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. 2004 Sep 9;561(Pt 1):195–203. doi: 10.1113/jphysiol.2004.068825

Effect of timing of iron supplementation on maternal and neonatal growth and iron status of iron-deficient pregnant rats

L Gambling 1, H S Andersen 1, A Czopek 1, R Wojciak 2, Z Krejpcio 2, H J McArdle 1
PMCID: PMC1665338  PMID: 15358806

Abstract

We have previously shown that maternal iron (Fe) deficiency not only reduces fetal size, but also increases blood pressure in the offspring when they are adults. In this paper we examine whether there are critical periods when supplementation reverses or fails to reverse the effect both on size and on expression of genes of Fe metabolism. We made dams Fe deficient, mated them and provided supplements of Fe in the diet from the beginning of gestation (0.5 days), from 7.5 days or from 14.5 days. Within 12 h of birth, dams and neonates were killed and tissues taken and examined. Fe deficiency throughout pregnancy reduces neonatal size. Supplementation from the beginning of the first, second or third week all reduced the effect. Maternal haematocrit was restored to normal levels only in animals given supplements for at least 2 weeks. In contrast, the neonates' Fe levels were normal in all supplemented groups. These results were mirrored in liver Fe levels and in transferrin receptor mRNA. Iron-responsive element (IRE)-regulated divalent metal transporter 1 (DMT1) increased in maternal and neonatal liver. Non-IRE-regulated DMT1 levels did not change in the maternal liver, but decreased in the neonatal liver. H and L ferritin mRNA levels also showed different patterns in the mother and her offspring. Finally, the neonatal size correlated with maternal Fe stores, and not with those of the fetus. The data demonstrate that Fe supplementation during pregnancy is most effective when given early, rather than later, in gestation.

Iron (Fe) deficiency in pregnancy has serious consequences for both the mother and her baby. In the immediate postnatal period, these include increased risk of low birth-weight, increased morbidity and mortality (Scholl & Hediger, 1994; Allen, 2000; Hamalainen et al. 2003). In the neonatal period, there is an increased risk of impaired motor development and coordination. In children, language development and scholastic achievement can be affected, there are significant psychological and behavioural effects and decreased physical activity (reviewed byn Kapil & Bhavna, 2002; Viteri & Gonzalez, 2002). As adults, the effects persist and can result in elevated blood pressure and cardiovascular problems (Crowe et al. 1995; Lewis et al. 2002; Gambling et al. 2003; Lisle et al. 2003).

Iron supplementation in appropriate cases therefore is clearly warranted. However, there is still debate whether supplementation should be universally provided. Additionally, neither the optimal time period nor the dose has been agreed. For example, World Health Organization have recommended supplementation of about 60 mg day−1 to all women from the second trimester onward (Stoltzfus & Dreyfuss, 1998; WHO, 2001). In contrast, Casanueva & Viteri (2003) suggest that 60 mg day−1 in normal women is too high, and results in haemoconcentration, decreased birth-weight and increased prematurity. The US Institute of Medicine (Viteri, 1998) recommends a flexible approach, ranging from no supplementation during the first two trimesters to 120 mg day−1, depending on a variety of parameters.

There is very little information about the mechanisms underpinning the differential effects of dose and timing. It is generally agreed that the mother uses both Fe stores and increased absorption to supply the developing fetus. However, how much each can or will contribute is not known. Nor is it clear how the mother will adapt to lower Fe status.

In order to examine these questions, we have developed a rat model of Fe deficiency during pregnancy. The pups born to Fe-deficient dams are smaller than control neonates, and have proportionately smaller livers. However, the degree of Fe deficiency in these pups is less than might be predicted. We have shown that the placenta up-regulates some of the proteins of Fe transport as a response to maternal deficiency. For example, transferrin receptor (TfR) levels increase markedly as deficiency increases (Gambling et al. 2001). Iron is transferred from the transferrin receptor in the endocytotic vesicle into the cell through a channel called divalent metal transporter 1 (DMT1). There are at least two isoforms of this protein. One is regulated by an iron-responsive element (IRE) and the other is not. The IRE form of the protein is also up-regulated in the placenta by maternal Fe deficiency. Consequently, the fetus accumulates Fe at the expense of its mother. How this is reflected in fetal liver metabolism of Fe is part of the subject of this paper. Additionally, we do not yet know whether there is an optimum or preferable time for supplementation. In this paper, therefore, we examine the effect of different time periods of supplementation on growth and development of the offspring.

