Molecular and Cellular Biochemistry

, Volume 396, Issue 1–2, pp 99–105 | Cite as

Hyperhomocysteinemia induced by methionine dietary nutritional overload modulates acetylcholinesterase activity in the rat brain

  • Dragan Hrnčić
  • Aleksandra Rašić -Marković
  • Tihomir Stojković
  • Milica Velimirović
  • Nela Puškaš
  • Radmila Obrenović
  • Djuro Macut
  • Veselinka Šušić
  • Vladimir Jakovljević
  • Dragan Djuric
  • Nataša Petronijević
  • Olivera Stanojlović


Methionine is the only endogenous precursor of homocysteine, sulfur—containing amino acid and well known as risk factor for various brain disorders. Acetylcholinesterase is a serine protease that rapidly hydrolyzes neurotransmitter acetylcholine. It is widely distributed in different brain regions. The aim of this study was to elucidate the effects of methionine nutritional overload on acetylcholinesterase activity in the rat brain. Males of Wistar rats were randomly divided into control and experimental group, fed from 30th to 60th postnatal day with standard or methionine-enriched diet (double content comparing to standard, 7.7 g/kg), respectively. On the 61st postnatal day, total homocysteine concentration was determined and showed that animals fed with methionine-enriched diet had significantly higher serum total homocysteine concentrations comparing to control rats (p < 0.01). Acetylcholinesterase activity has been determined spectrophotometrically in homogenates of the cerebral cortex, hippocampus, thalamus, and nc. caudatus. Acetylcholinesterase activity showed tendency to decrease in all examined brain structures in experimental comparing to control rats, while statistical significance of this reduction was achieved in the cerebral cortex (p < 0.05). Brain slices were stained with haematoxylin and eosin (H&E) and observed under light microscopy. Histological analysis of H&E-stained brain slices showed that there were no changes in the brain tissue of rats which were on methionine-enriched diet compared to control rats. Results of this study showed selective vulnerability of different brain regions on reduction of acetylcholinesterase activity induced by methionine-enriched diet and consecutive hyperhomocysteinemia.


Methionine Homocysteine Acetylcholinesterase Brain Rats 



This work was supported by the Ministry of Education, Science and Technological Development of Serbia (Grant #175032). We are grateful to reviewers for their constructive suggestions.


