Ellman method, cholinesterase activity

Procedure Cholinesterase activity was measured according to the modified biochemical methods developed for crude preparations (Gorunef ah, 1978), using Ellman reagent 5,5"-dithio-bis(p-nitrobenzoic acid) or its red analogue 2,2-dithio-bis-(p-phenyleneazo)-bis-(l-oxy-8-chlorine-3,6) -disulfur acid in the form of sodium salt, which interact with thiocholine salt (Roshchina 2001). Water extracts of vegetative microspores of horsetail (Equisetum arvense) or Hippeastrum hybridum microspores (150 mg of microspores in 30 ml for 1 h) were used. 

Methods for the detection of cholinesterase activity can generally be divided into four groups, electrometric, colorimetric,titrimetric and tintometric methods, with the last being the one mainly used in the field. The colorimetric method developed by Ellman et al. is the most frequently used procedure. Titrimetry is extremely accurate and precise, but rarely used because of its high cost and complexity. Electrometric methods are less sensitive than colorimetric methods and less accurate than titrimetric methods. 

The demand for routine measurements of cholinc-stcrase activities under field conditions resulted in the development of various field kits. The Test-mate Reid kit, which is currently widely used, is based on the Ellman method. It is a. self-contained portable device comprising a set of reagents and instmetions for use. The Test-mate kit measures AChE and BuChE activities and the hemoglobin content in a drop of blood the activities are automatically nonnalized to 25 C (Wilson, 2001), Oliveira et al (2002) tested three Test-mate kit models (on fetal bovine semm AChE) and compared the results with those measured on a standard spectrophotometer with a 96-wdl microplate reader. The results from the three models showed a discrepancy, and there was also no good agreement between the results of the three models and those obtained from the standard photometric measurement. The authors strongly recommend the use of cholinesterase standards when activities are measured with field kits. 

The activities of two enzymes have been used as biomarkers of effects for OPs, namely acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase, sometimes known as pseudocholinesterase (EC 3.1.1.8). The structure and function of these enzymes has been reviewed. " In humans the former is present in red blood cells and the latter in plasma, but such distribution is not true of all species. In dogs, both enzymes are present in plasma with a ratio of butyrylcholinesterase to acetylcholinesterase of 7 1, while in the rat, plasma cholinesterase activity comprises more acetylcholinesterase with a butyrylcholinesterase to acetylcholinesterase activity of 1 3 in males and 2 1 in females in neither blood compartment are the functions of the enzymes fully understood.Because of the possibility of confusion, the terms plasma cholinesterase and erythrocyte cholinesterase as synonyms for butyrylcholinesterase and acetylcholinesterase are to be deprecated, especially when used of enzymes in animals where serious confusion may result. It is often necessary to look in detail at animal studies to see what activity has been measured in each matrix. In particular, it is necessary to look at the substrate(s) used in the assay together with any inhibitors used. Methods for measuring acetylcholinesterase have been reviewed and acetylcholinesterase and butyrylcholinesterase activities can be measured separately. In almost all cases it is the enzyme activity, rather than protein concentration, that is measured and many of the procedures used are variants of the Ellman method. Correct storage of blood samples is important as reactivation of inhibited enzymes ex vivo can occur. 

The activity of cholinesterases is usually determined by Ellman s method (19). This assay can also be carried out in microtitre plates. Thirty microliters of the ChE dilution and 30 pL of DNTB are added to 210pL of40mM Britton-Robinson-I buffer pH 8.0. The reaction is started by addition of 30 pL of the appropriate acylthiocholine substrate. A microtiter plate reader is used to monitor the development of the absorption at 595 nm. The corresponding slope is used to calculate the esteratic activity. 

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