Novel sensitive high-performance liquid chromatographic method for assay of coumarin 7-hydroxylation |
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| Institution: | 1. Department of Food Science and Biotechnology, Daegu University, Gyeongsan 712-714, Republic of Korea;2. Food Consumption Safety Division, Ministry of Food And Drug Safety, Cheongju 361-709, Republic of Korea;3. Department of Agricultural Biotechnology, Center for Food and Bioconvergence, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul 151-921, Republic of Korea;4. Department of Physics, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea;1. Department of Natural Product Chemistry, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan;2. Department of Natural Product Chemistry, School of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan;1. Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan;2. Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, Japan;3. Department of Chemistry, University of Yangon, Yangon 11041, Myanmar;4. Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue University, 06 Ngo Quyen, Hue City, Viet Nam;5. Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan;1. Department of Food Science and Biotechnology, Daegu University, Gyeongsan 38453, Republic of Korea;2. Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Republic of Korea |
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| Abstract: | In this paper, a novel HPLC-based method with fluorometric detection of coumarin 7-hydroxylase is presented. The described method provides a time-effective, more sensitive and specific alternative to the previously used spectrofluorometric assay. Using the developed method, metabolism of coumarin in 11 samples of human liver microsomes was evaluated and 1790±690 pmol/min/nmol cytochrome P450 (CYP) activity was found. Kinetic parameters and linearity of coumarin 7-hydroxylation were studied in a reconstituted system consisting of recombinant CYP2A6 expressed in Escherichia coli, rat NADPH-CYP reductase and usual components. It was found that a 3.5 to 30 min time of incubation is suitable for estimation of coumarin 7-hydroxylase activity. Observed Km and Vmax values in the CYP2A6 reconstituted system were 1.48±0.37 μM and 3360±180 pmol product/min/nmol CYP, respectively. |
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