血浆氨酶促终点法测定的实验研究

Enzymatic End Point Assay for Determination of Ammonia in Plasma with GLDH NADH Reaction System.

介绍一种用谷氨酸脱氢酶 还原辅酶Ⅰ (GLDH NADH)为反应系统的血浆氨酶促终点比色测定法 .反应体系中加入 1 9kU/L的乳酸脱氢酶 (LDH) ,可排除内源性丙酮酸盐引起NADH消耗所致的吸光度持续下降对测定的干扰 .此法测定线性范围为 6～ 2 0 0 μmol/L ,回收率为 90 %～ 1 0 5% ,批内变异系数小于 5% ,常规条件下的变异系数小于 1 0 % . 30例健康志愿者清晨空腹静脉血浆氨水平为1 0～ 70 μmol/L .此法操作简单、迅速、精确 。

An improved enzymatic end point method for determination of plasma ammonia was reported. Semi micromethod were used with a total incubation volume 1 165 μl. Mix 125 μl EDTA Na 2 anticoagulation plasma and 1 000 μl PBS substrate solution containing 1 9 kU/L LDH (pH 8 0), then add 40 μl GLDH (≥12×10 4 U/L) to start the reaction. Incubation periods were shortened to 5 min. The limited detection for ammonia in plasma is 4 μmol/L, and this method is linear from 6 to 200 μmol/L. The method is accurate and reliable. The analytical recovery is from 90% to 105%. The within run CV% was≤5%. The routine condition variance of the method ranged from 5% to 10%. It was found that 1 9 kU/L LDH was sufficient to eliminate the decreace of NADH by endogenouse pyruvate and LDH in plasma. The normal fasting ammonia in plasma is 10～70 μmol/L.