Development of a method for extraction and assay of human erythrocyte acetylcholinesterase and pesticide inhibition

Abstract

A method of extracting membranes from red blood cells (RBCs) is described, which were in turn used to assay acetylcholinesterase (AChE) activity. The evidence for the enzyme activity was established by selective inhibition using 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide, tetraisopropyl pyrophosphoramide and neostigmine. Blood samples were exposed to three organophosphorus (dichlorvos, chlorpyrifos and diazinon) and two carbamate (carbaryl and carbofuran) pesticides. Afterwards AChE activities in RBC membranes were determined. The concentrations capable to inhibit the enzyme activity by 50% (IC₅₀) for the pesticides were 10.66 µM (dichlorvos), 21.42 µM (chlorpyrifos), 109.98 µM (carbaryl) and 5.44 µM (carbofuran). The results related to 20% enzyme inhibition (level used in the estimation of threshold limits for anticholinesterase compounds) were below those acceptable daily intake values enacted by relevant national and international regulations. These results suggest that the proposed AChE extraction from RBC and assay could be a suitable method for monitoring occupational exposure to pesticides.

Straightforward, Economical Procedures for Microscale Ellman’s Test for Cholinesterase Inhibition and Reactivation

“…In carrying out the Ellman's assays, special care should be taken with the toxicity of the employed inhibitors, as organophosphorus compounds are poisonous. Cholinesterases extraction from biological sources 18,19 may be an alternative to commercial sources in budget-tight laboratories. Stability of enzyme stock solutions must be considered, as well as the effect on cholinesterases by solvents in assayed compounds' solutions.…”

“…Such effects are relevant in measurements of Cholinesterase activity in tissue and blood samples in routine diagnosis. 5,6,9,10,[19][20][21] Interference caused by thiol residues found in these matrices requires different approaches for its minimization, such as sample dilution or previous treatment of biological matrix with DTNB. 5,6,10 Hemoglobin absorbance at 412nm could also be a potential source of error, which might require the use of a different wavelength or development of novel chromophores.…”