用肌酐亚氨水解酶偶联谷氨酸脱氢酶的肌酐酶法测定

摘要

Objective To develop an improved enzymatic method for assaying creatinine in serum and urine. Methods Cratinine iminhydrolase catalyzes the conversion of creatinine to N-methylhydantoin and ammonia,the later combines with 2-oxoglutarate and NADPH in the presence of glutamate dehydrogenase.Before beginning the reaction sequence,endogenous ammonia is removed by an “ammonia scavenger system”that involves the same auxiliary reaction.The NADPH and 2-Oxoglutarate consumed are restored through a reaction catalyzed by isocitrate dehydrogenase.The activity of this strictly magnesium-dependent enzyme is completely blocked by magnesium-complexing reagent that acts promptly whenever the reaction has started with creatinine iminohydrolase as startor.The reaction is monitored kinetically by measuring the NADPH decrease via its absorbance at 340nm.Results The method affords a simple,rapid,and sensitive assay with good precision and extended linearity.Test results compare closely with the HPLC precedure.The proposed method is not subject to interference from several metablites or from the drugs used commonly in clinics.Conclusion The method is easily automated and is suitable for routine work in clinical laboratories.%目的建立适合于自动化分析的人血清和尿肌酐的肌酐亚氨水解酶偶联谷氨酸脱氢酶指示系统的酶促动力学测定方法。方法在反应体系中加入异柠蒙酸脱氢酶及其作用底物异柠檬酸，再生由主反应前氨消耗的NADPH和α-酮戊二酸，其后加入镁离子络合剂CyDTA和肌酐亚氨水解酶的启动试剂启动反应，在此同时NADPH和α-酮戊二酸的再生反应被终止，测定NADPH于340 nm处吸光度的变化，计算样品中肌酐的浓度。结果本法测定线性范围为40～2000 μmol/L,批内和常规条件下的变异系数均小于5%，回收率为98.5%，与高效液相色谱法具有良好的相关性(r=0.9930)。乳糜、黄疸、溶血、酮体、乳酸盐、临床常用的治疗药物和高至2 mmol/L的氨对测定没有干扰。结论本法操作简便、精确，推荐作为常规测定方法。