Cholinesterase inhibition assays

Once analytical capability and rapid turn around time are available locally or on the farm, many crop management decisions will be guided by this type of analysis. One example available commercially allows state inspectors to rapidly check that pesticide containers have been properly rinsed before they are discarded. Certain states require a deposit on all restricted pesticide containers. Deposits are refunded only if the container being returned passes a rapid assay which confirms that the container has been properly rinsed. The tests currently used for this on-site analysis are based on immunoassays as well as a colorimetric cholinesterase inhibition assay (26). 

The physiological basis of action of organophosphates and carbamates is inhibition of acetylcholinesterase at the insect neuromuscular junction. Accordingly, for almost 30 years attempts have been made to analyse these pesticides using cholinesterase inhibition assays(14 >. Many of these assays have... 

Table VI. Analysis of pesticides used for treatment of stored grain using a cholinesterase inhibition assay...

Inhibition profiles were determined for phosphorothioate OP insecticides such as parathion, malathion, and diazinon (Figure 3). Because these compounds were only weakly inhibitory, the measured concentration range extended from 0.1 nM to 100 pM. The relative order of potency was malathion > diazinon > parathion. The commercially available oxidative transformation products of parathion and malathion (i.e., paraoxon and malaoxon) as well as dichlorvos, were also measured using this assay (Figure 4). The oxidative transformation products were significantly more potent AChE inhibitors than the parent compounds and showed inhibitory profiles comparable to dichlorvos. The cholinesterase inhibition assay yielded similar IC50 values for each of these compounds. Indeed, these compounds are typically reported to have inhibition constants within an order of magnitude of each other (16, 17). 

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. 

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). 

Mauriz, E., A. Calle, J.J. Manclus, et al. 2006. Single and multi-analyte surface plasmon resonance assays for simultaneous detection of cholinesterase inhibiting pesticides. Sens. Actuat. B Chem. 118 399M07. 

Choline bromide succinate, 998 Choline chloride, 466 Choline chloride carbamate, 427 Choline chloride succinate, 998 Choline citrate, 466 Choline dihydrogen citrate, 466 Choline salicylate, 965 Choline theophyllinate, 1011 Cholinesterase activity, assay kit, 1171 quantification in serum, 23 test for inhibition of, 6 Chondodendron tomentosum, 1057 Chromatographic performance, evaluation of, 189 Chromatography, adsorption, 204... 

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). 

Age-related differences in kinetic parameters of detoxification can partially explain the increased sensitivity of the young to acute exposure to chlorpyrifos and other OPs (Mortensen et ai, 1996 Moser et ai, 1998 Padilla et ai, 2000, 2004). Thus, in vitro assays show that differences in vivo are not due to intrinsic differences in sensitivity of the target enzyme (Mortensen el ai, 1998). Furthermore, differences in liver microsomal metabolism, which mediates activation and/or inactivation of some pesticides, do not adequately explain the increased sensitivity (Benke and Murphy, 1975 Brodeur and DuBoks, 1%7). Differences in detoxification pathways correlate better with age sensitivity. B-esterases (e.g., carboxylesterases) and A-esterase.s bind to and/or hydrolyze, and thus detoxify, some cholinesterase-inhibiting pesticides (Jokanovic et al., 1996 Maxwell, 1992). These pathways are much less well developed in the young, and maturation of these systems tracks the decreasing sensitivity to acute exposure to chlorpyrifos and other OPs (Attcrberry el ai, 1997 Benke and Murphy, 1975 Brodeur and DuBois, 1967 Chanda et ai, 1997, 2002 Mendoza, 1976 Mortensen et ai, 1996, 1998 ... 

IC50 Values Because of its stability, strong inhibitory effect and commercial availability, paraoxon has often been used as a reference compound for cholinesterase inhibition (7). Asa result of the widespread use of paraoxon as a reference inhibitor, we elected to compare the relative potency of compounds assayed to this inhibitor in the form of paraoxon equivalence. IC50 values and % inhibition relative to paraoxon (paraoxon equivalence) were determined for a wide range of cholinesterase inhibitors (Table III). The extent to which the data fit a four parameter curve fit or log-logit fit are also included as correlation coefficients. The compounds are placed in ascending order of calculated IC50 values. 

A wide range of bioanalytical assays based on cholinesterase inhibition have been reported over the past decade. IC,o values (molar concentration yielding 10% inhibition of the control activity and typically considered as the detection limit) that have been reported for paraoxon using AChE -based inhibition assays vary over the... 

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). 

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). 

With regard to assaying the inhibitory activity of extracts electro-chemically, one of the problems of assays using sensors based on cholinesterase was that considerable time, e.g. 30-45 min [46,47], could be needed for the activity of the enzyme electrode to fall below control levels. The time increased as the level of inhibition decreased. Such lengthy assays make any number of serial assays impractical. In previous work [48,49], it had been noted that if sensors were exposed to solution containing inhibitors and then allowed to dry, they could be... 

Acetylcholine perchlorate was used as the substrate for erythrocyte and brain cholinesterase. The washed erythrocytes from 2 ml. of blood were diluted with the solution mentioned above, to which saponin was added to lake the cells, and an aliquot was taken for assay. The results are expressed as micromoles of acetic acid produced per minute per milliliter of whole blood. Because we preferred to express erythrocyte activity in terms of whole blood, we adjusted each final value to its equivalent at a hematocrit of 50%. This calculation, involving the hematocrit obtained on each blood sample, serves to differentiate enzyme inhibition from the variability in activity associated with abnormal plasma-erythrocyte volume ratios. 

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. 

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. 

---