The cross-talk of iron and ascorbate -Complexation and intracellular regulation of transport in human enterocytes

Doctoral thesis, 2011

Iron deficiency is a serious health issue in developing countries and also in vulnerable groups of the industrialized world. There are several ways to address this issue. The present approach was to learn more about the basic mechanisms of iron uptake in the presence of ascorbate, one of the best enhancers of iron absorption. In a future perspective, the new insights may be used to optimize iron absorption by harnessing the endogenous system of the body. We initiated the work by investigating iron complexation by ascorbate (and other food factors) in a system mimicking the duodenal lumen. The complexation was measured by differential pulse anodic stripping voltammetry. After this study we moved on to study uptake mechanisms in human intestinal cells. We used the Caco-2 cell model to study iron and ascorbate interactions on the cellular level. We investigated the expression of iron and ascorbate transport-related proteins (NRAMP2, DCYTB, ferritin, ferroportin, and SVCT1) by Western blot and ELISA. The results showed that ascorbic acid chelates aqueous Fe2+ and stabilizes hydrated Fe3+ at proximal duodenal pH. This effect may explain the enhancing effect on iron intestinal uptake. However, we also observed that ascorbic acid (ascorbate at pH 7.4) was affecting iron uptake on the intracellular level. Intracellular ascorbate increased iron-dependent ferritin formation as much as 50%, indicating the importance of the intracellular effects. In addition, we observed ascorbate effects on the protein expression of NRAMP2, DCYTB, and ferroportin. The NRAMP2 and DCYTB expression in ascorbate-treated cells was initially up-regulated, 2 h after an iron dose. If valid in vivo, this window can be used to optimize iron absorption. In addition, we observed that iron-replete cells were more efficient in absorbing ascorbate than their iron-deficient counterparts. This was explained by an increased expression of the ascorbate transporter SVCT1 in the presence of iron. Concluding this thesis, the results from cell-cultures are extrapolated to human, and a possible strategy for improving iron absorption is suggested.