Cholinesterases methods

Kralj, N. (1967). Morphologic and histochemical studies on the nervous system of tapeworms revealed by the cholinesterase method (Taenia hydatigena, Dipylidium caninum and Moniezia expansa). Veterinarski Arhiv, 37 277-86. 

Augustinsson, K.B. (1971). Determination of activity of cholinesterases. Methods Biochem. Analyt. 217 (Suppl.) 217-73. 

Similarly to quantitative determination of high surfactant concentrations, many alternative methods have been proposed for the quantitative determination of low surfactant concentrations. Tsuji et al. [270] developed a potentio-metric method for the microdetermination of anionic surfactants that was applied to the analysis of 5-100 ppm of sodium dodecyl sulfate and 1-10 ppm of sodium dodecyl ether (2.9 EO) sulfate. This method is based on the inhibitory effect of anionic surfactants on the enzyme system cholinesterase-butyryl-thiocholine iodide. A constant current is applied across two platinum plate electrodes immersed in a solution containing butyrylthiocholine and surfactant. Since cholinesterase produces enzymatic hydrolysis of the substrate, the decrease in the initial velocity of the hydrolysis caused by the surfactant corresponds to its concentration. Amounts up to 60 pg of alcohol sulfate can be spectrometrically determined with acridine orange by extraction of the ion pair with a mixture 3 1 (v/v) of benzene/methyl isobutyl ketone [271]. 

Organophosphates, such as methyl parathion, are known to inhibit cholinesterase activity. A method has been developed to measure the extent of this inhibition and relate it to organophosphate exposure (EPA 1980d Nabb and Whitfield 1967). In this EPA-recommended method, blood is separated into plasma and red blood cell fractions. The fractions are treated with saline solution, brought to pH 8 with sodium hydroxide, and dosed with acetylcholine perchlorate. The ensuing acetic acid releasing enzyme reaction... 

Nabb DP, Whitfield F. 1967. Determination of cholinesterase by an automated pH-stat method. Arch Environ Health 15 147-154. 

TharrD. 1998. Rapid assessment of organophosphate-induced cholinesterase depression A comparison of laboratory and field kit methods to detect human exposure to organophosphates. Appl Occup Environ Hyg 13 265-268. 

Oxime carbamates are generally applied either directly to the tilled soil or sprayed on crops. One of the advantages of oxime carbamates is their short persistence on plants. They are readily degraded into their metabolites shortly after application. However, some of these metabolites have insecticidal properties even more potent than those of the parent compound. For example, the oxidative product of aldicarb is aldicarb sulfoxide, which is observed to be 10-20 times more active as a cholinesterase inhibitor than aldicarb. Other oxime carbamates (e.g., methomyl) have degradates which show no insecticidal activity, have low to negligible ecotoxicity and mammalian toxicity relative to the parent, and are normally nondetectable in crops. Therefore, the residue definition may include the parent oxime carbamate (e.g., methomyl) or parent and metabolites (e.g., aldicarb and its sulfoxide and sulfone metabolites). The tolerance or maximum residue limit (MRL) of pesticides on any food commodity is based on the highest residue concentration detected on mature crops at harvest or the LOQ of the method submitted for enforcement purposes if no detectable residues are found. For example, the tolerances of methomyl in US food commodities range from 0.1 to 6 mg kg for food items and up to 40 mg kg for feed items. ... 

Altstein, M., Segev, G., Aharonson, N., Ben-Aziz, O., Turniansky, A. and Avnir, D. (1998) Sol-gel-entrapped cholinesterases A microtiter plate method for monitoring anticholinesterase compounds. Journal of Agricultural and Food Chemistry, 46, 3318-3324. 

The design and implementation of a portable fiber-optic cholinesterase biosensor for the detection and determination of pesticides carbaryl and dichlorvos was presented by Andreou81. The sensing bioactive material was a three-layer sandwich. The enzyme cholinesterase was immobilized on the outer layer, consisting of hydrophilic modified polyvinylidenefluoride membrane. The membrane was in contact with an intermediate sol-gel layer that incorporated bromocresol purple, deposited on an inner disk. The sensor operated in a static mode at room temperature and the rate of the inhibited reaction served as an analytical signal. This method was successfully applied to the direct analysis of natural water samples (detection and determination of these pesticides), without sample pretreatment, and since the biosensor setup is fully portable (in a small case), it is suitable for in-field use. 

Procedure Cholinesterase activity in analyzed tissue or the matrix (biotest with immobilized AChE) is determined in the incubation media [consisting of substrate ATCh - 34 mmol maleate buffer 0.1 M, pH = 6.0- 6.5 ml sodium citrate 0.1 M - 0.5 ml CuS045H20 0.03M -1.0 ml distilled H20 (or inhibitor in variant with toxin analyzed) -1.0 ml potassium ferricyanide 0.005 M -1 ml.] Volume of incubation media in one test - 400 mcl. As a blank (control sample), a treatment of the exposure without the substrate is used. If inhibitory effects of allelochemical (or any toxin) are analyzed, before the substrate addition the sample was preliminary exposed to allelochemical inhibitor. Two methods for the AChE-biotests may be recommended (i) in microcells ( stationary conditions ) and (ii) in flowing columns-reactors ( dynamic conditions ). 

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. 

Karnovsky, M. and Roots, L.A. (1964). A direct colouring method for cholinesterases. Journal of Histochemistry and Cytochemistry 12 219-225. 

