大鼠肝细胞液体外甲基化代谢丹酚酸A的酶促反应动力学

Enzyme kinetic studies on in vitro methylation metabolism of salvianolic acid A by rat liver cytosol

目的筛选大鼠肝细胞液作为儿茶酚-O-甲基转移酶(COMT)供体,建立丹酚酸A(SAA)体外孵育甲基化代谢的酶促反应。方法采用HPLC-UV法检测孵育体系中底物SAA的浓度,根据底物消耗量计算SAA的体外酶促代谢反应速度和动力学参数。结果在大鼠肝细胞液体外温孵体系中,SAA的酶促甲基化代谢反应呈现饱和动力学特征,符合Michaelis-Menten曲线,最大反应速率(Vmax)和米氏常数(Km)分别为30.4 nmol·mol-1·min-1,0.179 mmol·L-1。结论大鼠肝脏的COMT酶对SAA有较强的亲和力,可催化SAA发生快速、完全的甲基化代谢,大鼠肝细胞液与SAA在控制条件下体外温孵是高收率、选择性制备SAA甲基化代谢产物的一种有效手段。

Objective In the present study, rat liver cytosol was selected as donor of eatechol - O - methyhransferase （COMT） to develop an incubation system for in vitro enzyme - catalyzed methylation metabolism of SAA. Methods The contents of unmetabolized SAA left in the incubation system were determined by specific and reliable HPLC - UV assay. According to the substrate consumption, the enzyme reaction rate was calculated and the enzyme kinetic analysis of the reaction was conducted. Results Under the optimal in vitro incubation conditions, the metabolic elimination of SAA fit in with Michaelis - Menten curve and the maximum reaction rate （Vmax） was revealed as 30.4nmol·mol^-1·min^-1,0.179mmol·L^-1 with a Km value of 0. 179 mmol·L^-1. Conclusion These results indicated that COMT in rat liver has fairly high affinity for SAA and this polyphenol compound may undergo remarkable first - pass metabolism in vivo. The present study also suggested that in vitro incubation with rat liver cytosol would be a satisfactory biochemical pathway to obtain methylation metabolites of SAA with high yield and selectivity.