Methods

Experimental diets

The experimental diets were based on a dried egg albumin diet and conformed to American Institute of Nutrition guidelines for laboratory animals (Williams & Mills, 1970). Iron sulphate was added to achieve levels of added Fe of 50 (control diet), 7.5 (Fe-deficient diet) or 75 mg kg−1 (Fe-supplemented diet). Dietary ingredients were purchased from Mayjex Ltd (Chalfont-St Peter, UK), BDH Chemicals (Poole, UK) or Sigma (Poole, UK).

Experimental animals

Experiment was performed using 40 weanling female rats of the Rowett Hooded Lister strain. Animals were housed in cages under constant conditions (temperature 22°C; humidity 55%; 12 h:12 h light:dark illumination photoperiod). All the animals were fed control diet (50 mg Fe (kg diet)−1) for 2 weeks, before being randomized into two groups. The first group, eight animals, remained on the control diet throughout the experiment, including during pregnancy, whilst the remaining 32 animals were placed on the Fe-deficient diet (7.5 mg Fe (kg diet)−1) for 4 weeks before mating. All the rats were mated with males of the same strain. Mating was confirmed by detection of a vaginal plug, and this day was denoted as day 0.5. At 0.5, 7.5 and 14.5 days gestation, separate groups of eight rats were taken from the deficient diet and given the supplemented diet until parturition. The remaining eight rats continued on the Fe-deficient diet. The pups were killed within 12 h of birth by decapitation. The dams were killed by stunning and cervical cord dislocation. All experimental procedures were approved and conducted in accordance with the UK Animals (Scientific Procedures) Act 1986.

Sample collection

The number of neonates (male and female) was counted. Maternal and neonatal blood samples were collected. Maternal livers as well as neonatal livers, hearts, lungs, kidneys and spleens, taken from six neonates (3 male and 3 female), chosen from each mother at random, were rapidly dissected, weighed and frozen in liquid nitrogen before being stored at –70°C.

Haematological measurements

Maternal and neonatal haematocrit were measured by drawing blood into capillary tubes, which were then centrifuged in a high-speed haematocrit centrifuge (Universal 32R, Hettich; Scientific Laboratory Supplies, Coatbridge, UK) and read in a microhaematocrit reader.

Atomic absorption spectrophotometric analyses

For determination of total Fe content in tissues, the heat-dried liver samples were treated with nitric acid (Ultrapure; Merck, Poole, UK). The total Fe content in these samples was determined by graphite furnace atomic spectrophotometry (Aanalyst 600, Perkin Elmer, Beaconsfield, UK). Standards and quality controls were included as appropriate.

Primers for real-time RT-PCR

Complementary DNA PCR primers for the TfR, DMT1 IRE and non-IRE forms, ferritin H and ferritin L were designed using Primer Express software (version 1.5, Applied Biosystems, Warrington, UK) from the DNA sequences GenBank Accession numbers M58040, AF008439, AF029757, NM_012848 and XM_216824, respectively. The primers were as follows: TfR forward primer 1757–1779 bp, reverse primer 1818–1838 bp; DMT1 IRE forward primer 2168–2188 bp, reverse primer 2228–2247 bp; DMT1 non-IRE form forward primer 1650–1673 bp, reverse primer 1700–1722 bp; ferritin H forward primer 585–606 bp, reverse primer 632–656 bp; and ferritin L forward primer 140–156 bp and reverse primer 14–35 bp. The primer sets had a calculated annealing temperature of 58°C. Primers were ordered from MWG Biotech, Munich, Germany.

DNase treatment

Total RNA was prepared by use of TRI reagent (Helena Biosciences, Sunderland, UK) according to the manufacturer's instructions. To eliminate genomic DNA contamination total RNA samples were treated with ribonuclease (RNase)-free Dnase 1 Amplification Grade (Invitrogen Ltd, Paisley, UK), before complementary DNA synthesis. Ten micrograms of total RNA was added to a total volume of 20 μl containing 2 μl 10 × reaction buffer and 1 μl Dnase 1. The total RNA was incubated for 10 min at 25°C, followed by precipitation of the RNA by adding 5 μl 3 m sodium acetate pH 4.8, 55 μl isopropanol and 30 μl diethyl pyrocarbonate (DEPC)-treated water. RNA was precipitated at −20°C for at least 1 h, then centrifuged, washed in 75% ethanol and the pellet dissolved in DEPC-treated water. RNA concentrations were estimated by Agilent analysis (Agilent 2100 Bioanalyser, Agilent Technologies, Stockport, UK).

