Cholinesterase activity assays

Cholinesterase Activity Assay Kit colonmefric method for plasma or serum... 

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Most hospitals and forensic laboratories are capable of performing cholinesterase activity assays. Two examples of the usefulness of acetylcholinesterase and butyryl-cholinesterase activity assays are given below. 

The activity of the cholinesterases can be determined directly using traditional spectrophotometric methods and also electrochemical techniques. Electrochemical methods for cholinesterase activity assay that are based on pH-shift potentiometry have been described (5-11). Conventional pH electrodes (7-11) and pH sensitive field effect transistors (5, 6) were employed as transducers coupled with cholinesterase enzymes. The main disadvantage of the pH-shift based method is a strong requirement for low buffer capacity of the sample. In addition, the sensitivity of pH based analytical techniques, in general, is less than that based on amperometric assay. The theoretical threshold of pH based assay methods is as low as 58 mV per decade of analyte concentration. Ion-selective membranes (12) and mediator-assisted potentiometry (13) have also been proposed for assays of cholinesterase inhibitors. 

Blood specimens of approximately 5 mL were collected on two separate days during the week preceding the study. Additional blood specimens of approximately 5 mL each were collected approximately 24 and 48 hr after the start of the study. These blood specimens were drawn and assayed for plasma cholinesterase activity by personnel from the Michigan Division Medical Department of The Dow Chemical Company. 

The activity of allelochemicals inhibitors of cholinesterase was assayed as listed below ... 

Shiotsuka RN. 1988. Pilot assay to assess cholinesterase activity in rats exposed by inhalation to technical grade disulfoton. Study No. 88-941-AG. Report No. 98358. Mobay Corporation, Corporate Toxicology Department, Stilwell, Kansas. 

Cholinesterase Activity. Cholinesterase activity was assayed by automatic, continuous, alkali titration of acid released from the substrate, a method previously utilized by Wilson (12), Main and Dauterman (6), Shellenberger et al. (8), and others. 

TABLE G-1 The Elhnan Assay of Cholinesterase Activities in Plasma from 18 Rats... 

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. 

Kolf-Clauw M, Jez S, Ponsart C, Delamanche IS. Acetyl- and pseudo-cholinesterase activities of plasma, erythrocytes, and whole blood in male beagle dogs using Elhnan s assay. Vet Hum Toxicol 2000 42 216-9. 

The cholinesterases are generally accepted as being synthesized in the liver, and the assay of cholinesterase first became of interest to the clinician and to the clinical chemist as a test of liver function. Low serum cholinesterase activities are found in acute hepatitis, acute cirrhosis, and in liver metastases—that is, in those conditions where the hepatic synthesis of the protein is impaired. The synthesis of several other proteins is also reduced in such conditions, so that cholinesterase assay has been largely superseded as a test of liver function by measurements related to such proteins as albumin and prothrombin. Nevertheless, cholinesterase still has a place in the assessment of hepatic and other diseases, as discussed in Section 5.2. 

However, the situation concerning silent cholinesterase variants is rather complex, and some conclusions that have been drawn may be incorrect. Some reports have stated that no enzyme activity and no cholinesterase-like protein could be detected in the silent sera studied, but it is clear that whether or not any cholinesterase activity is observed depends on a number of factors. If the gene and all of its alleles are absent, no protein product will be produced, and the phenotype will be silent under all assay conditions. The same holds true if a gene is present... 

In summary, the situation concerning the silent or near silent cholinesterase variants is complex. There are at least three cholinesterase variants which are most importantly characterized by a severe quantitative deficiency in cholinesterase activity. Family studies indicate that these three variant are alleles of the gene for usual cholinesterase, Ey, although more studies are needed in this area. There are indications that a variety of other near-silent cholinesterase subtypes exist. Cases of true silence, because the gene is absent or because it produces no protein product, appear to be very rare. Many cases reported to have no cholinesterase activity have subsequently been found to have at least a trace of activity when studied imder more sensitive assay conditions or with a different substrate. 

In the protocol of Fcaster el ui (2004), three substrates are used ATCh, BTCh, and PTCh. The cholinesterase activities in whole blood are measured separately with each substrate, and the activities of AChE and BuChE are calculated by using the so-called sensitivity coefficients. These are determined prior to the assay by applying selective AChE and BuChE inhibitors to reference samples in order to evaluate the contribution of each enzyme to the hydrolysis of each of the three substrates. 

The use of enzyme activity assays in the presence and absence of inhibitors has some merit and can be automated. Cholinesterase variants have been detected by Kalow and Genest (1957) and Harris and Whittaker (1961) using dibucaine and fluoride inhibition, respectively they have provided a means of obtaining both qualitative information (type of variant) and quantitative information (activity of cholinesterase in the sample) about the enzyme in serum. 

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