Skip to main content
Log in

Ascorbic acid uptake affects ferritin, Dcytb and Nramp2 expression in Caco-2 cells

  • Published:
European Journal of Nutrition Aims and scope Submit manuscript



Ascorbic acid (vitamin C) enhances iron uptake in human intestinal cells. It is commonly believed that the enhancement is due to the capacity of ascorbic acid to reduce ferric iron to ferrous iron. Other suggestions have recently been made about the effects of ascorbic acid on the cellular metabolism of iron. These effects must be investigated for several reasons. One important issue is to study whether ascorbic acid has effects on iron metabolism in the absence of extracellular iron in the intestinal lumen.

Aim of the study

The aim of this investigation was to determine whether cellular uptake of ascorbic acid affects iron acquisition in the Caco-2 cell line. The possible event was investigated by studying the expression of the iron storage protein ferritin, the iron uptake protein Nramp2 and a duodenal ferric reductase Dcytb after incubating ascorbic acid deficient or ascorbic acid fed cells with iron and/or ascorbic acid.


The above stated interactions were studied in the human Caco-2 cell model. Cell lysates were collected and subjected to SDS-PAGE and Western blotting. The blotted samples were stained with specific antibodies (Rabbit α-human-Nramp2 and Goat α-human Dcytb) against the respective proteins and the bands achieved were analysed by reflective density measurements. The cellular ferritin content was analysed with a commercial kit and the intracellular ascorbic acid concentration was measured by HPLC.


The results indicate that ascorbic acid uptake induces both iron independent and iron dependent ferritin formation, but the effect on iron dependent ferritin expression was significantly greater (470% compared to 19%). Western Blot analyses revealed a long term down-regulating effect of ascorbic acid on iron independent and iron dependent Nramp2 and Dcytb expression. However, the down-regulation of Dcytb was in general more extensive than that of Nramp2 (31–50% compared to 8–29%). In a second study of short term Nramp2 and Dcytb expression, the results suggested that both proteins were significantly up-regulated by ascorbic acid, regardless of intracellular ascorbic acid status. However, the impact of iron alone on Nramp2 up-regulation seems to be greater in the absence of ascorbic acid.


The influence of intracellular ascorbic acid status on ferritin formation must be considered in iron uptake studies in Caco-2 cells. This could be a cause of diverging inter-laboratory results. The long term down-regulation of Nramp2 and Dcytb seems to correlate with results of human studies, where long term ascorbic acid supplementation does not affect iron status. Similarly, the short term up-regulation of Nramp2 and Dcytb seems to agree with the improvement in iron uptake shown in humans when single doses of ascorbic acid were administrated. These results are important for the understanding of the impact of ascorbic acid on iron status and will hopefully lead to further investigations on the matter.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others


  1. Atanasova BD, Li ACY, Bjarnason I, Tzatchev KN, Simpson RJ (2005) Duodenal ascorbate and ferric reductase in human iron deficiency. Am J Clin Nutr 81:130–133

    CAS  Google Scholar 

  2. Beers MF, Johnson RG, Scarpa A (1986) Evidence for an ascorbate shuttle for the transfer of reducing equivalents across chromaffin granule membranes. J Biol Chem 261(6):2529–2535

    CAS  Google Scholar 

  3. Cook JD, Reddy MB (2001) Effect of ascorbic acid intake on nonheme-iron absorption from a complete diet. Am J Clin Nutr 73:93–98

    CAS  Google Scholar 

  4. Daruwala R, Song J, Koh WS, Rumsey SC, Levine M (1999) Cloning and functional characterization of the human sodium-dependent vitamin C transporters hSVCT1 and hSVCT2. FEBS Lett 460:480–484

    Article  CAS  Google Scholar 

  5. Dupic F, Fruchon S, Bensaid M, Loreal O, Brissot P, Borot N, Roth MP, Coppin H (2002) Duodenal mRNA expression of iron related genes in response to iron loading and iron deficiency in four strains of mice. Gut 51:648–653

    Article  CAS  Google Scholar 

  6. Fairweather-Tait S, Lynch S, Hotz C, Hurrell R, Abrahamse L, Beebe S, Bering S, Bukhave K, Glahn R, Hambidge M, Hunt J, Lonnerdal B, Miller D, Mohktar N, Nestel P, Reddy M, Sandberg A-S, Sharp P, Teucher B, Trinidad T (2005) The usefulness of in vitro models to predict the bioavailability of iron and zinc: a consensus statement from the harvestPlus expert consultation. Int J Nutr Res 75(6):371–374

