Cholinesterase activity assays methods

Cholinesterase Activity Assay Kit colonmefric method for plasma or serum... 

The activity of the cholinesterases can be determined directly using traditional spectrophotometric methods and also electrochemical techniques. Electrochemical methods for cholinesterase activity assay that are based on pH-shift potentiometry have been described (5-11). Conventional pH electrodes (7-11) and pH sensitive field effect transistors (5, 6) were employed as transducers coupled with cholinesterase enzymes. The main disadvantage of the pH-shift based method is a strong requirement for low buffer capacity of the sample. In addition, the sensitivity of pH based analytical techniques, in general, is less than that based on amperometric assay. The theoretical threshold of pH based assay methods is as low as 58 mV per decade of analyte concentration. Ion-selective membranes (12) and mediator-assisted potentiometry (13) have also been proposed for assays of cholinesterase inhibitors. 

Cholinesterase Activity. Cholinesterase activity was assayed by automatic, continuous, alkali titration of acid released from the substrate, a method previously utilized by Wilson (12), Main and Dauterman (6), Shellenberger et al. (8), and others. 

Exposure to a toxic dose of OP results in inhibition of acetylcholinesterase and butyrylcholinesterase activities. The most common method to measure OP exposure is to assay acetylcholinesterase and butyrylcholinesterase activities in blood using a spectrophotometric method (EUman et al, 1961 Wilson et al, 2005 Worek et al, 1999). The drawbacks of activity assays are that they do not identily the OP. They show that the poison is a cholinesterase inhibitor but do not distinguish between nerve agents, OP pesticides, carbamate pesticides, and tightly bound, noncovalent inhibitors like tacrine and other anti-Alzheimer drugs. In addition, low-dose exposure, which inhibits less than 20% of the cholinesterase, carmot be determined by measuring acetylcholinesterase and butyrylcholinesterase activity because individual variability in activity levels is higher than the percent inhibition. 

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. 

Direct kinetic assays are the only valid methods for the measurement of activators and inhibitors and calibration plots of the percentage activation or inhibition by known amounts of the substance can be made. Examples of inhibition assays include the quantitation of organophosphorus pesticides using the inhibition of cholinesterase (EC 3.1.1.7) while manganese can be measured in amounts as low as 1 X 10-12 mol using its activating effect on isocitrate dehydrogenase (EC 1.1.1.41). 

Fluorogenic Substrates, Guilbault and Kramer (20) pubhshed a method using a fluorometric assay for anticholinesterase compoimds. The substrates used were nonfluorescent compounds, the acetyl and butyl esters of 1- and 2-naphthol, which are hydrolyzed by cholinesterase to highly fluorescent materials. The rate of change of fluorescence was related to enzyme activity, and inhibition was measured by decreased rate of change in the production of fluorescence. 

Mosca A, Bonora R, Ceriotti F, Franzini C, Lando G, Patrosso MC, et al. Assay using succinyldithiochohne as substrate the method of choice for the measurement of cholinesterase catalytic activity in serum to diagnose sucdnylchoMne sensitivity. Clin Chem Lab Med 2003 41 317-22. 

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. 

Mosca, A., Bonora. R., Cerimti, R Franzini, C Lando, G Patross. M.C., ZaninoUo, M., and Panteghini, M, (2003). Assay using succinyldithiocholine as sub.strate The method of choice for the measurement of cholinesterase catalytic activity in serum lo diagno.se. succinyldicholine. sensitivity. CHn. Chem. Lab. Med. 41,317-322. 

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