Expanding the scope of application of clinical biochemical reagent kits for determining metabolite concentrations and enzyme activities and optimizing protocols of their using

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription or Fee Access

Abstract

Introduction. The main consumers of commercial reagent kits for the study of metabolites and enzymes are clinical laboratories. Therefore, protocols for their using and calculating indices were developed mainly for blood and urine analysis. However, it is regularly necessary to determine metabolites concentration and enzymes activity to solve other tasks.

Purpose of the work – as a case study estimation of glucose concentration and amylase activity in hemolymph plasma of the gastropod mollusc Achatina fulica to demonstrate how protocols for using some clinical reagent kits for determining metabolites concentration and enzymes activity can be optimized and how the scope of their application can be expanded.

Material and Methods. The hemolymph plasma of the gastropod mollusc A. fulica served as the object of the study. Glucose concentration and amylase activity were determined in hemolymph plasma using clinical biochemical reagent kits from Spinreact (Spain). The standard and optimized protocols for determining glucose concentration are described. An optimized protocol for determining amylase activity has been described previously.

Results. A. fulica is often used as a convenient model in experimental and practical developments of different areas of focus. The knowledge of diagnostically significant biochemical indices of hemolymph plasma of mollusks makes it possible to monitor the "state of health" of animals during their cultivation in laboratories and practical using. In contrast to the original protocol for determining glucose concentration, full calibration experiments were performed, with sample analysis time reduced to one minute. The optimized protocols for using reagent kits proposed in the article were tested to determine glucose concentration and amylase activity in the hemolymph plasma of A. fulica (n=16). The values were 0,42 ± 0,07 mM and 27,8 ± 0,6 IU/L, respectively.

Conclusion. As a case study estimation of glucose concentration and amylase activity in hemolymph plasma of the gastropod mollusc Achatina fulica the effectiveness of the proposed ways of optimization of protocols for using some clinical reagent kits was demonstrated. The optimized protocols are suitable for determining the concentration of such metabolites as glucose and lactate as well as for analyzing the kinetics of various enzymes with direct measurements of their activity. The performed optimization allowed to expand the scope of application of biochemical reagent kits used in medicine, as well as to significantly reduce the time of analysis.

Full Text

Restricted Access

About the authors

N. M. Malygina

St. Petersburg State University; S.M. Kirov Military Medical Academy

Author for correspondence.
Email: n.m.malygina@mail.ru
ORCID iD: 0000-0001-9049-3969

Post-graduate Student, Department of Biochemistry, Faculty of Biology, teacher of the Department of Clinical Biochemistry and Laboratory Diagnostics

Russian Federation, 7-9 Universitetskaya Emb., St Petersburg, 199034; 6, Akademika Lebedeva ul., St. Petersburg, 194044

A. Y. Lianguzov

St. Petersburg State University

Email: andrey.lyanguzov@spbu.ru
ORCID iD: 0000-0003-4803-9070

Ph.D. (Biol.), Senior Specialist of the Observatory of Environmental Safety Resource Center of the St. Petersburg State University Science Park

Russian Federation, 7-9 Universitetskaya Emb., St Petersburg, 199034

T. A. Petrova

St. Petersburg State University

Email: t.a.petrova@spbu.ru
ORCID iD: 0000-0002-0213-5457

Ph.D. (Biol.), Associate Professor, Specialist of the Observatory of Environmental Safety Resource Center of the St. Petersburg State University Science Park

 

Russian Federation, 7-9 Universitetskaya Emb., St Petersburg, 199034

A. M. Ivanov

St. Petersburg State University; S.M. Kirov Military Medical Academy

Email: iamvma@mail.ru
ORCID iD: 0000-0002-8899-7524

Dr.Sc. (Med.), Professor, Corresponding Member of the RAS, Head of Department of Laboratory Medicine, Medical Institute; Head of the Department of Clinical Biochemistry and Laboratory Diagnostics President of the Association of Laboratory Specialists and Organizations «Federation of Laboratory Medicine» of Russian Federation

Russian Federation, 7-9 Universitetskaya Emb., St Petersburg, 199034; 6, Akademika Lebedeva ul., St. Petersburg, 194044

