Asia Pacific Journal of Medical Toxicology

Clarifying an Electrometric Method for Determining Blood Cholinesterase Activity: A Scientific Letter

Document Type : Letter to Editor

Author

Fouad Kasim Mohammad

1Pharmacology and Toxicology Department of Physiology, Biochemistry and Pharmacology College of Veterinary Medicine, University of Mosul, Mosul, Iraq

https://doi.org/10.22038/apjmt.2022.19924
Abstract
Abstract
 
In this scientific letter, a modified electrometric method is clarified for rapid and accurate determination of blood cholinesterase (ChE) activity in man and various animal species. Our electrometric method for ChE determination is a modified one that we refined and developed after several years of research and validations in various animal species as well as in man. The developed method has been applied in many research projects on poisoning with organophosphate and carbamate insecticides. Given the importance of the subject of ChE biomonitoring, and to further clarify the assay technique of the modified electrometric method, a brief and a concise description of the procedure would be beneficial for researchers of limited resources. The enzymatic (ChE) reaction mixture consisted of 3 ml of distilled water, 0.2 ml of plasma, erythrocytes or whole blood, and 3 ml of barbital-phosphate buffer (pH 8.1). The initial pH (pH1) of the mixture is measured with a pH meter, and thereafter 0.1 ml of the substrate acetylcholine iodide (7.1%) or acetylthiocholine iodide (7.5%) is added. The mixture is incubated at 37 ºC for 30 min in most animal species or for 20 min in man; the pH2 of the reaction mixture is measured again. The activity of blood ChE is calculated as follows:
ChE activity (D pH/20 min-in man) = (pH1 – pH2) - D pH of blank (no blood sample).
 
 

Keywords

Cholinesterase Method
Organophosphates
Carbamates
Insecticide poisoning

Monitoring blood (whole blood, plasma, serum or erythrocytes) cholinesterase (ChE) activity is used to assess the exposure of man and animals to organophosphate and carbamate insecticides (1-3). Usually a reduction of blood ChE activity by 20-30% from baseline values is an indication of exposure to ChE inhibiting pesticides (2,3). Enzyme inhibition by > 50% calls for immediate healthcare measures (1-3). Several methods are available to measure ChE activity in the blood and nervous tissues (3,4). A modified electrometric method is available for measuring blood and brain ChE activity that is a simple procedure which does not need elaborate equipment and was adopted by many researchers (5-27). However, a published article erroneously referenced this modified electrometric method mentioned above (28). This prompted us to clarify and elucidate few key points about the method. The original electrometric Michel method has two incubation periods for a total of more than one hour at 25 oC (29), whereas our electrometric method is mainly characterized by a single incubation period of 20 min in man (5) and 30 min or even more in different animal species at 37 oC (6-27).

Our electrometric method of ChE determination is a modified one that we refined and developed after several years of research and validations in various animal species (6-27) as well as in man (5,25). We also applied the developed method on many research projects on poisoning with organophosphate and carbamate insecticides (6-26). For review of differences in animal species and man, see published references (5,18,23-26,30).

Because of the importance of the subject of ChE biomonitoring (1-4) and to further clarify the assay technique of the modified electrometric method, here is a brief but concise description of the procedure. It is hoped that it would be beneficial for researchers of limited resources.

Chemicals: 7.5% aqueous solution of acetylthiocholine iodide (or 7.1% acetylcholine iodide), sodium barbital, potassium dihydrogen phosphate, sodium chloride, and double distilled water.

Barbital-Phosphate buffer: 1.237 g sodium barbital, 0.163 g potassium dihydrogen phosphate and 35.07 g sodium chloride/ L distilled water, pH 8.1.

Equipment: Centrifuge, Water bath, pH meter

Procedure:

  1. Collect venous blood samples in heparinized test tubes.
  2. Separate the plasma from erythrocytes by centrifugation.
  3. To a 10-ml beaker add 3 ml distilled water, 0.2 ml plasma, erythrocytes or even whole blood.
  4. Add 3 ml of barbital-phosphate buffer solution.
  5. Measure the pH of the mixture (pH1).
  6. Add 0.10 ml of 7.5% acetylthiocholine iodide (or 7.1% acetylcholine iodide) to the mixture.
  7. Incubated at 37 ºC for 30 min for most animal species; 20 min in man.
  8. Measure the pH of the mixture (pH2).
  9. Calculate cholinesterase activity:

ChE activity (DpH/30 min or 20 min) = (pH1 – pH2) - D pH of blank

The blank is without sample. The unit of activity is Δ pH/30 or 20 min.