Reverse transcription

First strand complementary DNA was synthesized by priming with hexamers using the Taqman RT Reagent Kit (Applied Biosystems, Stockport, UK). Reverse transcription was performed in 20 μl reactions using 200 ng of Dnase-treated RNA. Reverse transcription was performed by addition of 12.3 μl RT mix, such that the final concentration was 1 × buffer, 5.5 mm MgCl2, 2 mm dNTP, 2.5 μm hexamer primer, 8 U RNase inhibitor and 25 U Multiscribe™ RT. The mixture was incubated at 25°C for 10 min, incubated at 48°C for 30 min and heated to 95°C for 5 min, and then finally cooled to 4°C. To determine the presence of contaminating cDNA, reverse transcription was omitted in the cDNA synthesis reaction. A specific product was never detected (data not shown).

Real-time quantitative PCR

Real-time PCR amplification and analysis was performed using a 7700 Sequence Detection System (Applied Biosystems) and ABI prism software version 1.9 (Applied Biosystems). Reactions were performed in 25 μl final volume with 300 nm primers and 5 μl cDNA. Magnesium chloride, nucleotides, buffer and Taq DNA polymerase were included in the SYBR Green Master Mix (Applied Biosystems). The PCR amplification was performed according to the manufacturer's instructions and included heating to 50°C for 5 min and denaturation at 95°C for 10 min, followed by 40 cycles with 95°C for 15 s and 60°C for 1 min. To confirm amplification specificity the PCR products from each primer pair were subjected to gel electrophoreses (2.5% Agarose-1000, Invitrogen, with ethidium bromide). For all primer pairs only one product of correct size was detected (data not shown).

Standard curves were generated from increasing amounts of cDNA made from maternal liver control RNA. The cycle threshold (CT) values were used to calculate and plot a linear regression line by plotting the logarithm of template concentration (x axis) against the CT value (y axis). These regression lines were used to calculate the expression level (nanograms of total RNA) for unknown samples.

Statistical analyses

For each dam, the data from the the pups were averaged and recorded as a single point. One-way ANOVA was used to determine statistical significance between multiple data sets and verified by the least significant difference (LSD) test. Linear regression test was used to determine statistical significance between continuous variables. Significance was assumed at P = 0.05. All analyses were carried out using Statistica 5.0 (Statsoft, Polska, Krakow, Poland). The results are presented as means ± s.e.m.

The change in expression in mRNA levels between the control group and the treated groups is presented as a percentage of the control, with the average of the controls set to 100%. Each group contained amplified cDNAs from three plates, and the average of these measurements was used to calculate group mean and s.e.m. Significant differences (P < 0.05) between treated and control groups were determined using Student's unpaired t test, two tailed (GraphPad Instat, version 3.05).

Results

The duration of Fe deficiency had no effect on maternal growth, or the number of neonates. In contrast, neonatal weight was altered by the treatment (Table 1). Those animals born to deficient mothers were significantly smaller, with proportionately smaller livers, lungs and spleens, but with larger hearts (P < 0.05). The offspring of mothers supplemented at different times were intermediate between control and deficient offspring. Interestingly, however, the lungs of those animals whose mothers were supplemented were significantly larger than either control or deficient offspring (Table 1).

Table 1.

