Cholinesterases standards

The demand for routine measurements of cholinc-stcrase activities under field conditions resulted in the development of various field kits. The Test-mate Reid kit, which is currently widely used, is based on the Ellman method. It is a. self-contained portable device comprising a set of reagents and instmetions for use. The Test-mate kit measures AChE and BuChE activities and the hemoglobin content in a drop of blood the activities are automatically nonnalized to 25 C (Wilson, 2001), Oliveira et al (2002) tested three Test-mate kit models (on fetal bovine semm AChE) and compared the results with those measured on a standard spectrophotometer with a 96-wdl microplate reader. The results from the three models showed a discrepancy, and there was also no good agreement between the results of the three models and those obtained from the standard photometric measurement. The authors strongly recommend the use of cholinesterase standards when activities are measured with field kits. 

The current gold standard of treatment for cognitive symptoms includes pharmacologic management with a cholinesterase inhibitor and/or an N-methyl-D-aspartate (NMDA) antagonist. 

These systems have been used in many bioanalytical applications. A Prospekt system coupled with MS quantitated eserine N-oxide, a cholinesterase inhibitor, in human plasma for low level (4.5 mg) oral administration pharmacokinetic studies (Pruvost et al. 2000). After conditioning of the SPE cartridge (PLRP-S, Spark) with methanol (5 mL/min, 0.5 min) and water (5 mL/min, 0.5 min), a volume of 250 jj.L plasma plus internal standard was injected and washed (water, 1 mL/min, 3 min). The analytes were flushed out with 80 20 ammonium acetate (20 mM, pH 3.5 adjusted with formic acid) and acetonitrile (0.3 mL/min) and separated on a Zobax SB-CN column (150 x 2.1 mm inner diameter, 5 jim). A calibration range of 25 pg/mL to 12.5 ng/mL was achieved with a run time of 10.5 min. 

The effect of a standard dose of succinylcholine lasts only about 10 min. It is often given at the start of anesthesia to facilitate intubation of the patient. As expected, choUnesterase inhibitors are unable to counteract the effect of succinylcholine. In the few patients with a genetic deficiency in pseudocholinesterase (= nonspecific cholinesterase), the succinylcholine effect is significantly prolonged. 

Neuromuscular blockade produced by succinylcholine and mivacurium can be prolonged in patients with a genetically abnormal variant of plasma cholinesterase. The dibucaine number is a measure of the ability of a patient to metabolize succinylcholine and can be used to identify at-risk patients. Under standardized test conditions, dibucaine inhibits the normal enzyme by 80% and the abnormal enzyme by only 20%. Many genetic variants of plasma cholinesterase have been identified, although the dibucaine-related variants are the most important. Given the rarity of these genetic variants, plasma cholinesterase testing is not a routine clinical procedure. 

The pesticides are detected electrochemically by measuring the degree of inhibition of cholinesterase on the screen-printed electrodes. The degree of inhibition can be thought of as the ratio of the response of electrodes (to substrate) exposed to pesticide (standard or wool extract) to that of electrodes not exposed to pesticide or exposed to a blank (extract from wool which has not been exposed to pesticide). An efficient and convenient way of doing this is to expose the SPCEs to standards and wool extracts prior to measurement of inhibition in the electrochemical cell. 

It may be possible to use an array of electrodes containing various enzymes in combination with multivariate statistical analyses (principal component analysis, discriminant analysis, partial least-squares regression) to determine which pesticide(s) the SPCE has been exposed to and possibly even how much, provided sufficient training sets of standards have been measured. The construction methods for such arrays would be the same as described in this protocol, with variations in the amounts of enzyme depending on the inhibition constants of other cholinesterases for the various pesticides of interest. 