  1. 1.
    Finkelstein JD (1998) The metabolism of homocysteine: pathways and regulation. Eur J Pediatr 157(Suppl. 2):S40–S44PubMedCrossRefGoogle Scholar
  2. 2.
    Selhub J (1999) Homocysteine metabolism. Annu Rev Nutr 19:217–246. doi: 10.1146/annurev.nutr.19.1.217 PubMedCrossRefGoogle Scholar
  3. 3.
    Hoffer LJ (2004) Homocysteine remethylation and trans-sulfuration. Metabolism 53(11):1480–1483. doi: 10.1016/j.metabol.2004.06.003 PubMedCrossRefGoogle Scholar
  4. 4.
    McCully KS (1969) Vascular pathology of homocysteinemia: implications for thepathogenesis of arteriosclerosis. Am J Pathol 56(1):111–112PubMedPubMedCentralGoogle Scholar
  5. 5.
    Djurić D, Jakovljević V, Rašić-Marković A, Đurić A, Stanojlović O (2008) Homocysteine, folic acid and coronary artery disease: possible impact on prognosis and therapy. Indian J Chest Di Allied Sci 50:39–48Google Scholar
  6. 6.
    Herrmann W, Obeid R (2011) Homocysteine: a biomarker in neurodegenerative diseases. Clin Chem Lab Med 49(3):435–441. doi: 10.1515/CCLM.2011.084 PubMedGoogle Scholar
  7. 7.
    Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg IH, D’Agostino RB, Wilson PW, Wolf PA (2002) Plasma homocysteine as a risk factor for dementia and Alzheimer’s disease. N Engl J Med 346:476–483. doi: 10.1056/NEJMoa011613 PubMedCrossRefGoogle Scholar
  8. 8.
    Duan W, Ladenheim B, Cutler RG, Kruman II, Cadet JL, Mattson MP (2002) Dietary folate deficiency and elevated homocysteine levels endanger dopaminergic neurons in models of Parkinson’s disease. J Neurochem 80:101–110. doi: 10.1046/j.0022-3042.2001.00676.x PubMedCrossRefGoogle Scholar
  9. 9.
    Sachdev P (2004) Homocysteine, cerebrovascular disease and brain atrophy. J Neurol Sci 226:25–29. doi: 10.1016/j.jns.2004.09.006 PubMedCrossRefGoogle Scholar
  10. 10.
    Stanojlović O, Rašić-Marković A, Hrnčić D, Šušić V, Macut Dj, Radosavljević T, Djurić D (2009) Two types of seizures in homocysteine thiolactone–treated adult rats, behavioral and encephalographic study. Cell Mol Neurobiol 29:329–339. doi: 10.1007/s10571-008-9324-8 PubMedCrossRefGoogle Scholar
  11. 11.
    Jakubowski H (2004) Molecular basis of homocysteine toxicity in humans. Cell Mol Life Sci 61:470–487. doi: 10.1007/s00018-003-3204-7 PubMedCrossRefGoogle Scholar
  12. 12.
    Perla-Kajan J, Twardowski T, Jakubowski H (2007) Mechanisms of homocysteine toxicity in humans. Amino Acids 32:561–572PubMedCrossRefGoogle Scholar
  13. 13.
    Troen AM (2005) The central nervous system in animal models of hyperhomocysteinemia. Prog Neuropsychopharmacol Biol Psychiatry 29:1140–1151. doi: 10.1016/j.pnpbp.2005.06.025 PubMedCrossRefGoogle Scholar
  14. 14.
    Hrnčić D, Rašić-Marković A, Krstić D, Macut D, Djuric D, Stanojlović O (2010) The role of nitric oxide in homocysteine thiolactone-induced seizures in adult rats. Cell Mol Neurobiol 30:219–231. doi: 10.1007/s10571-009-9444-9 PubMedCrossRefGoogle Scholar
  15. 15.
    Rasić-Marković A, Stanojlović O, Hrnčić D, Krstić D, Čolović M, Šusić V, Radosavljević T, Djuric D (2009) The activity of erythrocyte and brain Na+/K+ and Mg2+-ATPases in rats subjected to acute homocysteine and homocysteine thiolactone administration. Mol Cell Biochem 327:39–45PubMedCrossRefGoogle Scholar
  16. 16.
    Prado MA, Reis RA, Prado VF, de Mello MC, Gomez MV, de Mello FG (2002) Regulation of acetylcholine synthesis and storage. Neurochem Int 41(5):291–299. doi: 10.1016/S0197-0186(02)00044-X PubMedCrossRefGoogle Scholar
  17. 17.
    Gralewicz S (2006) Possible consequences of acetylcholinesterase inhibition in organophosphate poisoning. Discussion continued. Med Pr 57(3):291–302PubMedGoogle Scholar
  18. 18.
    Schulpis K, Kalimeris K, Bakogiannis C, Tsakiris T, Tsakiris S (2006) The effect of in vitro homocystinuria on the suckling rat hippocampal acetylcholinesterase. Metab Brain Dis 21(1):21–28. doi: 10.1007/s11011-006-9001-x PubMedCrossRefGoogle Scholar
  19. 19.
    Stefanello FM, Zugno AI, Wannmacher CM, Wajner M, Wyse AT (2003) Homocysteine inhibits butyrylcholinesterase activity in rat serum. Metab Brain Dis 18(3):187–194. doi: 10.1023/B:MEBR.0000020189.89585.3b PubMedCrossRefGoogle Scholar
