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Department of Pediatrics, McMaster University, Hamilton ON L8N 3Z5, Canada.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • infant piglets iron absorption • calcium: iron interactions • adaptation • high calcium diet.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Cook et al. 1991,
Dawson-Hughes et al. 1986, Hallberg et al. 1991 and 1992b). Based on
these findings, it was suggested that infants fed a diet with a high
calcium content may be at risk for developing iron deficiency (Barton
et al. 1983
, Hallberg et al. 1992b).
The issue of calcium:iron interactions is important for premature
infants. Supplementation of calcium and phosphorus is common practice
for premature infants fed their mother's milk or formulas designed for
premature infants during the early neonatal period to prevent
osteopenia (Schanler 1995
). Furthermore, to optimize growth and bone
mineralization calcium and phosphorus supplementation of formulas that
are intended for feeding to premature infants after discharge from
hospital is gaining interest (Chan 1993
, Lucas et al. 1992
). Of
particular relevance is the fact that the premature infant has a
marginal iron status at birth and is more vulnerable to becoming iron
deficient compared to an infant born at term (Friel et al. 1990
).
To extrapolate to premature infants the results of calcium:iron
interaction studies of adult humans or animals require caution for
several reasons. The meals consumed in adult human or animal studies
are more complex in composition than the liquid formulas or breast milk
consumed by infants. Complex diets may contain specific components,
such as fiber, which partially account for the reduction in iron
absorption. The subjects in adult studies are in an iron replete
status. Premature infants have high iron needs and thus might have a
greater efficiency of absorption compared to adults. Further, most of
the reported studies were performed with single test meals, ignoring
the possibility that a subject may adapt its iron metabolism to
counteract the inhibition of iron absorption by a diet high in calcium.
Finally, the source of supplemental calcium may be important (Prather and Miller 1992
). Studies in adults have used calcium chloride
(Hallberg et al. 1992b
), calcium carbonate, calcium citrate, hydroxy
apatite or calcium phosphate (Cook et al. 1991
, Dawson-Hughes et al. 1986
, Hallberg et al. 1991
). Premature infants, however, are usually
supplemented with calcium tribasic or calcium gluconate and potassium
phosphate. More recently, calcium glycerophosphate
(CaGP)5
was used in mother's milk fortifiers (Schanler and Abrams 1995
, Wauben et al. 1998
).
There is no information as to whether calcium supplementation inhibits
iron absorption or alters iron status in infant populations. Because
calcium supplementation of premature infants is maintained over a
prolonged period of time, it is important to determine if continuous
exposure to a high calcium diet inhibits iron absorption. In previous
studies, the infant piglet was used as a model for premature infants to
study mineral metabolism (Atkinson et al. 1993
). The benefits of using
the piglet as a model for infant nutrition research is that gut
structure and functions are similar to that of humans (Moughan and Rowan 1989
). Further, the digestive system in the newborn piglet is
less developed at birth than the human newborn infant (Moughan et al. 1992
) making the piglet a more appropriate model for the preterm infant
than other animal models.
To address the issue of calcium:iron interactions in infant populations, we hypothesized that, when consumed over a prolonged period of time, a diet high in calcium will not inhibit iron absorption or compromise iron status. Our objectives were to study in infant piglets the influence of a diet high in calcium on 1) in vivo iron absorption, 2) in vitro iron uptake at intestinal absorption sites and 3) iron status and tissue iron stores.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Eighteen 3–5-d-old male Yorkshire piglets were removed from the sows
at the Swine Research Facility, Arkell Farms (Guelph, Ontario, Canada)
and transported to the McMaster University Central Animal Facility. All
procedures were in agreement with the Guide for the Care and Use of
Laboratory Animals (Canadian Council on Animal Care 1993
). Upon
arrival, the piglets were randomly assigned to a regular piglet formula
(NC) (n = 9) or to a high calcium piglet formula (HC)
(n = 9). At that time an iron dextran injection
providing 100 mg elemental iron was administered intramuscularly. Iron
concentration in the experimental diets was adjusted in combination
with the modified iron dextran dose to achieve a marginal state of iron
repletion (defined as a hemoglobin concentration below the normal
range) to simulate iron status of the premature infant.
