Acetylcholinesterase activity assays

In cases where the mode of action is the strong or irreversible inhibition of an enzyme system, the assay may measure the extent of inhibition of this enzyme. This may be accomplished by first measuring the activity of the inhibited enzyme and then making comparison with the uninhibited enzyme. This practice is followed when studying acetylcholinesterase inhibition by organophosphates (OP). Acetylcholinesterase activity is measured in a sample of tissue of brain from an animal that has been exposed to an OP. Activity is measured in the same way in tissue samples from untreated controls of the same species, sex, age, etc. Comparison is then made between the two activity measurements, and the percentage inhibition is estimated. 

Most hospitals and forensic laboratories are capable of performing cholinesterase activity assays. Two examples of the usefulness of acetylcholinesterase and butyryl-cholinesterase activity assays are given below. 

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. 

Fonnum, F. (1969) Radiochemical micro-assays for the determination of choline acetyltransferase and acetylcholinesterase activities. Biochem. J., 115,465-472. 

Worek, R, Eyer, R, Thiermann, H., 2012. Determination of acetylcholinesterase activity by the Ellman assay a versatile tool for in vitro research on medical countermeasures against organophosphate poisoning. Drug Test Anal. 4, 282-291. 

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. 

Compound 10 was evaluated for anti-bacterial activity and acetylchohnesterase (AChE) inhibitory activities. This compound was foimd to be inactive in antibacterial assay and exhibited AChE inhibitory activity with an ICj, value of 67 pM. Acetylcholine serves as a neurotransmitter in the central and peripheral nervous system. Acetylcholinesterase (AChE) stops the function of acetylcholine by its... 

In the following example we describe the implementation of a mass spectromet-ric assay for acetylcholinesterase (AChE) [22]. AChE plays an important role in the nervous system. This enzyme rapidly hydrolyzes the active neurotransmitter acetylcholine into the inactive compounds choline and acetic acid. Amongst others, low levels of acetylcholine in the synaptic cleft are associated with Alzheimer s disease [23, 24]. Patients afflicted by this disease may benefit from inhibition of AChE activity thereby increasing ACh level. 

I. K. Rhee, N. Appels, T. Luijendijk, H. Irth, R. Verpoorte Determining acetylcholinesterase inhibitory activity in plant extracts using a fluorimetric flow assay. Phytochem Anal 2003, 14, 145-149. 

In another report, several acetylcholinesterase (AChE) inhibitors, including tacrine, edrophonium, tetramethyl- and tetraethyl-ammonium chloride, carbofu-ran, and eserine were assayed on a chip [1045]. AChE converted the substrate, acetylthiocholine, to thiocholine. This product reacted with coumarinylphenyl-maleimide (CPM) to form thiocholine-CPM (a thioether) for LIF detection. Since the acetonitrile solvent used to dissolve CPM inhibited AChE activity, the CPM solution was added after the enzymatic reaction [1045]. Another enzyme inhibition assay using a peptide substrate was performed on a PMMA chip [1046]. 

The EMMIA system was developed by Ngo and Lenhoff (N3, N4). In this assay, enzyme activity is modulated by an enzyme modulator which is coupled to antigen (free form) but not by the complex of enzyme modulator-antigen and antibody (bound form). As shown in Fig. 2 and Table 6, in an enzyme inhibitor immunoassay, an enzyme inhibitor is used as a negative modulator. For example, the reaction mixture for measuring thyroxine consists of acetylcholine inhibitor-thyroxine conjugate [I-Ag], acetylcholinesterase [E], unlabeled thyroxine [Ag], and antithyroxine antibody [Ab]. When the amount of unlabeled thyroxine, which binds to antibody [Ab Ag], is increased, the free form of acetylcholine inhibitor-thyroxine conjugate [I-Ag] increases, and the enzyme activity decreases. Therefore, the enzyme activity is inversely proportional to the concentration of unlabeled thyroxine. A tetrazyme kit (Abbott) is now available for measuring thyroxine. 

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