Assessment of iron availability using combined in vitro digestion and Caco-2 cell culture

Abstract

A model for the rapid assessment of iron availability was developed that combines in vitro digestion with iron uptake by Caco-2 cell monolayers. In this method, samples (beef, ascorbic acid, or citric acid) were adjusted to pH 2, labeled with 59Fe, and subjected to pepsin digestion (pH 2, 37°C) for 1 h to simulate gastric digestion. Next, a dialysis bag (12,000–14,000 molecular weight cutoff) containing 150 mM PIPES buffer (pH 6.7) was placed in the digest and incubation continued for 30 min. Then, a pancreatin-bile mixture was added, and incubation was continued for an additional 2 h. The contents of the dialysis bag were removed and an aliquot applied to Caco-2 cell monolayers. After a 60 min incubation, iron that was non-specifically bound to the surface of the monolayer was removed by rinsing with a solution containing bathophenanthrolinedisulfonic acid and sodium dithionite. Cells were then counted for 59Fe activity to measure uptake. Beef and ascorbic acid enhanced Caco-2 cell iron uptake, whereas citric acid had no effect. These results compare favorably with literature reports of human studies and suggest that a dialyzable factor(s) less than 14,000 daltons, released during beef digestion, was responsible for the iron absorption-enhancing properties of beef. We believe that this system will be useful for studying basic mechanisms of iron absorption and for in vitro estimation of iron bioavailability.