Relative bioavailability of two iron fortificants, electrolytic Fe and ferric orthophosphate, was related to that of the reference ferrous sulfate with in vitro and rat model depletion-repletion methods in four laboratories to compare values directly with those obtained in a parallel human study. In vitro testing was performed on Fe compounds with both solubility and dialysis in a simulated in vitro gastrointestinal digestion system. Two depletion-repletion techniques, hemoglobin-regeneration efficiency (HRE) and an official method of the Association of Official Analytical Chemists (AOAC), were examined. AOAC relative biological values (RBV) of electrolytic Fe were 0.66 and 0.78 and of FePO4 were 0.25 and 0.34. HRE values were 0.78 and 0.58 for electrolytic Fe and FePO4, respectively. When compared with FeSO4 in a radiolabeled farina-based meal fed to humans, the RBV of FePO4 was 0.25 and electrolytic Fe 0.75. Results obtained with the AOAC method serve as the most reliable prediction of Fe bioavailability in the human although in vitro dialysis is a promising screening technique.
Eight laboratories conducted a test for the estimation of the bioavailability of iron from 4 sources, using depleted male albino rats. Ferrous sulfate was used as the reference standard. Ferric orthophosphate was found to have a relative biological value of 11 (range 6–22), an old sample of hydrogen-reduced iron 27 (range 15–41), and ferric citrate 96 (range 75–125). Good results were obtained with a simplified basal diet prepared without ingredients that had previously contributed variable quantities of iron. There was no apparent advantage in using the change in hemoglobin during the repletion period instead of the final homoglobin value as the criterion of response to iron supplements. Several statistical treatments of the data yielded similar conclusions regarding relative biological values of the iron sources.
A collaborative study of the hemoglobin repletion test was conducted with male weanling rats. The rats were depleted for 4 weeks on a low-iron casein based diet. They were then divided into 13 comparable groups of at least 8 animals each. One group continued on the basal diet. Three groups were given different levels of each of 4 samples. Sample 1 was FeS04.7H20, used as the reference standard. Samples 2 and 3 were from the same lot of electrolytically reduced iron separated into fractions of different particle size by nitrogen elutriation, and were 7—10 and 27—40 /tm, respectively. Sample 4 was a mixture of several lots of food grade ferric orthophosphate. After 2 weeks on the test diets, individual blood samples were drawn for hemoglobin determinations. The data were analyzed by the parallel lines technique, and relative biological values, vs. FeS04 = 100, together with 95% confidence limits were calculated. The relative biological values were as follows: reduced iron, 7—10 /mi = 63.5 ± 10.9; reduced iron, 27— 40 /im = 3 7 . 9 ± 1 2 . 2 ; ferric orthophosphate = 44.5±4.8. This revised method for measuring bioavailability of iron has been adopted as official first action to replace the original method, 39.A15-39.A17.
Study was continued on the animal hemoglobin repletion test for measuring bioavailability of iron. With most iron sources, similar results were obtained with chicks and with rats. An important exception was noted with reduced iron. Seventeen samples were tested for solubility in dilute HCl and for bioavailability when fed to chicks and rats. The chicks utilized all samples reasonably well, but the rats were not able to efficiently utilize samples with particle sizes greater than 325 mesh. The mean relative biological value (vs. FeSO4 = 100) for the 17 samples was 50.5 ±8.3 when tested with chicks and 32.3±17.7 when tested with rats. Some, but not all, of the differences could be explained on the basis of particle size distribution. Similar relative biological values were obtained when the sample of reduced iron was added directly to the test diet or when it was used to enrich flour which was subsequently baked into bread and the bread was then added to the test diet. Plasma iron responses in human volunteers following test doses of the reduced iron samples were more closely related to the rat data, and this suggested that rats were the preferred species to measure bioavailability for man. The rat data also measured bioavailability of iron for guinea pigs.
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