Human cytochromes P450 mediating phenacetin O-deethylation in vitro: Validation of the high affinity component as an index of CYP1A2 activity

Phenacetin O-deethylation, widely used as an index reaction for cytochrome P450 1A2 (CYP1A2) activity, displays biphasic kinetics in human liver micro somes. CYP1A2 has been identified as contributing to the high affinity comp onent, but is not verified as the sole contributor to the high affinity pha se. In addition, the human CYP isoforms accounting for the low affinity pha se have not been identified. We have used heterologously expressed human CY P isoforms to identify, kinetically characterize, and predict the relative contribution of the major human liver CYP isoforms mediating phenacetin O-d eethylation. CYP1A2 (K-m 31 mu M) is the only high affinity phenacetin O-de ethylase in human liver microsomes, while CVPs 2A6 (K-m 4098 mu M), 2C9 (K- m 566 mu M), 2C19 (K-m 656 mu M), 2D6 (K-m 1021 mu M), and 2E1 (K-m 1257 mu M) all contribute to the low affinity phase of the reaction. Considering t he relative abundance of the various CYPs in human liver, CYP1A2 accounts f or 86% of net reaction velocity at a substrate concentration of 100 mu M, w hile CYP2C9 becomes the primary phenacetin O-deethylase at substrate concen trations of 865 mu M and higher and accounts for 31% of the net V-max of th e reaction. Predictions from kinetic studies on heterologously expressed CY Ps are consistent with chemical inhibition studies on human liver microsome s with sulfaphenazole and alpha-naphthoflavone that suggest a greater role for CYP2C9, and a smaller role for CYP1A2, at higher substrate concentratio ns. Thus CYP1A2 is the only high affinity human liver phenacetin O-deethyla se, thereby validating the use of the high affinity component as an index o f CYP1A2 activity in human liver microsomes.