红细胞醛糖还原酶荧光法的建立及意义

THE DETERMINATION AND SIGNIFICANCE OF ACTIVITIES OF ERYTHROCYTE ALDOSE REDUCTASE

目的建立一种准确测定醛糖还原酶（ＡＲ）活性的方法。方法利用ＮＡＤＰＨ、ＤＬ－甘油醛、磷酸缓冲液和适量红细胞溶血液组成的反应体系，以荧光法测定了红细胞ＡＲ活性，并对酶反应最佳条件进行了实验探讨。结果①血红蛋白浓度在１～１０μｌ范围内，随着所加量的增加，反应后的ＮＡＤＰＨ消耗也逐渐增加，两者之间有很好的相关性；②反应温度对测定结果的影响不很明显；③以ＮＡＤＰＨ作辅酶时测得酶活性约为ＮＡＤＰＨ的１／３，这两种辅酶测得的荧光读数有良好的相关性（ｒ＝０．９２）；④ｐＨ６．０～６．５时ＮＡＤＰＨ的消耗最大；⑤批内变异系数（ＣＶ）５．２７％；批间变异系数（ＣＶ）８．９８％；⑥该法测定２０例正常对照和４０例糖尿病人红细胞ＡＲ结果表明，糖尿病人ＡＲ活性显著高于正常人（Ｐ＜０．０１），且ＡＲ活性与血糖值正相关（Ｐ＜０．０１）。结论荧光法灵敏、准确、稳定、特异，为深入研究ＡＲ与慢性并发症（ＤＣＣ）的发生、发展的关系，提供了一个可靠的手段。

Objective The increasing activity of aldose reductase has been suspected to relative to the developement of diabetic chronic complications(DCC).The aim of this study is to establish an acurate method to measure the activities of erythrocyte aldose reductase.Method By utilizing a reacting system consisting of NADPH,DLglyceraldehyde,phosphate buffer and erythrocyte hemolysite,a method of flurorescence analysis was established and optimum reaction were investigated.Result Optimum pH was 6.0～6.5,optimum reacting time was 5 minites.Withrun and betweenrun(CV)were 5.27% and 8.98%,respectively.The activities of erythrocyte aldose reductase were determined with this method in 40 cases of diabetic patients and 20 cases of nondiabetic control subjects.The results showed that the activities of erythrocyte aldose reductase were significantly increased in diabetic compared with control(P<0.01) ,and the aldose reductase activity apparently correlaed with plasma glucose levels(P<0.01).Conclusion Flurorescence analysis is a sensitive,accurate,stable and specific method,the determination of erythrocyte aldose reductase activity is an useful method in the study of DCC.