Experimental diets.
The composition of the liquid piglet formulas is shown in Table 1
. The high calcium concentration in HC was achieved by adding CaGP to
the diet. The increase in the molar calcium:iron ratio in HC from NC
(relative increase calcium:iron ratio of 1.3; calcium:iron ratio
NC = 230 and HC = 540) was similar to the increase in molar
calcium:iron ratio in premature infants fed mother's milk with
additional calcium (relative increase calcium:iron ratio of 1.3;
calcium:iron ratio of preterm mother's milk ~400; and preterm
mother's milk with calcium supplements ~935), but was somewhat
greater than the increase in the calcium:iron ratio when term infant
formulas are supplemented with calcium (relative increase calcium:iron
ratio of 0.4; calcium:iron ratio of term formula ~540 and term
formula plus additional calcium ~780).
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After arrival at our Central Animal Facilities the piglets were weaned to the regular liquid piglet formula (NC). At 4–5 d of age when the piglets tolerated feeds of 400 mL/(kg · d), the HC formula was introduced to piglets assigned to that group. Weaning to the experimental diets was achieved by 7 d of age. The experimental diets were then fed for 2–2.5 wk and piglets were killed at 21–24 d of age.
Growth and iron status. Piglets were weighed prior to each morning feeding. Weight was determined to 1 g (Sartorius, Goettingen, Germany). Length was measured at three time points during the protocol by measuring snout to rump length when piglets were anesthetized for blood sampling. Length was measured with a non-stretchable tape measure. Blood samples from food-deprived piglets were collected in dry heparin by using an internal jugular blind-stab technique while piglets were under light anesthesia (isofluorane gas Aerrane; Anaquest, Ontario) at 6, 12 and 21 d of age to determine hemoglobin (Hb) and hematocrit concentrations (Kodak Ektachem Analyzer, model 700XR, Rochester, NY). Prior to necropsy, while piglets were anesthetized, blood was collected by cardiac puncture to determine serum iron concentrations.
In vivo iron absorption.
After the experimental diets were fed for a minimum of 5 d, iron
absorption from the experimental diets was determined over a 6-d period
using two radiotracers (Davidson et al. 1990
). In this method the iron
absorption from the diet (in the presence of calcium and iron) is
determined with one radiotracer (59Fe), and the absorption
of iron from a reference dose (in the absence of calcium) is determined
with another radiotracer (55Fe). This reference dose is
given to determine the variation in optimal iron absorption capacity
between piglets.
Iron absorption was determined, in the absence of calcium, from a 5 mL
reference dose consisting of an aqueous solution containing 3 mg
ferrous sulphate, 30 mg ascorbic acid and 111 MBq 55Fe.
55Fe absorption was calculated from incorporation of
55Fe into red blood cells 6 d post dosing adjusting
for isotopic decay, assuming a blood volume of 90 mL/kg body weight
(Talbot & Swenson 1970
) and assuming that 80% of absorbed iron was
incorporated into red blood cells (Davidson et al. 1990
).
55Fe was quantified in whole blood samples after digestion
and decolorization by the modified method of Eakens and Brown (1966)
.
The samples were counted using a Phillips PW4700 scintillation counter
(Phillips, The Netherlands) with an efficiency of approximately 20%.
Iron absorption from the experimental diets was determined with the
tracer 59Fe. NC or HC formula (25 mL) was labeled with
74–148 MBq 59Fe and equilibrated over a 3-h period. Both
the 55Fe and 59Fe doses were administered by
oral gavage after a period of 9 h where food was withheld and
followed by a 3-h period where food was withheld. 59Fe
absorption from the diet was determined 6 d post dosing by whole
body counting using iodide crystals (Engineered by the Department of
Nuclear Medicine, McMaster University, Hamilton, ON). Whole body counts
of 59Fe activity were performed by positioning the piglet a
fixed distance below a lead shielded sodium iodide crystal. Gamma ray
spectra were collected during the scans and the signals from the
detector were analyzed in a nuclear data multichannel analyzer system.