  20. 20.
    Scherer EB, da Cunha AA, Kolling J, da Cunha MJ, Schmitz F, Sitta A, Lima DD, Delwing D, Vargas CR, Wyse AT (2011) Development of an animal model for chronic mild hyperhomocysteinemia and its response to oxidative damage. Int J Dev Neurosci 29(7):693–699. doi: 10.1016/j.ijdevneu.2011.06.004 PubMedCrossRefGoogle Scholar
  21. 21.
    Petrović M, Fufanović I, Elezović I, Čolović M, Krstić D, Jakovljević V, Đurić D (2010) The effect of homocysteine thiolactone on acetylcholinesterase activity in rat brain, blood and brain. Ser J Exp Clin Res 11(1):19–22Google Scholar
  22. 22.
    Darvesh S, Walsh R, Martin E (2007) Homocysteine thiolactone and human cholinesterases. Cell Mol Neurobiol 27(1):33–48. doi: 10.1007/s10571-006-9114-0 PubMedCrossRefGoogle Scholar
  23. 23.
    Whittaker VP, Barker LA (1972) The subcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles. Method Neurochem 2:1–52Google Scholar
  24. 24.
    Ellman GL, Courtney KD, Anres V Jr, Feather-Stone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95PubMedCrossRefGoogle Scholar
  25. 25.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  26. 26.
    De Bree A, Verschuren WM, Kromhout D, Kluijtmans LA, Blom HJ (2002) Homocysteine determinants and the evidence to what extent homocysteine determines the risk of coronary heart disease. Pharmacol Rev 54(4):599–618. doi: 10.1124/pr.54.4.599 PubMedCrossRefGoogle Scholar
  27. 27.
    Pexa A, Herrmann M, Taban-Shomal O, Henle T, Deussen A (2009) Experimental hyperhomocysteinaemia: differences in tissue metabolites between homocystine and methionine feeding in a rat model. Acta Physiol (Oxf) 197(1):27–34. doi: 10.1111/j.1748-1716.2009.01981.x CrossRefGoogle Scholar
  28. 28.
    Dayal S, Arning E, Bottiglieri T, Böger RH, Sigmund CD, Faraci FM, Lentz SR (2004) Cerebral vascular dysfunction mediated by superoxide in hyperhomocysteinemic mice. Stroke 35(8):1957–1962PubMedCrossRefGoogle Scholar
  29. 29.
    Blaise SA, Nédélec E, Schroeder H, Alberto JM, Bossenmeyer-Pourié C, Guéant JL, Daval JL (2007) Gestational vitamin B deficiency leads to homocysteine-associated brain apoptosis and alters neurobehavioral development in rats. Am J Pathol 170(2):667–679PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Fukada S, Shimada Y, Morita T, Sugiyama K (2006) Suppression of methionine-induced hyperhomocysteinemia by glycine and serine in rats. Biosci Biotechnol Biochem 70(10):2403–2409PubMedCrossRefGoogle Scholar
  31. 31.
    Matté C, Mackedanz V, Stefanello FM, Scherer EB, Andreazza AC, Zanotto C, Moro AM, Garcia SC, Gonçalves CA, Erdtmann B, Salvador M, Wyse AT (2009) Chronic hyperhomocysteinemia alters antioxidant defenses and increases DNA damage in brain and blood of rats: protective effect of folic acid. Neurochem Int 54(1):7–13PubMedCrossRefGoogle Scholar
  32. 32.
    Schweinberger BM, Schwieder L, Scherer E, Sitta A, Vargas CR, Wyse AT (2014) Development of an animal model for gestational hypermethioninemia in rat and its effect on brain Na+, K+-ATPase/Mg2+-ATPase activity and oxidative status of the offspring. Metab Brain Dis 29(1):153–160. doi: 10.1007/s11011-013-9451-x PubMedCrossRefGoogle Scholar
  33. 33.
    Kolling J, Scherer EB, Siebert C, Marques EP, Dos Santos TM, Wyse AT (2014) Creatine prevents the imbalance of redox homeostasis caused by homocysteine in skeletal muscle of rats. Gene 545(1):72–79. doi: 10.1016/j.gene.2014.05.005 PubMedCrossRefGoogle Scholar
  34. 34.
    Lipton SA, Kim WK, Choi YB, Kumar S, D’Emilia DM, Rayudy PV, Arnelle DR, Stamler JS (1997) Neurotoxcity associated with dual actions of Homocysteine at the N-methyl-d-aspartate receptor. Proc Natl Acad Sci USA 94:5923–5928. doi: 10.1073/pnas.94.11.5923 PubMedCrossRefPubMedCentralGoogle Scholar
  35. 35.
    Lazarewicz JW, Ziembowicz A, Matyja E, Stafiej A, Zieminska E (2003) Homocysteine-evoked 45Ca release in the rabbit hippocampus is mediated by both NMDA and group I metabotropic glutamate receptors: in vivo microdialysis study. Neurochem Res 28:259–269PubMedCrossRefGoogle Scholar
