A population approach to enzyme characterization and identification: Application to phenacetin O-deethylation

Purpose. To determine the enzyme kinetics (EK) and identify the human cytochrome(s) P450 (CYP) involved in the deethylation of phenacetin to acetaminophen using a population-based method. Methods. A sparse data set was generated from incubations containing human liver microsomes (n = 19) with phenacetin. Estimates of the EK parameters were obtained by fitting the concentration-velocity data to Michaelis-Menten models by using nonlinear mixed effects modeling. Relationships between the EK parameters and the CYP activities determined for these liver microsomes were examined. Results. A two-enzyme kinetic model with a saturated, low K-M enzyme and an unsaturated. high K-M enzyme capable of forming acetaminophen best Cit the data. The population estimates of the EK parameters were V-max1, 911 pmol/min/mg protein: K-M1, 11.3 muM and Cl-int2, 0.4 mul/min/mg. The coefficients of variation for interliver variability in V-max1 and residual error of the model were 39% and 15%, respectively. When the selective catalytic activities were examined as potential covariates. 7-ethoxyresorufin O-deethylation (CYP1A2) activity was found to be associated with the low K-M enzyme, however. the high K-M enzyme(s) could not be identified. Conclusions, The population approach characterized the EK parameters and identified the low K-M enzyme responsible for phenacetin O-deethylation as CYP1A2. Population modeling of EK provides valuable information on inter- and intraliver variability in CYP dependent activities.