    Article  CAS  Google Scholar 

  7. Fleming MD, Trenor CC, Su MA, Foernzler D, Beier DR, Dietrich WF, Andrews NC (1997) Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene. Nat Genet 16:383–386

    CAS  Google Scholar 

  8. Fleming MD, Romano MA, Su MA, Garrick LM, Garrick MD, Andrews NC (1998) Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for Nramp2 in endosomal iron transport. Proc Natl Acad Sci USA 95:1148–1153

    Article  CAS  Google Scholar 

  9. Glahn RP, Lee OA, Yeung A, Goldman MI, Miller DD (1998) Caco-2 Cell Ferritin Formation Predicts nonradiolabeled food iron availability in an in vitro digestion/Caco-2 cell culture model. J Nutr 128:1555–1561

    CAS  Google Scholar 

  10. Hallberg L, Brune M, Rossander L (1986) Effect of ascorbic acid on iron absorption from different types of meals. Studies with ascorbic-acid-rich foods and synthetic ascorbic acid given in different amounts with different meals. Hum Nutr Appl Nutr 40(2):97–113

    CAS  Google Scholar 

  11. MacDonald L, Thumser AE, Sharp P (2002) Decreased expression of vitamin C transporter SVCT1 by ascorbic acid in a human intestinal epithelial cell line. Br J Nutr 87:97–100

    Article  CAS  Google Scholar 

  12. Margolis S, Paule RC, Ziegler RG (1990) Ascorbic and dehydroascorbic acids measured in plasma preserved with dithiothreitol or metaphosphoric acid. Clin Chem 36(10):1750–1755

    CAS  Google Scholar 

  13. Martini LA, Tchack L, Wood RJ (2002) Iron treatment downregulates DMT1 and IREG1 mRNA expression in Caco-2 cells. J Nutr 132:693–696

    CAS  Google Scholar 

  14. McKie AT, Barrow D, Latunde-Dada GO, Rolfs A, Sager G, Mudaly E, Mudaly M, Richardson C, Barlow D, Bomford A, Peters TJ, Raja KB, Shirali S, Hediger MA, Farzaneh F, Simpson RJ (2001) An iron-regulated ferric reductase associated with the absorption of dietary iron. Science 291:1755–1759

    Article  CAS  Google Scholar 

  15. Moos T, Morgan EH (2004) The significance of the mutated divalent metal transporter (DMT1) on iron transport into the Belgrade rat brain. J Neurochem 88(1):233–245

    Article  CAS  Google Scholar 

  16. Plug CM, Dekker D, Bult A (1984) Complex stability of ferrous ascorbate in aqueous solution and its significance in iron absorption. Pharm World Sci 6(6):245–248

    CAS  Google Scholar 

  17. Sayers MH, Lynch SR, Jacobs P (1973) The effect of ascorbic acid supplementation on the absorption of iron in maize, wheat and soya. Br J Haematol 24:209–218

    Article  CAS  Google Scholar 

  18. Tandy S, Williams M, Leggett A, Lopez-Jimenez M, Dedes M, Ramesh B, Srai SK, Sharp P (2000) Nramp2 expression is associated with pH-dependent iron uptake across the apical membrane of human intestinal caco-2 cells. J Biol Chem 275(2):1023–1029

    Article  CAS  Google Scholar 

  19. Toth I, Rogers J, McPhee JA, Elliot SM, Abramson SL, Bridges KR (1994) Ascorbic acid enhances iron-induced ferritin translation in human leukemia and hepatoma cells. J Biol Chem 270(6):2846–2852

    Google Scholar 

  20. Yun S, Habicht J-P, Miller DD, Glahn RP (2004) An in vitro digestion/Caco-2 cell culture system accurately predicts the effects of ascorbic acid and polyphenolic compounds on iron bioavailability in humans. J Nutr 134:2717–2721

    CAS  Google Scholar 

Download references


We are very grateful to Dr. Stefan Allard and Anna Fermvik at the Department of Nuclear Chemistry at Chalmers University of Technology in Sweden for their assistance with γ-counting of the ferritin samples. This study was funded by the Swedish Council for Environment, Agricultural Sciences and Spatial Planning (Formas) (Grant no. 222-2004-1889) and by the Dr. Per Håkansson Foundation.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Nathalie M. Scheers.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Scheers, N.M., Sandberg, AS. Ascorbic acid uptake affects ferritin, Dcytb and Nramp2 expression in Caco-2 cells. Eur J Nutr 47, 401–408 (2008).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: