荧光法动态观察糖尿病大鼠红细胞醛糖还原酶活性

目的比较测定醛糖还原酶(AR)活性的两种荧光法(碱终止法和酸终止法)的差异,应用其中较好的一种动态观察糖尿病(DM)大鼠红细胞AR活性。方法SD大鼠随机等分为四组正常对照组和DM20、40、60d组,用辅酶(NADPH或NADP)、DL甘油醛、磷酸缓冲液等组成各自的反应体系,比较碱终止法和酸终止法测定AR活性的灵敏度和重复性,择其优者测定各组大鼠红细胞AR活性。结果NADP和NADPH浓度相同时酸终止法所测荧光值较高;辅酶浓度在0～400μmol/L时标准曲线呈线性;酸终止法标准曲线的批内和批间变异系数分别为0.83%和2.28%,碱终止法为17.88%和16.69%,两法所测标本的批内和批间变异系数分别为4.25%、7.12%和1.13%、13.07%;高氯酸可消除血红蛋白对荧光的影响;DM组AR活性较正常对照增高(P<0.05),且随病程延长活性增加,但DM组间AR活性无统计学差异;血糖与AR活性正相关(r=0.873)。结论酸终止法较碱终止法简便、灵敏、准确、稳定。DM大鼠红细胞AR活性增高,并随病程延长有增加的趋势;提示AR活性的动态观察有可能作为糖尿病并发症发展进程的监测指标。

Dynamic observation of blood aldose reductase activity on diabetic rats by the fluorimetry method

Objective By comparing the differences between two fluorimetry methods (acid-stopping and alkali-stopping methods) on aldose reductase (AR) activity determination to investigate the dynamical changes of erythrocyte AR activity on diabetic rats. Methods SD rats were randomly divided into four groups: control group and three diabetic groups after STZ inducing for 20,40,60 days. Sensitivity and reduplication of two methods were compared. Then AR activity was assayed with the better method. Results When the concentrations of NADP and NADPH were equal, the fluorescent intensity tested by acid-stopping method was higher than that by alkali-stopping method; the standard curve presented linearity at 0~400 mmol/L of coenzyme. The coefficient of variation (CV) of standard curve of acid-stopping and alkali-stopping methods were 0.83% vs 2.28% and 17.88% vs 16.69% respectively; the CV of sample in the two methods were 4.25% vs 7.12% and 1.13% vs 13.07% respectively. Perchloric acid could eliminate the influence of hemoglobin on fluorescent value. AR activity was significantly increased in diabetic groups compared with that of the control (P<0.05) and increased during the course of diabetes. AR activity without statistical difference in DM group was correlated with blood glucose (r=0.873). Conclusions Acid-stopping analysis is a more simple, sensitive, accurate and stable method . AR activity with an increasing tendency during the course of diabetes development suggests that the dynamic observation of AR activity may be used as the monitor index for DM progress.