Effect of duration of maternal Fe deficiency on maternal and neonatal growth

Control Deficient Supp.day 0.5 Supp.day 7.5 Supp.day 14.5
Maternal weight (g) 263±6a 258±7a 259±5a 258±5a 263±7a
Total no. of neonates 12.9±1.0a 13.1±0.9a 14.4±0.6a 13.3±0.7a 13.6±1.0a
Neonatal body weight (g) 5.40±0.13a 4.92±0.12b 5.32±0.16ab 5.47±0.17a 5.02±0.21ab
Neonatal liver (ROW; g (100 g)−1) 4.72± 0.08a 3.82± 0.16b 4.20± 0.14b 4.34± 0.2ab 4.25± 0.26ab
Neonatal heart (ROW; g (100 g)−1) 0.46± 0.02a 0.55± 0.04b 0.50± 0.02ab 0.48± 0.02ab 0.54± 0.03b
Neonatal lung (ROW; g (100 g)−1) 1.59± 0.05 1.42± 0.03 2.00± 0.06a 2.08± 0.03a 2.12± 0.11a
Neonatal kidney (ROW; g (100 g)−1) 1.08± 0.01a 1.02± 0.04 1.09± 0.03a 1.07± 0.04a 1.20± 0.12a
Neonatal spleen (ROW; g (100 g)−1) 0.22± 0.01a 0.19± 0.05a 0.24± 0.03a 0.23± 0.02a 0.36± 0.20a
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All results are presented as means ± s.e.m. Days indicate the time in gestation at which supplementation was started. Relative organ weights (ROW) were calculated as a percentage of total body weight (g (100 g)−1). Results sharing the same superscript are not significantly different.

Maternal haematocrit was decreased (P < 0.05) in dams fed the Fe-restricted diet to the end of experiment compared to the control group (Fig. 1A). Similar data were obtained for the haemoglobin levels in the maternal blood, except that the levels were also significantly decreased in those animals given supplements only in the last third of pregnancy (Fig. 1B). In the neonates, haematocrit was restored to control levels in all groups given Fe supplements (Fig. 2). Haemoglobin levels could not be recorded.

Figure 1. The effect of maternal Fe deficiency on maternal haematocrit (A) and haemoglobin (B).

Figure 1

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Animals were treated as described in the Methods and the results presented are the means ± s.e.m. of at least 6 animals per group. For dietary details see Methods section. *P < 0.05; **P < 0.01 when compared to the control value by one-way analysis of variance.

Figure 2. The effect of maternal Fe deficiency on haematocrit of neonatal rats.

Figure 2

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The results are the means ± s.e.m. of pups from at least 6 mothers per group. *P < 0.05; **P < 0.01 when compared to the control value by one-way analysis of variance.

These responses were mirrored in the measurements of Fe levels. In the maternal liver, Fe did not recover to control levels unless the supplementation was given for at least the last 2 weeks of gestation (Fig. 3A). In the neonate, liver Fe was at control levels in all supplemented animals, irrespective of the length of the supplementation period (Fig. 3B).

Figure 3. The effect of maternal Fe deficiency on liver Fe levels in the dam (A) and her neonates (B).

Figure 3

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The results are the means ± s.e.m. of at least 6 dams or the pups from 6 dams per group. *P < 0.05; **P < 0.01 when compared to the control value by one-way analysis of variance.

Examination of the genes involved in Fe metabolism in maternal and fetal liver also revealed these patterns. Figure 4A shows TfR levels in maternal liver and Fig. 4B shows levels in the liver of the neonates. Messenger RNA levels are significantly elevated in the mother in Fe-deficient animals and those supplemented for 1 week, decreasing to control levels only in those supplemented for 2 weeks and throughout pregnancy (Fig. 4A). In the neonates, levels are never significantly different from controls (Fig. 4B).

Figure 4. The effect of maternal Fe deficiency during pregnancy on TfR mRNA levels in maternal (A) and neonatal livers (B).

Figure 4

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Samples were taken and mRNA prepared, reverse transcribed and measured, as described in the Methods. The results are the means ± s.e.m. of livers from at least 6 dams and 6 neonates, each taken from a different litter. *P < 0.05; **P < 0.01 when compared to the control value by Student's unpaired t test.

Expression of the two DMT1 transcripts, IRE regulated and non-IRE regulated, showed different patterns in the maternal liver. The IRE-regulated transcript increased with increasing period of deficiency, while the non-IRE-regulated form did not change significantly (Fig. 5A). Different results were seen in the neonatal liver. The non-IRE-regulated form decreased as the period of deficiency increased, while the mRNA levels of the IRE-regulated form increased (Fig. 5B).

Figure 5. The effect of maternal Fe deficiency during pregnancy on DMT1 mRNA levels in maternal (A) and neonatal livers (B).

