Determination of acetylcholinesterase activity by the Ellman assay: A versatile tool for in vitro research on medical countermeasures against organophosphate poisoning

Abstract

Inhibition of acetylcholinesterase (AChE) is the main mechanism of action of organophosphorus compounds (OP), and AChE reactivators (oximes) are at present the only causal therapeutic approach. Being the key target of OP toxicity, AChE may serve as a valuable tool for diagnosis of OP exposure as well as for the investigation of the kinetics of interactions between OP and oximes. At present, the rapid, simple, and cheap spectrophotometric Ellman assay is widely used for diagnosis, therapeutic monitoring and in vitro kinetic investigations. Application of the assay for investigation of the interactions between AChE, inhibitors, and oximes requires the consideration of potential matrix effects (e.g. hemoglobin), side reactions (e.g. oximolysis of substrate) and other determinants (e.g. pH, temperature). By taking these factors into account, the Ellman assay allows the precise and reproducible determination of kinetic constants as a basis for the understanding of toxic OP effects and for the development of improved therapies against poisoning by OP. In addition, advanced applications of the Ellman assay, for example, in a dynamic in vitro model for the real-time activity determination of membrane-bound AChE, enables the proper investigation of relevant tissue, primarily respiratory muscle, and extends the applicability of this method.

Kinetic analysis of oxime-assisted reactivation of human, Guinea pig, and rat acetylcholinesterase inhibited by the organophosphorus pesticide metabolite phorate oxon (PHO)

“…Excess PHO was removed by dialysis, but re-inhibition of AChE by free oxime and/or phospho-oxime cannot be ruled out. These are well known challenges associated with oxime reactivation kinetics experiments (reviewed in [29]) that are difficult to eliminate completely. In addition, rapid reactivation was observed with some oximes, and a modified approach that permitted the evaluation of reactivation at shorter time points may have enabled more accurate estimation of the rate constants for some of the oximes evaluated in this study.…”

Small‐scale purification of butyrylcholinesterase from human plasma and implementation of a μLC‐UV/ESI MS/MS method to detect its organophosphorus adducts

“…Human BChE activity was measured by a modified Ellman assay . LC fractions or plasma were mixed with 10 mM PB (pH 7.4) and DTNB in a 96‐well microtiter plate prior to addition of BSCh for final concentrations of 1 mM for both reagents in a total volume of 230 μL.…”

“…Human BChE activity was measured by a modified Ellman assay. [28,29] LC fractions or plasma were mixed with 10 mM PB (pH 7.4) and DTNB in a 96-well microtiter plate prior to addition of BSCh for final concentrations of 1 mM for both reagents in a total volume of 230 μL. During reaction at 25°C the yellow product was monitored at 412 nm (ε 13,600 M À1 cm À1 ) in a Sunrise absorbance reader (Tecan, Crailsheim, Germany).…”

Assessment of food toxicology

“…As in vitro prescreening tools the human ether-a-go-gorelated gene (hERG) potassium ion channel [86][87][88] or acetylcholinesterase activity assay [89,90] are routinely used for a global assessment of cardiotoxicity or neurotoxicity, respectively. Other assays monitor the potential effects of toxicants which interfere with anti-inflammatory drugs.…”