Improved Bioavailability of Iron from Amino Acid Chelates in Fully Differentiated Human Intestinal Epithelial (Caco‐2) Cells
Authors: Doguer Caglar , Shay Neil , Collins James Forrest 
Published: November 2023

IntroductionHuman colonic carcinoma (Caco‐2) cells are a well‐established model of the human intestinal epithelium. When grown in post‐confluent culture, they take on a small intestinal epithelial cell‐like phenotype. This model system was utilized to test the hypothesis that mineral/amino acid chelates will have better bioavailability than mineral salts.MethodsCaco‐2 cells were plated on 0.4 mm pore size, 1.12 cm2 collagen‐coated transwell inserts (Corning) and grown for 20 days post‐confluence. Transepithelial electrical resistance (TEER) measurements >300 Ω cm2 and lack of phenol red flux confirmed the development of fully mature tight junctions. TEER Readings were taken prior to and after the uptake experiments to ensure that the metal compounds did not perturb monolayer integrity. In some experiments, tight junction integrity was also assessed by phenol red flux (which was essentially zero, indicating fully‐mature tight junctions). Metal‐ion chelates were obtained from a number of manufacturers for testing. Cells were treated with an iron chelator (deferoxamine [DFO]; 200 μM) or a zinc chelator (10 μM TPEN) for 24 hours, to induce iron or zinc deficiency, prior to the transport studies. No chelator was used for magnesium uptake studies. Equimolar amounts of the minerals (Fe, Zn or Mg) were added to the apical side of cells for transport studies. Cellular (uptake) and basolateral (efflux) mineral levels were subsequently quantified by atomic absorption spectrometry (for Fe and Zn) or by ICP/MS (Mg). Mineral levels were normalized by total protein content of cell lysates. Experiments were repeated 4 times, after extensive [SN1] pilot studies defined the correct conditions for uptake analysis (product concentrations, uptake time, etc.). Data were analyzed by one‐way ANOVA and only statistically significant results are reported below.ResultsAs compared to ferric ammonium citrate (an iron salt), iron uptake into cells and efflux from cells into the basolateral chamber was enhanced by the various metal/chelate forms. Increases in uptake from the metal‐ion chelates ranged from 3–10‐fold, while efflux was increased up to 5‐fold (as compared to FAC). Conversely, uptake of zinc and magnesium was not increased from the chelate forms, with uptake levels being no different than from ZnS04 or MgCl2. Efflux was also the same for all chemical forms of Zn and Mg.ConclusionsThe CaCo‐2 cell model is perhaps the best in vitro approach to assess relative bioavailability, although it does have limitations (e.g. lack of a mucus layer that exists in vivo). Nonetheless, this cell line was proven efficacious for assessing bioavailability of a number of chemical compounds. Our studies showed that iron was more bioavailable when it was derived from amino acid chelates than from a standard supplemental form of iron, ferric ammonium citrate. This is an important observation given the high worldwide prevalence of iron deficiency and the intense interest in developing superior forms of supplemental iron for clinical use. The next logical and necessary step is to confirm these observations in rodent models of iron deficiency.[SN1]This word is optional…Support or Funding InformationSupported by “Innophos Nutrition”