Iron Bioavailability from Multiple Biofortified Foods Using an In Vitro Digestion, Caco-2 Assay for Optimizing a Cyclical Menu for a Randomized Efficacy Trial

BACKGROUND: Inadequate nutritional status contributes to substantial losses in human health and productivity globally. A multiple biofortified food crop trial targeting iron, zinc, and vitamin A deficiencies among young children and their breastfeeding mothers is being conducted in India. OBJECTIVE: We sought to determine the relative iron bioavailability from biofortified and conventional crops and crop combinations representative of a cyclical menu using crops targeted for inclusion in the feeding trial. METHODS: Crops were procured from India, cooked, freeze-dried, and analyzed with an established in vitro digestion/Caco-2 iron bioavailability assay using a fixed sample weight. Crop proportions representative of meals planned for the human study were determined and combined such that samples included either all biofortified or all control crops. Crops were analyzed as single crops (n = 4) or crop combinations (n = 7) by variety (biofortified or control) in triplicate. The primary outcome was iron uptake measured by Caco-2 ferritin production normalized to total Caco-2 protein (nanograms of ferritin/milligrams of cell protein) analyzed for effects of crop variety and crop proportion using generalized linear models. RESULTS: Biofortified pearl millet alone demonstrated higher iron uptake than conventional varieties (5.01 ± 1.66 vs. 2.17 ± 0.96; P = 0.036). Addition of sweet potato or sweet potato + pulse improved iron uptake for all proportions tested in control varieties and select proportions for biofortified varieties (P ≤ 0.05). Two multiple crop combinations demonstrated modestly higher iron uptake from biofortified crops. CONCLUSIONS: Optimizing total iron delivery should consider matrix effects, processing, and promoters/inhibitors of iron absorption in addition to total iron concentration. Future directions include evaluating recipes as prepared for consumption and comparison against human iron bioavailability studies.