凝血酶时间法测定家兔尿液及胆汁中r-水蛭素

Determnation of r-Hirudin in Urine and Bile of Rabbits by Thrombin Time Method

建立了一种生物测定法---凝血酶时间法（TT法）以测定家兔尿液及胆汁中的r-水蛭素（r-Hirudin,rH）,从而为rH的排泄动力学研究提供测定方法学。本方法依据rH的抗凝血酶活性,以logTT延长率对rH浓度构建标准曲线。家兔尿液或胆汁10μL,依次加入40μL空白兔血浆、50μL Tris-HCl缓冲液（pH7.4）,以及50μL浓度为5U/mL的凝血酶溶液,由血凝仪记录凝血时间并计算凝血时间延长百分率,从标准曲线求出待测尿液及胆汁中的rH浓度。TT延长百分率（y）经对数转换与尿液或胆汁中rH浓度（x,mg/L）呈线性相关（r〉0.99）,线性范围0.49~2.50mg/L;检出限为0.49mg/L。高、中、低三种浓度rH尿及胆汁标准溶液日内及日间精密度（RSD）均〈10%,方法回收率〉95%,稀释回收率〉91.9%,室温及-20℃保存至少8h及半月内稳定。应用上述方法测定家兔静脉注射rH后尿及胆汁药物浓度以及研究尿药排泄动力学,均表明本研究建立的TT法精密度和回收率高,结果准确可靠,符合药动学要求。

A bioassay, thrombin time （Tr）, method was developed for determining r-hirudin （rH） in urine and bile of rabbits so as to provide a methodology for further studying rH urinary and biliary excretion pharmacokinetics. The rH level in urine and bile were measured based on antithrombin activity of rH. Tr was determined in a coagulometer following successive addition of 40 μL blank plasma of the rabbits, 50 μL pH 7.4 Tris-HCl buffer and 50 μL 5 U/mL thrombin solution to 10 μL urine and bile. The concentration of rH in urine and bile was read from the logTr prolongation rate-concentration calibration curve. An appropriate dilution with blank urine was inevitable if rH level in samples was higher than linear range. The logTr prolongation rate was found to be linearly related to the rH concentration in urine and bile in the range of 0.49 -2.50 mg/L （r 〉0.99）. The bioassay shows a good precision of 〈8.10% （intra-day RSD） and 〈9.07% （interday RSD） as well as high recovery of 95. 6% - 97. 7% （ method recovery） and 〉 91. 9% （ dilution recovery）, respectively, rH standard urine and bile samples were stable for at least 2 weeks at -20℃ and 8 h at room temperature. Using the method above mentioned, it was found that rH was undetectable in bile sample, the urinary excretion kinetic study of rH in rabbits dosed iv rH demonstrated an log （Xu∞ -Xut ）-t curve corresponding to the two-compartment model and the first-order kinetics with elimination half life of 65.4 min. rH in bile samples of rabbits receiving iv rH 1 mg/kg was undectable.