Molecular and Cellular Biochemistry

, Volume 451, Issue 1–2, pp 43–54 | Cite as

The effects of l-cysteine and N-acetyl-l-cysteine on homocysteine metabolism and haemostatic markers, and on cardiac and aortic histology in subchronically methionine-treated Wistar male rats

  • Sanja Kostić
  • Žarko Mićovic
  • Lazar Andrejević
  • Saša Cvetković
  • Aleksandra Stamenković
  • Sanja Stanković
  • Radmila Obrenović
  • Milica Labudović-Borović
  • Dragan Hrnčić
  • Vladimir Jakovljević
  • Dragan DjurićEmail author


Methionine is the precursor of homocysteine, a sulfur amino acid intermediate in the methylation and transsulfuration pathways; methionine-rich diets were used to induce hyperhomocysteinemia, and cardiovascular pathology was often observed. Other sulfur amino acids interfere with this metabolism, i.e., l-cysteine (Cys) and N-aceyl-l-cysteine (NAC), and probably also affect cardiovascular system. Their effects are controversial due to their ability to act both as anti- or pro-oxidant. Thus, this study aimed to elucidate their influence on levels of homocysteine, folate and vitamin B12, levels of different haemostatic parameters (fibrinogen, D-dimer, vWF Ag, vWF Ac) in rat serum or plasma as well as their effects on cardiac and aortic tissue histology in subchronically methionine-treated rats. Wistar albino rats were divided into 4 experimental groups: (a) control group (0.9% sodium chloride 0.1–0.2 mL/day) (n = 10) (K); (b) dl-methionine (0.8 mmol/kg/bw/day) (n = 10) (M); (c) dl-methionine (0.8 mmol/kg/bw/day) + l-cysteine (7 mg/kg/bw/day) (n = 8) (C); (d) dl-methionine (0.8 mmol/ kg/bw/day) + N-acetyl-l-cysteine (50 mg/kg/bw/day) (n = 8) (N). All substances were applied i.p., treatment duration 3 weeks. Lower levels of vitamin B12 in all the groups were found. Folate was reduced only in N group. Decreased fibrinogen was noted in C and N groups and increased D-dimer only in C. VWF activity was reduced in M and C groups. Deleterious effects in heart were observed, especially after Cys and NAC application. Aortic tissue remained unchanged. In conclusion, it could be said that sulfur amino acids have the significant impact on cardiovascular system in subchronically methionine-treated rats. This study points out the relevance of their complex interactions and deleterious effects mediated by either direct influence or procoagulant properties.


Sulfur amino acids Homocysteine Methionine Haemostasis Heart 



This work was supported by the Ministry of Education, Science and Technological Development of Republic of Serbia, grant number 175043 and by the COST Action [CA16225 RS] “Realizing the therapeutic potential of novel cardioprotective therapies (EUCARDIOPROTECTION).”

Compliance with ethical standards

Conflict of interest

No conflicts of interest, financial or otherwise, are declared by the authors.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in this study involving animals were in accordance with the ethical standards of the Faculty of Medicine University of Belgrade at which the study was conducted.


