Development and validation of a simple assay for the determination of cholinesterase activity in whole blood of laboratory animals

Abstract

Current methods for measuring acetylcholinesterase (AChE) activities in whole blood use butyrylcholinesterase (BChE)‐selective inhibitors. However, the poor selectivity of these inhibitors results in the inhibition of AChE activity to some degree, leading to errors in reported values. The goal of this study was to develop and validate a simple assay for measuring AChE and BChE activities in whole blood from humans as well as experimental animals. Blood was fractionated into plasma and erythrocytes, and cholinesterase activities were titrated against ethopropazine and (−)‐huperzine A to determine the lowest concentration of ethopropazine that inhibited BChE completely without affecting AChE activity and the lowest concentration of (−)‐huperzine A that inhibited AChE completely without interfering with BChE activity. Results indicate that 20 µm ethopropazine can be successfully used for the accurate measurement of AChE activity in blood from humans as well as animals. Use of (−)‐huperzine A is not required for measuring BChE activity in normal or ‘exposed’ blood samples. The method was validated for blood from several animal species, including mice, rats, guinea pigs, dogs, minipigs, and African green, cynomolgus and rhesus monkeys. This method is superior to all reported methods, does not require the separation of erythrocyte and plasma fractions, and is suitable for measuring cholinesterase activities in fresh or frozen blood from animals that were exposed to nerve agents or those that were administered high doses of BChE. The method is simple, direct, reproducible, and reliable and can easily be adapted for high‐throughput screening of blood samples. Published 2012. This article is a US Government work and is in the public domain in the USA.

In situ induced metal-enhanced fluorescence: A new strategy for biosensing the total acetylcholinesterase activity in sub-microliter human whole blood

“…On the other hand, the interferent is eliminated in the very low amount of blood samples (corresponds to 0.1 μL of the undiluted blood sample). The total AChE activity in the five human blood samples are also consistent with the AChE activity level in the human blood obtained using other methods (Worek et al, 1999;Sanz et al, 1991;Naik et al, 2013). These results indicate that this technique can reliably detect the total AChE activity assay in submicroliter human blood.…”

Distigmine Bromide Produces Sustained Potentiation of Guinea-Pig Urinary Bladder Motility by Inhibiting Cholinesterase Activity

“…Naik et al reported that more than 90% of plasma ChE activity arises from BChE in Hartley guinea-pig plasma and that this situation is similar in humans. 28) Therefore, any observed plasma ChE inhibitory effects are thought to reflect effects on BChE. Our present findings were consistent with a report by Yamamoto et al, which suggested that the inhibitory effects of distigmine on plasma ChE activities were independent of the dose administered.…”

Cholinergic signalling mechanisms and early implant healing phases in healthy versus generalized aggressive periodontitis patients: A prospective, case–control study

“…AChE and BChE activity levels were determined using a modified version of the biochemical assay developed by Naik, Liu, and Saxena () as previously described (Apatzidou et al, ).…”