Amino acids of the biliary system in rats with tetracycline-induced hepatosis and the use of milk phospholipids

Serhii Bryzhenko, Viktoriia Gryshchenko
Abstract

The relevance of the study is due to the high frequency of drug-induced liver injury in mammals and insufficient study of the issue of amino acid metabolism disorders in acute forms of hepatopathy of the corresponding genesis. In this regard, the study was aimed at identifying specific changes in the profile of free amino acids in the bile and liver of rats with tetracycline-induced fatty hepatosis, as well as determining the corrective effect of the phospholipid fraction of milk. The leading approach in studying this problem was an experiment on laboratory animals with modelling of fatty hepatosis due to the cytotoxic effect of tetracycline, followed by the selection of bile and liver samples, followed by studying the spectrum of free amino acids using thin-layer chromatography with ninhydrin staining. It was found that the general pattern of changes in the hepatobiliary system of diseased rats indicated a violation of bile acid conjugation processes and mitochondrial dysfunction, as well as a pronounced blockage of the use of free amino acids in metabolic processes. At the same time, the use of phospholipid-containing therapy in rats with tetracycline-induced fatty hepatosis activated the use of the intrahepatic reserve of amino acids involved in bile acid conjugation and antioxidant protection, in particular sulphur-containing representatives and the total fraction of glycine/serine/glutamine. It was determined that the bile of these rats also had a reduced content of glycine and taurine-containing amino acid fractions in the bile of these rats, with a simultaneous increase in the level of the total alanine/tyrosine/threonine and arginine/ornithine/lysine fractions, confirming the activation of detoxification processes in the liver involving free amino acids. It was found that the use of the phospholipid fraction of milk reduced the manifestations of disturbances in the intermediate metabolism of amino acids and contributed to the restoration of the content of leucine- and valine-containing fractions in the bile and liver of sick rats. The established patterns are of practical value for laboratory diagnosis and preclinical evaluation of hepatoprotective agents, since the amino acid profile of the hepatobiliary system can be used as a sensitive indicator of the severity of liver injury and as a criterion for determining the effectiveness of therapy

Keywords

chromatography; amino acid fractions; liver; bile; corrective therapy; tetracycline hydrochloride; laboratory diagnostics

