单探针与混合探针底物法在细胞色素P450酶底物酶促动力学中的比较研究

A comparative study of enzyme kinetics of single-probe and mixed-probe substrates on hepatic cytochrome P450

关键词:  细胞色素P450  肝微粒体  Cocktail  探针底物

Key words:  cytochrome P450  liver microsomes  Cocktail assay  probe substrates

摘要:

[目的] 研究5种细胞色素P450酶亚型的特异性探针底物非那西丁（CYP1A2）、右美沙芬（CYP2D6）、甲苯磺丁脲（CYP2C9）、奥美拉唑（CYP2C19）、咪达唑仑（CYP3A4）在人和大鼠肝微粒体中的酶动力学特征，比较两种探针底物法的适用性。[方法] 5种特异性探针底物分别经人或大鼠的肝微粒体代谢反应后，采用高效液相色谱法测定特异性探针底物的代谢速率，根据底物浓度-代谢速率曲线计算酶动力学常数。[结果] 单探针与混合探针两种方法在人肝微粒体中所得的V_max、K_m值具有一致性；而在大鼠肝微粒体中所得的K_m具有一定差异，表明该混合探针底物法在用于药物对大鼠肝微粒体中的细胞色素P450酶活性评价时可能会出现一定程度的影响。[结论] 在人肝微粒体中，两种探针底物法的V_max、K_m值具有一致性，因而混合探针底物法可代替单探针法用于细胞色素P450酶活性评价；在大鼠肝微粒体中，探针底物混合后可使得部分探针底物的K_m值升高，与酶亲和力下降，在细胞色素P450酶活性评价中可能出现假阴性结果。

Abstract:

[Objective] To compare the enzyme kinetics characteristics of cytochrome P450 isoform specific probe substrates phenacetin (CYP1A2), dextromethorphan (CYP2D6), tolbutamide (CYP2C9), omeprazole (CYP2C19) and midazolam (CYP3A4) in human and rat liver microsomes. Also to determine the applicability of the method of comparing two probe substrates. [Methods] Five specific probe substrates were treated with human or rat liver microsome to cause metabolic reactions. Metabolic rate of each specific probe substrates was determined by HPLC before calculation of kinetic parameters. [Results] In comparison, the V_max and K_m values from a single probe and mixed probe methods in human liver microsomes were consistent. However, K_m obtained from single probe substrate and mixed probe substrate methods were differences in rat liver microsomes, indicating that evaluation of CYP450 enzyme activity on drugs in the rat liver microsomes may occur to a certain extent with error by the mixed probe substrate method. [Conclusion] In human liver microsomes V_max, K_m values calculated by the two probe substrate methods are consistent, and thus mixed probe substrate method replace a single probe method for the evaluation of cytochrome P450 activity. In rat liver microsomes, K_m values of partial probe substrates are increased and the affinity of probe substrates to the enzymes is decreased after the probe mixed; therefore the possibility of false negative results exist in the cytochrome P450 activity evaluation.