Submitted to: Journal of Nutrition
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/12/2004
Publication Date: 7/1/2004
Citation: Glahn, R.P., Huh, E., Hotchkiss, A.T., Brouillette, J.N. 2004. Carbohydrate fractions from cooked fish promote iron uptake by caco-2 cells. Journal of Nutrition. 134:1681-1689. Interpretive Summary: The objective of this study was to isolate and characterize components from cooked meat (i.e. the "meat factor") that enhance iron absorption from food. Using various analytical and cell culture techniques, isolated fractions of meat samples were combined with iron and tested for enhancing effect. A fish haddock fillet was cooked and freeze-dried to be used as the muscle tissue of choice because of its low intrinsic iron content. It was demonstrated that the low pH of the stomach (pH 2.0) was the primary factor responsible for initiating the enhancing effect of fish on iron absorption. Subsequently, cooked fish samples were acidified to the pH of the stomach (pH 2.0) and incubated for 1 h to release the factor(s) from the fish. Similar experiments with chicken also showed the same effects. The liquid fraction of this acidic digest was then used as a starting material for the meat factor isolation procedures. Active fractions were found to be highly enriched with carbohydrates. Further analysis of purified samples indicated that specific carbohydrates contribute to the enhancing effect of meat on iron uptake in the intestine. These carbohydrates are possibly oligosaccharides originating from glycosaminoglycans in the extracellular matrix of muscle tissue.
Technical Abstract: The objective of this study was to isolate and characterize the meat factor(s) that enhances nonheme iron bioavailability using various analytical and in vitro cell culture techniques. Nonheme iron bioavailability was measured either via radiolabeled iron uptake or ferritin formation in Caco-2 cells. A cooked haddock fillet was lyophilized and used due to its low iron content. It was demonstrated that the low pH of the stomach (pH 2.0) was the primary factor responsible for initiating the enhancing effect of fish on nonheme iron uptake. Subsequently, cooked fish samples were titrated with HCl to pH 2.0 and incubated for 1 h to release the factor(s) from the fish. The supernatant of this acidic digest was then used for further isolation procedures. Fractions generated through Sephadex G-25 size exclusion were observed to increase Caco-2 cell iron uptake approximately 9 fold. Subsequent chromatography of these fractions via C18 reverse-phase HPLC were conducted, and enhancing activity was observed only in the "injection peak". This observation coupled with protein measurement and amino acid composition analysis revealed that the active fractions contained negligible amounts of proteins or amino acids. Active fractions were found to be highly enriched with carbohydrates. Subsequent anion-exchange chromatography yielded three active peaks that increased Caco-2 cell iron uptake 3.4 ~ 4.9 fold. Our results indicate that specific carbohydrates, possibly oligosaccharides originating from glycosaminoglycans in the extracellular matrix, contribute to the enhancing effect of meat on iron uptake by the enterocyte.