Suggested citation
Bryzhenko, S., & Gryshchenko, V. (2026). Amino acids of the biliary system in rats with tetracycline-induced hepatosis and the use of milk phospholipids. Ukrainian Journal of Veterinary Sciences, 17(1), 47-71. https://doi.org/10.31548/veterinary1.2026.47
References
  1. Al-Dalaen, S., Alzyoud, J., & Al-Qtaitat, A. (2016). The effects of L-arginine in modulating liver antioxidant biomarkers within carbon tetrachloride induced hepatotoxicity: Experimental study in rats. Biomedical and Pharmacology Journal, 9(1), 293-298. doi: 10.13005/bpj/938.
  2. Block, R.J., Durrum, E.L., & Zweig, G. (1958). A manual of paper chromatography and paper electrophoresis (2nd ed.). Cambridge: Academic Press.
  3. Bugajska, J., Berska, J., Pasternak, A., & Sztefko, K. (2023). Biliary amino acids and telocytes in gallstone disease. Metabolites, 13(6), article number 753. doi: 10.3390/metabo13060753.
  4. Colosimo, S., Bertoli, S., & Saffioti, F. (2023). Use of branched-chain amino acids as a potential treatment for improving nutrition-related outcomes in advanced chronic liver disease. Nutrients, 15(19), article number 4190. doi: 10.3390/nu15194190.
  5. Consden, R., Gordon, A.H., & Martin, A.J.P. (1944). Qualitative analysis of proteins: A partition chromatographic method using paper. Biochemical Journal, 38(3), 224-232. doi: 10.1042/bj0380224.
  6. Contarini, G., & Povolo, M. (2013). Phospholipids in milk fat: Composition, biological and nutritional properties. International Journal of Molecular Sciences, 14(2), 2808-2831. doi: 10.3390/ijms14022808.
  7. da Silva, R.P., Eudy, B.J., & Deminice, R. (2020). One-carbon metabolism in fatty liver disease and fibrosis: One-carbon to rule them all. The Journal of Nutrition, 150(5), 994-1003. doi: 10.1093/jn/nxaa032.
  8. Directive 2010/63/EU of the European Parliament and of the Council on the Protection of Animals Used for Scientific Purposes. (2010, September). Retrieved from https://eur-lex.europa.eu/eli/dir/2010/63/oj/eng.
  9. Eguchi, A., et al. (2025). Conjugated bile acids in serum reflect disease severity and predict survival in chronic liver disease of humans and rats. Scientific Reports, 15, article number 32911. doi: 10.1038/s41598-025-17560-7.
  10. Enomoto, H., et al. (2023). Branched chain amino acid-to-tyrosine ratio: Not only an indicator of the amino acid imbalance. Digestive Medicine Research, 6, article number 1. doi: 10.21037/dmr-22-29.
  11. Fontana, R.J., Liou, I., Reuben, A., Suzuki, A., Fiel, M.I., Lee, W., & Navarro, V. (2023). AASLD practice guidance on drug, herbal, and dietary supplement-induced liver injury. Hepatology, 77(3), 1036-1065. doi: 10.1002/hep.32689.
  12. Fotakis, C., Kalafati, I.-P., Amanatidou, A.I., Andreou, V., Matzapetakis, M., Kafyra, M., Varlamis, I., Zervou, M., & Dedoussis, G.V. (2023). Serum metabolomic profiling unveils distinct sex-related metabolic patterns in NAFLD. Frontiers in Endocrinology, 14, article number 1230457. doi: 10.3389/fendo.2023.1230457.
  13. Gregor, A., Pignitter, M., Fahrngruber, C., Bayer, S., Somoza, V., König, J., & Duszka, K. (2021). Caloric restriction increases levels of taurine in the intestine and stimulates taurine uptake by conjugation to glutathione. Journal of Nutritional Biochemistry, 96, article number 108781. doi: 10.1016/j.jnutbio.2021.108781.
  14. Gryshchenko, V.A., Musiychuk, V.V., Chernyshenko, V.O., Gornytska, O.V., & Platonova, T.M. (2019). Evaluation of biochemical indicators in blood plasma of rats with tetracycline-induced hepatosis and their correction by milk phospholipidsUkrainian Biochemical Journal, 91(1), 92-99. doi: 10.15407/ubj91.01.092.
  15. Hackman, R.H., & Lazarus, M. (1956). Quantitative analysis of amino acids using paper chromatography. Australian Journal of Biological Sciences, 9(2), 281-292. doi: 10.1071/BI9560281
  16. Holeček, M. (2024). Origin and roles of alanine and glutamine in gluconeogenesis in the liver, kidneys, and small intestine under physiological and pathological conditions. International Journal of Molecular Sciences, 25(13), article number 7037. doi: 10.3390/ijms25137037.
  17. Holeček, M., Mráz, J., & Tilšer, I. (1996). Plasma amino acids in four models of experimental liver injury in rats. Amino Acids, 10(3), 229-241. doi: 10.1007/BF00807325.
  18. Kanon, A.P., Spies, S.J., MacGibbon, A.K.H., & Fuad, M. (2024). Milk fat globule membrane is associated with lower blood lipid levels in adults: A meta-analysis of randomized controlled trials. Foods, 13(17), article number 2725. doi: 10.3390/foods13172725.
  19. Law of Ukraine No. 3447-IV “On the Protection of Animals from Cruelty”. (2006, February). Retrieved from https://zakon.rada.gov.ua/laws/show/3447-15#Text.
  20. Liu, J., Li, F., Zeng, Q., Hu, W., Yang, L., Luo, S., Li, D., & Deng, Y. (2025). Dysregulated bile acid metabolism drives lipid peroxidation and ferroptosis in NAFLD: Therapeutic potential for traditional Chinese medicine. Frontiers in Pharmacology, 16, article number 1669805. doi: 10.3389/fphar.2025.1669805.
  21. Liu, W.H., Liu, T.C., & Yin, M.C. (2008). Beneficial effects of histidine and carnosine on ethanol-induced chronic liver injury. Food and Chemical Toxicology, 46(5), 1503-1509. doi: 10.1016/j.fct.2007.12.013.
  22. Melnychuk, D.O., et al. (2015). Methods for investigating the functional state of the liver and biliary system. Kyiv: NUBiP.
