| 8080088 |
Rodrigues AD, Kukulka MJ, Surber BW, Thomas SB, Uchic JT, Rotert GA, Michel G, Thome-Kromer B, Machinist JM: Measurement of liver microsomal cytochrome p450 (CYP2D6) activity using [O-methyl-14C] dextromethorphan. Anal Biochem. 1994 Jun;219(2):309-20.
The activity of human liver microsomal cytochrome P4502D6 (CYP2D6) is readily estimated by following the O-demethylation of [O-methyl-14C] dextromethorphan. The basis of the assay is the quantitative measurement of [14C] formaldehyde (0.05-4.0 microM) after addition of NaOH to the microsomal incubates and extraction with methylene chloride. The assay is relatively simple, sensitive (limit of detection is approximately 5.0 pmol HCHO/h/mg microsomal protein) and does not require the use of HPLC or an internal standard. Formation of radiolabeled formaldehyde in human liver microsomes is linear for 20 min, up to a final protein concentration of 1.0 mg/ml. Furthermore, the O-demethylase activity in a panel of microsomes prepared from a series of human livers was significantly correlated with the immunochemically determined levels of CYP2D6 protein (r = 0.925, p < 0.001), and was inhibited (> 89%) by quinidine and lobeline. In addition, [O-methyl-14C]-dextromethorphan O-demethylation was exclusively catalyzed by cDNA-expressed CYP2D6 in microsomes prepared from human B-lymphoblast cells. The method is suitable for rapid screening of compounds as potential CYP2D6 cosubstrates and/or inhibitors. |
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