Baseline activity was measured immediately prior to tracer
administration, and subsequent activity was determined in quadruplicate
at 3 h on d 0 and d 1–6. The coefficient of variation for each
set of counts was generally <3%. Each measurement was adjusted for
background counts, and a reference 59Fe standard was used
to adjust for radioactivity decay. The fraction of the oral
59Fe dose retained by the piglet at a specific time was
determined as follows:
 |
where CPM is the average counts per minute of the piglet, BG is background count, ST is CPM of the reference 59Fe standard and time 0 is CPM on d 0.
In vitro iron uptake in brush border membrane vesicles.
After necropsy a segment of ~60 cm of the jejunum was removed.
Segments were rinsed and scraped while on ice to obtain mucosa for the
preparation of brush border membranes vesicles (BBMV). Intestinal BBMV
were obtained by employing a magnesium precipitation/differential
centrifugation method (Davidson and Lönnerdal 1988
, Kessler et al
1978
). Briefly, mucosa were homogenized in a hypo-osmotic buffer,
basolateral membranes and intracellular components were precipitated
with MgCl2, and differential centrifugation produced a
final pellet of purified brush border membrane fragments. This final
pellet was resuspended in a BBMV buffer solution (112 mmol NaCl/L, 100
mmol D-mannitol/L, 10 mmoltris-HEPES/L at pH 6.8). The prepared BBMV of
five piglets in each diet group were used in 59Fe uptake
studies the same day. To assess purity of BBMV, the specific activity
of sucrase was measured in the initial preparation and compared to the
final preparation, which contains BBMV. Sucrase activity was determined
by the method of Dahlqvist (1968)
. Protein content of the purified BBMV
was measured by the colorimetric method of Bradford (1976)
.
To perform iron uptake experiments, a modification of the procedure
outlined by Muir et al. (1984)
was used. Initial experiments were
performed to determine uptake kinetics, optimal pH, protein
concentration and iron concentration conditions for iron uptake by BBMV
of piglets. Iron uptake in response to different calcium:iron ratios in
the BBMV derived from piglets fed HC or NC was determined as follows:
500 µL of BBMV (containing between 450 and 750 µg protein) was
added to a 500 µL incubation solution (0–117 mmol
CaCl2/L, 0.25 mmol FeSO4/L, 37 MBq
59Fe and 5 mmol ascorbic acid/L at pH 6.8). A 20-fold molar
excess of ascorbate was added, and the incubation solution was
saturated with N2 to ensure that iron was maintained in the
iron(II) form. Isoosmolarity was maintained by adjustment of NaCl
concentration in the BBMV buffer solution. The maximum concentration of
CaCl2 that could be used to maintain isoosmolarity was 117
mmol/L. Thus the final molar calcium:iron ratios to which BBMV were
exposed were 0, 56, 112 and 235. After 1, 5, 10, 15 and 20 min
incubation, 50 µL of the BBMV-incubation solution was transferred in
triplicate to a Millipore filter (Type HA, 0.45µm, diameter 25 mm,
Millipore, Groton, CT), and 59Fe uptake was stopped by
addition of 100 µL ice cold stop solution (0.1 mmol
FeCl3/L, 100 µmol citrate/L at pH 7.0). Then the BBMV
were collected on the Millipore filters using a vacuum. The filters
were rinsed with 5 mmol EDTA/L to remove extracellular bound
59Fe. To determine iron uptake, the filters were counted
using a 
-counter (Minimax, auto-gamma, Packard-Canberra, Canada).
Iron uptake in response to different calcium:iron ratios was measured
in BBMV derived from seven piglets fed NC and five piglets fed HC.
Necropsy. Piglets were killed by a lethal, cardiac injection of sodium pentobarbital. Iron content in the body was determined by excising the liver, spleen, kidney, intestine and heart at necropsy. Organs were washed with saline and homogenized, lyophilized, ashed at 500°C (Thermolyne Furnace 30400, Sybron/Thermolyne Corporation, IW) and reconstituted in 10% nitric acid. Total iron in wet, digested serum and in organs was determined by flame atomic absorptiometry (Perkin Elmer, Norwalk, CT).
