Новий метод для визначення активності холінестерази

Mykola Ye. Blazheyevskіy; Olena V. Koval’ska; Vladislav V. Diadchenko

doi:10.24959/ophcj.21.234269

Authors

Mykola Ye. Blazheyevskіy National University of Pharmacy of the Ministry of Health of Ukraine, Ukraine https://orcid.org/0000-0002-8032-347X
Olena V. Koval’ska National University of Pharmacy of the Ministry of Health of Ukraine, Ukraine https://orcid.org/0000-0003-0113-7060
Vladislav V. Diadchenko National Technical University «Kharkiv Polytechnic Institute», Ukraine

DOI:

https://doi.org/10.24959/ophcj.21.234269

Keywords:

acetylcholinestherase; acetylcholine; photometric detection

Abstract

Aim. To develop a principally new method, which would allow achieving the necessary accuracy and reproducibility of the analysis results, for determining the activity of the blood cholinesterase; to create safe working conditions when performing the analysis.

Results and discussion. The kinetic method proposed for determining the activity of cholinesterase consists in photometric measurement of the rate of the enzymatic hydrolysis of the acetylcholine substrate (by its residue) in the phosphate buffer using p-phenetidine as an indicator. The rate of the enzymatic hydrolysis of acetylcholine was determined by the tangent of the inclination angle of the linear part of the kinetic curve in the А–t coordinates at a wavelength of 358 nm. The linear dependence of the conditional reaction rate (tgα) on the enzyme concentration was observed in the concentration range of 0.12 – 0.36 mg/mL. Metrological characteristics of the method developed were: RSD = 2.0 % (n = 5; P = 0.95), correctness 0.4 %. These values indicate that the method for determining the activity of blood cholinesterase is sensitive, reliable and reproducible.

Experimental part. The experiments on determining the rate of the enzymatic hydrolysis were repeated three times with a specific concentration of the enzyme. Using the data obtained the kinetic curves were constructed in the А–t coordinates; on their basis the tangents of the angles of inclination in min^-1 were calculated. The calibration graph was constructed using the average values of the tangents of the angles of inclination, which corresponded to a certain concentration of the solution of the working standard sample of the enzyme. The equation of the calibration dependence of tgα-enzyme concentration was calculated by the method of least squares and found to be tgα (min^-1) = –0.17с + 9.13 (r = 0.999).

Conclusions. As a result of the studies conducted, a new method for determining the activity of the cholinesterase enzyme has been developed. The method is characterized by a high sensitivity, reliability and reproducibility and provides safe working conditions when performing the analysis.

Supporting Agency

Стаття є фрагментом комплексних наукових робіт Національного фармацевтичного університету за темою «Органічний синтез та аналіз БАР, розробка лікарських засобів на основі синтетичних та напівсинтетичних субстанцій» (№ державної реєстрації: 0011U000943)

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References

Karasova, J. Z.; Maderycova, Z.; Tumova, M.; Jun, D.; Rehacek, V.; Kuca, K.; Misik, J. Activity of cholinesterases in a young and healthy middle-European population: Relevance for toxicology, pharmacology and clinical praxis. Toxicol. Lett. 2017, 277, 24 – 31. https://doi.org/10.1016/j.toxlet.2017.04.017.
Holas, O.; Musilek, K.; Pohanka, M.; Kuca, K. The progress in the cholinesterase quantification methods. Expert Opinion on Drug Discovery 2012, 7 (12), 1207 – 1223. https://doi.org/10.1517/17460441.2012.729037.
Ramsay, R. R.; Tipton, K. F. Assessment of Enzyme Inhibition: A Review with Examples from the Development of Monoamine Oxidase and Cholinesterase Inhibitory Drugs. Molecules 2017, 22 (7), 1192. https://doi.org/10.3390/molecules22071192.
Pohanka, M.; Hrabinova, M.; Kuca, K.; Simonato, J.-P. Assessment of Acetylcholinesterase Activity Using Indoxylacetate and Comparison with the Standard Ellman’s Method. International Journal of Molecular Sciences 2011, 12 (4), 2631 – 2640. https://doi.org/10.3390/ijms12042631.
Stepurska, K. V.; Soldatkin, O. O.; Peshkova, V. N.; Dzyadevych, S. V.; Soldatkin, A. P. Possibility of reactivation bioselective element acetylcholinesterase-based biosensor for inhibitory analysis of pesticide. Sensor Electronics and Microsystem Technologies 2013, 10 (1), 97 – 105. https://doi.org/10.18524/1815-7459.2013.1.112702.
Kalow, W.; Lindsay, H. A. A comparison of optical and manometric methods for the assay of human serum cholinesterase. Canadian journal of biochemistry and physiology 1955, 33 (4), 568 – 74. https://doi.org/10.1139/o55-071.
Kalow, W.; Genest, K. A method for the detection of atypical forms of human serum cholinesterase; determination of dibucaine numbers. Canadian journal of biochemistry and physiology 1957, 35 (6), 339 – 46. https://doi.org/10.1139/y57-041.
Hestrin, S. The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine, and its analytical application. The Journal of biological chemistry 1949, 180 (1), 249 – 61. https://doi.org/10.1016/S0021-9258(18)56740-5.
Яковлев, В. А. Кинетика ферментативного катализа; Наука: Москва, 1965; с. 144.
Hanker, J. S.; Gelberg, A.; Witten, B. Colorimetric Estimation of Some Cholinergic Esters: Application to the Demonstration of Acetylcholinesterase Activity. Journal of the American Pharmaceutical Association (Scientific ed.) 1958, 47 (10), 728 – 730. https://doi.org/10.1002/jps.3030471014.
Блажеєвський, М. Є.; Ковальська, О. В.; Дядченко, В. В. (Національний фармацевтичний університет). Спосіб визначення активності холінестерази крові. Патент України 117474, 26.06.2017.