Department of Toxicology, Faculty of Military Health Sciences, University of Defense, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic.
Chem Biol Interact. 2010 Sep 6;187(1-3):225-8. doi: 10.1016/j.cbi.2010.02.027. Epub 2010 Feb 24.
The method for automatic continual monitoring of acetylcholinesterase (AChE) activity in biological material is described. It is based on flexible system of plastic pipes mixing samples of biological material with reagents for enzyme determination; reaction product penetrates through the semipermeable membrane and it is spectrophotometrically determined (Ellman's method). It consists of sampling (either in vitro or in vivo), adding the substrate and flowing to dialyzer; reaction product (thiocholine) is dialyzed and mixed with 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) transported to flow spectrophotometer. Flowing of all materials is realised using peristaltic pump. The method was validated: time for optimal hydratation of the cellophane membrane; type of the membrane; type of dialyzer; conditions for optimal permeation of reaction components; optimization of substrate and DTNB concentrations (linear dependence); efficacy of peristaltic pump; calibration of analytes after permeation through the membrane; excluding of the blood permeation through the membrane. Some examples of the evaluation of the effects of AChE inhibitors are described. It was demonstrated very good uniformity of peaks representing the enzyme activity (good reproducibility); time dependence of AChE inhibition caused by VX in vitro in the rat blood allowing to determine the half life of inhibition and thus, bimolecular rate constants of inhibition; reactivation of inhibited AChE by some reactivators, and continual monitoring of the activity in the whole blood in vivo in intact and VX-intoxicated rats. The method is simple and not expensive, allowing automatic determination of AChE activity in discrete or continual samples in vitro or in vivo. It will be evaluated for further research of cholinesterase inhibitors.
描述了一种自动连续监测生物材料中乙酰胆碱酯酶 (AChE) 活性的方法。它基于一个灵活的塑料管系统，该系统混合了生物材料样本与酶测定试剂；反应产物穿透半透膜，然后通过分光光度法进行测定（埃勒曼法）。该方法包括取样（无论是在体外还是在体内）、加入底物并流动到透析器；反应产物（硫代胆碱）被透析并与 5,5'-二硫代双-2-硝基苯甲酸 (DTNB) 混合，然后输送到流动分光光度计。所有材料的流动都是通过蠕动泵实现的。该方法经过验证：纤维素膜最佳水合时间；膜的类型；透析器的类型；反应成分最佳渗透条件；底物和 DTNB 浓度的优化（线性依赖性）；蠕动泵的效率；分析物透过膜后的校准；排除血液透过膜的渗透。描述了一些评估 AChE 抑制剂作用的例子。结果表明，代表酶活性的峰具有很好的均匀性（良好的重现性）；VX 在体外对大鼠血液中 AChE 的抑制作用具有时间依赖性，从而可以确定抑制的半衰期，进而确定抑制的双分子速率常数；某些重激活剂对抑制的 AChE 的重激活，以及在完整和 VX 中毒大鼠体内在全血中进行的连续监测。该方法简单且成本低廉，允许在体外或体内离散或连续样本中自动测定 AChE 活性。它将进一步用于研究胆碱酯酶抑制剂。
