovine serum albumin in the medium which affects the enzymic activity.

2. Biphasic kinetics are observed for the O-deethylation of ethoxycoumarin in intact isolated rat hepatocytes. For the high-affinity component, K_m and V_max values are 5 μM and 43 pmol/min 10⁶ cells and for the low-affinity component are 414 μM and 915 pmol/min 10⁶ cells.

3. Addition of the substrate in dimethylformamide or omitting bovine serum albumin from the medium cause important changes in these kinetic parameters.