European Journal of Nutrition

, Volume 52, Issue 3, pp 1157–1167

Dietary folic acid intake differentially affects methionine metabolism markers and hippoccampus morphology in aged rats

  • Teresa Partearroyo
  • Julia Pérez-Miguelsanz
  • Natalia Úbeda
  • María Valencia-Benítez
  • Elena Alonso-Aperte
  • Gregorio Varela-Moreiras
Original Contribution



Folic acid (FA) is an emerging nutritional factor in the pathogenesis of diverse neurodegenerative disorders by still unknown mechanisms. The hippocampus is altered during the loss of cognitive abilities in humans and selectively affected when homocysteine increases. The aim was to evaluate the potential protective role of folic acid in the maintenance of biochemical markers related to the methionine cycle, as well as the integrity of the hippocampus as part of the brain in aged rats.


Male Sprague–Dawley rats (18 months old) were assigned to four different folic acid groups (0 mg FA/kg diet, deficient; 2 mg FA/kg diet, control; 8 mg FA/kg diet, moderate supplementation; 40 mg FA/kg diet, extra supplementation) for 30 days. We evaluated several parameters related to the methionine cycle. In addition, hippocampus areas were immunostained for specific neuronal markers and astrocytes.


Serum folate levels increased according to FA dietary level (p < 0.01). There was a significant increase in the serum homocysteine concentrations in the folic acid-deficient diet group (p < 0.01). However, brain S-adenosylmethionine and S-adenosylhomocysteine did not differ significantly between the folic acid groups. Consequently, the methylation ratio was also unchanged. The morphometric analysis did not show any differences in the number of neurons and astrocytes between groups, except when comparing the folic acid-deficient diet versus folic acid-supplemented diet in the striatum of the hippocampus.


Clearly, the dietary FA deficiency negatively affects the methionine metabolism biomarkers, while excessive supplementation seems to be unnecessary for optimal maintenance of the methylation cycle and hippocampus integrity.


Folic acid Supplementation Homocysteine S-adenosylmethionine S-adenosylhomocysteine DNA methylation Brain Hippocampus Aging 