Figure 5

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Both the non-IRE-regulated (□) and IRE-regulated forms (▪) were measured. Samples were taken and mRNA prepared, reverse transcribed and measured, as described in the Methods. The results are the means ± s.e.m. of livers from at least 6 dams and 6 offspring, each taken from a different litter. *P < 0.05; **P < 0.01 when compared to the control value by Student's unpaired t test.

We also measured levels of mRNA for H and L ferritin. In the mothers, there were no significant changes the levels in of either (Fig. 6A). In the neonates, levels of L ferritin mRNA did not change significantly, but H ferritin mRNA decreased with increasing times of deficiency (Fig. 6B).

Figure 6. The effect of maternal Fe deficiency during pregnancy on ferritin mRNA levels in maternal (A) and neonatal livers (B).

Figure 6

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Both L (□) and H ferritin (▪) were measured. Samples were taken and mRNA prepared, reverse transcribed and measured, as described in the Methods. The results are the means ± s.e.m. of livers from at least 6 dams and 6 offspring, each taken from a different litter. *P < 0.05; **P < 0.01 when compared to the control value by Student's unpaired t test.

Figure 7 examines the correlation between Fe levels in maternal and neonatal livers, and neonate birth-weight. Interestingly, a correlation is seen between maternal liver Fe levels and birth-weight (P < 0.0015, Fig. 7A) but not neonatal liver Fe and birth-weight (Fig. 7B).

Figure 7. Maternal and not neonatal liver Fe levels are related to neonatal birthweight.

Figure 7

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A, the liver Fe level of each mother is related to mean litter weight. Statistical analysis was carried out by linear regression. The dotted lines represent the 95% confidence limits. The slope of the line is significantly different from 0 and the data are correlated at P = 0.0015. B, mean litter liver Fe levels were measured and data correlated to mean litter weight as shown. The slope of the line is not significantly different from 0.

Discussion

The aim of this study was to determine the effect of the timing of moderate Fe supplementation of Fe-deficient rats during pregnancy on maternal growth, on pregnancy outcome and on Fe status in both mothers and their offspring. The data presented in this paper show that the severity of Fe deficiency had no effect on maternal growth and number of live neonates. This is in agreement with our previous study (Gambling et al. 2002) showing that maternal Fe deficiency does not affect viability and the number of fetuses.

The results of the present study also confirm our previous findings (Gambling et al. 2002) that neonatal body weight decreases with the severity of maternal Fe deficiency. Fetal growth restriction resulting from severe Fe deficiency has also been demonstrated by Crowe et al. (1995) in rats and Malhotra et al. (2002) in humans.

As might be expected, supplementation reverses the effect. The timing is important, however. Our data show that supplementation in the last third of pregnancy is not as effective as it is when given earlier. Although it is difficult to extrapolate directly from rats to humans, given the differences in timing of developmental milestones, the data support results published in human studies. For example, Hamalainen et al. (2003) demonstrated that maternal anaemia detected in the first trimester was associated with low-birth-weight infants, whereas the mid- and third-trimester anaemia groups did not show significantly different outcomes when compared with non-anaemic women. Additionally, Cogswell et al. (2003) have presented data suggesting that Fe supplementation, even in women of normal Fe status, during the first 28 weeks of pregnancy, but not during the latter half, results in a very significant increase in birth weight.

Even though the rat model has limitations, it also provides us with the possibility of identifying which organs are most susceptible to Fe deficiency. For example, it would seem, from our data, that pre- and early postimplantation embryos are most sensitive to maternal nutritional status. These developmental events occur proportionately much earlier in human pregnancy. Consequently, further studies are needed to identify the developmental windows, rather than the chronological windows, where Fe deficiency exerts its effects.

The distribution of Fe following supplementation is intriguing. The fact that fetal Fe levels recover before that of the mother indicates that Fe is preferentially diverted to the fetus as soon as it becomes available. The mechanism for this can be deduced from this and from our previous studies. Expression of the proteins of Fe transfer is significantly increased in the placenta during Fe deficiency (Gambling et al. 2002, 2003). The degree of increase is significantly greater than that seen in the maternal liver. Clearly, since the present study was carried out on neonates, we have no information on the degree of changes in TfR expression in the placenta. However, assuming they follow the same pattern as previously described, the Fe taken up in the supplemented animals will have been preferentially delivered to the fetus. This is substantiated because there is no significant difference in neonatal expression of the TfR in the liver. In the mother, in contrast, Fe levels did not returned to normal even following 2 weeks of supplementation.

