Details

Autor(en) / Beteiligte

• Rodrigues, A.D.
• Kukulka, M.J.
• Surber, B.W.
• Thomas, S.B.
• Uchic, J.T.
• Rotert, G.A.
• Michel, G.
• Thomekromer, B.
• Machinist, J.M.

Titel

Measurement of Liver Microsomal Cytochrome P450 (CYP2D6) Activity Using [O-methyl-14C] Dextromethorphan

Ist Teil von

• Analytical biochemistry, 1994-06, Vol.219 (2), p.309-320

Ort / Verlag

San Diego, CA: Elsevier Inc

Erscheinungsjahr

1994

Quelle

Elsevier ScienceDirect Journals

Beschreibungen/Notizen

• 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 μm) after addition of NaOH to the microsomal incubates and extraction with methylene chloride. The assay is relatively simple, sensitive (limit of detection is ∼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.