  36. 36.
    Zou CG, Zhao YS, Gao SY, Li SD, Cao XZ, Zhang M, Zhang KQ (2010) Homocysteine promotes proliferation and activation of microglia. Neurobiol Aging 31(12):2069–2079. doi: 10.1016/j.neurobiolaging.2008.11.007 PubMedCrossRefGoogle Scholar
  37. 37.
    Shi QS, Savage JE, Hufeisen SJ, Rauser L, Grajkowska E, Ernsberger P (2003) l-Homocysteine sulfinic acid and other acidic homocysteine derivates are potent and selective metabotropic glutamate receptor agonists. J Pharmacol Exp Ther 305:131–142. doi: 10.1124/jpet.102.047092 PubMedCrossRefGoogle Scholar
  38. 38.
    Hrnčić D, Rašić-Marković A, Krstić D, Dj Macut, Šušić V, Djuric D, Stanojlović O (2012) Inhibition of the neuronal nitric oxide synthase potentiates homocysteine thiolactone—induced seizures in adult rats. Med Chem 8(1):59–64. doi: 10.2174/157340612799278577 PubMedCrossRefGoogle Scholar
  39. 39.
    Ramakrishnan S, Sulochana KN, Lakshmi S, Selvi R, Angayarkanni N (2006) Biochemistry of homocysteine in health and diseases. Indian J Biochem Biophys 43:275–283PubMedGoogle Scholar
  40. 40.
    Zhou JF, Zhou W, Zhang SM, Luo YE, Chen HH (2004) Oxidative stress and free radical damage in patients with acute dipterex poisoning. Biomed Environ Sci 17:223–233PubMedGoogle Scholar
  41. 41.
    Tsakiris S, Angelogianni P, Schulpis KH, Stavridis JC (2000) Protective effect of l-phenylalanine on rat brain acetylcholinesterase inhibition induced by free radicals. Clin Biochem 33(2):103–106. doi: 10.1016/S0009-9120(99)00090-9 PubMedCrossRefGoogle Scholar
  42. 42.
    Vučević D, Petronijević N, Radonjić N, Rašić-Marković A, Mladenović D, Radosavljević T, Hrnčić D, Djuric D, Sušić V, Dj Macut, Stanojlović O (2009) Acetylcholinesterase as a potential target of acute neurotoxic effects of lindane in rats. Gen Physiol Biophys 8:18–24Google Scholar
  43. 43.
    Mikroulis AV, Psarropoulou C (2012) Endogenous ACh effects on NMDA-induced interictal-like discharges along the septotemporal hippocampal axis of adult rats and their modulation by an early life generalized seizure. Epilepsia 53(5):879–887. doi: 10.1111/j.1528-1167.2012.03440.x PubMedCrossRefGoogle Scholar
  44. 44.
    Sener U, Zorlu Y, Karaguzel O, Ozdamar O, Coker I, Topbas M (2006) Effects of common anti-epileptic drug monotherapy on serum levels of homocysteine, vitamin B12, folic acid and vitamin B6. Seizure 15(2):79–85PubMedCrossRefGoogle Scholar
  45. 45.
    Scherer EB, Loureiro SO, Vuaden FC, da Cunha AA, Schmitz F, Kolling J, Savio LE, Bogo MR, Bonan CD, Netto CA, Wyse AT (2014) Mild hyperhomocysteinemia increases brain acetylcholinesterase and proinflammatory cytokine levels in different tissues. Mol Neurobiol. (in press)Google Scholar
  46. 46.
    Massoulie J, Perrier N, Noureddine H, Liang D, Bon S (2008) Old and new questions about cholinesterases. Chem Biol Interact 175(1–3):30–44. doi: 10.1016/j.cbi.2008.04.039 PubMedCrossRefGoogle Scholar
  47. 47.
    Silman I, Sussman JL (2008) Acetylcholinesterase: How is structure related to function? Chem Biol Interact 175:3–10. doi: 10.1016/j.cbi.2008.05.035 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Dragan Hrnčić
    • 1
  • Aleksandra Rašić -Marković
    • 1
  • Tihomir Stojković
    • 2
  • Milica Velimirović
    • 2
  • Nela Puškaš
    • 3
  • Radmila Obrenović
    • 4
  • Djuro Macut
    • 4
  • Veselinka Šušić
    • 5
  • Vladimir Jakovljević
    • 6
  • Dragan Djuric
    • 1
  • Nataša Petronijević
    • 2
  • Olivera Stanojlović
    • 1
  1. 1.Institute of Medical Physiology “Richard Burian”Belgrade University Faculty of MedicineBelgradeSerbia
  2. 2.Institute of Clinical and Medical BiochemistryBelgrade University Faculty of MedicineBelgradeSerbia
  3. 3.Institute of Histology and Embryology “Aleksandar Dj. Kostic”Belgrade University Faculty of MedicineBelgradeSerbia
  4. 4.CCS, Faculty of MedicineUniversity of BelgradeBelgradeSerbia
  5. 5.Serbian Academy of Sciences and ArtsBelgradeSerbia
  6. 6.Department of Physiology, Faculty of Medical SciencesUniversity of KragujevacKragujevacSerbia

Personalised recommendations