Statistical analysis. The Student's t-test was used to determine differences between diet groups for in vivo iron absorption and tissue iron concentrations. A two-way ANOVA followed by the Student-Newman-Keuls multiple means test was applied to determine differences between diet groups for hemoglobin (Hb) concentrations and hematocrit (Hct) at different time points and for iron uptake at different calcium:iron ratios in BBMV. The statistical analysis were performed with SigmaStat Software (Jandel Scientific, San Rafael, CA). Data are expressed as the mean ± SD, unless stated otherwise. The level of significance for all tests was P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Initial body size and growth during the study did not differ between
diet groups. The iron dextran dose given intramuscularly combined with
a modified iron content in the liquid formula resulted in a marginal
iron repletion state in the piglets. As shown in Fig. 1
, the Hb concentration of all piglets was below the normal range but
above iron deficiency concentrations (Underwood 1977
) after adaptation
to the experimental diets. No differences between diet groups were
observed in Hb concentrations or Hct. Hb and Hct increased
significantly (P < 0.001) over time. No differences
were observed in serum iron at necropsy (24.6 ± 14.7 and
28.5 ± 12.9 mmol/L for NC and HC, respectively), but mean values
were in the lower range of normal values [17.7–53.2 mmol/L, (Pond and Houpt 1978
)] with 3 piglets fed NC and two fed HC being below the
normal range.
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In vivo iron absorption.
There were no significant differences in measurements of iron absorption from the diet between NC and HC piglets (55 ± 7 and 57 ± 17%, respectively). To control for variation in iron absorption capacity among piglets, 59Fe absorption from the diet was expressed as a ratio of 55Fe absorption. There were no differences between diet groups in the 59Fe/55Fe ratio, which was 0.66 ± 0.15 and 0.66 ± 0.17 for NC and HC, respectively.
In vitro iron uptake in brush border membrane vesicles.
Initial experiments, measuring Fe uptake at 4 and 37°C, determined
that iron uptake in BBMV was mediated by a facilitated transport
mechanism. Other conditions for optimal iron uptake in BBMV were pH:
6.75–7.00; protein concentration: 500–750 µg/500 µL incubation
solution; and optimal iron concentration: 0.10–0.50 mmol/L incubation
solution. Iron uptake in BBMV in response to incubation with
calcium:iron ratios of 56, 112 and 235 was significantly
(P < 0.001) suppressed after 20 min compared to
incubation with a calcium:iron ratio of 0 in both diet groups
(Fig. 2). No significant differences in iron uptake in BBMV were present between
HC and NC at any calcium:iron ratios (Fig. 2)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and iron absorption (Reddy and Cook, 1997
) were not reduced when
high calcium-containing meals or calcium supplements were consumed with
meals over a time period ranging from 5 d to 6 mo. These findings,
however, contradict earlier studies in adult humans and animals that
have demonstrated a distinct effect of calcium on both heme and
non-heme iron absorption in single test meals (Barton et al. 1983
, Cook
et al. 1991, Dawson-Hughes et al. 1986, Gleerup et al. 1995
, Hallberg
et al. 1991 and 1992b). Only two of these previous studies were
relevant to infant nutrition (Barton et al. 1993
, Hallberg et al.
1992a
). In these studies, human milk with and without added calcium was
fed to adult humans (Hallberg et al. 1992
) or to young rats (Barton et
al. 1993
). In the study by >Hallberg et al (1992)
, calcium was added to
human milk to simulate the higher calcium content of cow's milk. As a
result, the increase in the molar calcium:iron ratio from addition of
calcium to human milk was four-fold, from a calcium:iron ratio of
600–3000. In both of these studies iron absorption was significantly
reduced by addition of calcium to human milk. In contrast, the study of
Atkinson et al. (1993)
demonstrated that a five-fold increase in molar
calcium:iron ratio (from a calcium:iron of 2876 to 17742) of formulas
for iron replete piglets, which represent a better model for the human
neonate than adult humans or rats, did not significantly reduce iron
absorption when the high calcium diet was fed for a period of 5 d.