 

  1. References

    1.Vale A, Lotti M. Organophosphorus and carbamate insecticide poisoning. Handb Clin Neurol. 2015;131:149-68. doi: 10.1016/B978-0-444-62627-1.00010-X. PMID: 26563788.

     

    1. Wilson BW. Cholinesterase inhibition. In: Wexler P, editor. Encyclopedia of toxicology. 3rd ed. Amsterdam: Elsevier; 2014. p. 942-951. https://doi.org/10.1016/B978-0-12-386454-3.00116-0.

     

    1. Wilson BW, Arrieta DE, Henderson JD. Monitoring cholinesterases to detect pesticide exposure. Chem Biol Interact. 2005 Dec 15;157-158:253-6. doi: 10.1016/j.cbi.2005.10.043. Epub 2005 Nov 18. PMID: 16298353.

     

    1. Wilson B. Clinical enzymology. In: Loeb WF, Quimby FW, editors. The clinical chemistry of laboratory animals. Philadelphia: Taylor and Francis; 1999. p. 399-454.

     

    1. Mohammad FK, Alias AS, Ahmed OA. Electrometric measurement of plasma, erythrocyte, and whole blood cholinesterase activities in healthy human volunteers. J Med Toxicol. 2007 Mar;3(1):25-30. doi: 10.1007/BF03161035. PMID: 18072155; PMCID: PMC3550127.

     

    1. Mohammed FK, Al-Baggou B. Electrometric cholinesterase determination in poultry treated with dichlorvos and carbaryl. Online J Vet Res. 2005;9(1):1-5. Available from: http://onljvetres.com/chickencheabs2005.htm

     

    1. Abass KS, Mohammad FK. Validation of an electrometric method for cholinesterase measurement in the plasma and tissues of the chicken. 11th Scientific Congress, Faculty of Veteterinary Medicine, Assuit University. Assuit, Egypt: Assiut University; 2004. p. 241-259.

     

    1. Mohammad FK, Faris GA-M, Shindala MK. Comparative antidotal effects of diphenhydramine and atropine against dichlorvos-induced acute toxicosis in rats. Vet Arh. 2002;72(1):19-28.

     

    1. Mohammad FK, Mousa YJ, Al-Zubaidy MHI, Alias AS. Assessment of diphenhydramine effects against acute poisoning induced by the organophosphate insecticide dichlorvos in chicks. Hum Vet Med. 2012;4(1):6-13. Available from: http://www.hvm.bioflux.com.ro/docs/HVM_4.1.2.pdf

     

    1. Al-Baggou' BK, Mohammad FK. Antagonism of methomyl-induced toxicosis by diphenhydramine in rats. Environ Toxicol Pharmacol. 1999 Apr;7(2):119-25. doi: 10.1016/s1382-6689(99)00002-2. PMID: 21781916.

     

    1. Al-Zubaidy MH, Mousa YJ, Hasan MM, Mohammad FK. Acute toxicity of veterinary and agricultural formulations of organophosphates dichlorvos and diazinon in chicks. Arh Hig Rada Toksikol. 2011 Dec;62(4):317-23. doi: 10.2478/10004-1254-62-2011-2139. PMID: 22202465.

     

    1. Mohammad FK, Al-Badrany YM, Al-Jobory MM. Acute toxicity and cholinesterase inhibition in chicks dosed orally with organophosphate insecticides. Arh Hig Rada Toksikol. 2008 Sep;59(3):145-51. doi: 10.2478/10004-1254-59-2008-1873. PMID: 18796381.

     

    1. Al-Badrany YM, Mohammad FK. Effects of acute and repeated oral exposure to the organophosphate insecticide chlorpyrifos on open-field activity in chicks. Toxicol Lett. 2007 Nov 1;174(1-3):110-6. doi: 10.1016/j.toxlet.2007.09.001. Epub 2007 Sep 6. PMID: 17928175.

     

    1. Al-Baggou BK, Naser AS, Mohammad FK. Hydrogen peroxide potentiates organophosphate toxicosis in chicks. Hum Vet Med. 2011;3(2):142-9. Availablr from: http://www.hvm.bioflux.com.ro/docs/2011.3.142-149.pdf.