In AChE-based biosensors acetylthiocholine is commonly used as a substrate. The thiocholine produced during the catalytic reaction can be monitored using spectromet-ric, amperometric [44] (Fig. 2.2) or potentiometric methods. The enzyme activity is indirectly proportional to the pesticide concentration. La Rosa et al. [45] used 4-ami-nophenyl acetate as the enzyme substrate for a cholinesterase sensor for pesticide determination. This system allowed the determination of esterase activities via oxidation of the enzymatic product 4-aminophenol rather than the typical thiocholine. Sulfonylureas are reversible inhibitors of acetolactate synthase (ALS). By taking advantage of this inhibition mechanism ALS has been entrapped in photo cured polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ) to prepare an amperometric biosensor for... 

B. Bucur, A.F. Danet, and J.L. Marty Versatile method of cholinesterase immobilisation via affinity bonds using concanavalin A applied to the construction of a screen-printed biosensor. Biosens. Bioelectron. 20, 217-225 (2004). 

M. Altstein, G. Segev, N. Aharonson, O. Ben-Aziz, A. Turniansky, and D. Avnir, Sol-gel-entrapped cholinesterases a microtiter plate method for monitoring anti-cholinesterase compounds. J. Agric. Food Chem. 46, 3318-3324 (1998). 

Presently available methods to diagnose and biomonitor exposure to anticholinesterases, e.g., nerve agents, rely mostly on measurement of residual enzyme activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in blood. More specific methods involve analysis of the intact poison or its degradation products in blood and/or urine. These approaches have serious drawbacks. Measurement of cholinesterase inhibition in blood does not identify the anticholinesterase and does not provide reliable evidence for exposure at inhibition levels less than 20 %. The intact poison and its degradation products can only be measured shortly after exposure. Moreover, the degradation products of pesticides may enter the body as such upon ingestion of food products containing these products. 

Their specimen of cholinesterase was prepared from horse serum by the method of Stedman and Stedman,1 and the method of estimation was that of Ammon.2 The enzyme solution was placed in the right-hand flask of a Barcrofb manometer, in a total volume of 3 ml. of 0-2 per cent NaHC03 solution the gas phase was 5 per cent C02 in Na. The reaction, carried out at 20°, was started by adding a solution containing 2 mg. of acetylcholine chloride. The C02 output was usually linear until about 100 fi. had been produced. 

Effect of substrate concentration. In the following experiments the cholinesterase activities were measured by a continuous titration method. The digest of acetylcholine and horse-serum cholinesterase (total vol. 10 ml.), containing bromothymol blue and 0-0002 m phosphate, was titrated with 0-01 n NaOH to maintain the pH at 7-4. The titrations, which were carried out at 20°, were linear over a period of 10-15 min. The velocity was expressed as ml. 0-01 n NaOH/5 min. under the conditions used, it was proportional to the enzyme concentration. When an inhibitor was added, this was equilibrated with the enzyme, etc., for 5 min. at 20° before adding the substrate contained in a volume of 1 ml. 

True and pseudo-cholinesterase. The above serum preparations contained both the true and pseudo- cholinesterases of Mendel and Rudney.1 The effect of di-isopropyl phosphorofluoridate on these components was examined separately by means of the specific substrates described by Mendel, Mundel and Rudney,2 using the titration method described above. Phosphorofluoridate (5 x 10 8m) gave an inhibition of 57 per cent of the activity towards 00045m acetylcholine, 30 per cent of the activity towards 0-0005 m acetyl-/ methyl-choline, and 40 per cent of that towards 0-005 m benzoylcholine, after incubating the enzyme with the poison for 5 min. Thus in these experiments there appeared to be no appreciable difference in sensitivity of the true and pseudo-cholinesterases of horse serum to phosphorofluoridates. 

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). 

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. 

Journal reports by Bell and Gershon indicated that tetrahydroaminoacridine (THA), a cholinesterase inhibitor, was effective in reversing delirium induced by Ditran (JB-329) as a form of psychiatric treatment It is interesting that their use of Ditran for this purpose was similar to the atropine coma treatment method reported more than a decade earlier by Forrer, Miller et al. In our study, five subjects were given 5.0 mcg/kg of oral BZ on two occasions, 8-14 days apart 60 mcg/kg of THA was administered iv four hours after the time of the second BZ dose. We observed definite partial reversal of impairment soon after injection, but it was brief. An unexpected observation was the general tendency by the subjects to become impaired more rapidly and intensely by BZ on the second occasion - a finding that was later confirmed in a more careful study. 

Rapid advances in chemistry during the nineteenth and twentieth centuries, coupled with the success of mustard gas as a toxic weapon in World War I, attracted attention to the warfare potential of chemical agents. This led to support for research on lethal nerve agents during and immediately after World War II. The research was followed by the development of treatment methods, and prominent among these was the use of cholinesterase reactivators to reverse the lethal effects of anticholinesterase nerve gases. 

Severe cholinergic excess is a medical emergency, especially in rural communities where cholinesterase inhibitor insecticides are commonly used and in cultures where wild mushrooms are commonly eaten. The potential use of cholinesterase inhibitors as chemical warfare "nerve gases" also requires an awareness of the methods for treating acute poisoning (see Chapter 58). 

Both the nicotinic and the muscarinic effects of the cholinesterase inhibitors can be life-threatening. Unfortunately, there is no effective method for directly blocking the nicotinic effects of cholinesterase inhibition, because nicotinic agonists and antagonists cause blockade of transmission (see Chapter 27). To reverse the muscarinic effects, a tertiary (not quaternary) amine drug must be used (preferably... 

PAM is given to regenerate inhibited cholinesterase (acetylcholinesterase) enzyme at all affected sites (Schenker et al. 1992 Shankar 1967, 1978). The available information sufficiently satisfies the need for methods of reducing toxic effects. Therefore, further studies in this regard are not required. 

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