Interspecies differences in coumarin metabolism in liver microsomes examined by capillary electrophoresis

Abstract

1. A simple, rapid method was developed for studying xenobiotic metabolism by cytochrome P450 in liver microsome preparations. Capillary electrophoresis was used to separate the metabolite from the metabolic mixture.

2. Coumarin is metabolized to 7-hydroxycoumarin by a cytochrome P450 isoenzyme. Human, bovine, gerbil, mouse (Schofield, CD1), rat, rabbit, porcine, and cynomologus monkey microsomal preparations were investigated for coumarin metabolism by determining the content of 7-hydroxycoumarin present after metabolism.

3. Separation of 7-hydroxycoumarin from the reaction mixture was carried out in 50 mM phosphate buffer, pH 6.8, on a fused silica capillary at 25°C and 15 kV. The metabolic matrix consisted of an NADPH regeneration system, 205.5 μM coumarin, and the microsomal preparation. Standard curves were prepared in the microsomal preparation and the limit of quantification was 6.17 μM, with a linear range from 0 to 308.5 μM.

4. The reaction was initiated by the addition of the microsomes. An aliquot of the reaction mixture was removed at specific timed intervals over 2 h and injected directly onto a capillary electrophoresis column and the concentration of 7-hydroxycoumarin determined. The metabolism of coumarin to 7-hydroxycoumarin is greatest in human and monkey microsomes.