  1. 1.
    Driscoll I, Sutherland RJ (2005) The aging hippocampus: navigating between rat and human experiments. Rev Neurosci 16(2):87–121Google Scholar
  2. 2.
    West MJ (1993) Regionally specific loss of neurons in the aging human hippocampus. Neurobiol Aging 14(4):287–293CrossRefGoogle Scholar
  3. 3.
    Azcoitia I, Perez-Martin M, Salazar V, Castillo C, Ariznavarreta C, Garcia-Segura LM, Tresguerres JA (2005) Growth hormone prevents neuronal loss in the aged rat hippocampus. Neurobiol Aging 26(5):697–703CrossRefGoogle Scholar
  4. 4.
    Kruman II, Mouton PR, Emokpae R Jr, Cutler RG, Mattson MP (2005) Folate deficiency inhibits proliferation of adult hippocampal progenitors. NeuroReport 16(10):1055–1059CrossRefGoogle Scholar
  5. 5.
    Morris MS, Jacques PF, Rosenberg IH, Selhub J (2007) Folate and vitamin B-12 status in relation to anemia, macrocytosis, and cognitive impairment in older Americans in the age of folic acid fortification. Am J Clin Nutr 85(1):193–200Google Scholar
  6. 6.
    Strain JJ, Dowey L, Ward M, Pentieva K, McNulty H (2004) B-vitamins, homocysteine metabolism and CVD. Proc Nutr Soc 63(4):597–603CrossRefGoogle Scholar
  7. 7.
    Weikert C, Dierkes J, Hoffmann K, Berger K, Drogan D, Klipstein-Grobusch K, Spranger J, Möhlig M, Luley C, Boeing H (2007) B vitamin plasma levels and the risk of ischemic stroke and transient ischemic attack in a German cohort. Stroke 38(11):2912–2918CrossRefGoogle Scholar
  8. 8.
    Kruman II, Culmsee C, Chan SL, Kruman Y, Guo Z, Penix L, Mattson MP (2000) Homocysteine elicits a DNA damage response in neurons that promotes apoptosis and hypersensitivity to excitotoxicity. J Neurosci 20(18):6920–6926Google Scholar
  9. 9.
    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(7):476–483CrossRefGoogle Scholar
  10. 10.
    Haan MN, Miller JW, Aiello AE, Whitmer RA, Jagust WJ, Mungas DM, Allen LH, Green R (2007) Homocysteine, B vitamins, and the incidence of dementia and cognitive impairment: results from the Sacramento Area Latino Study on Aging. Am J Clin Nutr 85(2):511–517Google Scholar
  11. 11.
    Mattson MP, Shea TB (2003) Folate and homocysteine metabolism in neural plasticity and neurodegenerative disorders. Trends Neurosci 26(3):137–146CrossRefGoogle Scholar
  12. 12.
    Troen AM (2005) The central nervous system in animal models of hyperhomocysteinemia. Prog Neuropsychopharmacol Biol Psychiatry 29(7):1140–1151CrossRefGoogle Scholar
  13. 13.
    Kruman II, Kumaravel TS, Lohani A, Pedersen WA, Cutler RG, Kruman Y, Haughey N, Lee J, Evans M, Mattson MP (2002) Folic acid deficiency and homocysteine impair DNA repair in hippocampal neurons and sensitize them to amyloid toxicity in experimental models of Alzheimer’s disease. J Neurosci 22(5):1752–1762Google Scholar
  14. 14.
    Ho PI, Ortiz D, Rogers E, Shea TB (2002) Multiple aspects of homocysteine neurotoxicity: glutamate excitotoxicity, kinase hyperactivation and DNA damage. J Neurosci Res 70(5):694–702CrossRefGoogle Scholar
  15. 15.
    Shea TB, Rogers E (2002) Folate quenches oxidative damage in brains of apolipoprotein E-deficient mice: augmentation by vitamin E. Brain Res Mol Brain Res 108(1–2):1–6CrossRefGoogle Scholar
  16. 16.
    Tjiattas L, Ortiz DO, Dhivant S, Mitton K, Rogers E, Shea TB (2004) Folate deficiency and homocysteine induce toxicity in cultured dorsal root ganglion neurons via cytosolic calcium accumulation. Aging Cell 3(2):71–76CrossRefGoogle Scholar
  17. 17.
    Kado DM, Karlamangla AS, Huang MH, Troen A, Rowe JW, Selhub J, Seeman TE (2005) Homocysteine versus the vitamins folate, B6, and B12 as predictors of cognitive function and decline in older high-functioning adults: MacArthur Studies of Successful Aging. Am J Med 118(2):161–167CrossRefGoogle Scholar
  18. 18.
    Ravaglia G, Forti P, Maioli F, Martelli M, Servadei L, Brunetti N, Porcellini E, Licastro F (2005) Homocysteine and folate as risk factors for dementia and Alzheimer disease. Am J Clin Nutr 82(3):636–643Google Scholar
  19. 19.
