Cholinesterase Assays

Current measurements of cholinesterase are based upon the hydrolysis of an ester by an enzyme (Wills 1972 Whittaker 1986). The measurements for AChE and BuChE enzymes are sensitive to pH, temperature, and substrate concentration, and these factors must be considered in order to optimize the assay for the relevant species. There are basically four different principles for these assays, which include  

5-dithio-bis-2-nitrobenzoic acid (DTNB), produce a yellow color. The yellow-colored product has a large extinction coefficient and offers a highly sensitive method (Ellman et al. 1961). 

The potentiometric technique involves the measurement of pH, which falls as acetic acid released by hydrolysis of the acetylated enzyme occurs however, this method lacks sensitivity (Michel 1949). The radioisotopic method uses tritiated acetylcholine as substrate. The method is highly sensitive but requires a scintillation counter (Winteringham and Disney 1964 Johnson and Russel 1975). The manometric method is based upon the release of carbon dioxide evolved from the action of the bicarbonate in the system with the acid formed by hydrolysis of the ester (Augustinsson 1948). 

Some significant pitfalls are associated with the measurement of cholinesterase, and these include the timings of sample collection and analysis. The time between blood and tissue sampling and assay should be minimized to prevent further inhibition 

Plasma Cholinesterase Activities Measured Colorimetrically by Three Separate Substrate Methods in Four Species  

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Occupational air Air exposed to polystyrene strip with adsorbed cholinesterase assayed in cuvette with Ellman s reagent and acetylthiocholine UV absorbance (412 nm) 20 ppb (1-min exposure) No data Brown et al. 1984... 

Yager, J., H. McLean, M. Hudes and R.C. Spear. 1976. Components of variability in blood cholinesterase assay results. J. Occup. Med. 18 242-244. 

Halbrook, R.S., Guzman, C.E., Wilkinson, K.J., Watson, A.P., Mimro, N.B., Shugart, L.R. (1992). Rapid whole-blood cholinesterase assay with potential use for biological monitoring during chemical weapons disposal. J. Assoc. Off. Anal. Chem. Int. 75 549-53. 

There is a vast and continually growing literature on these two enzymes, but many papers have not distinguished clearly between them. The purpose of the present survey is to focus attention on human serum cholinesterase and its variants in the light of recent research, and to offer a critical assessment of reports of its physical and chemical properties and clinical and toxicological applications. The methodologies of cholinesterase assays and the older literature on the variant enzymes will not be considered in detail Relevant references may be found in several authoritative reviews (B15, D5, F4, G13, G16, H5, K5, LIO, L24, Ul, W35). Acetylcholinesterase, also, has been extensively reviewed (Nl, S22, S23, W29) and it will be discussed here only in order to make particular points of comparison with cholinesterase. 

The cholinesterases are generally accepted as being synthesized in the liver, and the assay of cholinesterase first became of interest to the clinician and to the clinical chemist as a test of liver function. Low serum cholinesterase activities are found in acute hepatitis, acute cirrhosis, and in liver metastases—that is, in those conditions where the hepatic synthesis of the protein is impaired. The synthesis of several other proteins is also reduced in such conditions, so that cholinesterase assay has been largely superseded as a test of liver function by measurements related to such proteins as albumin and prothrombin. Nevertheless, cholinesterase still has a place in the assessment of hepatic and other diseases, as discussed in Section 5.2. 

The best known clinical application of cholinesterase assay concerns the abnormally prolonged effect of the muscle relaxant succinylcholine that is found in a small proportion of patients. This compound, which was introduced into clinical medicine in the early 1950s (B29, B41, T47), owes its relaxant action to competition with acetylcholine for the receptors at the neuromuscular junction both cause depolarization of the muscle fibers, which contract. Acetylcholine is rapidly destroyed by acetylcholinesterase, so that repeated stimuli are applied to the muscle, causing a controlled contraction which persists as long as the nerve is stimulated. When, however, succinylcholine is administered, it is not destroyed by acetylcholinesterase, and its action persists until a large proportion of the dose has been hydrolyzed in the plasma. After initial contraction, the muscle fibers passively elongate to give the relaxation required by the anesthetist. 

Wilson, B. W. et al. 1996. Eactors in standardizing automated cholinesterase assays. Journal of Toxicology and Environmental Health 48 187-195. 

Due to the wide interindividual variations of AChE and BuChE activities in human blood (see Section V), a single cholinesterase assay does not give information about the absorption of a cholinesterase inhibitor. To calculate the degree of cholinesterase inhibition due to absorption of an inhibitor, one has to know the AChE and BuChE activities before the exposure of an individual to OPs or CMs (preexposure activities). 

The inhibition of brain cholinesterase is a biomarker assay for organophosphorous (OP) and carbamate insecticides (Chapter 10, Section 10.2.4). OPs inhibit the enzyme by forming covalent bonds with a serine residue at the active center. Inhibition is, at best, slowly reversible. The degree of toxic effect depends upon the extent of cholinesterase inhibition caused by one or more OP and/or carbamate insecticides. In the case of OPs administered to vertebrates, a typical scenario is as follows sublethal symptoms begin to appear at 40-50% inhibition of cholinesterase, lethal toxicity above 70% inhibition. 

Blood specimens of approximately 5 mL were collected on two separate days during the week preceding the study. Additional blood specimens of approximately 5 mL each were collected approximately 24 and 48 hr after the start of the study. These blood specimens were drawn and assayed for plasma cholinesterase activity by personnel from the Michigan Division Medical Department of The Dow Chemical Company. 

