The application of a reference interval to assess the normal variability of several antioxidant system parameters in liver and muscle of the three-spined stickleback

A.A. Kochneva, A.L. Rabinovich, D.L. Lajus, S.R. Kurpe and I.V. Sukhovskaya

Proceedings of the Zoological Institute RAS, 2024, 328(4): 707–725 · https://doi.org/10.31610/trudyzin/2024.328.4.707

Full text

Abstract

When conducting research, reproducibility and correct assessment of the results obtained are important tasks that require the use of modern methods and approaches. To assess the state of biological systems, significance testing of the null hypothesis with respect to the assumed values of the “norm” is often used. To assess the state of biological systems, a common approach is to test the significance of the null hypothesis in relation to the assumed values of the “norm”. Recently, however, the dichotomous approach of confirming or rejecting the null hypothesis has been criticized by data analysts due to the lack of validation of methods for assessing p-level significance and the biological validity of the hypotheses under investigation. Consequently, a novel methodology for identifying the “normal” state of biological systems was proposed and developed into the theory of reference values (RV). Furthermore, the concepts of reference interval (RI) and reference values have become prevalent in various research fields. Our study evaluated the relative intervals of several antioxidant system (AOS) parameters (glutathione S-transferase, catalase, guaiacol-dependent peroxidase, superoxide dismutase) in the liver and muscles of male three-spined stickleback Gasterosteus aculeatus L. in the Seldianaya Inlet of White Sea during the spawning period. The three-spined stickleback is a species that is abundant in the White Sea and has been the subject of numerous studies on population genetics, evolutionary biology, and ecology. AOS plays a role in the defense of the organism against the negative influence of exogenous and endogenous factors causing oxidative stress. Its indicators are used as biomarkers of the physiological state of the organism. The activity of AOS enzymes in stickleback samples from other biotopes of the White Sea (Sukhaya Salma Strait and Koliushkovaya Lagoon) was also compared with each other and relatively calculated RI. Stickleback from the Koliushkovaya Lagoon and the Sukhaya Salma Strait differed in liver superoxide dismutase activity, and the mean values of enzyme activity were included in the RI. To assess the reproducibility of statistical tests, the effect of stickleback sample size on the result of comparing samples was modeled using t-criterion, and the type of dependence and sample sizes at 50% probability of a positive test were determined. The data obtained on biomarkers of oxidative stress in male G. aculeatus may prove useful for monitoring the state of stickleback populations with the aim of developing this species as a model object for ecological studies of both the White Sea ecosystem and the fish population in this reservoir.

Key words

antioxidant system, White Sea, muscles, liver, reference interval, three-spined stickleback, Gasterosteus aculeatus

Submitted May 17, 2024 · Accepted November 11, 2024 · Published online December 25, 2024

References

Bakhvalova A.E., Ivanova T.S., Ivanov M.V., Demchuk A.S., Movchan E.A. and Lajus D.L. 2016. Long-term changes in the role of threespine stickleback Gasterosteus aculeatus in the White Sea: predatory fish consumption reflects fluctuating stickleback abundance during the last century. Evolutionary Ecology Research, 17(3): 317–334.

Beers R.F. and Sizer I.W. 1952. A spectrophotometric method for measuring the breakdown of hydrogenperoxide by catalase. The Journal of biological chemistry, 195: 133–140. https://doi.org/10.1016/s0021-9258(19)50881-x

Bell M.A. and Foster S.A. 1994. The evolutionary biology of the three-spine stickleback. Oxford University Press, Oxford, New York, Tokyo, 571 p.