     

    1. Al-Zubaidy MH, Mohammad FK. Metoclopramide protection of diazinon-induced toxicosis in chickens. J Vet Sci. 2007 Sep;8(3):249-54. doi: 10.4142/jvs.2007.8.3.249. PMID: 17679771; PMCID: PMC2868131.

     

    1. Mohammad FK, Mousa Y J, Hasan MM. Acute toxicity and neurobehavioral effects of diphenhydramine in chicks. J Poult Sci. 2012;49(1):51-6. https://doi.org/10.2141/jpsa.011050. Available from: https://www.jstage.jst.go.jp/article/jpsa/49/1/49_011050/_pdf/-char/en.

     

    1. Mohammad FK, Faris GA, Al-Kassim NA. A modified electrometric method for measurement of erythrocyte acetylcholinesterase activity in sheep. Vet Hum Toxicol. 1997 Dec;39(6):337-9. PMID: 9397501.

     

    1. Mohammad FK, Al-Baggou B, Alias A, Faris GA-M. Application of an electrometric method for measurement of in vitro inhibition of blood cholinesterases from sheep, goats and cattle by dichlorvos and carbaryl. Vet Med. 2006;51(2):45-50. https://doi.org/10.17221/5516-VETMED.

     

    1. Mohammad FK, Al-Baggou BK, Naser AS, Fadel MA. In vitro inhibition of plasma and brain cholinesterases of growing chicks by chlorpyrifos and dichlorvos. J App Anim Res. 2014;42(4): 423-8. https://doi.org/10.1080/09712119.2013.875912.

     

    1. Alias AS, Mohammad FK. Electrometric measurement of plasma and tissue cholinesterase activities of four wild birds in Iraq. J Biol Res 2005 Dec;4:197-202. Available from: http://www.jbr.gr/papers20052/10-Ashraf%20S.%20Alias.pdf

     

    1. Alias AS, Al-Zubaidy MH, Mousa YJ, Mohammad FK. Plasma and whole brain cholinesterase activities in three wild bird species in Mosul, IRAQ: In vitro inhibition by insecticides. Interdiscip Toxicol. 2011 Sep;4(3):144-8. doi: 10.2478/v10102-011-0022-x. PMID: 22058655; PMCID: PMC3203916.

     

    1. Faris GA-M, Mohammad FK. Cholinesterase inhibition by dichlorvos and diphenhydramine in mice. Iraqi J Vet Sci. 1996;9(1):7-13.

     

    1. Mohammad FK, Al-Zubaidy MHI, Alias AS. Electrometric determination of erythrocyte, plasma and whole blood cholinesterase activities in sheep, goats and cattle and their in vitro inhibition by anticholinesterase insecticides. J Pharmacol Toxicol. 2007; 2:131-141.

     

    1. Mohammad FK, Faris GA-M, Alias AS, Al-Baggou' BKh. Blood cholinesterase activities in cattle, sheep and goats measured by a modified electrometric method. J. Anim Vet Adv. 2005; 4: 923-926.

     

    1.  Ahmed O, Mohammad F. A simplified electrometric technique for rapid measurement of human blood cholinesterase activity. Internet J Toxicol. 2004; 2(1). Avaulable from: https://print.ispub.com/api/0/ispub-article/6803

     

    1. Mohammad FK. Review of a practical electrometric method for determination of blood and tissue cholinesterase activities in animals. VetScan. 2007;2(2):1-12. Available from: https://journal.vetscan.co.in/index.php/vs/article/view/21.

     

    1. Mohammad FK. Laboratory guide in toxicology. 2nd ed. Columbia, MO: Mizzou Media, University of Missouri; 2010. 3rd ed. Available from: https://in.b-ok.as/book/16382014/9cfdef.

     

    28.Jamshidzade A, Nicknahad H, Mohammadi Bardbori A, Talati M. Comparative measurement of serum acetyl cholinesterase enzyme using three different methods. Iranian J Toxicol. 2009; 2 (4):268-272. URL: http://ijt.arakmu.ac.ir/article-1-63-en.html.

     

    1.  Michel HO. An electrometric method for the determination of red blood cell and plasma cholinesterase activity. J Lab Clin Med. 1949;34:1564–1568. 

     

    30. Mohammad FK, St Omer VE. Modifications of Michel's electrometric method for rapid measurement of blood cholinesterase activity in animals: a minireview. Vet Hum Toxicol. 1982 Apr;24(2):119

Home

Guide for Authors

Submit Manuscript

Reviewers

Contact Us