Figure 2 represented a log-probit plot of the observed inhibition of purified bovine erythrocyte acetylcholinesterase as a function of concentration for several of the transformation products of aminocarb. The observation that these inhibition curves are parallel suggests a similar mechanism of interaction for the various derivatives. The parameter I5f. (the concentration of inhibitor required to achieve 50% inhibition oi the enzyme activity) for each of the inhibitors were calculated and are recorded in Table 1. These values are reported relative to the parent compound aminocarb = 1. Also included in Table 1 are the relative toxicities of several of these products to house crickets (Acheta domesticus). It had been our intention to develop bioassay tests using the target insect itself, the eastern spruce budworm (Choristoneura fumiferana). However, spray tower results were quite variable and it was considered that genetic variability of the stock culture made the production of uniform test batches difficult to achieve. Using the house crickets, an LD q of 130-155 ppm for aminocarb standard was observed over the course of more than 25 bioassays. Also included in Table 1 are observations by Abdel-Wahab and Casida (19) using human plasma or house fly head cholinesterases. 

Long-term exposure to azinphos-methyl, above the average 8-hour standard set by the Occupational Safety and Health Administration (OSHA), can impair concentration and memory, and cause headache, irritability, nausea, vomiting, muscle cramps, and dizziness. Cholinesterase inhibition from exposure to azinphos-methyl may persist for 2 to 6 weeks. Repeated exposure to small amounts may... 

Henderson, J., M. O Malley, J. Sanborn, M. Verder-Carlos and B. Wilson (1998). Comparison of Acetylcholinesterase Assays Run Under Conditions Specified by the Standard Ellman Method and Conditions Specified by a Commercial Cholinesterase Reagent Kit, Department of Pesticide Regulation, Worker Health and Safety Branch Report HS-1752, Sacramento, CA, USA. 

Buccafusco, J.J., Heithold, D.L., Chon, S.H. (1990). Long-term behavioral and learning abnormalities produced by the irreversible cholinesterase inhibitor soman effect of a standard pretreatment regimen and clonidine. Toxicol. Lett. 52 319-29. 

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). 

Bambuterol has been reported to alter the metabolism of mivacurium (17). Bambuterol has a dose-dependent inhibitory effect on plasma cholinesterase activity and prolongs the effects of suxamethonium. Bambuterol 10 mg was given to 28 patients 2 hours before an elective operation requiring general anesthesia. The patients given bambuterol had a 67-97% fall in plasma cholinesterase activity, leading to reduced clearance of mivacurium. This resulted in a shorter onset and a 3- to 4-fold prolongation of action of the neuromuscular blockade produced by standard doses of mivacurium. 

Phenol Red. The first publications of an automated procedure for the measurement of cholinesterase inhibitors are those of Winter 14) and Winter and Ferrari (15). The method employed an Autoanalyzer instrumental system wherein the extracts containing the insecticide were incubated with a standard cholinesterase solution at 37 °G. A continuous sample from the incubation bath is buffered and mixed with acetylcholine iodide. After a second incubation, the acetic acid released by the action of the uninhibited cholinesterase is measured colorimetrically, using phenol red as the indicator. More recently, Fischl et al. 16) reported a method for rapid detection of organic phosphate pesticides in serum. Strips of filter paper were impregnated with a buffered acetylcholine substrate solution containing phenol red as an indicator. When no inhibition is present, the acid released from the action of cholinesterase turns the paper yellow. When cholinesterase has been inhibited, the paper turns pink-to-violet. 

Russian workers have prepared for pharmacological studies simple carbamates of trachelanthamidine (68) by treatment with the appropriate isocyanate. Some cholinesterase inhibitors based on the standard decamethonium model have been prepared. Decamethylene bistrachelanthamine dibromide (69) showed marked curare-like activity in cats and rabbits. New procedures for the iodometric determination of platyphylline bitartrate and for the colorimetric determination of trichodesmine and incanine in the organs of poisoned animals have been described. 

The US EPA Acute Dietary population adjusted dose (PAD) is 0.0007 mg kg day fenthion. This standard is based on plasma cholinesterase inhibition in monkey (NOAEL = 0.07mgkg day ), divided by a composite uncertainty factor of 100 (lOx interspecies, lOx intraspecies), and a lx factor for Food Quality Protection Act (FQPA). 

---