Berner D. and Amrhein V. 2022. Why and how we should join the shift from significance testing to estimation. Journal of Evolutionary Biology, 35(6): 777–787. https://doi.org/10.1111/jeb.14009

Catteau A., Bado-Nilles A., Beaudouin R., Tebby C., Joachim S., Palluel O., Turiès C., Chrétien N., Nott K., Ronkart S., Geffard A. and Porcher J.M. 2021. Water quality of the Meuse watershed: Assessment using a multi-biomarker approach with caged three-spined stickleback (Gasterosteus aculeatus L.). Ecotoxicology and Environmental Safety, 208: 111407. https://doi.org/10.1016/j.ecoenv.2020.111407

Ceriotti F. and Henny J. 2008. “Are my laboratory results normal?” Considerations to be made concerning reference intervals and decision limits. eJIFCC, 19(2): 106–114. Available at: https://pmc.ncbi.nlm.nih.gov/articles/PMC4975205/

Ceriotti F., Hinzmann R. and Panteghini M. 2009. Reference intervals: the way forward. Annals of Clinical Biochemistry, 46(Pt 1): 8–17. https://doi.org/10.1258/acb.2008.008170

Chance B. and Maehly A.C. 1955. Assay of Catalase and Peroxidase. Methods in Enzymology, 2: 764–775. https://doi.org/10.1016/S0076-6879(55)02300-8

Clinical & Laboratory Standards Institute (CLSI). 2008. Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline – Third Edition. CLSI document C28-A3. Wayne, PA: Clinical and Laboratory Standards Institute, 59 p.

Demchuk A., Ivanov M., Ivanova T., Polyakova N., Golovin P. and Lajus D. 2018. Feeding of the threespine stickleback Gasterosteus aculeatus (Linnaeus, 1758) in spawning grounds. Transactions of KarRC RAS, 4: 42–58. [In Russian]. https://doi.org/10.17076/them818

Dixon W.J. 1953. Processing Data for Outliers. Biometrics, 9(1): 74–89. https://doi.org/10.2307/3001634

Dorgham A.S., Golovin P.V., Ivanova T.S., Ivanov M.V., Saveliev P.D. and Lajus D.L. 2018. Morphological variation of threespine stickleback (Gasterosteus aculeatus) on different stages of spawning period. Transactions of KarRC RAS, 4: 59–73. [In Russian]. https://doi.org/10.17076/them819

Finnegan D. 2022. Reference Intervals. R package version 1.3.0. Available from: https://CRAN.R-project.org/package=referenceIntervals (accessed 24 October 2023)

Fridovich I. 1975. Superoxide Dismutases. Annual Review of Biochemistry, 44: 147–159. https://doi.org/10.1146/annurev.bi.44.070175.001051

Friedrichs K.R., Harr K.E., Freeman K.P., Szladovits B., Walton R.M., Barnhart K.F. and Blanco-Chavez J. 2012. ASVCP reference interval guidelines: determination of de novo reference intervals in veterinary species and other related topics. Veterinary Clinical Pathology, 41: 441–453. https://doi.org/10.1111/vcp.12006

GOST R 53022.3-2008. 2009. Clinical laboratory technologies. Requirements of quality of clinical laboratory tests. Part 3. Assessment of laboratory tests clinical significance. [In Russian]. Available from: https://protect.gost.ru/document1.aspx?control=31&baseC=6&page=0&month=8&year=2009&search=&id=174413 (accessed 24 October 2023)

Gräsbeck R. and Saris N-E. 1969. Establishment and use of normal values. Explore the current issue of Scandinavian Journal of Clinical and Laboratory Investigation, 26(Suppl. 110): 62–63.

