Measurement of liver microsomal cytochrome p450 (CYP2D6) activity using [O-methyl-14C]dextromethorphan.
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.
Rodrigues
A D
AD
Drug Metabolism Department, Abbott Laboratories, Abbott Park, Illinois 60064.
Kukulka
M J
MJ
Surber
B W
BW
Thomas
S B
SB
Uchic
J T
JT
Rotert
G A
GA
Michel
G
G
Thome-Kromer
B
B
Machinist
J M
JM
D001402:B-Lymphocytes; D002250:Carbon Radioisotopes; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019389:Cytochrome P-450 CYP2D6; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000096:biosynthesis / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D003915:Dextromethorphan / Q000138:chemical synthesis / Q000378:metabolism*; D005557:Formaldehyde / Q000032:analysis; D006801:Humans; D007118:Immunoassay; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000032:analysis* / Q000096:biosynthesis / Q000378:metabolism; D011865:Radioisotope Dilution Technique; D013379:Substrate Specificity; D014162:Transfection
D002250:Carbon Radioisotopes; D018076:DNA, Complementary; D005557:Formaldehyde; D003915:Dextromethorphan; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019389:Cytochrome P-450 CYP2D6