Cholinesterase, activity determinations

Coye MJ, Lowe JA, Maddy KT. 1986. Biological monitoring of agricultural workers exposed to pesticides I. Cholinesterase activity determinations. J Occup Med 28 619-627. 

Winter, G.D. (1960). Cholinesterase activity determination in an automated analysis system. Ann. NY Acad. Sci. 87 629-35. 

Stoytcheva M, Zlatev R, Valdez B, Magnin JP, Velkova Z (2006) Electrochemical sensor based on Arthrobacter globiformis for cholinesterase activity determination. Biosens Bioelectron 22(l) l-9. doi 10.1016/j.bios.2005.11.013... 

Individuals with hereditary low plasma cholinesterase levels (Kalow 1956 Lehman and Ryan 1956) and those with paroxysmal nocturnal hemoglobinuria, which is related to abnormally low levels of erythrocyte acetylcholinesterase (Auditore and Hartmann 1959), would have increased susceptibility to the effects of anticholinesterase agents such as methyl parathion. Repeated measurements of plasma cholinesterase activity (in the absence of organophosphate exposure) can be used to identify individuals with genetically determined low plasma cholinesterase. 

Levels of ACHE and PCHE vary in healthy people because of genetic differences or under specific physio-pathological conditions inter-individual coefficients of variation of cholinesterase activity have been determined to be about 15 to 25% for PCHE and 10 to 18% for RBC-ACHE. Corresponding figures for intra-individual variations are 6% and 3 to 7%, respectively (Dillon and Ho, 1987). 

The cholinesterase to determine the toxic activity may be chosen (i) in pure form of commercial enzyme from animals in a water buffer solution or using biosensors, enzyme preparation impregnated into a rigid matrix that significantly activates the enzymic activity and (ii) in the form of crude extracts from plant or animal tissues. 

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 ). 

The cholinesterase activity is determined in the presence of alllelochemicals-alkaloids (Fig. 4) and reactive oxygen species ozone and peroxides (Table 1). 

Although some steroids have been reported to reduce the toxic effects of some insecticides, the steroid ethylestrenol decreased the rate of recovery of depressed cholinesterase activity in disulfoton- pretreated rats (Robinson et al. 1978). The exact mechanism of this interaction was not determined. Ethylestrenol alone caused a small decrease in cholinesterase activity, and, therefore, resulted in an additive effect. Rats excreted less adrenaline and more noradrenaline when given simultaneous treatments of atropine and disulfoton compared with rats given disulfoton alone (Brzezinski 1973). The mechanism of action of disulfoton on catecholamine levels may depend on acetylcholine accumulation. In the presence of atropine, the acetylcholine effect on these receptors increases the ability of atropine to liberate catecholamines. 

Most of the signs and symptoms resulting from diazinon poisoning are due to the inhibition of an enzyme called acetylcholinesterase in the nervous system. This enzyme is also found in your red blood cells and a similar enzyme (serum cholinesterase) is found in blood plasma. The most common test for exposure to many organophosphorus insecticides, including diazinon, is to determine the level of cholinesterase activity in the red blood cells or plasma. This test requires only a small amount of blood and is routinely available in your doctor s office. It takes time for this enzyme to completely recover to normal levels following exposure. Therefore, a valid test may be conducted a number of days following the suspected exposure. This test indicates only exposure to an insecticide of this type. It does not specifically show exposure to diazinon. 

Other chemicals or disease states may also alter the activity of this enzyme. There is a wide range of normal cholinesterase activity in the general population. If you have not established your normal or baseline value through a previous test, you might have to repeat the test several times to determine if your enzyme activity is recovering. 

This method of pesticide detection is based on the inhibition of cholinesterase activity. This is a non-specific measurement and as such cannot determine which pesticide the enzyme electrode had been exposed to. This protocol has described a method for extraction and detection of diazinon, but other organophosphate pesticides can be substituted (e.g. chlorfenvinphos) bearing in mind that the enzyme has different inhibition constants for other pesticides and quantities may need to be altered. 

The Department has developed methods for monitoring the exposure of workers exposed to organophosphate and carbamate pesticides. These methods utilize the determination of plasma and red blood cell cholinesterase activities and urinary alkyl phosphates. Studies are reported vrti ich show that these methods have proven useful in evaluating the safety effectiveness of closed-transfer systems and in determining reentry times for field workers. 

For adequate toxicologic evaluation, determinations should be made on lipids, hormones, acid/base balance, methemoglobin, and cholinesterase activity. In other words, additional clinical biochemistry may be resorted to wherever necessary to arrive at meaningful conclusions in association with the observed effects. Urinalysis, although not required on a routine basis, should be performed whenever there is an indication based on observed toxicity. 

Exposure to organophosphate pesticides is often measured by determination of alkyl phosphate or phenol metabolites in the urine. Determination of blood cholinesterase activity can be a valuable indicator of exposure if pre-exposure cholinesterase activity is known (3, 5). Since normal cholinesterase levels... 

