Differentiation of cytochrome P-450 activities with scoparone as substrate. A rapid one-step HPLC-analysis

A direct and highly sensitive high performance liquid chromatography (HPLC) method for measuring cytochrome P-450 activities in biological probes, with the substrate scoparone, is described. Using 10-100 microliter of a supernatant of an incubation mixture of scoparone (0.1 mmol/l), microsomal fractions (1 nmol P-450/ml), MgCl2 (2 mmol/l), and NADPH (4 mmol/l), the products scopoletin and isoscopoletin as well as the substrate scoparone were separated by HPLC on an ODS-Hypersil RP-18 column. The compounds were detected by a UV-spectrophotometer (345 nm) and quantified by the aid of an external calibration curve. The limits of detection were 10 pmol for the products scopoletin and isoscopoletin. This method allows the direct quantification of P-450 activities and the simultaneous differentiation between "3-methylcholanthren-like" or "phenobarbital-like" induction states by estimation of the scopoletin: isoscopoletin ratios.