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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。
OBJECTIVE: To compare the difference between the substrate elimination method and the traditional method for the determination of the production of phenacetin (CYP1A2) and dextromethorphan (CYP2D6), which is a specific substrate of cytochrome P450 enzyme (CYP) Eliminate the feasibility of the law. Methods: A quantitative method for the determination of substrate and its metabolites was established based on HPLC. The initial rate of elimination (“substrate consumption”) and the initial rate of formation of the product at different initial concentrations of substrate were measured. ( “Product generation method ”); Enzyme kinetic parameters Km and Vmax were calculated using linear regression and curve fitting. RESULTS: Km and Vmax of phenacetin (CYP1A) were similar to those of product formation method by substrate elimination method. However, when measuring CYP2D activity, dextromethorphan was metabolized by other isozymes of mouse liver microsomes at the same time. Apparent parameters are quite different. Conclusion: For the specific probe drugs, the results of the two methods are consistent. The substrate depletion method is a reliable and simple method to determine microsomal enzyme kinetic parameters simply by selecting the appropriate substrate probe and data processing protocol.