Interindividual variability in catalytic activity and immunoreactivity of three major human liver cytochrome P450 isozymes

Objective: Interindividual variations in immunoreactivity and function of three major human drug metabolising P450 monooxygenases has been investigated in liver microsomes from 42 Caucasians (kidney donors or liver biopsies). Methods: Diclofenac 4'-hydroxylation, dextromethorphan O-demethylation and midazolam 1'-hydroxylation, measured by HPLC in incubates, were used as probes to determine CYP2C9, CYP2D6 and CYP3A4 function kinetics, respectively. Immunoquantification of the three isoforms was achieved by Western blotting, using rabbit polyclonal antibodies raised against human CYP2C9 and human CYP3A4, and mouse monoclonal antibody raised against human CYP2D6. Results: Diclofenac 4'-hydroxylation exhibited Michaelis-Menten kinetics with k(M) = 3.4 mu mol . l(-1) and V-max = 45 nmole . mg(-1)P . h(-1). Relative immunoreactivity of CYP2C9 was correlated with V-max and CL(int). Dextromethorphan O-demethylation in EM (extensive metabolisers) liver microsomes also showed Michaelis-Menten kinetics, with k(M) = 4.4 mu mol . l(-1) and V-max = 5.0 nmol . mg(-1)P . h(-1). Relative immunoreactivity of CYP2D6 was correlated with V-max and CL(int). Midazolam 1'-hydroxylation also exhibited Michaelis-Menten kinetics with k(M) = 3.3 mu mol . l(-1) and V-max = 35 nmol mg(-1)P . h(-1). Relative immunoreactivity of CYP3A4 was correlated with V-max and CL(int). Immunoreactivity and function were correlated for each isozyme, but there was no cross correlation between isozymes. Conclusion: The velocity of metabolite formation (V-max) by the three major human drug metabolising P450 monooxygenases is correlated with their immunoreactivity in liver microsomes. Interindividual variation was much larger for V-max than k(M). Interindividual variability was more pronounced for CYP2D6, probably due to the presence of several different functional alleles in the population of extensive metabolisers.