    Smith AD (2002) Homocysteine, B vitamins, and cognitive deficit in the elderly. Am J Clin Nutr 75(5):785–786Google Scholar
  20. 20.
    Walzem RL, Clifford AJ (1988) Folate deficiency in rats fed diets containing free amino acids or intact proteins. J Nutr 118:1089–1096Google Scholar
  21. 21.
    Partearroyo T, Ubeda N, Alonso-Aperte E, Varela-Moreiras G (2010) Moderate or supranormal folic Acid supplementation does not exert a protective effect for homocysteinemia and methylation markers in growing rats. Ann Nutr Metab 56(2):143–151CrossRefGoogle Scholar
  22. 22.
    Roncales M, Achon M, Manzarbeitia F, Maestro de las Casas C, Ramirez C, Varela-Moreiras G, Perez-Miguelsanz J (2004) Folic acid supplementation for 4 weeks affects liver morphology in aged rats. J Nutr 134(5):1130–1133Google Scholar
  23. 23.
    Achon M, Alonso-Aperte E, Varela-Moreiras G (2002) High dietary folate supplementation: effects on diet utilization and methionine metabolism in aged rats. J Nutr 6(1):51–54Google Scholar
  24. 24.
    Achón M, Alonso-Aperte E, Reyes L, Úbeda N, Varela-Moreiras G (2000) High-dose folic acid supplementation in rats: effects on gestation and the methionine cycle. Br J Nutr 83:177–183Google Scholar
  25. 25.
    Achon M, Reyes L, Alonso-Aperte E, Ubeda N, Varela-Moreiras G (1999) High dietary folate supplementation affects gestational development and dietary protein utilization in rats. J Nutr 129(6):1204–1208Google Scholar
  26. 26.
    Alonso-Aperte E, Varela-Moreiras G (1996) Brain folates and DNA methylation in rats fed a choline deficient diet or treated with low doses of methotrexate. Int J Vit Nut Res 66(3):232–236Google Scholar
  27. 27.
    Varela-Moreiras G, Selhub J (1992) Long-term folate deficiency alters folate content and distribution differentially in rat tissues. J Nutr 122(4):986–991Google Scholar
  28. 28.
    Maldonado E, Murillo J, Barrio C, Del Río A, Pérez-Miguelsanz J, López-Gordillo Y, Partearroyo T, Paradas I, Maestro C, Martínez-Sanz E, Varela-Moreiras G, Martínez-Álvarez C (2011) Occurrence of cleft-palate and alteration of Tgf-β(3) expression and the mechanisms leading to palatal fusion in mice following dietary folic-acid deficiency. Cells Tissues Organs 194:406–420CrossRefGoogle Scholar
  29. 29.
    Fell D, Benjamin LE, Steele RD (1985) Determination of adenosine and S-adenosyl derivatives of sulfur amino acids in rat liver by high-performance liquid chromatography. J Chromatogr 345:150–156CrossRefGoogle Scholar
  30. 30.
    Christman JK, Weich N, Schoenbrun B, Schneideman N, Acs G (1980) Hypomethylation of DNA during differentiation of Friend erythroleukemia cells. J Cell Biol 86:366–379CrossRefGoogle Scholar
  31. 31.
    von Bohlen Und Halbach O (2007) Immunohistological markers for staging neurogenesis in adult hippocampus. Cell Tissue Res 329(3):409–420CrossRefGoogle Scholar
  32. 32.
    Rasband WS (1997–2006) Imagen, US National Instituteof Health, Bethesda, Maryland, USA,
  33. 33.
    West MJ, Slomianka L, Gundersen HJ (1991) Unbiased stereological estimation of the total number of neurons in thesubdivisions of the rat hippocampus using the optical fractionator. Anat Rec 231(4):482–497CrossRefGoogle Scholar
  34. 34.
    Clifford AJ, Heid MK, Muller HG, Bills ND (1990) Tissue distribution and prediction of total body folate of rats. J Nutr 120:1633–1639Google Scholar
  35. 35.
    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(1):101–110CrossRefGoogle Scholar
  36. 36.
    Mahfouz MM, Kummerow FA (2004) Vitamin C or vitamin B6 supplementation prevent the oxidative stress and decrease of prostacyclin generation in homocysteinemic rats. Int J Biochem Cell Biol 36:1919–1932CrossRefGoogle Scholar
  37. 37.
    Varela-Moreiras G, Pérez-Olleros L, García-Cuevas L, Ruiz-Roso B (1994) Effects of ageing on folate metabolism in rats fed a long term folate deficient diet. Int J Vitam Nutr Res 64:294–299Google Scholar
  38. 38.