Gräsbeck R., Siest G., Wilding P., Williams G.Z. and Whitehead T.P. 1978. IFCC Expert Panel on the Theory of Reference Values. Provisional recommendation on the theory of reference values. Part 1. The concept of reference values. Clinica Chimica Acta, 87: 459F–65F. https://doi.org/10.1093/clinchem/25.8.1506

Gross J. and Ligges U. 2015. Nortest: Tests for Normality. R package version 1.0-4. Available from: https://CRAN.R-project.org/package=nortest

Habig W.H., Pabst M.J. and Jakoby W.B. 1974. GlutathioneS-Transferases. The first enzymatic step inmercapturic acid formation. The Journal of biological chemistry, 249: 7130–7139. https://doi.org/10.1016/S0021-9258(19)42083-8

Henny J., Petitclerc C., Fuentes-Arderiu X., Petersen P.H., Queraltó J.M., Schiele F. and Siest G. 2000. Need for revisiting the concept of reference values. Clinical Chemistry and Laboratory Medicine, 38(7): 589–95. https://doi.org/10.1515/CCLM.2000.085

Henny J., Vassault A., Boursier G., Vukasovic I., Mesko Brguljan P., Lohmander M., Ghita I., Andreu F.A., Kroupis C., Sprongl L., Thelen M.H., Vanstapel F.J., Vodnik T., Huisman W., Vaubourdolle M., on behalf of the Working Group Accreditation and ISO/CEN standards (WG-A/ISO) of the EFLM. 2016. Recommendation for the review of biological reference intervals in medical laboratories. Clinical Chemistry and Laboratory Medicine, 54(12): 1893–1900. https://doi.org/10.1515/cclm-2016-0793

Horn P.S., Feng L., Li Y. and Pesce A.J. 2014. Effects of outliers and nonhealthy individuals on reference interval estimation. Clinical Chemistry, 47: 2137–2145. https://doi.org/10.1093/clinchem/47.12.2137

Hoseinifar S.H., Yousefi S., Van Doan H., Ashouri G., Gioacchini G., Maradonna F. and Carnevali O. 2021. Oxidative Stress and Antioxidant Defense in Fish: The Implications of Probiotic, Prebiotic, and Synbiotics. Reviews in Fisheries Science & Aquaculture, 29(2): 198–217. https://doi.org/10.1080/23308249.2020.1795616

Hrubec T.C., Cardinale J.L. and Smith S.A. 2000. Hematology and plasma chemistry reference intervals for cultured tilapia (Oreochromis hybrid). Veterinary Clinical Pathology, 29(1): 7–12. https://doi.org/10.1111/j.1939-165x.2000.tb00389.x

Jan K., Ahmed I. and Dar N.A. 2021. Haematological and serum biochemical reference values of snow trout, Schizothorax labiatus habiting in river Sindh of Indian Himalayan region. The Journal of Fish Biology, 98(5): 1289–1302. https://doi.org/10.1111/jfb.14661

Joy S., Alikunju A.P., Jose J., Sudha H.S.H., Parambath P.M., Puthiyedathu S.T. and Philip B. 2017. Oxidative stress and antioxidant defense responses of Etropluss uratensis to acute temperature fluctuations. The Journal of Thermal Biology, 70(Pt B): 20–26. https://doi.org/10.1016/j.jtherbio.2017.10.010

Krupnova M., Ivanova T. and Nemova N. 2019. The activity of lysosomal proteases in the organs of female threespine stickleback (Gasterosteus aculeatus Linnaeus) in the spawning period. Transactions of KarRC RAS, 6: 37–43. [In Russian]. https://doi.org/10.17076/eb886

Lajus D., Ivanova T., Rybkina E., Lajus J. and Ivanov M. 2021. Multidecadal fluctuations of threespine stickleback in the White Sea and their correlation with temperature. ICES Journal of Marine Science, 78(2): 653–665. https://doi.org/10.1093/icesjms/fsaa192

Lajus D.L., Golovin P.V., Yurtseva A.O., Ivanova T.S., Dorgham A.S. and Ivanov M.V. 2019. Fluctuating asymmetry as an indicator of stress and fitness in stickleback: a review of the literature and examination of cranial structures. Evolutionary Ecology Research, 20(1): 83–106.

