Cholinesterases activity measurements

Procedure Allelochemical and a compound belonging to natural artificial pesticides and medicinal drugs is preliminary added into the reaction media (see section Add). The difference in cholinesterase activity (measured as shown in sections 15.3) between a control (without the substance added) and the experimental variant is estimated. The results are compared with the effects of the cholinesterase inhibitors neostigmine and physostigmine. 

Plasma Cholinesterase Activities Measured Colorimetrically by Three Separate Substrate Methods in Four Species ... 

Gage, J. C. 1967. The significance of blood cholinesterase activity measurements. Residue Reviews 18 159-173. 

III. GENERAL OUTLINE OF CHOLINESTERASE ACTIVITY MEASUREMENTS IN HUMAN BLOOD... 

Simeon, V. (1967). Methods for cholinesterase activity measurements (in Croatian], Arft. Hig. Rada Totsikol. 18,29-39. 

NGF unprimed cells cultured without serum in the presence of chloipyrifos (10 pM), trichloropyridinol (10 ptAf j. or chlorpyrifos-oxon (10 nAf). with or without NGF 24-168 hr, NGF primed cells given NGF 1 week in serum-free medium and then replaied and treated with OP with or without NGF 24 hr. ncurite outgrowth and cholinesterase activities measured. 

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. 

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... 

Serat WF, Mengle DC (1973) Quality control in the measurement of blood cholinesterase activities among persons exposed to pesticides. Bull Environ Contam Toxicol 9 24-27. 

Hydraulic fluids themselves cannot be measured in blood, urine, or feces, but certain chemicals in them can be measured. Aliphatic hydrocarbons, which are major components of mineral oil hydraulic fluids and polyalphaolefin hydraulic fluids, can be detected in the feces. Certain components of organophosphate ester hydraulic fluids leave the body in urine. Some of these fluids inhibit the enzyme cholinesterase. Cholinesterase activity in blood can be measured. Because many other chemicals also inhibit cholinesterase activity in blood, this test is not specific for organophosphate ester hydraulic fluids. This test is not available at most doctor s offices, but can be arranged at any hospital laboratory. See Chapters 2 and 6 for more information. 

Wills JH. 1972. The measurement and significance of changes in the cholinesterase activities of erythrocytes and plasma in man and animals. CRC Crit Rev Toxicol (March) 153-202. 

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. 

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. 

Disulfoton and its breakdown products can be measured in the blood, urine, feces, liver, kidney, or body fat of exposed people. In cases of occupational or accidental exposure to disulfoton, the breakdown products are often measured in the urine. The breakdown products are relatively specific for disulfoton and a few other similar organophosphate pesticides and can be detected in urine for up to one week after people were last exposed. Because disulfoton inhibits cholinesterase in blood and in blood cells, inhibition of this enzyme activity may also suggest exposure to disulfoton. Cholinesterase activity in blood and in blood cells may remain inhibited for as long as 1-2 weeks after the last exposure. Because other organophosphate pesticides also inhibit cholinesterase activity in blood and blood cells, this test is not specific for disulfoton. The measurement of cholinesterase in blood and blood cells and the amount of disulfoton breakdown products in the urine cannot always predict how much disulfoton you were exposed to. Your doctor can send samples of your blood or urine to special laboratories that perform these tests. Chapters 2 and 6 provide more information about medical tests. 

Because cholinesterase inhibition is a very sensitive biomarker for other chemicals, it is not always conclusive evidence of disulfoton exposure. However, depression of cholinesterase activity can alert a physician to the possibility of more serious neurological effects. Erythrocyte acetylcholinesterase activity more accurately reflects the degree of synaptic cholinesterase inhibition in nervous tissue, while serum cholinesterase activity may be associated with other sites (Goldfrank et al. 1990). In addition, a recent study showed that after rats received oral doses of disulfoton for 14 days, acetylcholinesterase levels in circulating lymphocytes correlated better with brain acetylcholinesterase activity than did erythrocyte cell cholinesterase activities during exposure (Fitzgerald and Costa 1993). However, recovery of the activity in lymphocytes was faster than the recovery of activity in the brain, which correlated better with the activity in erythrocytes. Animal studies have also demonstrated that brain acetylcholinesterase depression is a sensitive indicator of neurological effects (Carpy et al. 1975 Costa et al. 1984 Schwab and Murphy 1981 Schwab et al. 1981, 1983) however, the measurement of brain acetylcholinesterase in humans is too invasive to be practical. 

It frequently invalidates the measurement of blood cholinesterase activity as a diagnostic index of poisoning. 

In the final study to be mentioned in regard to the possible penetration of pyrldinium oximes into brain, anesthetized, atropi-nized cats were given intravenous injections of sarin at 27/tg/kg. 12 Thirty minutes later, to allow clearance of unreacted sarin from the tissues, some cats received saline injections into one common carotid artery and others received similar injections of 1 at 15 mg/kg. The cholinesterase activity of cerebral cortex was measured. In animals given 1, nearly 20% of the cholinesterase in their cerebral cortices that had been inhibited by sarin was calculated to have been reactivated by the oxime. 

Another class of enzymes that has found wide application in the biosensor field in the last decades is that of the cholinesterases which have been mainly used for the detection of pesticides. For the amperometric detection of cholinesterase activity, both the substrates acetylcholine and acetylthiocholine have been extensively used [6-9], the latter being preferred because this avoids the use of another enzyme, choline oxidase, which is usually coupled with acetylcholinesterase. However, the amperometric measurement of thiocholine, produced by... 

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. 

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