应用TT法测定家兔尿液中重组水蛭素浓度的方法学研究

Determination of r-hirudin in urine of rabbits by thrombin time method

目的:建立一种生物测定法——凝血酶时间法(TT 法),以测定家兔尿液中的重组水蛭素(rH),进而研究其尿药排泄动力学。方法:依据 rH 的抗凝血酶活性,采用 TT 法测定家兔尿中的 rH 浓度。取家兔尿液10μL,依次加入空白血浆40 μL、pH 7.4的 Tris-HCl 缓冲液50μL,以及5 u·mL^(-1)的凝血酶溶液50μL,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中的 rH 浓度。结果:TT 延长百分率(Y)经对数转换与尿液中 rH 浓度(X,μg·mL^(-1))呈线性相关,线性方程为 Y=0.7598X+1.0098,r=0.9956,线性范围0.49～2.50μg·mL^(-1),最低检测限为0.49μg·mL^(-1)。高、中、低3种浓度 rH 尿液标准溶液测得的日内精密度 RSD 小于8.10%,日间精密度 RSD 小于9.07%,回收率为95.6%～97.7%,稀释回收率大于95.6%(5～200倍稀释)和91.9%(500倍稀释)。水蛭素尿液标准溶液冰冻(-20℃)保存至少半月内稳定,0～4℃及室温保存至少8 h 内稳定,-20℃及室温反复冻融3次稳定。3只家兔 iv rH 1.0 mg·kg^(-1)后不同时间段收集尿液,应用 TT 法测定其 rH 浓度,经亏量法求得 rH 的消除半衰期为65.4 min,其 log(X_(u∞)-X_(ut))-t 作图符合尿药排泄动力学曲线。结论:应用 TT 法测定家兔尿中的rH 浓度,方法特异性高、精密度好、回收率高、线性好,结果准确可靠,可用于家兔尿中 rH 浓度的测定和尿药排泄动力学的研究。

Objective：To develop a bioassay,i, e. thrombin time（TT） method,for determining r-hirudin（rH） in urine of rabbits so as to provide a methodology for further studying rH urinary excretion pharmacokinetics. Methods： The rH level in urine was measured by means of anti - thrombin activity of rH. TT was determined in a coagulometer following successive addition of 40 μL blank plasma of the rabbits,50 μL Tris - HC1 buffer（pH 7. 4）and 50 μL5 u · mL^-1 thrombin solution to 10 μL urine. The concentration of rH in urine was read from the logTT prolongation rate - concentration calibration curve. An appropriate dilution with blank urine was inevitable if rH level in urine samples was higher than linear range. Results：The logTt prolongation rate was found to be linearly related to the rH concentration in urine over the range of 0. 49 - 2. 50 μg · mL^- 1 （ r = 0. 9956）. The bioassay showed a good precision of 〈 8. 10% （ within - day RSD） and 〈9. 07% （ between - day RSD） as well as high recovery of 95. 6% -97. 7% （ method recovery） and 〉90. 0% （dilution recovery）, rH standard urine samples were stable for at least 2 weeks at -20 ℃ and 8 h at room temperature. Application of the above method to 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 rain. Conclusion ：The TY method developed by us is well suitable for measurement of rH concentration in urine and for study of its urinary excretion pharmacokinetics in rabbits.