| Autor: |
Rodrigues AD; Drug Metabolism Department, Abbott Laboratories, Abbott Park, Illinois 60064., Kukulka MJ, Surber BW, Thomas SB, Uchic JT, Rotert GA, Michel G, Thome-Kromer B, Machinist JM |
| Jazyk: |
angličtina |
| Zdroj: |
Analytical biochemistry [Anal Biochem] 1994 Jun; Vol. 219 (2), pp. 309-20. |
| DOI: |
10.1006/abio.1994.1271 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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