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 via radiolabeled iron uptake or ferritin formation in Caco-2 cells. Fish haddock fillet was cooked and lyophilized 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 nonheme iron uptake. Subsequently, cooked fish samples were titrated with HCl to pH 2.0 and incubated for 1 h without digestive enzymes to release the factor(s) from the fish. The supernatant of this acidic digest was then used as a starting material for the meat factor isolation procedures. Fractions generated through Sephadex G-25 size exclusion increased 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 highly enriched with carbohydrates. Subsequent chromatography via high performance anion exchange chromatography with pulsed amperometric detection yielded 3 active peaks that increased Caco-2 cell iron uptake 3.4- to 4.9-fold. Our results indicate that specific carbohydrates contribute to the enhancing effect of meat on iron uptake by the enterocyte. These carbohydrates may be oligosaccharides originating from glycosaminoglycans in the extracellular matrix of muscle tissue.

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