MEASUREMENT OF LIVER MICROSOMAL CYTOCHROME-P450 (CYP2D6) ACTIVITY USING [O-METHYL-C-14] DEXTROMETHORPHAN

The activity of human liver microsomal cytochrome P4502D6 (CYP2D6) is readily estimated by following the O-demethylation of [O-nethyl-C-14]d extromethorphan. The basis of the assay is the quantitative measuremen t of [C-14]formaldehyde (0.05-4.0 mu m) after addition of NaOH to the microsomal incubates and extraction with methylene chloride. The assay is relatively simple, sensitive (limit of detection is similar to 5.0 pmol HCHO/h/mg microsomal protein) and does not require the use of HP LC or an internal standard. Formation of radiolabeled formaldehyde in human liver microsomes is linear for 20 min, up to a final protein con centration of 1.0 mg/ml. Furthermore, the O-demethylase activity in a panel of microsomes prepared from a series of human livers was signifi cantly correlated with the immunochemically determined levels of CYP2D 6 protein (r = 0.925, p < 0.001), and was inhibited (>89%) by quinidin e and lobeline. In addition, [O-methyl-C-14]dextromethorphan O-demethy lation was exclusively catalyzed by cDNA-expressed CYP2D6 in microsome s prepared from human B-lymphoblast cells. The method is suitable for rapid screening of compounds as potential CYP2D6 cosubstrates and/or i nhibitors. (C) 1994 Academic Press, Inc.