HPLC method for the determination of ethoxyresorufin-o-deethylase (EROD) activity in bovine liver microsomes

Nowadays, an increasing interest in farm animals drug metabolism has been recorded, for their possible exposure to chemicals and the use of drugs which might result in residues for consumers. The cytochrome P4501A (CYP1A) is mainly involved in the biotrasformation of contaminants and the mostly used assay for the measurement of its catalytical activity is the Burke’s fluorometric O-deethylation of ethoxyresorufin (EROD). In veterinary species such a method is not always as sensitive, due to the low P450 content found either in the liver than in extrahepatic tissues. Aim of the present study was to validate an EROD HPLC isocratic method with fluorescence detection, by using pooled bovine liver microsomes. The method was found to be more sensitive than that above reported in terms of resorufin detection limit (0,022 pmol, mean value of 20 blank samples plus 3 S.D.). Intra-day and inter-day precisions (CV%) were less than 10%. The Michaelis–Menten plot kinetic parameters were 0,2276±0,05148 μM (km) and 0,4883±0,03455 nmol/min mg prot (Vmax). The Eadie–Hofstee plot analysis pointed out how EROD, in cattle, follows a typical monophasic kinetic pattern. Moreover, confirmatory western blot detected proteins cross-reacting with a polyclonal anti-rabbit CYP1A1/2 antibody. Ongoing studies are actually running in our lab aiming to positively apply this method also in the veal calf, which is known to possess lower amounts of liver P450 compared to beef cattle; furthermore, confirmatory chemical inhibition studies, by using the CYP1A inhibitor α-naphthoflavone, are envisaged.