  1. 1.
    Yang M, Vousden KH (2016) Serine and one-carbon metabolism in cancer. Nat Rev Cancer 16:650–662Google Scholar
  2. 2.
    Chin K, Toue S, Kawamata Y, Watanabe A, Miwa T, Smriga M et al (2015) A 4-week toxicity study of methionine in male rats. Int J Toxicol 34(3):233–241Google Scholar
  3. 3.
    Banecka-Majkutewicz Z, Sawuła W, Kadziński L, Węgrzyn A, Banecki B (2012) Homocysteine, heat shock proteins, genistein and vitamins in ischemic stroke—pathogenic and therapeutic implications. Acta Biochem Pol 59(4):495–499Google Scholar
  4. 4.
    Deutz NE, Simbo SY, Ligthart-Melis GC, Cynober L, Smriga M, Engelen MP (2017) Tolerance to increased supplemented dietary intakes of methionine in healthy older adults. Am J Clin Nutr 106(2):675–683Google Scholar
  5. 5.
    Mazza A, Cicero AF, Ramazzina E, Lenti S et al (2016) Nutraceutical approaches to homocysteine lowering in hypertensive subjects at low cardiovascular risk: a multicenter, randomized clinical trial. J Biol Reg Homeost Agents 30:921–927Google Scholar
  6. 6.
    Palazhy S, Kamath P, Vasudevan DM (2015) Elevated oxidative stress among coronary artery disease patients on statin therapy: a cross sectional study. Indian Heart J 67:227–232Google Scholar
  7. 7.
    Refsum H, Ueland PM, Nygård O, Vollset SE (1998) Homocysteine and cardiovascular disease. Annu Rev Med 49:31–62Google Scholar
  8. 8.
    Jacobson WD (1998) Homocysteine and vitamins in cardiovascular disease. Clin Chem 44(8 Pt 2):1833–1843Google Scholar
  9. 9.
    Castro R, Rivera I, Blom HJ, Jakobs C, Tavares de Almeida I (2006) Homocysteine metabolism, hyperhomocysteinaemia and vascular disease: an overview. J Inherit Metab Dis 29(1):3–20Google Scholar
  10. 10.
    Ueland PM, Mansoor MA, Guttormsen AB et al (1996) Reduced, oxidized and protein-bound forms of homocysteine and other aminothiols in plasma comprise the redox thiols status—a possible element of the extracellular antioxidant defence system. J Nutr 126:1281S–1281S4SGoogle Scholar
  11. 11.
    Ueland PM (1995) Homocysteine species as components of plasma redox thiol status. Clin Chem 41(3):340–342Google Scholar
  12. 12.
    Dudman NPB, Hicks C, Wang J, Wilcken DEL (1991) Human arterial endothelial cell detachment in vitro: its promotion by homocysteine homocysteine and cysteine. Atherosclerosis 91(1–2):77–83Google Scholar
  13. 13.
    Saez G, Thornalley PJ, Hill HAO, Hems R, Bannister JV (1982) The production of free radicals during the autooxidation of cysteine and their effect on isolated rat hepatocytes. Biochem Biophys Acta 719(1):24–31Google Scholar
  14. 14.
    Mills BJ, Weiss MM, Lang CA, Liu MC, Ziegler C (2000) Blood glutathione and cysteine changes in cardiovascular disease. J Lab Clin Med 135(5):396–401Google Scholar
  15. 15.
    Jacob N, Bruckert E, Giral P, Foglietti MJ, Turpin G (1999) Cysteine is a cardiovascular risk factor in hyperlipidemic patients. Atherosclerosis 146(1):53–59Google Scholar
  16. 16.
    El-Khairy L, Ueland PM, Nygård O et al (1999) Lifestyle and cardiovascular disease risk factors as determinants of total cysteine in plasma: the Hordaland Homocysteine Study. Am J Clin Nutr 70(6):1016–1024Google Scholar
  17. 17.
    El-Khairy L, Ueland PM, Refsum H, Graham IM, Vollset SE (2001) Plasma total cysteine as a risk factor for vascular diseases. Circulation 103(21):2544–2549Google Scholar