Experiment 2. Cholinesterase as a sensor on the cell surface A target of the allelochemical may also be a surface sensor-cholinesterase (Fig. 10). If after the staining with Red analogue of Ellman reagent the blue colour is absent in the allelochemical treated microspore, possible target is the enzyme (Roshchina, 2001a,b) as for alkaloid berberine tested. If after the treatment by the test allelochemical, the colour is absent or light, the compound inhibits the enzyme (also see biochemical assay in Chapter 11). 

The activity of allelochemicals inhibitors of cholinesterase was assayed as listed below ... 

Gorun, V., Proinov, I., Baltescu, V., Balaban, G. and Barzu, O. (1978). Modified Ellman procedure for assay of cholinesterases in crude enzymatic preparations. Analytical Biochemistry 86 324-326. 

Johnson CD, Russell RL. A rapid, simple radiometric assay for cholinesterase suitable for multiple determinations. Anal. Biochem. 64 229-238, 1975. 

Support Function Protective Clothing for Hazardous Chemicals Operations - NFPA 1993. Quincy, MA. Technical Bulletin - Assay Techniques for Detection of Exposure To Sulfur Mustard, Cholinesterase Inhibitors, Terrorism Incident Annex to the Federal Response Plan. Washington, D.C. 1995. 

The cholinesterase-inhibiting activity of the phosphorofluoridates was compared quantitatively with that of eserine sulphate thus. To 0-2 ml. of heparinized human plasma was added 05 ml. of a solution containing either eserine or the phosphorofluoridate in varying concentrations then the mixture was kept at room temperature for 10 min. before 1 /tg. of acetylcholine in 1 c.c. saline solution was added. After 5 min. at room temperature, the mixture was made up to 10 ml. with frog saline containing eserine 1/100,000, which at once stopped the action of any cholinesterase not yet inactivated. The solution was then assayed for acetylcholine on the frog rectus-muscle preparation. 

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). 

Shiotsuka RN. 1988. Pilot assay to assess cholinesterase activity in rats exposed by inhalation to technical grade disulfoton. Study No. 88-941-AG. Report No. 98358. Mobay Corporation, Corporate Toxicology Department, Stilwell, Kansas. 

Simple alkyl or aryl thioesters are commonly assayed as substrates of hydrolases, witness the hydrolysis of phenyl thioesters by horse serum carbox-ylesterase [150], For most substrates investigated, e.g., phenyl thioacetate, phenyl thiopropionate, and phenyl thiobutyrate (7.66, R = Me, Et, and Bu, respectively), kcat values were found, which were a few times larger than those of corresponding nitrophenyl esters, whereas the affinities were lower by approximately one order of magnitude. Methyl and phenyl esters of various linear thioacids were also found to be good substrates of mammalian liver carboxylesterases and serum cholinesterases [151]. 

Frolich and colleagues (1998) analyzed ACh in human CSF by different methods, which included thermospray/mass spectroscopy, HPLC/mass spectroscopy, HPLC-EC Pt electrode and gas chromatogra-phy/mass spectroscopy (GC/MS). An SPE extraction was used for cleanup and concentration. Samples were run with and without the IMER to rule out any interference by physostigmine, a cholinesterase inhibitor, in the HPLC-EC assay. HPLC-EC and GC-MS gave data correlations with similar sensitivities, but the HPLC-EC values were 39% lower. Analysis using thermospray/mass spectroscopy and HPLC/ mass spectroscopy did not provide adequate sensitivity and the data obtained were inconsistent. 

Additional novel analytical techniques include coating a polystyrene strip with cholinesterase, exposing the strip to an atmosphere (passive sampling), then immersing the strip in a cuvette with reagent for assay... 

Methods for Determining Biomarkers of Exposure and Effect. Section 2.6.1 reported on biomarkers used to identify or quantify exposure to diazinon. Some methods for the detection of the parent compound in biological samples were described above. The parent chemical is quickly metabolized so the determination of metabolites can also serve as biomarkers of exposure. The most specific biomarkers will be those metabolites related to 2-isopropyl-6-methyl-4-hydroxypyrimidine. A method for this compound and 2-(r-hydroxy-l -methyl)-ethyl-6-methyl-4-hydroxypyrimidine in dog urine has been described by Lawrence and Iverson (1975) with reported sensitivities in the sub-ppm range. Other metabolites most commonly detected are 0,0-diethylphosphate and 0,0-diethylphosphorothioate, although these compounds are not specific for diazinon as they also arise from other diethylphosphates and phosphorothioates (Drevenkar et al. 1993 Kudzin et al. 1991 Mount 1984 Reid and Watts 1981 Vasilic et al. 1993). Another less specific marker of exposure is erythrocyte acetyl cholinesterase, an enzyme inhibited by insecticidal organophosphorus compounds (see Chapter 2). Methods for the diazinon-specific hydroxypyrimidines should be updated and validated for human samples. Rapid, simple, and specific methods should be sought to make assays readily available to the clinician. Studies that relate the exposure concentration of diazinon to the concentrations of these specific biomarkers in blood or urine would provide a basis for the interpretation of such biomarker data. 

Enzymatic techniques have also been employed in the analysis of these compounds. The toxicity of carbamate insecticides is due to the inhibition of the enzyme acetylcholine esterase, so the determination of these compounds can be achieved by enzyme inhibition (2,83,119), bioassay (118,167), or enzyme-linked immunosorbent assay (ELISA) (168-171). In the detection of carbamates by fluorimetric enzyme inhibition, the effluent from a reversed-phase chromatographic column was incubated with cholinesterase, which was introduced via a postcolumn reagent delivery pump. Then, the resulting partially inhibited cholinesterase was reacted with N-methyl indoyl acetate to produce a fluorophore and a reduction in the baseline fluorescence (172). 

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