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| A comparative study of enzyme kinetics of single-probe and mixed-probe substrates on hepatic cytochrome P450 |
| Hits 2640 Download times 3014 Received:August 11, 2013 |
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| DOI
10.11656/j.issn.1673-9043.2013.04.05 |
| Key Words
cytochrome P450;liver microsomes;Cocktail assay;probe substrates |
| Author Name | Affiliation | E-mail | | MA Ye-tao | Faculty of Chinese Materia Medica, Tianjin University of TCM, Tianjin 300193, China | | | FENG Shan | Faculty of Chinese Materia Medica, Tianjin University of TCM, Tianjin 300193, China | | | ZHAO Jin-yi | Faculty of Chinese Materia Medica, Tianjin University of TCM, Tianjin 300193, China | | | YAN Jing-jing | Faculty of Chinese Materia Medica, Tianjin University of TCM, Tianjin 300193, China | | | HE Xin | Faculty of Chinese Materia Medica, Tianjin University of TCM, Tianjin 300193, China
Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin 300193, China | hexintn@yahoo.com |
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| Abstract
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| [Objective] To compare the enzyme kinetics characteristics of cytochrome P450 isoform specific probe substrates phenacetin (CYP1A2), dextromethorphan (CYP2D6), tolbutamide (CYP2C9), omeprazole (CYP2C19) and midazolam (CYP3A4) in human and rat liver microsomes. Also to determine the applicability of the method of comparing two probe substrates. [Methods] Five specific probe substrates were treated with human or rat liver microsome to cause metabolic reactions. Metabolic rate of each specific probe substrates was determined by HPLC before calculation of kinetic parameters. [Results] In comparison, the Vmax and Km values from a single probe and mixed probe methods in human liver microsomes were consistent. However, Km obtained from single probe substrate and mixed probe substrate methods were differences in rat liver microsomes, indicating that evaluation of CYP450 enzyme activity on drugs in the rat liver microsomes may occur to a certain extent with error by the mixed probe substrate method. [Conclusion] In human liver microsomes Vmax, Km values calculated by the two probe substrate methods are consistent, and thus mixed probe substrate method replace a single probe method for the evaluation of cytochrome P450 activity. In rat liver microsomes, Km values of partial probe substrates are increased and the affinity of probe substrates to the enzymes is decreased after the probe mixed; therefore the possibility of false negative results exist in the cytochrome P450 activity evaluation. |
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