  18. 18.
    Wilcken DE, Wilcken B (1976) The pathogenesis of coronary artery disease. A possible role for methionine metabolism. J Clin Invest 57:1079–1082Google Scholar
  19. 19.
    Csontos C, Rezman B, Foldi V, Bogar L et al (2011) Effect of N-acetylcysteine treatment on the expression of leukocyte surface markers after burn injury. Burns 37(3):453–464Google Scholar
  20. 20.
    Palacio JR, Markert UR, Martinez P (2011) Anti-inflammatory properties of N-acetylcysteine on lipopolysaccharide-activated macrophages. Inflamm Res 60(7):695–704Google Scholar
  21. 21.
    Xu CC, Yang SF, Zhu LH, Cai X, Sheng YS, Zhu SW, Xu JX (2014) Regulation of N-acetyl cysteine on gut redox status and major microbiota in weaned piglets. J Anim Sci 92(4):1504–1511Google Scholar
  22. 22.
    Speidl WS, Nikfardjam M, Niessner A et al (2007) Mild hyperhomocysteinemia is associated with a decreased fibrinolytic activity in patients after ST-elevation myocardial infarction. Thromb Res 119(3):331–336Google Scholar
  23. 23.
    Di Minno MN, Tremoli E, Coppola A, Lupoli R, Di Minno G (2010) Homocysteine and arterial thrombosis: challenge and opportunity. Thromb Haemost 103(5):942–961Google Scholar
  24. 24.
    D’Angelo A, Selhub J (1997) Homocysteine and thrombotic disease. Blood 90(1):1–11Google Scholar
  25. 25.
    Liapi C, Zarros A, Theocharis S, Al-Humadi H et al (2009) The neuroprotective role of l-cysteine towards the effects of short-term exposure to lanthanum on the adult rat brain antioxidant status and the activities of acetylcholinesterase, (Na+, K+)- and Mg2+-ATPase. Biometals 22(2):329–335Google Scholar
  26. 26.
    Akbulut S, Elbe H, Eris C, Dogan Z et al (2014) Cytoprotective effects of amifostine, ascorbic acid and N-acetylcysteine against methotrexate- induced hepatotoxicity in rats. World J Gastroenterol 20(29):10158–10165Google Scholar
  27. 27.
    Bradbury P, Gordon KC (1982) Connective tissues and stains. In: Bancroft JD, Stevens A (eds) Theory and practice of histological techniques, 2nd edn. Churchill Livingstone, Edinburgh, pp 122–144Google Scholar
  28. 28.
    Baszczuk A, Kopczyński Z (2014) Hyperhomocysteinemia in patients with cardiovascular disease. Postepy Hig Med Dosw (Online) 68:579–589Google Scholar
  29. 29.
    Kaul S, Zadeh AA, Shah PK (2006) Homocysteine hypothesis for atherothrombotic cardiovascular disease. Not Valid J Am Coll Cardiol 48(5):914–923Google Scholar
  30. 30.
    Waldmann A, Koschizke JW, Leitzmann C, Hahn A (2005) German vegan study: diet, life-style factors, and cardiovascular risk profile. Ann Nutr Metab 49(6):366–372Google Scholar
  31. 31.
    Pawlak R (2015) Is vitamin B12 deficiency a risk factor for cardiovascular disease in vegetarians? Am J Prev Med 48(6):e11–e26Google Scholar
  32. 32.
    Troen AM, Lutgens E, Smith DE, Rosenberg IH, Selhub J (2003) The atherogenic effect of excess methionine intake. Proc Natl Acad Sci USA 100(25):15089–15094Google Scholar
  33. 33.
    Ekim M, Ekim H, Yilmaz KY et al (2015) Study on relationships among deep vein thrombosis, homocysteine & related B group vitamins. Pak J Med Sci 31(2):398–402Google Scholar
  34. 34.
    Sule AA, Chin TJ, Khien H (2012) Recurrent unprovoked venous thromboembolism in a young female patient with high levels of homocysteine. Int J Angiol 21:95–98Google Scholar
