Letter to the editor

Ellman's method is still an appropriate method for measurement of cholinesterases activities

Seyed Vahid Shetab-Boushehri1[*],2

1Department of Toxicology & Pharmacology, School of Pharmacy, International Campus, Iran University of Medical Sciences, Tehran, Iran

2Razi Drug Research Center, Iran University of Medical Sciences, Tehran, Iran

EXCLI J 2018;17:Doc798

 

Dear Editor,

It has been recently argued that Ellman's method has not sufficient accuracy for measurement of cholinesterases (ChEs) activities (Komersová et al., 2007[3]; Sinko et al., 2007[6]; Pohanka et al., 2011[5]; Dingova et al., 2014[1]). This method was first introduced and described by Ellman and his colleagues and is based on thiocholine-derivative (acetyl- or butyryl) hydrolysis by ChEs (true or pseudo) and reaction of resulting thiocholine with thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and formation of 5-thio-2-nitrobenzoic acid (TNB) anion. Measurement of TNB ion absorption at 410 nm for pseudo-ChE and 440 nm for reticulocytic acetyl-ChE indirectly determines ChE activity (Ellman et al., 1961[2]). Some authors proposed that indoxylacetate is better substrate than acetylthiocholine (ATCH) as it does not react with oxime antidotes and thiol used for Ellman's method (Pohanka et al., 2011[5]). Moreover, DTNB which is unstable, interacts with free sulfhydryl groups in the sample, and may affect cholinesterase activity (Dingova et al., 2014[1]). Other researchers showed that oximes react with ATCH (oximolysis), producing thiocholine and consequently TNB ion (Sinko et al., 2007[6]). Some authors also mentioned that when the concentration of DTNB is far higher than the concentration of ATCH, hydrolysis rate of ATCH is decreased resulting in a lower measured ChE activity (Komersová et al., 2007[3]).

Although above-mentioned problems have been stated about Ellman's method, each of them has its own logical solution. First, by simple appropriate dilution of sample, the thiol- and oxime-containing compounds in the sample are also diluted and hence the extent of reaction of DTNB with these substances is considerably reduced (Mohammadi et al., 2017[4]). Second, molar attenuation coefficients of indoxylacetate and TNB are 3900 M-1.cm-1 (Pohanka et al., 2011[5]) and 13600 M-1.cm-1 (Ellman et al., 1961[2]) respectively. Thus, the detection limit for measurement of ChE activity by DTNB is about 3.5 times lower than that by indoxylacetate. Third, for DTNB concentrations of 0.2-0.598 mM, the best ratio of DTNB/ATCH concentrations is 1.25-3.74 which results in optimum rate of ATCH hydrolysis by ChE (Komersová et al., 2007[3]).

 

References

1. Dingova D, Leroy J, Check A, Garaj V, Krejci E, Hrabovska A. Optimal detection of cholinesterase activity in biological samples: Modifications to the standard Ellman’s assay. Anal Biochem. 2014;462:67-75.
2. Ellman GL, Courtney KD, Andres V Jr, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961;7:88-90.
3. Komersová A, Komers K, Cegan A. New findings about Ellman's method to determine cholinesterase activity. Z Naturforsch C. 2007;62:150-4.
4. Mohammadi H, Jalilian J, Karimi MY, Shetab-Boushehri SV. In vitro cysteine reactivates organophosphate insecticide dichlorvos-inhibited human cholinesterases. Sultan Qaboos Univ Med J. 2017;17:e293-e300.
5. Pohanka M, Hrabinova M, Kuca K, Simonato J-P. Assessment of acetylcholinesterase activity using indoxylacetate and comparison with the standard Ellman’s method. Int J Mol Sci. 2011;12:2631-40.
6. Sinko G, Calić M, Bosak A, Kovarik Z. Limitation of the Ellman method: cholinesterase activity measurement in the presence of oximes. Anal Biochem. 2007;370:223-7.
 
 
 

[*] Corresponding Author:

Seyed Vahid Shetab-Boushehri, Department of Toxicology & Pharmacology, School of Pharmacy, International Campus, Iran University of Medical Sciences, Tehran, Iran; Tel.: +98 2186702030, Fax: +98 21 88622523, PO Box: 14665-354, eMail: shetab.v@iums.ac.ir, vshetab@gmail.com