Supplementary Material for: Novel Iron-Whey Protein Microspheres Protect Gut Epithelial Cells from Iron-Related Oxidative Stress and Damage and Improve Iron Absorption in Fasting Adults

<p><b><i>Background:</i></b> Iron food fortification and oral iron formulations are frequently limited by poor absorption, resulting in the widespread use of high-dose oral iron, which is poorly tolerated. <b><i>Methods:</i></b> We evaluated novel iron-denatured whey protein (Iron-WP) microspheres on reactive oxygen species (ROS) and viability in gut epithelial (HT29) cells. We compared iron absorption from Iron-WP versus equimolar-dose (25 mg elemental iron) ferrous sulphate (FeSO<sub>4</sub>) in a prospective, randomised, cross-over study in fasting volunteers (<i>n</i> = 21 per group) dependent on relative iron depletion (a ferritin level ≤/>30 ng/mL). <b><i>Results:</i></b> Iron-WP caused less ROS generation and better HT29 cell viability than equimolar FeSO<sub>4</sub>. Iron-WP also showed better absorption with a maximal 149 ± 39% increase in serum iron compared to 65 ± 14% for FeSO<sub>4</sub> (<i>p</i> = 0.01). The response to both treatments was dependent on relative iron depletion, and multi-variable analysis showed that better absorption with Iron-WP was independent of baseline serum iron, ferritin, transferrin saturation, and haemoglobin in the overall group and in the sub-cohort with relative iron depletion at baseline (<i>p</i> < 0.01). <b><i>Conclusions:</i></b> Novel Iron-WP microspheres may protect gut epithelial cells and improve the absorption of iron versus FeSO<sub>4</sub>. Further evaluation of this approach to food fortification and supplementation with iron is warranted.</p>