  35. 35.
    Sharma GS, Kumar T, Dar TA, Singh LR (2015) Protein N-homocysteinylation: from cellular toxicity to neurodegeneration. Biochim Biophys Acta 1850(11):2239–2245Google Scholar
  36. 36.
    Ozkan Y, Ozkan E, Simsek B (2002) Plasma total homocysteine and cysteine levels as cardiovascular risk factors in coronary heart disease. Int J of Cardiol 82(3):269–277Google Scholar
  37. 37.
    Lentz SR, Sadler JE (1991) Inhibition of thrombomodulin surface expression and protein C activation by the thrombogenic agent homocysteine. J Clin Invest 88:1906–1914Google Scholar
  38. 38.
    Fryer RH, Wilson BD, Gubler DB, Fitzgerald LA, Rodgers GM (1993) Homocysteine, a risk factor for premature vascular disease and thrombosis, induces tissue factor activity in endothelial cells. Arterioscler Thromb 13:1327–1333Google Scholar
  39. 39.
    Nappo F, De Rosa N, Marfella R, De Lucia D et al (1999) Impairment of endothelial functions by acute hyperhomocysteinemia and reversal by antioxidant vitamins. JAMA 281:2113–2118Google Scholar
  40. 40.
    Schreiner PJ, Wu KK, Malinow MR, Stinson VL, Szklo M, Nieto FJ, Heiss G (2002) Hyperhomocyst(e)inemia and hemostatic factors: the atherosclerosis risk in communities study. Ann Epidemiol 12(4):228–236Google Scholar
  41. 41.
    Gerdes VEA, Kremer Hovinga HA, Ten Cate H et al (2004) Homocysteine and markers of coagulation and endothelial cell activation. J Thromb Haemost 2:445–451Google Scholar
  42. 42.
    Nagaraja D, Noone ML, Bharatkumar VP, Christopher R (2008) Homocysteine, folate and vitamin B12 in puerperal cerebral venous thrombosis. J Neurol Sci 272(1–2):43–47Google Scholar
  43. 43.
    Sofi F, Marcucci R, Bolli P, Giambene B, Sodi A, Fedi S, Menchini U, Gensini GF, Abbate R, Prisco D (2008) Low vitamin B6 and folic acid levels are associated with retinal vein occlusion independently of homocysteine levels. Atherosclerosis 198(1):223–227Google Scholar
  44. 44.
    Zhou K, Zhao R, Geng Z, Jiang L, Cao Y, Xu D (2012) Association between B-group vitamins & venous thrombosis: systematic review & metaanalysis of epidemiological studies. J Thromb Thrombolysis 34(4):459–467Google Scholar
  45. 45.
    Hron G, Lombardi R, Eichinger S, Lecchi A, Kyrle PA, Cattaneo M (2007) Low vitamin B6 levels & the risk of recurrent venous thromboembolism. Haematol 92:1250–1253Google Scholar
  46. 46.
    Diaz DE, Tuesta AM, Ribo MD, Belinchon O, Marchena PJ, Bruscas MJ, Val E (2005) Low level of vitamin B12 & venous thromboembolic disease in elderly men. J Intern Med 258:244–249Google Scholar
  47. 47.
    Sagristá ML, García AE, Africa De Madariaga M et al (2002) Antioxidant and pro-oxidant effect of the thiolic compounds N-acetyl-l-cysteine and glutathione against free radical-induced lipid peroxidation. Free Radic Res 36:329–340Google Scholar
  48. 48.
    Mansoor MA, Bergmark C, Svardal AM et al (1995) Redox status and protein binding of plasma homocysteine and other aminothiols in patients with early-onset peripheral vascular disease. Arterioscler Thromb Vascu Biol 15:232–240Google Scholar
  49. 49.
    Preibisch G, Küffner C, Elstner EF (1993) Biochemical model reactions on the prooxidative activity of homocysteine. Z Naturf C 48(1–2):58–62Google Scholar
  50. 50.
    Starkebaum G, Harlan JM (1986) Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine. J Clin Invest 77:1370–1376Google Scholar
