摘要
目的建立一种基于NADP+衍生物荧光的高灵敏度的体外测定醛糖还原酶(aldose reductase,AR)活性的HPLC方法。方法从大鼠的肾脏中分离纯化AR,以DL-甘油醛为底物,加入NADPH启动酶促反应。以C18柱分离NADP+衍生物,流动相为乙腈∶水(75∶25),流速1.0 ml.min-1,柱温25℃,荧光检测激发波长370 nm,发射波长465 nm。测定NADP+衍生物的稳定性以及NADPH、DL-甘油醛、pH、温度对AR活性的影响。结果 NADP+衍生物稳定性好,保留时间为(1.4±0.01)min,附近无干扰峰,其浓度与荧光强度线性关系良好(r=0.9994),最低检测限为25 pmol。日内和日间精密度均小于10%。结论本方法操作简单、灵敏度高、特异性强,可用于体外AR的活性的测定。
Aim To establish a sensitive HPLC method for the determination of aldose reductase（AR） activity,based on the HPLC analysis of NADP＋ derivative fluorescence formed by AR.Methods AR was isolated and purified from rat kidney.The mobile phase was acetonitrile-water（75 ∶ 25） delivered at a flow rate of 1.0 ml.min-1 by isocratic reversed-phase elution on C18 column.The temperature was controlled at 25 ℃ and the fluorescence of NADP＋ derivative was monitored at 370 nm /465 nm.The effect of NADPH,DL-glyceraldehyde,pH,and temperature on AR activities was studied.Results The NADP＋ derivative was stable,whose retention time was（1.4 ± 0.01） min with no interference peaks.A good linearity was observed（r = 0.9994） with the detection limit at 25 pmol.Both the inter-day and intraday precision was less than 10%.Conclusion The method is simple,sensitive and credible and can be used for the determination of AR activities.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2012年第10期1472-1475,共4页
Chinese Pharmacological Bulletin
基金
澳门大学研究基金[No MYRG161(Y2-L2)-ICMS11-CXP]
作者简介
陈修平（1977-），男，博士，助理教授，研究方向：药理学，Tel：0853-83974873，E—mml：xpchen@umac．mo；
石京山（1959-），男，博士，教授，研究方向：药理学，E-mNl：shijs@zmc．edu．cn
