The 4 AA formulation specifically increased iron flux in duodenal epithelial organ cultures in the Ussing chamber. Net ⁵⁹Fe flux and conductance were quantified in the presence and absence of combinations of AAs. Flux was significantly higher in the 4 AA group as compared with controls (A); *P < .05; n = 12 for control, n = 11 for 4 AA. Conductance was significantly lower initially and after 30 and 60 minutes in the 4 AA group compared with control fluxes (B); *P < .05, **P < .01. Flux was also measured in the presence of single AAs from the 4 AA formulation (n = 3-5) (C). ⁵⁹Fe flux was then compared between 4 AAs and 3 AAs (excludes Gln) (D); n = 11 for 4 AAs, n = 7 for 3 AAs. Conductance was also measured during ⁵⁹Fe flux studies with 3 AAs and 4 AAs (E); **P < .01; n = 16 for 3 AAs, n = 18 for 4 AAs. ⁵⁹Fe flux was also assessed in the presence of 5 AAs that did not increase DMT1 expression in the loop assays. No change was noted when comparing to control samples (F); n = 12 for control and n = 8 for 5 AAs. These 5 AAs also tightened the mucosal barrier (G); *P < .05, **P < .01; n = 16 for control and 5 AAs. ⁵⁹Fe flux data were analyzed by nonparametric Student’s t test, while conductance data were analyzed by 1-way ANOVA followed by Sidak’s multiple comparison test.