Biphasic kinetics of quaternary ammonium glucuronide formation from amitriptyline and diphenhydramine in human liver microsomes.

The tricyclic antidepressant amitriptyline and the H1-receptor antagonist diphenhydramine are conjugated in human liver microsomes fortified with UDP-glucuronic acid at their tertiary amino groups with the formation of quaternary ammonium glucuronides. The kinetics of the reactions were found to be biphasic with apparent KM1 and KM2 values of 1.4 microM and 311 microM for amitriptyline and 2.6 microM and 1180 microM for diphenhydramine in four liver samples. Vmax1 values varied between 2 and 17 pmol-mg protein-1.min-1 for the two substrates and Vmax2 values between 80 and 740 pmol-mg protein-1.min-1. A close correlation existed between amitriptyline and diphenhydramine glucuronidation rates in microsomes from seven livers at concentrations corresponding to 10-40% of KM2. At low concentrations, diphenhydramine competitively inhibited the glucuronidation of amitriptyline. Vmax/K(M) values of the high-affinity UDP-glucuronosyltransferase(s) (UGTs) exceed those of the low-affinity enzyme(s) severalfold, such that the former should make the major contribution to N-glucuronidation of the drugs at therapeutic concentrations in vivo.