荧光法测定醛糖还原酶活性的优化

Optimization of fluorimetric method for aldose reductase activity

目的找出优化NADP生成荧光法测定红细胞醛糖还原酶(AR)活性的方案。方法通过研究血红蛋白浓度、激发荧光水浴时间以及NADP溶液贮存时间对荧光强度的影响,优化红细胞AR活性测定的NADP生成法;以此方法测定正常及糖尿病大鼠AR活性。结果 NADP生成法的精确度受血红蛋白影响,在终止反应后的NADP溶液中加入250μl6%高氯酸沉淀血红蛋白可消除此影响;水浴激发荧光时间5、30、60min,荧光值变异系数无差异;在激发荧光前将反应体系生成的NADP溶液贮存于-20℃的条件下至少可以稳定保存4w。糖尿病大鼠AR活性明显高于正常对照组(P<0.05)。结论优化的NADP生成法可以更准确测定红细胞AR活性。

Objective To optimize the the NADP-generating fluorimetric method for aldose reductase （AR） activity. Methods The effect of haemoglobin ,the time of aqueous bath arousing fluorescence and storage time of NADP solution on the fluorescent value was ob- served. It was optimized that the NADP-generating fluorimetry assayed erythroeytes AR activity. AR activity of normal and diabetic mellitus rat were examined. Results The accuracy of this assay was influenced by hemoglobin concentration which could be eliminated by adding 250 μl 6 % perchloric acid to precipitate hemoglobin as the reaction was terminated. It had no difference for the variation coefficient of fluores- cence value when the time that the fluorescence was provocated through waterbath was 5,30,60 min. NADP solution could be stored in the condition of - 20℃ for 4 weeks at least. AR activity was higher in diabetic rats than that of normal rats （ P 〈 0. 05 ）. Conclusions Opti- mized NADP-generating fluorimetry can measure activity of erythrocyte AR accurately.