title: [O-ethyl 14C]phenacetin O-deethylase activity in human liver microsomes.
abstract: The activity of human liver microsomal cytochrome P450 1A2 (CYP1A2) is readily estimated by following the O-deethylation of [O-ethyl 14C]phenacetin (PODase). The basis of the assay is the quantitative measurement of [14C]acetaldehyde, remaining in the supernatant of assay incubates, after extraction of unmetabolized [O-ethyl 14C]phenacetin with charcoal. In the presence of native human liver microsomes (K(m) = 54 +/- 27 microM; 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 microM; 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 < 0.001; N = 11) with CYP1A2-selective 7-ethoxyresorufin O-deethylase activity, and was markedly inhibited (> or = 92%) by furafylline (FURA, IC50 = 0.4 microM) and 7,8-benzoflavone (ANF, IC50 = 0.1 microM), two well known CYP1A2 inhibitors. Inhibitors selective for other forms of CYP (e.g. CYP3A, CYP2C, CYP2D6, CYP2E1) elicited a marginal effect (< or = 17% inhibition) at relatively high concentrations (> or = 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 14C]phenacetin as substrate.
authors.affiliation: Drug Metabolism Department, Abbott Laboratories, USA.
authors.forename: A D
authors.initials: AD
authors.lastname: Rodrigues
authors.forename: B W
authors.initials: BW
authors.lastname: Surber
authors.forename: Y
authors.initials: Y
authors.lastname: Yao
authors.forename: S L
authors.initials: SL
authors.lastname: Wong
authors.forename: E M
authors.initials: EM
authors.lastname: Roberts
mesh_terms: D002250:Carbon Radioisotopes; D019388:Cytochrome P-450 CYP1A2 / Q000032:analysis*; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology*; D010615:Phenacetin / Q000378:metabolism*
chemical_list: D002250:Carbon Radioisotopes; D019388:Cytochrome P-450 CYP1A2; D010615:Phenacetin