Nathalie M. Scheers Ann-Sofie Sandberg Ascorbic acid uptake affects ferritin, Dcytb and Nramp2 expression in Caco-2 cells Received: 20 February 2008 Accepted: 11 September 2008 Published online: 24 September 2008 N.M. Scheers (&) Æ A.-S. Sandberg Dept. of Chemical and Biological Engineering/Food Science Chalmers University of Technology 412 96 Goteborg, Sweden Tel.: +46-31/772-3821 Fax: +46-31/772-3830 E-Mail: nathalie.scheers@chalmers.se j Abstract Background Ascorbic acid (vitamin C) enhances iron uptake in human intestinal cells. It is commonly believed that the enhancement is due to the capac- ity of ascorbic acid to reduce ferric iron to ferrous iron. Other sug- gestions 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 metabo- lism 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 ascor- bic 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 incu- bating ascorbic acid deficient or ascorbic acid fed cells with iron and/or ascorbic acid. Methods 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 a-human- Nramp2 and Goat a-human Dcytb) against the respective pro- teins and the bands achieved were analysed by reflective density measurements. The cellular ferri- tin content was analysed with a commercial kit and the intracel- lular ascorbic acid concentration was measured by HPLC. Results 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 indepen- dent and iron dependent Nramp2 and Dcytb expression. However, the down-regulation of Dcytb was in general more extensive than that of Nramp2 (31–50% com- pared to 8–29%). In a second study of short term Nramp2 and Dcytb expression, the results sug- gested that both proteins were significantly up-regulated by ascorbic acid, regardless of intra- cellular ascorbic acid status. However, the impact of iron alone on Nramp2 up-regulation seems to be greater in the absence of ascorbic acid. Conclusions 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- ORIGINAL CONTRIBUTION Eur J Nutr (2008) 47:401–408 DOI 10.1007/s00394-008-0741-8 EJN 741