【摘要】 目的:以细胞色素P450酶(CYP)的特异性底物非那西丁(CYP1A2)和右美沙芬(CYP2D6)为探针,比较底物消除法和传统产物生成法测定的差异,评价底物消除法的可行性。方法:基于高效液相色谱技术建立底物及其特征代谢物的定量分析方法,测量底物在不同起始浓度下的消除初速率("底物消耗法")和形成产物的初速率("产物生成法");采用线性回归法和曲线拟合法求算酶动力学参数Km和Vmax。结果:采用底物消除法测得非那西丁(CYP1A)的Km和Vmax与产物生成法相近;但测定CYP2D活性时,因右美沙芬同时被小鼠肝微粒体其它同工酶代谢,所得表观参数有较大差别。结论:对特异性强的探针药物,两方法结果一致。只要选择合适底物探针和数据处理方案,底物消耗法是一种可靠、简便测定微粒体酶动力学参数的方法。更多还原

【Abstract】 Objective:To evaluate the practicability of substrate deletion approach by comparing enzyme kinetic parameters obtained from this new approach and the traditional product formation method,using phenacetin and dextromethorphan as substrate probe for cytochrome P450(CYP) 1A2 and 2D6,respectively.Methods: A high-performance liquid chromatographic method was used for monitoring the concentrations of the remaining substrate and its metabolite formation at various reaction time points during the rat liver microsome incubation.The Km and Vmax were calculated by both linear regression and curve fitting for the Michaelis-Menten kinetics.Results: The similar kinetic constants are obtained for CYP1A applying phenacetin as probe.However,they varied significantly for probing the CYP2D activities using dextromethorphan as substrate that is metabolized by several isoenzymes at the same time.Conclusion: The substrate deletion approach is in good overall agreement with the product formation method for probe substrates with strong specificity,and it can be used as a robust and efficient protocol to determine Km and Vmax using microsomes only by choosing the suitable substrate as well as the data analysis processing.更多还原