Measurement of liver microsomal cytochrome p450 (CYP2D6) activity using [O-methyl-14C]dextromethorphan

Anal Biochem. 1994 Jun;219(2):309-20. doi: 10.1006/abio.1994.1271.


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.

Publication types

  • Comparative Study

MeSH terms

  • B-Lymphocytes
  • Carbon Radioisotopes
  • Cell Line
  • Chromatography, High Pressure Liquid / methods
  • Cytochrome P-450 CYP2D6
  • Cytochrome P-450 Enzyme System / analysis*
  • Cytochrome P-450 Enzyme System / biosynthesis
  • Cytochrome P-450 Enzyme System / metabolism
  • DNA, Complementary / metabolism
  • Dextromethorphan / chemical synthesis
  • Dextromethorphan / metabolism*
  • Formaldehyde / analysis
  • Humans
  • Immunoassay
  • Isotope Labeling / methods
  • Kinetics
  • Magnetic Resonance Spectroscopy / methods
  • Microsomes, Liver / enzymology*
  • Mixed Function Oxygenases / analysis*
  • Mixed Function Oxygenases / biosynthesis
  • Mixed Function Oxygenases / metabolism
  • Radioisotope Dilution Technique
  • Substrate Specificity
  • Transfection


  • Carbon Radioisotopes
  • DNA, Complementary
  • Formaldehyde
  • Dextromethorphan
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • Cytochrome P-450 CYP2D6