建立用生色底物法研究重组水蛭素在大鼠血浆中的药动学

Determination of recombinant hirudin in plasma of rats by chromogenic substrate assay

目的：建立用生色底物法（chromogenic substrate assay,CSA）测定大鼠血浆中重组水蛭素（re-combinant hirudin,rH）含量的方法,并用该法研究rH在大鼠血浆中的药动学。方法：以TGPApNA（tosyl-gly-cyl-polyl-arginine-para-nitraniline）为底物,凝血酶（thrombin,Th）能水解TGPApNA释放出对-硝基苯胺（para-nitraniline,pNA）,pNA在405nm处有特异性吸收,通过测定反应体系中未被rH中和的Th量,间接测定rH的活性。结果：CSA法能有效地测定大鼠血浆中rH的含量;rH的浓度在3.125~40 ng.mL-1范围内呈线性关系;日间和日内精密度相对标准偏差均符合要求。应用上述方法测定大鼠iv 2.0 mg.kg-1的rH后不同时间的血药浓度,结果表明该药符合二室模型一级动力学过程。结论：用生色底物法测定大鼠血浆中rH具有准确、可靠、重现性好的特点,可用于rH药动学的研究。

Objective： To develop a chromogenic substrate assay for determining the concentrations of the recombinant hirudin（rH） in plasma of rats.Methods： Because thrombin can specifically bind to rH and can hydrolyze chromogenic substrate（tosyl-glycyl-polyl-arginine-para-nitraniline） to release para-nitraniline with specific absorption at 405nm.Thus,it was used to detect the concentration of rH in plasma of rats.Results： The method could effectively and accurately detect the concentration of rH in plasma of rats.The linear range of rH was 3.125~40 ng·mL-1.The RSD of intra-day and inter-day was satisfactory.Applying the method,the concentration of the rH in plasma of rats was detected at different time points after iv 2.0 mg·kg-1.The results showed that the change in drug concentration met two-compartment model.Conclusion： Chromogenic substrate assay is an accurate and reproducible method and can be used in pharmacokinetic study to detect rH concentration in plasma of rats.