In one sense, the results are not surprising. Iron is critical for rapidly developing fetal and neonatal organ systems. Iron is prioritized to haemoglobin synthesis in red blood cells when Fe supply does not meet Fe demand. Therefore, non-haem-containing tissues such as skeletal muscle, heart and brain will become Fe deficient before signs of Fe deficiency anaemia (Rao & Georgieff, 2002).

The effects on the two forms of DMT1 are also interesting. There are data to suggest that regulation of DMT1 by Fe is altered at different stages in gestation. Lonnerdal and colleagues have shown that there is an apparent developmental regulation of DMT1 in rats (Leong et al. 2003), with greater sensitivity in the gut occurring at postnatal day 20 rather than day 10. In one sense, this is not surprising, since the duodenum is not exposed to the Fe-deficient environment directly until birth. In contrast, as we have previously shown (Gambling et al. 2001) and confirmed in the present study and in Lonnerdal's paper (Leong et al. 2003), liver DMT1 levels are increased in Fe deficiency in the pre- and early postnatal period.

The effect on the non-IRE-regulated DMT1 has not previously been reported. The function of this transcript is not clear, and at present we have no obvious explanation. Ke et al. (2003) have suggested that the two isoforms both respond to Fe deficiency in the heart, while Tchernitchko et al. (2002) suggest that there are other promoters and/or regulators involved in induction of the different isoforms. Clearly, there is much yet to be elucidated.

The effect on ferritin mRNAs was also intriguing. There was no significant change in the maternal liver, or in L ferritin in the neonate, while the H ferritin level significantly decreased both in neonates born from Fe-deficient mothers and in those born from mothers supplemented for 1 week only. H and L ferritin are independently regulated proteins with both transcriptional and translational regulation in response to cellular Fe level. Han et al. (2000) studied the H:L ferritin ratio and mRNA levels in various regions of the brain of male Fe-deficient, control and Fe-supplemented rats. They found that ferritin H and L subunits within the brain respond differently to Fe status. On the basis of experiments with synthetic ferritin heteropolymers, Levi et al. (1994) concluded that the ferritins with high L:H chain ratios are the most efficient in incorporating Fe (Levenson & Fitch, 2000). The data we have obtained in this study would seem to support that observation.

What is most surprising is that there is no correlation between neonatal Fe levels and neonatal size. Instead, the relationship appears to between maternal Fe and the neonate. This suggests that the mother rather than factors deriving from the fetus determines size at birth. There are other data to support these observations. In sheep, Wallace et al. (2002) have shown that placental growth is regulated by maternal nutrition and this, in turn, regulates fetal growth. Various hormone treatments, such as parathyroid hormone-related protein in rats (Wlodek et al. 2004), dexamethasone in sheep (Kerzner et al. 2002) or leptin in sheep (Thomas et al. 2001) have all been shown to alter fetal growth (see Reynolds et al. 2004 for a comprehensive review). At this stage, we are not certain which hormone mediates the effect in anaemic rats, but it is clear that the data may have considerable relevance in understanding the relationship between maternal anaemia and pregnancy outcome in humans.

In summary, therefore, our data show that the developing fetus is very sensitive to maternal Fe status, and that the period of supplementation is critical in reversing the effects of maternal anaemia. Anaemia during pregnancy is endemic in many parts of the world and is a significant concern even in developed countries. It is well established that supplementation is of limited value outside of well-controlled experimental studies. Our data may provide an explanation for this, in that if it is given at an inappropriate time, it will be less effective at reversing the consequences of maternal anaemia. Certainly the data would suggest that early supplementation will be more efficacious than during the last trimester. The mechanisms underlying these observations clearly remain to be determined, but a better understanding will be very valuable in developing the appropriate strategies for treatment of mothers suffering from anaemia during pregnancy.

Acknowledgments

This work was supported by the European Union (QLK1-1999-00337), The International Copper Association and Scottish Executive Environmental and Rural Affairs Department. We are grateful for the invaluable assistance of the staff of the Biological Resources Unit and Lynne Beattie for technical assistance, and to Ann White for proofreading and secretarial assistance.

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