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