Lajus D.L., Golovin P.V., Zelenskaia A.E., Demchuk A.S., Dorgham A.S., Ivanov M.V., Ivanova T.S., Murzina S.A., Polyakova N.V., Rybkina E.V. and Yurtseva A.O. 2020a. Threespine stickleback of the White Sea: population characteristics and role in the ecosystem. Contemporary Problems of Ecology, 2: 167–183. [In Russian]. https://doi.org/10.15372/SEJ20200203

Lajus D.L., Lysenko L.A., Kantserova N.P., Tushina E.D., Ivanova T.S. and Nemova N.N. 2020b. Spatial heterogeneity and temporal dynamics of protein degrading activity and life-history traits in threespine stickleback Gasterosteus aculeatus. International Aquatic Research, 12(3): 161–170. https://doi.org/10.22034/iar.2020.1894323.1019

Lee J.W., Choi H., Hwang U.K., Kang J.C., Kang Y.J., Kim K.I. and Kim J.H. 2019. Toxic effects of lead exposure on bioaccumulation, oxidative stress, neurotoxicity, and immune responses in fish: A review. Environmental Toxicology and Pharmacology, 68: 101–108. https://doi.org/10.1016/j.etap.2019.03.010

Lessells C.K. 2008. Neuroendocrine control of life histories: what do we need to know to understand the evolution of phenotypic plasticity? Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 363(1497): 1589–98. https://doi.org/10.1098/rstb.2007.0008

Lushchak V.I. 2016. Contaminant-induced oxidative stress in fish: a mechanistic approach. Fish Physiology and Biochemistry, 42(2): 711–47. https://doi.org/10.1007/s10695-015-0171-5

Lysenko L., Kantserova N., Tushina E., Poliakova N., Lajus D. and Nemova N. 2018. The White Sea threespine stickleback, Gasterosteus aculeatus, differentiation at the beginning of spawning by the activity of calcium-dependent proteases and population characteristics. Transactions of KarRC RAS, 5: 79–88. [In Russian]. https://doi.org/10.17076/eco700

Marchand A., Tebby C., Beaudouin R., Catteau A., Porcher J.M., Turiès C. and Bado-Nilles A. 2020. Reliability evaluation of biomarker reference ranges for mesocosm and field conditions: Cellular innate immunomarkers in Gasterosteus aculeatus. Science of The Total Environment, 698: 134333. https://doi.org/10.1016/j.scitotenv.2019.134333

Marchand A., Tebby C., Beaudouin R., Hani Y.M.I., Porcher J.M., Turies C. and Bado-Nilles A. 2019. Modelling the effect of season, sex, and body size on the three-spined stickleback, Gasterosteus aculeatus, cellular innate immunomarkers: A proposition of laboratory reference ranges. Science of The Total Environment, 648: 337–349. https://doi.org/10.1016/j.scitotenv.2018.07.381

Marchand A., Tebby C., Catteau A., Turiès C., Porcher J.M. and Bado-Nilles A. 2021. Application in a biomonitoring context of three-spined stickleback immunomarker reference ranges. Ecotoxicology and Environmental Safety, 223: 112580. https://doi.org/10.1016/j.ecoenv.2021.112580

Martínez-Álvarez R.M., Morales A.E. and Sanz A. 2005. Antioxidant defenses in fish: Biotic and abiotic factors. Reviews in Fish Biology and Fisheries, 15(1–2): 75–88. https://doi.org/10.1007/s11160-005-7846-4

McKinnon J.S. and Rundle H.D. 2002. Speciation in nature: the threespine stickleback model systems. Trends in Ecology & Evolution, 17: 480–488. https://doi.org/10.1016/S0169-5347(02)02579-X

National Committee for Clinical Laboratory Standards (NCCLS). 2000. How to Define and Determine Reference Intervals in the Clinical Laboratory; Approved Guideline – Second Edition. NCCLS document C28-A2. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087–1898, USA, 35 p.

