Ferritin-protein levels in Caco-2 cells following exposure to LM Fe(III) poly oxo-hydroxide (nano Fe), Fe(III) maltol (FeM) or Fe(II) sulphate-ascorbate (FeSO₄ + AA).

<p><b>A</b>, Ferritin-protein regulation in differentiated and undifferentiated cells. ***, <i>p</i>=0.0003. Cells were incubated for 1 h with 200 μM Fe plus a further 23 h in fresh, non-supplemented MEM to allow for ferritin formation. <b>B</b>, Phase distribution of Fe in the BSS uptake medium: i.e. fractional percentage of microparticulate (black bars), nanoparticulate (red bars) and soluble Fe (open bars) for each Fe material. Values are mean ± s.d. of 3 independent experiments. <b>C</b>, Effect of LM Fe(III) poly oxo-hydroxide particle dispersion (in BSS medium, closed bars) or agglomeration (in MEM medium, open bars) on ferritin-protein levels in differentiated cells: the LM Fe(III) poly oxo-hydroxide was dispersed in its nano-form (99 ± 2% nano) using BSS or agglomerated (97 ± 2% microparticulate) using MEM. Data are mean of 3 independent experiments (each experiment with 3 replicate wells). FeM: soluble iron control, Fe(III) maltol. ***, <i>p</i>=0.0002 for the comparison between BSS and MEM. <b>D</b>, TEER changes in differentiated Caco-2 cell monolayer at different time points during incubation with BSS supplemented with LM Fe(III) poly oxo-hydroxide (open circles) or non-supplemented BSS control (closed inverted triangles). Incubations were for 3 h with 200 μM Fe (measurements at 1, 2 & 3 h) plus a further 21 h in fresh, non-supplemented MEM (24-h). Values are expressed as a percentage of the initial measurement and are shown as mean ± s.d. of 3 independent experiments (each experiment with 3 replicate wells). Experimental points are connected with a solid line to aid visualization and not because a linear relationship is assumed between time and TEER measurement. Detailed methodology is available in the Methods Section and in Methods S1.