In this review a broad overview of historical and current methods for the assessment of iron bioavailability was given. These methods can be divided into iron solubility studies, iron absorption studies, endpoint measures, and arithmetic models. The pros and cons of all methods were discussed. First, studies on in vitro and in vivo iron solubility have been described. The disadvantages of iron solubility include the impossibility of measuring absorption or incorporation of iron. Furthermore, only the solubility of nonheme iron, and not heme iron, can be studied. Second, we focused on iron absorption studies (either with the use of native iron, radioiron or stable iron isotopes), in which balance techniques, whole-body counting or postabsorption plasma iron measurements can be applied. In vitro determination of iron absorption using intestinal loops or cell lines, was also discussed in this part. As far as absorption studies using animals, duodenal loops, gut sacs or Caco-2 cells were concerned, the difficulty of extrapolating the results to the human situation seemed to be the major drawback. Chemical balance in man has been a good, but laborious and expensive, way to study iron absorption. Whole-body counting has the disadvantage of causing radiation exposure and it is based on a single meal. The measurement of plasma iron response did not seem to be of great value in determining nutritional iron bioavailability. The next part dealt with endpoint measures. According to the definition of iron bioavailability, these methods gave the best figure for it. In animals, the hemoglobin-repletion bioassay was most often used, whereas most studies in humans monitored the fate of radioisotopes or stable isotopes of iron in blood. Repletion bioassays using rats or other animals were of limited use because the accuracy of extrapolation to man is unknown. The use of the rat as a model for iron bioavailability seemed to be empirically based, and there were many reasons to consider the rat as an obsolete model in this respect. The double-isotope technique was probably the best predictor of iron bioavailability in humans. Disadvantages of this method are the single meal basis and the exposure to radiation (as far as radioisotopes were used). Finally, some arithmetic models were described. These models were based on data from iron bioavailability studies and could predict the bioavailability of iron from a meal.

The influence of high CaCO, intake on the bioavailability of Fe from FeSO, was assessed during Fe repletion of rats with Fe-deficiency-induced anaemia. Fe-deficient rats with a mean blood haemoglobin concentration of 4.1 mmol/l were fed on purified Fe-adequate diets containing either 6.2 or 25.0 g CaCO,/kg (ten rats per group). Haemoglobin repletion after 14 d was significantly depressed by high CaCO, intake (9.5 v. 9.8 mmol/l for high and low CaCO, intake respectively; P = 0.03), as was apparent Fe retention (367 v. 552 pg/d during days S7, P < 0.001 ; 146 Y. 1% pg/d during days 19-21, P < 0.001). The concentration of Fe in the liquid phase of the proximal half of the small intestine was significantly lower in the high-CaCO, group (3.71 v. 520 pg/g digesta; P = 0-02). Mucosal uptake and mucosal transfer of Fe were determined with orally administered %Fe and Cr as a non-absorbable marker. Mucosal transfer was significantly diminished by CaCO, loading (90 v. 100% of mucosal uptake; P = OM), whereas mucosal uptake was not. 59Fe retention values at 14 d after administration were not significantly different (57.6 v. 51.9%; P = 0.14).

New Zealand White rabbits were used to investigate the influence of increasing dietary P concentrations on growth performance, mineral balance, kidney calcification and bone development. The minimum dietary P requirement of 0·22 % (National Research Council) is usually exceeded in commercial natural-ingredient chows, leading to undesirable kidney calcifications. In order to study the optimal dietary P level, rabbits were fed semi-purified diets with four different P levels (0·1, 0·2, 0·4, and 0·8 %; w/w) at a constant dietary Ca concentration (0·5 %) during an 8-week period. Body weight and growth were not influenced by the dietary P level. During two periods (days 20 -23 and 48 -51), faeces and urine were collected quantitatively for the analysis of Ca, Mg and P and balances were calculated. Increased dietary P intake caused increased urinary and faecal P excretion and P apparent absorption and retention. Faecal Ca excretion increased with higher dietary P levels, whereas urinary Ca excretion reacted inversely. The apparent absorption of Ca became reduced at higher dietary P concentrations, but Ca retention was unchanged. The response of Mg was in a similar direction to that of the Ca balance. Kidney mineral content increased with higher dietary P levels, indicating the presence of calcified deposits. Nephrocalcinosis became more severe in kidney cortex and medulla at increasing dietary P levels, as was confirmed by histological analysis. Femur bone length was not differentially influenced by dietary P. Bone density (g/cm 3 ) of the femur diaphysis became significantly lower at the 0·8 % dietary P level as compared with the 0·2 % P group only. The bone Mg content was significantly increased on the 0·8 % P diet, both in the diaphysis and epiphysis. Plasma P concentration increased and plasma Ca decreased with higher dietary P levels, whereas plasma Mg levels were unaffected. The present study shows that the current recommended minimum dietary P level of 0·2 % for rabbits, as advised by the National Research Council in 1977, leads to a normal growth and bone development, but also causes some degree of kidney calcifications at a dietary Ca level of 0·5 %. As the dietary P level of 0·1 % virtually prevented kidney calcification and at the same time did not give evidence for any deleterious effects on growth and bone development, this indicates that the current recommended dietary P level for rabbits should be regarded as a maximum advisable concentration, and that a lower P level may be more optimal.

SummaryAn improved and sensitive method for studying iron absorption in mice with alterations in body iron stores is described. Mice with varying iron status were given a double isotopelabelled test dose containing S9Peand SICr as a non-absorbable indicator, via an oroesophageal needle. Using a whole-body counter it was possible to measure in vivo the initial mucosal iron uptake and long-term iron retention and to calculate mucosal iron transfer. A significant difference was demonstrated between normal and both anaemic and dietary iron-loaded mice with regard to the various steps of iron absorption. When mice were tested twice for iron absorption, the results were highly reproducible. In conjunction with other parameters, the method described is useful in studying the mechanism and the regulation of iron absorption in mice.

We studied Fe absorption from FeSO 4 in rats with Fe deficiency-induced anaemia that were given an Fe-sufflcient purified diet without or with ascorbic acid (10-4 g/kg diet). Attention was focused on mucosal Fe uptake as measured in vivo by a double-isotope technique. Haemoglobin repletion and liver Fe levels were not affected when the ascorbic acid-supplemented diet was given, but apparent Fe absorption and retention of orally administered 59 Fe were significantly enhanced. The distribution of Fe between liquid and solid phases of contents of both the stomach and the proximal intestine was not affected by the feeding of the ascorbic acid, but ascorbic acid significantly enhanced mucosal Fe uptake. It is concluded that ascorbic acid in the diet raises mucosal Fe uptake through a mechanism independent of the intestinal Fe solubility.