[O-ethyl 14C]phenacetin O-deethylase activity in human liver microsomes.

The activity of human liver microsomal cytochrome P450 1A2 (CYP1A2) is readily estimated by following the O -deethylation of [ O -ethyl 14 C]phenacetin (PODase). The basis of the assay is the quantitative measurement of [ 14 C]acetaldehyde, remaining in the supernatant of assay incubates, after extraction of unmetabolized [ O -ethyl 14 C]phenacetin with charcoal. In the presence of native human liver microsomes ( K m = 54 ± 27 μM; V max = 14 ± 2.3 nmol/hr/mg; mean ± SD; N = 3 different livers) and human B-lymphoblastoid cell microsomes containing cDNA-expressed CYP1A2 ( K m = 46 μM; V max = 55 nmol/hr/nmol CYP), PODase activity conformed to monophasic Michaelis-Menten kinetics. Furthermore, PODase activity in a panel of microsomes prepared from a series of human livers was significantly correlated ( r = 0.91; p N = 11) with CYP1A2-selective 7-ethoxyresorufin O -deethylase activity, and was markedly inhibited (≥ 92%) by furafylline (FURA, IC 50 = 0.4 μM) and 7,8-benzoflavone (ANF, IC 50 = 0.1 μM), two well known CYP1A2 inhibitors. Inhibitors selective for other forms of CYP ( e.g. CYP3A, CYP2C, CYP2D6, CYP2E1) elicited a marginal effect (≤ 17% inhibition) at relatively high concentrations (≥ 10· K i ). It is concluded that the inhibition of human liver microsomal CYP1A2 activity can be readily determined by using a charcoal-based radiometric method employing [ O -ethyl 14 C]phenacetin as substrate.