Peng Wen Xing, Wang Lian Sheng, Li Huan De, Abd El-Aty A M, Chen Guo Lin, Zhou Hong Hao
Department of Clinical Pharmacy of the Second Xiangya Hospital, Central South University, Changsha, China.
Clin Chim Acta. 2003 Aug;334(1-2):77-85. doi: 10.1016/s0009-8981(03)00194-3.
In vitro studies with rats and human liver microsomes (HLM) demonstrated that daidzein is readily metabolized to mono-hydroxylated compounds. In this study, daidzein mono-hydroxylated metabolites was investigated using human liver microsomes to identify the cytochrome P450 (CYP) isoform(s) involved in this metabolic pathway.
Kinetic analysis for the formation rates 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), were performed using human liver microsomes (HLM) and recombinant enzymes at substrate concentrations ranging from 0.5 to 400 micromol/l. 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.
Michaelis-Menten kinetic parameters were best fitted to a one-component enzyme kinetic model. The mean K(m) (micromol/l) and V(max) (micromol/g min) values (+/-S.D.) were 26.86 (10.45) and 4.76 (2.07), 53.83 (22.25) and 2.29 (1.04), 51.48 (29.32) and 2.21(0.82), 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) substantially inhibited the formation rates 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 micromol/l, respectively. The IC(50) of estrogen for the formation of 7,3',4'-THI, 6,7,4'-THI and 7,8,4'-THI was 51, 60 and 64 micromol/l, respectively. The IC(50) of MAB-1A2 for the formation of the mono-hydroxylated products was 1 micromol/l, 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.
Daidzein mono-hydroxylated products are principally metabolized by CYP1A2 in human.
对大鼠和人肝微粒体（HLM）进行的体外研究表明，大豆苷元很容易代谢为单羟基化化合物。在本研究中，使用人肝微粒体对大豆苷元单羟基化代谢产物进行研究，以鉴定参与该代谢途径的细胞色素P450（CYP）同工酶。
在底物浓度范围为0.5至400微摩尔/升的条件下，使用人肝微粒体（HLM）和重组酶对大豆苷元单羟基化代谢产物（包括7,8,4'-三羟基异黄酮（7,8,4'-THI）、7,3',4'-三羟基异黄酮（7,3',4'-THI）和6,7,4'-三羟基异黄酮（6,7,4'-THI））的生成速率进行动力学分析。应用九种针对不同CYP同工酶的选择性抑制剂或底物探针来筛选负责大豆苷元单羟基化代谢的同工酶。
米氏动力学参数最适合单组分酶动力学模型。7,8,4'-THI、7,3',4'-THI和6,7,4'-THI生成速率的平均K(m)（微摩尔/升）和V(max)（微摩尔/克·分钟）值（±标准差）分别为26.86（10.45）和4.76（2.07）、53.83（22.25）和2.29（1.04）、51.48（29.32）和2.21（0.82）。CYP1A2特异性抑制剂呋拉茶碱、雌激素以及抗人CYP1A2单克隆抗体（MAB-1A2）显著抑制单羟基化代谢产物的生成速率。呋拉茶碱对7,3',4'-THI、6,7,4'-THI和7,8,4'-THI生成的IC(50)分别为1.0、0.9和0.8微摩尔/升。雌激素对7,3',4'-THI、6,7,4'-THI和7,8,4'-THI生成的IC(50)分别为51、60和64微摩尔/升。MAB-1A2对单羟基化产物生成的IC(50)为1微摩尔/升，但包括香豆素（CYP2D6）、磺胺苯唑（CYP2C9/10）、奥美拉唑（CYP2C19）、奎尼丁（CYP2D6）、二乙基二硫代氨基甲酸盐（CYP2E1）、三乙酰竹桃霉素（CYP3A4）和酮康唑（CYP3A4）在内的其他选择性抑制剂或底物探针与人肝微粒体共同作用时均未产生此效果。
在人体内，大豆苷元单羟基化产物主要由CYP1A2代谢。
