血清山梨醇脱氢酶活力测定的方法学研究

Study on the Method of Determinating Serum Sorbitol Dehydrogenase Activity

目的 建立一种简易、快速的山梨醇脱氢酶 (SDH)活性测定法。方法 用双试剂法进行测定 ,波长 340nm ,温度37℃ ,血清 :试剂 =1∶15 ,第一次孵育时间 2min ,加入第二试剂后 ,用延迟时间 30s,读数间隔时间 15s,读数次数 6次。结果 最适 pH为 8 8,SDH对山梨醇的Km为 1.2 4mmol/L ,山梨醇 :NAD+=6∶1。批内CV(n =2 0 )为 2 7%～ 4.2 % ;批间CV为 3 1%～ 8.3 %。在 90U/L以下线性良好 ,参考值上限为 12 .5U/L。结论 该方法简易、快速、灵敏 ,线性范围宽 ,干扰因素少 ,所需样品量少。

Objective To develop a simpler and quicker sorbitol dehydrogenase (SDH) activity determination method. Method It was based on conversion of sorbitol to fructose with simultaneous reduction of NAD + in Tris-Hcl buffer (pH 8.8) at 37 ℃. Dual-reagent method was implemented. The wavelength measures 340 nm and the serum and reagent ratio is 1∶15. The 1 st incubation lasted 2 mins. After the addition of the 2 nd reagent the incubation extended for 30 s. Reading interval lasted 15 s and readings total 6 times. Result The study indicated that the optimal pH was 8.8 and the Km for sorbitol was 1.24 mmol/L and that sorbitol to NAD + ratio was 6∶1. It also showed that with-run and between-run precision (CV) measures 2.7% ～4.2% and 3.1%～8.3% respectively and that SDH activity was well linear beneath 90 U/L. The serum SDH activity of 50 healthy persons was (8.3±2.1)U/L. There was no noticeable difference between sexes. Conclusion The method is worth popularizing for its simplicity, quickness, sensitivity, wider linear scope, fewer interfering factors and less samples.