(P11-019-23) An Optimized Amino Acid Formulation Stimulates Iron Absorption in Iron-Deficient Mice and Membrane Localization of DMT1 in Caco-2 Cells

Objectives: The goal of this study is to improve iron absorption using an optimized formulation of four amino acids (4AA-O) including aspartate, glutamate, glutamine and glycine. We hypothesized that these 4AA-O stimulate iron absorption during diet-induced iron deficiency in mice to a greater extent compared to a previous formulation (4AA), and that this process involves DMT1 trafficking to the plasma membrane of enterocytes.

Methods: Following Ussing chamber experiments to determine optimum concentrations of the four amino acids, in vivo radiotracer assays were conducted using ⁵⁹Fe to assess the comparative efficacy of the 4AA-O to the 4AA formulation and to control formulations prepared in electrolyte buffer or water. Eight-week-old male Swiss Webster mice fed a low iron diet for 10 days were fasted for 2 hours, then mice were given 2.5 µCi of ⁵⁹Fe by oral gavage in a solution of either 4AA-O in water or electrolyte buffers and sacrificed after 1 hour. In vitro, immunofluorescence staining of DMT1 was conducted in Caco2 cells to evaluate the localization of DMT1 in intestinal cells in the presence or absence of 4AA-O.

Results: Outcomes of radiotracer studies showed a significant increase in ⁵⁹Fe absorption in iron-deficient mice treated with 4AA-O compared to the electrolyte formulation and to the 4AA formulation. The preparation of the 4AA-O in water led to a further 2-fold increase in iron absorption compared to 4AA-O prepared in electrolyte buffer. Preliminary confocal imaging experiments showed an increase in DMT1 expression at the plasma membrane of Caco2 cells administered 4AA-O. Long-term iron supplementation studies are ongoing to determine the long-term efficacy of this formulation in preclinical mouse models of iron deficiency.

Conclusions: These findings show that the 4AA-O leads to further increase in iron absorption in iron-deficient mice via changes in localization of DMT1 from intracellular vesicles to the plasma membrane. This study lays a foundation for the potential use of the 4AA-O as an improved oral iron supplement to treat iron deficiency.

Funding Sources: This investigation was funded by grant DK074867 from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (JFC, PI), and grant DK109717 from NIDDK and the Office of Dietary Supplements (to JFC, PI).