|Laparra, Jose Moises -|
|Barbera, Reyes -|
|Alegria, Amparo -|
|Miller, Dennis -|
Submitted to: Journal of Food Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 1, 2009
Publication Date: August 11, 2009
Citation: Laparra, J., Barbera, R., Alegria, A., Glahn, R.P., Miller, D.D. 2009. Purified glycosaminoglycans from cooked haddock may enhance Fe uptake via endocytosis in a Caco-2 cell culture model. Journal of Food Science. 74(6):H168-173. Interpretive Summary: Our lab has developed a model for studying mineral absorption from different foods and food combinations. Food samples undergo a simulated digestion and are placed over Caco-2 cells, which act as a mimic of the intestinal lining. An increase in cell ferritin (an iron storage protein) formation was used as a measure of Fe uptake. Our past in vitro studies have indicated that meat enhances iron absorption, specially some carbohydrates such as glycosaminoglycans(GAGS) located in the extracellular matrix of muscle tissues. However, the accepted iron uptake mechanism(s) described to date would not explain the GAGs enhancing effect on Fe uptake. This study evaluates the cellular metabolic response(s) and regulation of the main accepted iron transporters in mammal cells. The obtained data will help to improve our understanding of the potential coordinated mechanism(s) present in mammal cells to maintain iron homeostasis. The latter will allow us to design more effective dietary supplements in complying the nutritional requirements on Fe uptake.
Technical Abstract: This study aims to understand the enhancing effect of glycosaminoglycans (GAGs), such as chondroitin/dermatan structures, on Fe uptake to Caco-2 cells. High sulfated GAGs were selectively purified from cooked haddock. An in vitro digestion/Caco-2 cell culture model was used to evaluate Fe uptake (cell ferritin formation) from a Fe+3-containing solution, and Fe+3/ascorbic acid (AA) and Fe+3/GAGs mixtures. Mitochondria (MTT test) and endosomal/lysosomal activities (neutral red uptake, NR), intracellular accumulation of reactive oxygen species and GSH concentration were monitored as biomarkers of the changes of cellular metabolism. Changes in mRNA expression of Fe transporters, divalent metal transporter-1 (DMT1) and duodenal cytochrome-B (DcytB), were also evaluated. The Fe uptake from Fe+3/GAGs mixture was up to 1.8-fold higher than from Fe+3 alone. Both Fe+3 alone and Fe+3/AA mixture produced highest increase in MTT conversion. In contrast, cell cultures exposed to the Fe+3/GAGs mixture exhibited highest NR uptake values. All Fe-containing solutions tested caused a sharp intramitochondrial accumulation of reactive oxygen species. Cell cultures exposed to the Fe+3/GAGs mixture exhibited a more preserved (by 8%) intracellular GSH concentration compared to cultures exposed to Fe+3 or Fe+3/AA mixture. In addition to cell responses, the mRNA expression of Fe transporters may suggest that Fe could also be internalized into cells by endocytosis in addition to via DMT1 in Fe+3/GAGs mixtures. These aspects need to be confirmed in in vivo experiments to better establish nutritional interventional strategies.