Validation of a HPLC method for the detrmination of cytochrome P450 2C (CYP2C) activity in bovine liver microsomes.

Drug metabolizing enzymes, particularly those belonging to the cytochrome P450 superfamily (P450), influence the metabolic fate of most drugs and chemicals. Therefore, a body of literature concerning the role played by P450 isoforms in human drug metabolism has been published; by contrast, fewer and often contradictory data have been obtained in veterinary species. About 20% of drugs clinically used in humans are metabolised by CYP2C, an isoform consisting of four genes (CYP2C8, CYP2C9, CYP2C18, CYP2C19) and showing relevant genetic polymorphisms. The antidiabetic agent tolbutamide (TBT) has been proved to be a suitable and sensitive substrate of CYP2C9 activity, which represents the CYP2C isoform mostly expressed in the human liver. Aim of the present study was to verify the usefulness of TBT as a marker substrate of CYP2C9 activity also in cattle, by using a High Performance Liquid Chromatography (HPLC) method with UV–vis detection and pooled bovine liver microsomes. The detection limit of the metabolite 4–hydroxyl-TBT was 0,11 μM (mean value of 20 blank samples plus 3 S.D.). The method was both accurate (recoveries >70%) and precise (CV% <15%). The Michaelis–Menten plot kinetic parameters, obtained with a single–component fitting equation, were 1018 ± 91,72 μM (km) and 0,089 ± 0,0039 nmol/min mg prot (Vmax). Moreover, the Eadie – Hofstee plot analysis demonstrated that TBT hydroxylase activity in bovine liver microsomes exhibit a monophasic kinetic pattern (R2=0.915). Finally, proteins cross-reacting with polyclonal anti-human CYP2C8/9/19 and anti-rat CYP2C12 were found by using an immunoblotting approach. Present results demonstrate that TBT, despite the high km value obtained, might be used as a marker substrate of CYP2C9 activity in cattle, too.