  23. Miyazaki, T., Ueda, H., Ikegami, T., & Honda, A. (2023). Upregulation of taurine biosynthesis and bile acid conjugation with taurine through FXR in a mouse model with human-like bile acid composition. Metabolites, 13(7), article number 824. doi: 10.3390/metabo13070824.
  24. Moore, S., & Stein, W.H. (1948). Photometric ninhydrin method for use in the chromatography of amino acids. Journal of Biological Chemistry, 176(1), 367-388. doi: 10.1016/S0021-9258(18)51034-6.
  25. Morine, Y., et al. (2022). Essential amino acids as diagnostic biomarkers of hepatocellular carcinoma based on metabolic analysis. Oncotarget, 13, 1286-1298. doi: 10.18632/oncotarget.28306.
  26. Nguyen, M.T., Lian, A., Guilford, F.T., & Venketaraman, V. (2025). A literature review of glutathione therapy in ameliorating hepatic dysfunction in non-alcoholic fatty liver disease. Biomedicines, 13(3), article number 644. doi: 10.3390/biomedicines13030644.
  27. Order of the Ministry of Education and Science, Youth and Sports of Ukraine No. 249 “On Approval of the Procedure for Conducting Research and Experiments on Animals by Scientific Institutions”. (2012, March). Retrieved from https://zakon.rada.gov.ua/laws/show/z0416-12#Text.
  28. Ozsoy, Y., Coskun, T., Yavuz, K., Ozbilgin, K., Var, A., & Ozyurt, B. (2011). The effects of L-arginine on liver damage in experimental acute cholestasis: An immunohistochemical study. HPB Surgery, 2011, article number 306069. doi: 10.1155/2011/306069.
  29. Pan, S., Fan, M., Liu, Z., Li, X., & Wang, H. (2021). Serine, glycine and one-carbon metabolism in cancer (Review). International Journal of Oncology, 58(2), 158-170. doi: 10.3892/ijo.2020.5158.
  30. Partridge, S.M. (1948). Filter-paper partition chromatography of sugars: I. General description and application to the qualitative analysis of sugars in apple juice, egg white and foetal blood of sheep. Biochemical Journal, 42(2), 238-250. doi: 10.1042/bj0420238.
  31. Patent of Ukraine No. 86516. (2009). Veterinary bioactive addidition of liposomal form and method of reparative therapy in hepatology. Retrieved from https://sis.nipo.gov.ua/uk/search/detail/422804/.
  32. Rom, O., et al. (2020). Glycine-based treatment ameliorates NAFLD by modulating fatty acid oxidation, glutathione synthesis, and the gut microbiome. Science Translational Medicine, 12(572), article number eaaz2841. doi: 10.1126/scitranslmed.aaz2841.
  33. Skat-Rørdam, J., Lykkesfeldt, J., Gluud, L.L., & Tveden-Nyborg, P. (2025). Mechanisms of drug induced liver injury. Cellular and Molecular Life Sciences, 82(1), article number 213. doi: 10.1007/s00018-025-05744-3.
  34. Stofan, M., & Guo, G.L. (2020). Bile acids and FXR: Novel targets for liver diseases. Frontiers in Medicine, 7, article number 544. doi: 10.3389/fmed.2020.00544.
  35. Tajiri, K., & Shimizu, Y. (2018). Branched-chain amino acids in liver diseases. Translational Gastroenterology and Hepatology, 3, article number 47. doi: 10.21037/tgh.2018.07.06.
  36. Tan, N.Y.T., et al. (2024). The glutamate-serine-glycine index as a biomarker to monitor the effects of bariatric surgery on non-alcoholic fatty liver disease. Journal of the ASEAN Federation of Endocrine Societies, 39(2), 54-60. doi: 10.15605/jafes.039.02.20.
  37. Thakur, S., Kumar, V., Das, R., Sharma, V., & Mehta, D.K. (2024). Biomarkers of hepatic toxicity: An overview. Current Therapeutic Research, Clinical and Experimental, 100, article number 100737. doi: 10.1016/j.curtheres.2024.100737.
  38. Trammell, S.A.J., et al. (2023). Identification of bile acid-CoA:amino acid N-acyltransferase as the hepatic N-acyl taurine synthase for polyunsaturated fatty acids. Journal of Lipid Research, 64(9), article number 100361. doi: 10.1016/j.jlr.2023.100361.
  39. Trillos-Almanza, M.C., Martinez Aguilar, M., Buist-Homan, M., Bomer, N., Arevalo Gomez, K., de Meijer, V.E., van Vilsteren, F.G.I., Blokzijl, H., & Moshage, H. (2024). Branched-chain amino acids and their metabolites decrease human and rat hepatic stellate cell activation. Molecular Biology Reports, 51, article number 1116. doi: 10.1007/s11033-024-10027-4.
  40. Williams, R.J., & Kirby, H. (1948). Paper chromatography using capillary ascent. Science, 107(2784), 481-483. doi: 10.1126/science.107.2784.481.
  41. Wu, Z., et al. (2022). Milk fat globule membrane attenuates acute colitis and secondary liver injury by improving the mucus barrier and regulating the gut microbiota. Frontiers in Immunology, 13, article number 865273. doi: 10.3389/fimmu.2022.865273.
  42. Zhang, Q., Ye, L., Xin, F., Zhou, J., Cao, B., Yan, D., & Linxi, Q. (2021). Milk fat globule membrane supplementation during suckling ameliorates maternal high fat diet-induced hepatic steatosis in adult male offspring of mice. The Journal of Nutrition, 151(6), 1487-1496. doi: 10.1093/jn/nxab026.
  43. Zhang, Y., Zhan, L., Zhang, L., Shi, Q., & Li, L. (2024). Branched-chain amino acids in liver diseases: Complexity and controversy. Nutrients, 16(12), article number 1875. doi: 10.3390/nu16121875.
  44. Zhao, J., Zeng, J., Zhu, C., Li, X., Liu, D., Zhang, J., Li, F., Targher, G., & Fan, J.-G. (2023). Genetically predicted plasma levels of amino acids and metabolic dysfunction-associated fatty liver disease risk: A Mendelian randomization study. BMC Medicine, 21, article number 469. doi: 10.1186/s12916-023-03185-y.
  45. Zhou, A.L., & Ward, R.E. (2024). Dietary milk polar lipids modulate gut barrier integrity and lipid metabolism in C57BL/6J mice during systemic inflammation induced by Escherichia coli lipopolysaccharide. Journal of Dairy Science, 107(10), 7578-7593. doi: 10.3168/jds.2024-24759.