    Achón M, Varela-Moreiras G, Alonso-Aperte E (2000) High dietary folate supplementation in weanling rats affects dietary protein metabolism. Implications in the methionine cycle. In: Mato JM, Caballero A (eds) workshop on methinine metabolism: molecular and clinical implications. Master Line 2000: 303–317Google Scholar
  39. 39.
    Selhub J, Jacques F, Wilson PWF (1993) Vitamin status and intake as primary determinations of homocysteinemia in an elderly population. JAMA 270:2693–2698CrossRefGoogle Scholar
  40. 40.
    Balaghi M, Horne DW, Wagner C (1993) Hepatic one-carbon metabolism in early folate deficiency in rats. Biochem J 291:145–149Google Scholar
  41. 41.
    Niculescu MD, Zeisel SH (2002) Diet, methyl donors and DNA methylation: interactions between dietary folate, methionine and choline. J Nutr 132(8 Suppl):2333S–2335SGoogle Scholar
  42. 42.
    Brandeis M, Ariel M, Cedar H (1993) Dynamics of DNA methylation during development. BioEssays 15(11):709–713CrossRefGoogle Scholar
  43. 43.
    Friso S, Choi SW (2002) Gene–nutrient interactions and DNA methylation. J Nutr 132(8S):2382S–2387SGoogle Scholar
  44. 44.
    Laird PW, Jaenisch R (1994) DNA methylation and cancer. Human molecular genetic 3:1487–1495Google Scholar
  45. 45.
    James SJ, Pogribny IP, Pogribna M, Miller BJ, Jernigan S, Melnyk S (2003) Mechanisms of DNA damage, DNA hypomethylation, and tumor progression in the folate/methyl-deficient rat model of hepatocarcinogenesis. J Nutr 133(11 Suppl 1):3740S–3747SGoogle Scholar
  46. 46.
    Pogribny IP, Basnakian AG, Miller BJ, Lopatina NG, Poirier LA, James SJ (1995) Breaks in genomic DNA and within the p53 gene are associated with hypomethylation in livers of folate/methyl-deficient rats. Cancer Res 55(9):1894–1901Google Scholar
  47. 47.
    Varela-Moreiras G, Ragel C, Peréz de Miguelsanz J (1995) Choline deficiency and methotrexate treatment induces marked but reversible changes in hepatic folate concentrations, serum homocysteine and DNA methylation rates in rats. J Am Coll Nutr 14(5):480–485Google Scholar
  48. 48.
    Kovacheva VP, Mellott TJ, Davison JM, Wagner N, Lopez-Coviella I, Schnitzler AC, Blusztajn JK (2007) Gestational choline deficiency causes global and Igf2 gene DNA hypermethylation by up-regulation of Dnmt1 expression. J Biol Chem 282(43):31777–31788CrossRefGoogle Scholar
  49. 49.
    Mulder C, Schoonenboom NS, Jansen EE, Verhoeven NM, van Kamp GJ, Jakobs C, Scheltens P (2005) The transmethylation cycle in the brain of Alzheimer patients. Neurosci Lett 386(2):69–71CrossRefGoogle Scholar
  50. 50.
    Siegmund KD, Connor CM, Campan M, Long TI, Weisenberger DJ, Biniszkiewicz D, Jaenisch R, Laird PW, Akbarian S (2007) DNA methylation in the human cerebral cortex is dynamically regulated throughout the life span and involves differentiated neurons. PLoS One 2(9):e895CrossRefGoogle Scholar
  51. 51.
    Balk EM, Raman G, Tatsioni A, Chung M, Lau J, Rosenberg IH (2007) Vitamin B6, B12, and folic acid supplementation and cognitive function: a systematic review of randomized trials. Arch Intern Med 167(1):21–30CrossRefGoogle Scholar
  52. 52.
    Raman G, Tatsioni A, Chung M, Rosenberg IH, Lau J, Lichtenstein AH, Balk EM (2007) Heterogeneity and lack of good quality studies limit association between folate, vitamins B-6 and B-12, and cognitive function. J Nutr 137(7):1789–1794Google Scholar
  53. 53.
    Obeid R, Kostopoulos P, Knapp JP, Kasoha M, Becker G, Fassbender K, Herrmann W (2007) Biomarkers of folate and vitamin B12 are related in blood and cerebrospinal fluid. Clin Chem 53(2):326–333CrossRefGoogle Scholar
  54. 54.
    Durga J, van Boxtel MP, Schouten EG, Kok FJ, Jolles J, Katan MB, Verhoef P (2007) Effect of 3-year folic acid supplementation on cognitive function in older adults in the FACIT trial: a randomised, double blind, controlled trial. Lancet 369(9557):208–216CrossRefGoogle Scholar
  55. 55.
    Mattson MP, Magnus T (2006) Ageing and neuronal vulnerability. Nat Rev Neurosci 7(4):278–294CrossRefGoogle Scholar
  56. 56.