It is difficult, however, to extrapolate any of these studies to the
human neonate. A four- to five-fold increase in calcium:iron ratio by
supplementing calcium in the diet is greater than would be observed in
clinical practice for infants by the addition of calcium to formulas or
breast milk.
During infancy the need for iron is high, and it was shown that the
efficiency of iron absorption from human milk is greater in infants
(49%) than in adults (21–23%) (McMillan et al. 1976
, Oski and Landaw 1980
, Saarinen et al. 1977
). Thus a high efficiency of iron absorption
in the infant piglets, possibly also as a result of their marginal iron
status, may explain in part why the inhibitory effect of calcium on
iron absorption was not observed in vivo. It is also possible that
after adaptation to a diet high in calcium, an adaptive response to the
inhibition of iron by calcium occurs, possibly involving upregulation
of iron transfer accross the brush border membrane. In adult humans,
the findings of Minihane and Fairweather-Tait (1998)
suggested an
adaptive response to the reduction in absorbable iron in the presence
of calcium. Most previous studies, which have investigated calcium:iron
interactions using single test meals, have ignored the possibility of
the presence of an adaptation in iron metabolism in response to
continuous inhibition of iron uptake by calcium.
The location(s) within the enterocyte where calcium may interact with
iron uptake remains speculative. The inhibitory mechanism of calcium on
iron absorption may involve transfer from the mucosal cell into the
circulation (Barton et al. 1993
, Hallberg et al. 1991a, Wienk et al.
1996
). Alternatively, calcium may compete for iron binding sites on the
intestinal shuttle protein mobilferrin, which could interfere with
intestinal iron uptake and intracellular transport (Conrad et al. 1993
,
Wolf and Wesling-Resnick 1994
). From the measurements of iron uptake in
BBMV, it appears that calcium and iron do compete for uptake by the
enterocyte. No differences, however, were found in iron uptake between
BBMV from HC and NC piglets, suggesting that an adaptation to
counteract the inhibitory effect of calcium on iron did not occur by
upregulating iron binding sites on the brush border membrane. However,
other possible sites of calcium:iron interactions or adaptation were
not investigated in our in vitro experiments. Further, it is possible
that the number of piglets used for BBMV experiments was too small.
Another explanation for this may be that in vitro the chelation of iron
on the inside of the membrane of BBMV is a reversible process in the
presence of chelating agents such as EDTA (Muir et al. 1984
). EDTA was
used for removal of extracellular bound 59Fe in the BBMV
experiments. In vivo, however, the chelation of iron inside the
enterocyte is likely irreversible, as iron is further metabolized by
intracellular mechanisms and is not available for transport out of the
enterocyte. Future studies will be needed to investigate the specific
sites of calcium:iron interactions and possible adaptation mechanisms.
In summary, this is the first report addressing calcium:iron interactions in an appropriate infant-animal model for extrapolation to the issues related to nutrition of premature infants. A diet high in calcium did not inhibit iron absorption and it can be speculated that there may be an adaptive response to the inhibition of iron absorption by calcium to meet the increased iron needs in the presence of a high calcium diet. The specific mechanisms of such an adaptation remains to be determined. At amounts currently used in premature infant diets, calcium supplementation will likely not compromise iron status in early neonatal life.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Dairy Farmers of Canada.
2 Presented in part at the 9th
International meeting of trace elements in man and animals, May 1996,
Banff, AB, Canada [Wauben, I., Incitti, F., Webber, C., Atkinson S.
(1997). Calcium does not inhibit iron absorption after adaptation to a
high calcium diet in the infant piglet model. In: Trace Elements In Man
And Animals TEMA-9 (Fischer, P., L'Abbe M., Cockell K., Gibson R.,
eds.), pp. 15–16. NCR Research Press, Ottawa]
3 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
5 Abbreviations used: BBMV, brush border membrane
vesicles; CaGP, calcium glycerophosphate; Hb, hemoglobin; HC, high
calcium diet; Hct, hematocrit; NC, normal calcium diet.
Manuscript received August 17, 1998. Initial review completed October 7, 1998. Revision accepted November 19, 1998.
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