Measurement of iron absorption in milk-fed calves using a dual stable isotope technique

Fractional absorption of an oral dose of iron was determined in calves receiving an irondefi cient diet with and without supplementation of iron. Iron supplements were Fe-equivalent doses of oral ferrous sulphate or parenteral gleptoferron. Animals receiving no supplementation showed haematological results suggestive of mild anaemia. Forms of iron supplementation did not differ signifi cantly in their ability to prevent the onset of anaemia. Iron fractional absorption determined using a dual stable isotope labelling method was 60, 39 and 18% in the iron defi cient calves, and the calves supplemented with parenteral iron and oral iron, respectively.


INTRODUCTION
Dual stable isotopic labelling has been used to measure the fractional absorption of minerals in humans (e.g., Kastenmayer et al., 1994;Walczyk et al., 1997).We have adapted this technique in the development of a pre-ruminant model to study the effects of oral vs parenteral supplementation on the regulation of iron metabolism.

MATERIAL AND METHODS
Eighteen pre-ruminant calves from 2 sires were used to develop an animal model of contrasting states of iron absorption over an experimental period of 68 d.The diet consisted of 100% reconstituted milk powder (increasing concentration: 14 to 20% milk solids; 1.7 mg Fe/kg DM).The calves were 8.7 (SD 2.6) days old and 43.7 (SD 5.7) kg liveweight (LW) when treatments started.Treatments consisted of no Fe supplementation (Fe-defi cient), Parenteral Fe (600 mg of iron as gleptoferron: Gleptosyl TM intramuscular every 21 d) and Oral Fe (100 mg of Fe as iron sulphate per kg milk solids).The two Fe treatments were planned to provide the equivalent of 34 mg of physiologically available Fe per kg of LW gain.Blood samples were collected weekly and LW of the calves recorded twice weekly.Blood samples were analysed for haematological parameters using a Coulter Counter.Iron status of the calves was assessed using haemoglobin (Hb) concentrations in blood.On d 47, blood volumes were estimated using the Evan's Blue dilution method and haematocrit to estimate the mass of the blood Fe pool and calculate optimal loads of iron isotopes.On d 48, four animals in each treatment group were dosed with 57 Fe (oral) and 58 Fe (intravenous).A week later (d 55), erythrocytes were collected and analysed for iron isotope concentrations by dynamic reaction cell ICP-MS.Ratios of 57 Fe/ 58 Fe in blood cells were used to calculate the fractional absorption of orally-dosed iron.Animals were slaughtered on d 62 and 63 of the experiment (n=9 each d), liver and longissimus dorsi muscle collected and analysed for total iron concentration.
Statistical analysis was conducted using the GLM and MIXED procedures of SAS v 8.0 (Hb concentration data were analysed using the "repeated" option with auto-regressive variance-covariance structure and 'time' effect).Treatment, sire and their interaction were included as main effects.All animal manipulations were approved by the Crown Research Institutes Palmerston North Campus Animal Ethics Committee.

RESULTS
Overall daily LW gain was not affected by treatments (0.7 kg/d).Plasma volumes (% of LW) measured on day 47 were 8.2 (range 6.6 to 10.3), 9.9 (range 6.0 to 16.9) and 8.8 (range 6.4 to 15.4) for the Control, Parenteral Fe and Oral Fe, respectively.Actual iron intake was 40 and 28 mg Fe per kg LW gain for the parenteral and oral iron groups, respectively.
There were no signifi cant differences in the blood Hb concentration of calves receiving parenteral or oral iron supplementation.Clinical data from weekly blood samples showed that the Fe-defi cient group developed signs compatible with low iron status (mild hypochromic anaemia: Figure 1).Signifi cant differences between the Fe-defi cient and Fe supplemented groups were detected as early as 28 d of the experimental period.
Fe defi ciency resulted in greater dietary Fe absorption compared to Fesupplemented animals (P=0.02).Iron defi cient calves had a lower hepatic Fe concentration (P=0.01) that was 64 and 17% of those measured in orally-and parenterally-supplemented calves, respectively).Fe-defi cient calves had lower Fe IRON ABSORPTION IN MILK-FED CALVES muscular concentration (P=0.01) that was 71 and 75% of the concentrations in Oral Fe and Parenteral Fe groups, respectively (Table 1).

DISCUSSION
To our knowledge, this is the fi rst report of fractional absorption of Fe measured in calves using the dual stable isotope labelling method.Our results confi rm previous indirect estimates of absorption in calves consuming Fe-defi cient diets or receiving oral Fe-supplementation with contrasting Fe status (NRC, 2001 and references therein).
Although the parenteral and oral Fe dose rates were equivalent on a LW gain basis, it appears that the injected amount may have been just suffi cient to ).The hepatic Fe concentration measured at the end of the experiment (d 63) suggested that the body appeared to manage the higher Fe load of parenteral dose by storing it in the liver, presumably as inert haemosiderin.The difference in mean and range in fractional absorption in the Fe-supplemented animals suggested that the parenteral administration of Fe is not as effective as its oral equivalent in establishing a feedback signal to the gut to reduce the fractional absorption of Fe from the diet.

CONCLUSIONS
The dual stable isotope method for estimation of fractional absorption of iron provides sensible estimates of iron absorption in milk-fed calves.While parenteral iron seems adequate to prevent signs of sub-clinical anaemia, there is indication that the body stores a sizeable proportion of the parenteral dose in the liver, with incomplete feedback to the gastrointestinal tract.Given the clear functional difference in Fe absorption, the animal model described herein appears suitable to assess the effect of iron supplementation on the genome and proteome involved in Fe absorption in the pre-ruminant animal.

Table 1 .
Fractional absorption of iron and hepatic iron concentration in calves receiving three iron regimes.Means with different letter within column are different (LSD α = 0.01)