异育银鲫肝微粒体体外恩诺沙星N-脱乙基酶酶促反应动力学初步研究

建立了异育银鲫肝微粒体体外孵育恩诺沙星(enrofloxacin,EF)的酶促反应体系,应用RP-HPLC法检测其主要代谢产物环丙沙星(ciprofloxacin,CF),以间接反映恩诺沙星N-脱乙基酶的活性。采用二氯甲烷提取、正已烷去脂提取有效成分,样品回收率均大于81.4%,含CF或EF的肝微粒体样品能在4℃下稳定至少72 h,日内变异系数不超过3.51%,日间变异系数不超过3.71%,EF和CF准确度分别小于8.1%、6.5%,检测限(信超比为3)分别为0.15μmol/L、0.3μmol/L。恩诺沙星在银鲫肝微粒体中代谢符合一级消除方程C=140e-0.072t,消除速率常数(K)为0.072/h,消除半衰期(T1/2)为9.627 h。用Origin Lab软件的Hyperbl模型进行拟和,以方程y=P1.x/(P2+x)表示米氏常数Vmax、Km两者的关系,求得Vmax为0.2602±0.0147nmol/mg.min,Km为53.8985±10.2257μmol/L,内在清除率(Clint)约为0.0048 mL/(min.mg)。恩诺沙星消除率和环丙沙星转化率的研究结果提示,恩诺沙星在银鲫肝微粒体中的代谢以N-脱乙基反应为主,提示可能还通过其它途径生成另外代谢产物。而酶动力学研究数据(Vmax,Clint值)表明,恩诺沙星N-脱乙基酶与底物结合力不强,酶促反应强度处于中等水平。据此,笔者推测恩诺沙星在银鲫肝微粒体中的代谢速度较为缓慢。

Metabolism of enrofloxacin and detection of metabolic enzyme activity in the liver microsome of johnny carp (Carasius auratus gibelio)

An enzymatic reaction system with enrofloxacin(EF) in the liver microsome of johnny carp(Carasius auratus gibelio) was established.A reliable method for the determination of EF-N deethylase activity using major metabolite ciprofloxacin(CF) by reversed phase high performance liquid chromatography(RP-HPLC) was reported.The medicine was extracted by dichloromethane and the liver microsome was degreased by N-hexane.Sample average recovery was higher than 81.4%,CF or EF were stable at 4 ℃ for at least 72 h in spiked liver microsome samples.The inter-day variation precision and intra-day variation precision were lower than 3.51% and 3.71% respectively.The accuracy of EF and CF were lower than 8.1% and 6.5% respectively,the detection limit for EF and CF(signal-to-noise ratio of 3) were 0.15 and 0.3 μmol/L.The data of the metabolism studies were consistant with first-order elimination equation C=140e-0.07 2 t,the elimination rate constant(K) and elimination half life(T1/2) were 0.072 h and 9.627 h respectively.The Michaelis-Menten parameters such as Km and Vmax were estimated by analyzing Eadie-Hofstee plots using the software Origin Lab,the equation y = P1·x/(P2 + x) was applied to demonstrate the relationship between Km and Vmax.The maximum reaction rate(Vmax) reached 0.260 2 nmol/mg/min,with a michaelis constant(Km) of 53.898 5 μmol/L and the internal clearance rate(Cl int) of 0.004 8 mL/(min·mg) could be calculated.According to the results of elimination coefficient for EF and transformation efficiency for CF,EF might have other metabolites than the primary N-deethylation in the liver microsome of carasius auratus gibelio.Nevertheless,the affinity between EF-deethylase and the substrate was moderate.According to the enzyme-kinetic parameters the conclusion that the intensity for enzymatic reaction and the metabolism for EF was low was drawn out.