Noble J.E. and Bailey M.J. 2009. Quantitation of Protein. Methods in Enzymology, 463: 73–95. https://doi.org/10.1016/S0076-6879(09)63008-1

Nussey D.H. 2005. Selection on Heritable Phenotypic Plasticity in a Wild Bird Population. Science, 310(5746): 304–306. https://doi.org/10.1126/science.1117004

Östlund-Nilsson S., Mayer I. and Huntingford F.A. 2007. Biology of the three-spine stickleback. Boca Raton: CRC Press, 392 p.

Ozarda Y. 2016. Reference intervals: current status, recent developments and future considerations. Biochemia Medica, 26(1): 5–16. https://doi.org/10.11613/BM.2016.001

Poole T. 1997. Happy animals make good science. Laboratory Animals, 31: 116–124. https://doi.org/10.1258/002367797780600198

R Core Team. 2020. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Available from: https://www.R-project.org/

Rabinovich A., Vysotskaya R., Lyubartsev A., Quirke N. and Lobaskin V. 2017. Assessing the state of an organism and toxicity of substances using biochemical indicators. Transactions of KarRC RAS, 9: 84–97. [In Russian]. https://doi.org/10.17076/eco634

RStudio Team. 2019. RStudio: Integrated Development for R. RStudio, Inc., Boston, MA Available from: http://www.rstudio.com/

Sanchez W., Aït-Aïssa S., Palluel O., Ditche J.M. and Porcher J.M. 2007. Preliminary investigation of multi-biomarker responses in three-spined stickleback (Gasterosteus aculeatus L.) sampled in contaminated streams. Ecotoxicology, 16(2): 279–87. https://doi.org/10.1007/s10646-006-0131-z

Shahjahan M., Islam M.J., Hossain M.T., Mishu M.A., Hasan J. and Brown C. 2022. Blood biomarkers as diagnostic tools: An overview of climate-driven stress responses in fish. Science of the Total Environment, 843: 156910. https://doi.org/10.1016/j.scitotenv.2022.156910

Smirnov L., Sukhovskaya I. and Kochneva A. 2019b. Variability of some antioxidant defense parametersand concentration of protein in the larvae of the three-spined stickleback (Gasterosteus aculeatus) in White Sea in the summer. Principles of the Ecology, 2: 98–109. [In Russian]. https://doi.org/10.15393/j1.art.2019.8542

Smirnov L., Sukhovskaya I., Borvinskaya E. and Lajus D. 2019a. The variability of some parameters of antioxidant protection in the muscle and liver of the three-spined stickleback (Gasterosteus aculeatus) in the white sea during the spawning period. Transactions of KarRC RAS, 12: 55–66. [In Russian]. https://doi.org/10.17076/eb1055

Solberg H.E. 1987. International Federation of Clinical Chemistry (IFCC). Approved recommendation on the theory of reference values. Part 5. Statistical treatment of collected reference values. Clinica Chimica Acta, 170: S13–S32. https://doi.org/10.1016/0009-8981(87)90151-3

Stoliar O. and Lushchak V. 2012. Environmental Pollution and Oxidative Stress in Fish. In: V. Lushchak (Ed.). Oxidative Stress – Environmental Induction and Dietary Antioxidants. InTech, Rijeka, Croatia: 131–166. https://doi.org/10.5772/38094

Sukhovskaya I., Borvinskaya E., Smirnov L. and Nemova N. 2010. Comparative analysis of the methods for determination of protein concentration – spectrophotometry in the 200–220 nm range and the bradford protein assay. Transactions of KarRC RAS. The “Experimental Biology” series, 2: 68–71. [In Russian].

Vysotskaya R., Buoy E., Krupnova M., Nemova N. and Lajus D. 2021. Participation of acid hydrolases in the adaptations of juvenile three spine sticklebacks Gasterosteus aculeatus L. of the White Sea. Transactions of KarRC RAS, 11: 69–79. [In Russian]. https://doi.org/10.17076/eb1504