大豆苷元在人肝微粒体中的单羟化代谢机制

Mechanism of mono-hydroxylation of daidzein in human liver microsomes

目的 探讨大豆苷元在人肝微粒中羟基化代谢所涉及的肝细胞色素P4 5 0 (CYP)同工酶 ,为研究其在人体内的代谢提供基础。方法 通过分析大豆苷元在肝微粒体中和重组CYP酶中形成的单羟化代谢物的酶促动力学 ,分析其酶学模型 ,然后用不同CYP同工酶选择性抑制剂或底物进行抑制实验 ,初步筛选出介导大豆苷元单羟化代谢所涉及的CYP同工酶。结果 代谢物的形成动力学符合米氏方程单酶模型。CYP1A2选择性抑制剂呋喃茶碱和CYP1A2单克隆抗体均能明显抑制 3种单羟化代谢物的形成。而其他CYP选择性的抑制剂对 3种代谢物的形成没有或较小产生抑制作用。用重组酶实验得出相同结果。结论 体外肝微粒体研究表明 ,大豆苷元的单羟基代谢主要由CYP1A2所介导。

Aim To identify the cytochrome P450 (CYP) isoform (s) involved in daidzein mono-hydroxylated metabolites using human liver microsomes. Methods Kinetic analysis of the rates of formation of mono-hydroxylated metabolites of daidzein, including 7,8,4′-trihydroxyisoflavone (7,8,4′-THI), 7,3′,4′-trihydroxyisoflavone (7,3′,4′-THI) and 6,7,4′-trihydroxyisoflavone (6,7,4′-THI), was performed using human liver microsomes (HLM) and recombinant enzymes at substrate concentrations ranging from 0.5 to 400 μmol·L -1. Nine selective inhibitors or substrate probes specific for different CYP isoforms were applied for screening the isoform (s) responsible for mono-hydroxylated metabolism of daidzein. Results Michaelis-Menten kinetic parameters were best fitted to one-component enzyme kinetic model. The mean K m ( μmol·L -1) and V max ( μmol·g -1·min -1) values were 27±10 and 4.8±2.1, 54±22 and 2.3±1.0, 51±29 and 2.2±0.8, for the formation rates of 7,8,4′-THI, 7,3′,4′-THI, and 6,7,4′-THI, respectively. Furafylline, the CYP1A2 specific inhibitor, estrogen, and monoclonal antibody raised against human CYP1A2 (MAB-1A2) apparently inhibited the formation of mono-hydroxylated metabolites, The IC 50 of Fur for the formation of 7,3′,4′-THI, 6,7,4′-THI and 7,8,4′-THI was 1.0, 0.9 and 0.8 mol·L -1, respectively. The IC 50 of estrogen for the formation of 7,3′,4′-THI, 6,7,4′-THI and 7,8,4′-THI were 51, 60 and 64 mol·L -1, respectively. The IC 50 of MAB-1A2 for the formation of the mono-hydroxylated products was 1 mol·L -1, but neither other selective inhibitor nor substrate probes, including coumarin (CYP2D6), sulphaphenzole (CYP2C9/10), omeprazole (CYP2C19), quinidine (CYP2D6), diethyldithiocarbamate (CYP2E1), troleandomycin (CYP3A4) and keteconazole (CYP3A4), did so with human liver microsomes. Conclusion The in vitro studies indicated that daidzein mono-hydroxylated products were principally metabolized by CYP1A2 in human.