终点平衡尿酸酶法与动力学尿酸酶法联用测定尿酸

Integration of the kinetic uricase method with the equilibrium uricase method for uric acid assay

目的：考察终点平衡尿酸酶法和动力学尿酸酶法联用测定尿酸的可行性和线性范围。方法：在25℃下1.2 ml硼酸钠缓冲液（0.20 mol/L,pH 9.2）中用293 nm吸收跟踪尿酸酶反应;测定加入5μl尿酸酶液前反应体系吸收,加入5μl苛求芽孢杆菌尿酸酶液后记录5.0 min内吸收变化。终点平衡法以尿酸酶反应前吸收为起点而尿酸完全氧化后吸收为本底,二者之差为尿酸净吸收且同尿酸浓度成正比;动力学法分析尿酸酶反应过程预测本底。分析模拟尿酸酶反应曲线,探索从终点平衡法转换为动力学法的尿酸浓度界限值;实验测定联用法的可行性和线性范围。结果：分析40 U/L苛求芽孢杆菌尿酸酶模拟反应曲线,发现两种方法转换的尿酸浓度界限值可为5.0μmol/L;分析实验反应曲线证实此界限值可行。在40 U/L尿酸酶时终点平衡法测量范围1.0~6.0μmol/L;联用法测量范围1.5~60μmol/L且对30μmol/L黄嘌呤有抗性;在5.0μmol/L以下变异系数小于10%而5.0μmol/L以上可小于5%。结论：这种联用法测定尿酸成本更低、线性范围更宽且效率高。

Objective：To examine the feasibility and the linear range for serum uric acid assay by integrating a kinetic uricase method with the equilibrium uricase method.Methods：Uricase reaction curves were monitored within 5.0 min by absorbance at 293 nm in 1.2 ml borate buffer（0.20 mol/L,pH 9.2） at 25℃;the absorbance at 293 nm of a reaction solution minus 5 μl uricase solution was measured as the initial absorbance,the absorbance after the addition of 5 μl solution of recombinant Bacillus fastidiosus uricase to complete the reaction was measured as the background absorbance.The difference between the initial absorbance and the background absorbance should be proportional to final levels of uric acid.By the kinetic uricase method,the background absorbance was predicted by kinetic analysis of uricase reaction curve rather than being determined by experimentation.The threshold of a uric acid level for changing from the equilibrium uricase method to the kinetic uricase method was estimated by simulation analysis.The threshold and the linear range of the integration strategy were examined by experimentation.Results：The threshold was 5.0 μmol/L uric acid in reaction solutions by simulation at 40 U/L uricase,and was supported by experimentation.The equilibrium uricase method determined 1.0 to 6.0 μmol/L uric acid.The integration strategy determined uric acid from 1.5 to 60 μmol/L with resistance to final 30 μmol/L xanthine;CV was below 10% for uric acid below 5.0 μmol/L and was lower than 5% for uric acid over 5.0 μmol/L.Conclusion：The integration strategy for uric acid assay had wider linear range,lower cost and practical analysis efficiency.