Cholinesterase techniques

Shute, C. C. D. and Lewis, P. R. (1961) The use of cholinesterase techniques combined with operative procedures to follow nervous pathways in the brain. Bibl. anat. (Basel), 2, 34-49. 

Biosensors ai e widely used to the detection of hazardous contaminants in foodstuffs, soil and fresh waters. Due to high sensitivity, simple design, low cost and real-time measurement mode biosensors ai e considered as an alternative to conventional analytical techniques, e.g. GC or HPLC. Although the sensitivity and selectivity of contaminant detection is mainly determined by a biological component, i.e. enzyme or antibodies, the biosensor performance can be efficiently controlled by the optimization of its assembly and working conditions. In this report, the prospects to the improvement of pesticide detection with cholinesterase sensors based on modified screen-printed electrodes are summarized. The following opportunities for the controlled improvement of analytical characteristics of anticholinesterase pesticides ai e discussed ... 

Hunt, K.A. and M.J. Hooper. 1993. Development and optimization of reactivation techniques for carbamate-inhibited brain and plasma cholinesterases in birds and mammals. Anal. Biochem. 212 335-343. 

Hunt, K.A., M.J. Hooper, and E.E. Littrell. 1995. Carbofuran poisoning in herons diagnosis using cholinesterase reactivation techniques. Jour. Wildl. Dis. 31 186-192. 

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. 

Francis placed strips of the retina from different animals in sodium sulphate to precipitate the cholinesterase in situ. Some strips were then incubated with acetylthiocholine, while others were kept in D.F.P. solution before the incubation. The tissues after preliminary washings were then treated with appropriate reagents so as to precipitate the copper derivative of thio-choline. The sections ultimately obtained showed dark deposits at those points where the enzyme was present, and deposits were absent if D.F.P. had destroyed the enzyme. As a result of the application of this technique, Francis was able to establish that for all the animals examined, except the frog, true cholinesterase was present only at the inner synaptic layer. 

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... 

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). 

Method. A diagram of the apparatus is shown in Fig.4.29. Any suitable liquid chromatograph may be used. The AutoAnalyzer is modified such that the liquid sampler is fitted to the end of the chromatographic column. The proportioning pump is by-passed. The set-up of the AutoAnalyzer is the same as that for normal measurements of cholinesterase. The application of this technique to the determination of CGA 18809 in plum-leaf extract is shown in Fig.4.30. A comparison is made with UV analysis of the same extract. The limit of detection for CGA 18809 is c . 20 ng at a 3 1 signal to noise ratio. The relative inhibitions of several organophosphates and carbamates are compared in Table 4.9. Diazoxon may be detected in low picogram quantities. 

Tests for specific classes of chemicals include the measurement of transmitter stimulated adenyl cyclase and Na/K-ATPase for chemicals that affect receptor function or cholinesterase inhibition for organophosphates or carbamates. Electrophysiological techniques may detect chemicals such as DDT or pyrethroids, which affect impulse transmission. 

Stansley, W. Field results using cholinesterase reactivation techniques to diagnose acute anticholinesterase poisoning in birds and fish. Arch. Environ. Contam. Toxicol. 25 315-321, 1993. 

Fig. 2.8. Nervous system of the tetrathyridium larva of Mesocestoides sp. based on histochemical (for cholinesterase) and histological techniques. (After Hart, 1967.)...

The understandable correlation among careless technique, absenteeism, cholinesterase inhibition, and elevated average urinary PNP levels suggested that the latter was a highly reliable biological index of chronic parathion exposure and one that could ultimately predict chronic parathion toxicity. 

The other case history was an example of the complete opposite. Here, low urine concentrations were observed in spite of obvious excessive exposure and moderate cholinesterase decline. This was a 49-year-old agricultural sprayman whose techniques were atrocious and protection was minimal. In spite of these practices, however, urine concentrations were always below the 1-p.p.m. level of PNP. It was also noted that, although not drinking excessively, over an 80-day period he failed to concentrate his urine above isotonic levels, suggesting some underlying urinary concentration defect. Correction of his urine to osmotic levels observed in other spraymen would have raised his PNP level to 5 p.p.m. Subsequent evaluation confirmed the presence of proximal and distal renal tubular dysfunction. 

A method to set REIs would account for the rate of dermal absorption, the rate of foliar contact and the rate of change in cholinesterase. These factors were used in the Popendorf and Leffingwell (1982) Unified Field Model for determining REIs. This model also accounts for the relative rate of DFR dissipation, and differences in potency based on the dermal LD50 of the pesticide. The Unified Field Model is an elegant technique that takes into account many variables affecting exposure and cholinesterase inhibition as a response. Ultimately, the rate of cholinesterase inhibition, and not a fixed level of inhibition, is the primary... 

It is important that the preoperative examination is carried out meticulously and that the tumour can be adequately assessed by imaging techniques regarding its intrahepatic location, size, extent and segmental affiliation. When participation of vascular or biliary structures or involvement of extrahepatic organs is suspected, additional investigations are called for. The hepato-logical status is determined as follows enzymatic and mesenchymal activities, cholestasis and jaundice, functional test values (e. g. cholinesterase, albumin, coagula-... 

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