Abstract

The aim of this study was to evaluate the (3S)-3-hydroxylation and the N-oxidation of quinidine as biomarkers for cytochrome P-450 (CYP)3A4 activity in human liver microsome preparations. An HPLC method was developed to assay the metabolites (3S)-3-hydroxyquinidine (3-OH-Q) and quinidine N-oxide (Q-N-OX) formed during incubation with microsomes from human liver and from Saccharomyces cerevisiae strains expressing 10 human CYPs. 3-OH-Qformation complied with Michaelis-Menten kinetics (mean values ofV_max and K_m: 74.4 nmol/mg/h and 74.2 μM, respectively).Q-N-OX formation followed two-site kinetics with mean values of V_max,K_m andV_max/K_m for the low affinity isozyme of 15.9 nmol/mg/h, 76.1 μM and 0.03 ml/mg/h, respectively. 3-OH-Q and Q-N-OXformations were potently inhibited by ketoconazole, itraconazole, and triacetyloleandomycin. Isozyme specific inhibitors of CYP1A2, -2C9, -2C19, -2D6, and -2E1 did not inhibit 3-OH-Q orQ-N-OX formation, withK_i values comparable with previously reported values. Statistically significant correlations were observed between CYP3A4 content and formations of 3-OH-Q andQ-N-OX in 12 human liver microsome preparations. Studies with yeast-expressed isozymes revealed that only CYP3A4 actively catalyzed the (3S)-3-hydroxylation. CYP3A4 was the most active enzyme in Q-N-OXformation, but CYP2C9 and 2E1 also catalyzed minor proportions of theN-oxidation. In conclusion, our studies demonstrate that only CYP3A4 is actively involved in the formation of 3-OH-Q. Hence, the (3S)-3-hydroxylation of quinidine is a specific probe for CYP3A4 activity in human liver microsome preparations, whereas the N-oxidation of quinidine is a somewhat less specific marker reaction for CYP3A4 activity, because the presence of a low affinity enzyme is demonstrated by different approaches.