ABSTRACT
OBJECTIVE: To characterize the metabolism of genistin and study its enzymatic kinetics in rat liver microsome by HPLC-MS. METHODS: Genistin was incubated with rat liver microsomal incubation system. HPLC-MS method was used to characterize the metabolites. A metabolite generation method was established for quantitative analysis of genistein with sulfamethoxazole as internal standard. The enzyme kinetics parameters Vmax and Km was calculated by the GraphPad Prism 5.0 software. RESULTS: The metabolites in vitro incubation system was identified as genistein. The optimal time in rat liver microsomes incubation time of 40 min, the optimal protein concentration of 1 mg · mL-1, a substrate concentration of 50 μmol · L-1. The enzyme kinetics parameters of genistein were as follows; Vmax=(0.1042 ± 0.0033) μmol · min-1 · mg(pro)-1, Km=(28.96 ± 2.80) μmol · L-1. CONCLUSION: The results indicate that genistin can be metabolited as the form of hydroxylation in rat liver microsome. Metabolite generation method is a reliable and simple method for determination of kinetic parameters of hepatic microsomal enzymes, and enzyme kinetic parameters obtained of genistin provide important parameters for further study.