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A sample of reduced iron was separated by air elutriation into several fractions based on particle size; and the relative biological values (RBVs) of 3 fractions containing fine, medium, and coarse particles were determined with rats and chicks, using diets containing either skim milk powder or casein as the major source of protein. The RBV of reduced iron for rats was shown to be dependent on particle size by curative and prophylactic assay procedures, with the source of protein having no marked effect. In contrast to rats, chicks utilized reduced iron to a higher degree, especially the iron from coarse particles. Prophylactic assays with rats indicated that a 3–4 week feeding period from weaning at 21 days of age gave RBVs which were not significantly different from those obtained by repletion of hemoglobin of anemic animals. After 5 weeks on the test diets, however, the rats lost their ability to distinguish between coarse and medium particles of reduced iron when fed with a diet containing casein as the source of protein.

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Elemental iron powders produced by different processes were separated by nitrogen elutriation into fractions based on particle size. The fractions containing fine particles (7–10 μm) of electrolytic iron, hydrogen (H2)- and carbon monoxide (CO)-reduced iron, and the whole sample of carbonyl iron (92% <11 μm) were blended into freshly milled wheat flour and stored at about 21°C. Storing the enriched flour up to 9 months had no effect on the relative biological value (RBV) of iron for rats from each type of iron supplement. Baking bread with flour containing electrolytic, H2-reduced, and carbonyl iron after storage for 10 months also had no effect on the RBV of the iron powders. There was no change in the RBV of iron for rats from flour enriched with electrolytic iron (200 mg/kg) and ascorbic acid (200 and 1665 mg/kg flour) after storage for 1 week and 3 months. The decreases in RBV due to increase in size of particles (7–10 vs. 20–26 μm) were significant for H2-reduced iron and CO-reduced iron but not for electrolytic iron and carbonyl iron. The RBVs of elemental iron powders for rats were similar to the values obtained with humans, based on iron absorption after an overnight fast.

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