References

  1. Spinreact. https://www.spinreact.com. Дата обращения: 16.05.24.
  2. Trinder P. Determination of blood glucose using an oxidase-peroxidase system with a non-carcinogenic chromogen. Journal of Clinical Pathology. 1969; 22:158–161. doi: 10.1136/jcp.22.2.158.
  3. R open programming language. Data obrashheniya: 16.05.24; https://cran.r-project.org.
  4. Малыгина Н.М., Петрова Т.А., Лянгузов А.Ю. и др. Как повысить точность оценки начальной скорости ферментативной реакции. Доклады Академии наук. 2019; 488(2): 225–228. [Malygina N.M., Petrova T.A., Lianguzov A.Y. et al. An approach to better estimation of the initial velocity of enzyme reactions. Doklady Biochemistry and Biophysics. 2019; 488(2): 311–312. (In Russ.)]. doi: 10.1134/S1607672919050053.
  5. Юдина Н.Ю., Козлова Т.Н., Харькова А.С. Кинетические параметры реакции окисления D-глюкозы в присутствии арабинозы. Известия Тульского государственного уни-верситета. Естественные науки. 2022; 3: 42–52. [Yudina N.Yu., Kozlova T.N., Khar'kova A.S. Kinetic parameters of the oxidation reaction D-glucose in the presence of arabinose. Izvestiya Tul'skogo gosudarstvennogo universiteta. Estestvennye nauki. 2022; 3: 42–52. (In Russ.)] doi: 10.24412/2071-6176-2022-3-42-52.
  6. Brockelman C.R., Sithithavorn P.Z. Carbohydrate reserves and hemolymph sugars of the African giant snail, Achatina fulica in relation to parasitic infection and starvation. Zeitschrift für Parasitenkunde. 1980; 62: 285–291. doi: 10.1007/BF00926568.
  7. Nowakowska A., Caputa M., Rogalska J. Seasonal changes in cryoprotectants concentrations in Helix pomatia snails. Journal of Physiology and Pharmacology. 2006; 57(Suppl. 8): 93–105. doi: 10.1007/s00360-010-0539-x.
  8. Vassilev N., Simova S., Dangalov M. et al. 1H NMR based study of metabolites profiling of garden snail Helix lucorum hemolymph. Bulgarian Chemical Communications. 2021; 53(Special Issue A): 49–56. doi: 10.34049/bcc.53.А.0009.
  9. Borges E., Vuaden F.C., Cognato G.P. et al. Effects of star-vation on haemolymphatic glucose levels, glycogen contents and nucleotidase activities in different tissues of Helix aspersa (Muller, 1774) (Mollusca, Gastropoda). Journal of Experimental Zoology Part A: Comparative Experimental Biology. 2004; 301A: 891–897. doi: 10.1002/jez.a.121.
  10. Pinheiro J., Amato S.B. Eurytrema coelomaticum (Digenea, Dicrocoeliidae): the effect of infection on carbohydrate con-tents of its intermediate snail host, Bradybaena similaris (Gastropoda, Xanthonychidae). Memórias do Instituto Oswaldo Cruz. 1994; 89(3): 407–410. doi: 10.1590/s0074-02761994000300020.
  11. Tunholi V.M., Tunholi-Alves V.M., Monteiro C.O. et al. Biolo-gical, biochemical and histological features of Bradybaena simi-laris (Gastropoda: Pulmonata) infected by Heterorabditis indica (Rhabditida: Heterorhabditidae) strain LPP1. Experimental Para-sitology. 2017; 179: 28–35. doi: 10.1016/j.exppara.2017.06.004.
  12. Martins F.G., Lima M.G., Castro R.N. et al. Bulimulus te-nuissimus (mollusca) as a new potential host of Angio-strogylus cantonensis (nematoda), a histological and meta-bolic study. Journal of Invertebrate Pathology. 2018; 154: 65–73. doi: 10.1016/j.jip.2018.04.003.
  13. Reddy D.C.S., Jayaram V., Sowjanya K. et al. Dial variations in the levels of blood glucose and hepatopancreatic glycogen in the slug, Laevicaulis alte (Ferussac 1821). Experientia. 1978; 34: 606. doi: 10.1007/bf01936985.
  14. Лянгузов А.Ю., Малыгина Н.М., Петрова Т.А. О методи-ческих ошибках оценивания начальных скоростей фермен-тативных реакций и способах их коррекции (на примере лактатдегидрогеназы). Биофизика. 2023; 68(6): 1117–1121. [Lianguzov A.Yu., Malygina N.M., Petrova T.A. On metho-dological errors in estimating the initial velocities of enzyme-catalyzed reactions and approaches to their correction: a case study with lactate dehydrogenase. Biophysics. 2023; 68(6): 924–928. (In Russ.)]. doi: 10.1134/S0006350923060118.
  15. Ziętek J., Guz L., Winiarczyk S. et al. Study on establishing normal ranges of chosen biochemical parameters of haemolymph of Cornu aspersum maxima and Cepaea nemoralis gastropods. Polish Journal of Veterinary Sciences. 2018; 21(3): 445–449. doi: 10.24425/122617.

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Figure. Calibration plot for the determining glucose concentration by the glucose oxidase method by the absorbance level of incubation medium in one minute

Download (117KB)
Indexing metadata

Copyright (c) 2024 Russkiy Vrach Publishing House