  51. 51.
    Wu XY, Luo AY, Zhou YR, Ren JH (2014) N-acetycysteine reduces oxidative stress, nuclear factor-kB activity and cardiomyocyte apoptosis in heart failure. Mol Med Rep 10(2):615–624Google Scholar
  52. 52.
    Hashem SI, Perry CN, Bauer M, Han S et al (2015) Brief report: oxidative stress mediates cardiomyocyte apoptosis in a human model of danon disease and heart failure. Stem Cells 33(7):2343–2350Google Scholar
  53. 53.
    Wilder T, Ryba DM, Wieczorek DF et al (2015) N-acetylcysteine reverses diastolic dysfunction and hypertrophy in familial hypertrophic cardiomyopathy. Heart Circ Physiol 309(10):H1720–H1730Google Scholar
  54. 54.
    Eleftheriadou I, Grigoropoulou P, Moyssakis I et al (2013) The effect of hyperhomocysteinemia on aortic distensibility in healthy individuals. Nutrition 29(6):876–880Google Scholar
  55. 55.
    Jiang Y, Zhang H, Sun T, Wang J, Sun W, Gong H et al (2012) The comprehensive effects of hyperlipidemia and hyperhomocysteinemia on pathogenesis of atherosclerosis and DNA hypomethylation in ApoE–/– mice. Acta Biochim Biophys Sin 44(10):866–875Google Scholar
  56. 56.
    Hofmann MA, Lalla E, Lu Y, Gleason MR, Wolf BM, Tanji N, Ferran LJ Jr, Kohl B, Rao V, Kisiel W, Stern DM, Schmidt AM (2001) Hyperhomocysteinemia enhances vascular inflammation and accelerates atherosclerosis in a murine model. J Clin Invest 107:675–683Google Scholar
  57. 57.
    Zhou J, Moller J, Danielson CC, Bentzon J, Ravn HB, Austin RC, Falk E (2001) Dietary supplementation with methionine and homocysteine promotes early atherosclerosis but not plaque rupture in apoE-deficient mice. Arterioscl Thromb Vasc Biol 21:1470–1476Google Scholar
  58. 58.
    Lentz SR (2005) Mechanisms of homocysteine-induced atherothrombosis. J Thromb Haemost 3:1646–1654Google Scholar
  59. 59.
    Mecif KO, Bouguerra SA, Benazzoug Y (2017) Plasma and Aorta Biochemistry and MMPs Activities in Female Rabbit Fed Methionine Enriched Diet and Their Offspring. J Nutr Metab 2017:2785142Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Sanja Kostić
    • 1
  • Žarko Mićovic
    • 2
  • Lazar Andrejević
    • 3
  • Saša Cvetković
    • 3
  • Aleksandra Stamenković
    • 4
  • Sanja Stanković
    • 5
  • Radmila Obrenović
    • 5
  • Milica Labudović-Borović
    • 6
  • Dragan Hrnčić
    • 1
  • Vladimir Jakovljević
    • 7
    • 8
  • Dragan Djurić
    • 1
    Email author
  1. 1.Faculty of Medicine, Institute of Medical Physiology “Richard Burian”University of BelgradeBelgradeSerbia
  2. 2.Military Health DepartmentMinistry of DefenceBelgradeSerbia
  3. 3.Clinic of Gynecology and ObstetricsFaculty of Medical Science University of Pristina - Kosovska MitrovicaKosovska MitrovicaSerbia
  4. 4.St. Boniface Hospital Research Center, Institute of Cardiovascular SciencesUniversity of ManitobaWinnipegCanada
  5. 5.Centre of Medical BiochemistryClinical Centre of SerbiaBelgradeSerbia
  6. 6.Faculty of Medicine, Institute of Histology and Embryology “Aleksandar Dj. Kostic”University of BelgradeBelgradeSerbia
  7. 7.Department of Physiology, Faculty of Medical SciencesUniversity of KragujevacKragujevacSerbia
  8. 8.Department of Human Pathology1st Moscow State Medical University IM SechenovMoscowRussian Federation

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