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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Fourteen experiments were concluded on the effect of protein and carbohydrate sources in the assay diets on the relative biological values (RBV) of food grade sodium iron pyrophosphate (NaFePP) and ferric orthophosphate (FePO4) vs. ferrous sulfate (FeSO4) for rats and chicks, using the response in blood hemoglobin to graded levels of supplemental iron as the assay criterion. Two trials were made with volunteers to estimate the RBV of NaFePP and FePO4 for humans, based on the increase in plasma iron 2 hours after ingestion of 100 mg of iron of a test dose following an overnight fast. The RBV of iron from NaFePP and FePO4 for rats were consistently lower when the basal diet contained nonfat dried milk (9 to 16 and 28, respectively, vs. FeSO4=100) than when casein was used (25 to 28 and 49, respectively). The addition of 22% beta-lactose, replacing sucrose in a diet containing casein fed to rats, significantly reduced the RBV of iron from NaFePP from 25 to 15; the addition of 22% alpha-lactose or 40% glucose in place of sucrose had no effect on the RBV of this iron source. The RBV of iron from NaFePP and FePO4 for chicks were 4 to 6 and 4, respectively, with the source of dietary protein having no effect. Very low RBV of iron from NaFePP and FePO4 (4.3 and 5.9, respectively, vs. FeSO4=100) were also obtained with human subjects.