To determine the effect on the skin and on the cholinesterase activity of the blood of persons engaged in handling the Vaporizers, 10 volunteers were selected for investigation six men and four women. Before they handled the Vaporizer, blood was drawn by venipuncture from each subject, and the cholinesterase activity of the plasma and erythrocytes was determined. Two of the volunteers were used in a preliminary trial, one in handling the Vaporizer for 30 minutes, and the other by having the Vaporizer fixed to the volar aspect of the forearm for 30 minutes. Another sample of blood was drawn 24 hours after the experimental procedures (Table I). 

The subjects were divided arbitrarily into two groups, A and B. Members of group A handled the Vaporizer for 30 minutes each day in the manner required for household or farm use. A portion of Vaporizer, 2Vz by 5 inches, was affixed with adhesive tape in direct contact with the bare skin of the volar surface of the forearm of each member of group B, for 30 minutes of each of 5 successive days. Fresh Vaporizers were applied on the first and third of the 5 successive days. Cholinesterase activity was determined before the first period of contact and after the second and fifth (Tables I and II). 

The results of this investigation indicate no detectable inhibition of the cholinesterase activity of either plasma or erythrocytes as a result of handling Vapona Resin Vaporizers under the conditions of this experiment or of maintaining Vaporizers in direct contact with the skin for 30 minutes per day on 5 successive days. The fluctuations in cholinesterase activity were well within the normal range of day-to-day variation. The second determination of the cholinesterase activity of the plasma of subject 6 was unusually high. No explanation can be offered for this unusually high value, except to note that the specimen was Very milky (the probable result of lipemia) and that it was taken on the day following the death of the subjects mother. 

Just before and at intervals during the 6 months of the experimental regimen, blood was drawn from the subjects to determine cholinesterase activity. Table III presents the results over the entire period. 

Plasma or serum cholinesterase (pseudocholinesterase) is inhibited by a munber of compounds and can also be decreased in ftie presence of liver impairment. Erythrocyte cholinesterase (true cholinesterase) reflects more accurately the cholinesterase status of the central nervous system. However, pseudocholinesterase activity responds more quickly to an inhibitor and returns to normal more rapidly than eiythrocyte-cholinesterase activity. Thus, measurement of pseudocholinesterase activity is quite adequate as a means of diagnosing acute exposure to organophosphorus compounds, but cases of illness which may be due to chronic exposure to these compounds should also be investigated by determining the erydirocyte-cholinesterase activity. A colorimetric method for this purpose has been reported (K.-B. Augustinsson et ah, Clinica chim. Acta, 1978, 89, 239-252). 

Normal values for cholinesterase in serum are 1900 to 4000 milliunits/ml. Commercial kits for the determination of cholinesterase activity are available (see Reagent Appendix). 

Determination of cholinesterase activity is based on a number of principles. In general, an enzyme is added to the buffered or unbuffered mixture and the enzymatic reaction is initiated by adding the substrate. Different parts of the reaction mixture are determined (continually or noncontinuaUy), i.e. unhydrolyzed substrate or reaction products, either directly or indirectly (Augustinsson, 1971 Holmstedt, 1971 Witter, 1963). The conditions must be... 

For practical purposes (individual and interindividual variation), determination of individual norm activity is recommended (this approach is better than that of calculating the decrease from an average value) as well as separate determination of cholinesterases, the red blood cell AChE and plasma BuChE. The activity determined in the whole human blood corresponds to about 10% of BuChE and 90% of AChE. This is different to rats where this ratio is 29% of BuChE and 71% of AChE (Bajgar, 1972). Erythrocyte AChE activity seems to be more useful for these purposes than BuChE activity in plasma. 

Konickova, J., Wadso, T. (1971). Use of flow microcalorimetry for the determination of cholinesterase activity and its inhibition by organophosphorus pesticides. Acta Chem. Scand. 25 2360-82. 

Kusu, F., Tsuneta, T., Takamura, K. (1990). Fluorimetric determination of pseudo cholinesterase activity in postmortem blood samples. J. Forensic Sci. 35 1330. ... 

Michel, H.O. (1949). An electrometric method for the determination of red blood cell and plasma cholinesterase activity. J. Lab. Clin. Med. 34 1564-8. 

Another laboratory value that is often obtained in these exposures is serum pseudoeholinesterase. Serum pseudocholinesterase activities are often assessed as normal in children because the reference standards may not be reliable when assessing children. To add to the complexity, the normal range of serum cholinesterase activity is wide (Sofer et al, 1989). Authors have described the limitations of this measurement in determining therapy for children. In fact, it is recommended that a therapeutic and diagnostic trial of atropine should be given whenever there is any possibility of intoxication with these chemicals (Sofer et al, 1989). 

Sevelova, L., Bajgar, J., Saxena, A., Doctor, B.P. (2004). Protective effect of equine hutyrylcholinesterase in inhalation intoxication of rats with sarin determination of blood and brain cholinesterase activities. Inhal. Toxicol. 16 531-6. 

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