    Ehninger D, Kempermann G (2008) Neurogenesis in the adult hippocampus. Cell Tissue Res 331(1):243–250CrossRefGoogle Scholar
  57. 57.
    Mercier F, Kitasako JT, Hatton GI (2002) Anatomy of the brain neurogenic zones revisited: fractones and the fibroblast/macrophage network. J Comp Neurol 451(2):170–188CrossRefGoogle Scholar
  58. 58.
    Palmer TD, Willhoite AR, Gage FH (2000) Vascular niche for adult hippocampal neurogenesis. J Comp Neurol 425(4):479–494CrossRefGoogle Scholar
  59. 59.
    Kuhn HG, Dickinson-Anson H, Gage FH (1996) Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci 16(6):2027–2033Google Scholar
  60. 60.
    Parent JM, Yu TW, Leibowitz RT, Geschwind DH, Sloviter RS, Lowenstein DH (1997) Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus. J Neurosci 17(10):3727–3738Google Scholar
  61. 61.
    Kempermann G, Kuhn HG, Gage FH (1997) More hippocampal neurons in adult mice living in an enriched environment. Nature 386(6624):493–495CrossRefGoogle Scholar
  62. 62.
    Scott BW, Wang S, Burnham WM, De Boni U, Wojtowicz JM (1998) Kindling-induced neurogenesis in the dentate gyrus of the rat. Neurosci Lett 248(2):73–76CrossRefGoogle Scholar
  63. 63.
    Uda M, Ishido M, Kami K, Masuhara M (2006) Effects of chronic treadmill running on neurogenesis in the dentate gyrus of the hippocampus of adult rat. Brain Res 1104(1):64–72CrossRefGoogle Scholar
  64. 64.
    Young D, Lawlor PA, Leone P, Dragunow M, During MJ (1999) Environmental enrichment inhibits spontaneous apoptosis, prevents seizures and is neuroprotective. Nat Med 5(4):448–453CrossRefGoogle Scholar
  65. 65.
    Ra SM, Kim H, Jang MH, Shin MC, Lee TH, Lim BV, Kim CJ, Kim EH, Kim KM, Kim SS (2002) Treadmill running and swimming increase cell proliferation in the hippocampal dentate gyrus of rats. Neurosci Lett 333(2):123–126CrossRefGoogle Scholar
  66. 66.
    Kim SH, Kim HB, Jang MH, Lim BV, Kim YJ, Kim YP, Kim SS, Kim EH, Kim CJ (2002) Treadmill exercise increases cell proliferation without altering of apoptosis in dentate gyrus of Sprague-Dawley rats. Life Sci 71(11):1331–1340CrossRefGoogle Scholar
  67. 67.
    Chen Z, Schwahn BC, Wu Q, He X, Rozen R (2005) Postnatal cerebellar defects in mice deficient in methylenetetrahydrofolate reductase. Int J Dev Neurosci 23(5):465–474CrossRefGoogle Scholar
  68. 68.
    Rabaneda LG, Carrasco M, López-Toledano MA, Murillo-Carretero M, Ruiz FA, Estrada C, Castro C (2008) Homocysteine inhibits proliferation of neuronal precursors in the mouse adult brain by impairing the basic fibroblast growth factor signaling cascade and reducing extracellular regulated kinase 1/2-dependent cyclin E expression. FASEB J 22(11):3823–3835CrossRefGoogle Scholar
  69. 69.
    Kronenberg G, Harms C, Sobol RW, Cardozo-Pelaez F, Linhart H, Winter B, Balkaya M, Gertz K, Gay SB, Cox D, Eckart S, Ahmadi M, Juckel G, Kempermann G, Hellweg R, Sohr R, Hörtnagl H, Wilson SH, Jaenisch R, Endres M (2008) Folate deficiency induces neurodegeneration and brain dysfunction in mice lacking uracil DNA glycosylase. J Neurosci 28(28):7219–7230CrossRefGoogle Scholar
  70. 70.
    Troen AM, Shukitt-Hale B, Chao WH, Albuquerque B, Smith DE, Selhub J, Rosenberg J (2006) The cognitive impact of nutritional homocysteinemia in apolipoprotein-E deficient mice. J Alzheimers Dis 9(4):381–392Google Scholar
  71. 71.
    Troen AM, Shea-Budgell M, Shukitt-Hale B, Smith DE, Selhub J, Rosenberg IH (2008) B-vitamin deficiency causes hyperhomocysteinemia and vascular cognitive impairment in mice. Proc Natl Acad Sci USA 105(34):12474–12479CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Teresa Partearroyo
    • 1
  • Julia Pérez-Miguelsanz
    • 2
  • Natalia Úbeda
    • 1
  • María Valencia-Benítez
    • 2
  • Elena Alonso-Aperte
    • 1
  • Gregorio Varela-Moreiras
    • 1
  1. 1.Departamento de Ciencias Farmacéuticas y de la Alimentación, Facultad de FarmaciaSan Pablo CEU UniversityMadridSpain
  2. 2.Departamento de Anatomía y Embriología Humana, I. Facultad de MedicinaComplutense UniversityMadridSpain

Personalised recommendations