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ORIGINAL RESEARCH COMMUNICATION |
1 From the Departments of Nutrition (W-IL and BL) and Internal Medicine (CLB and BL), University of California, Davis, and the Department of Pharmacology and Toxicology, Uppsala Biomedical Center, Uppsala, Sweden (JT).
2 Supported by intramural research grants. 3 Reprints not available. Address correspondence to B Lönnerdal, Department of Nutrition, University of California, One Shields Avenue, Davis, CA 95616. E-mail: bllonnerdal{at}ucdavis.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Objective: The objective was to investigate at a molecular level the regulation of iron absorption during infancy in a rat pup model. We examined the developmental expression of DMT1 and FPN1 and the effects of iron supplementation on their expression and on iron absorption and utilization during infancy.
Design: Rat pups were given daily oral doses of 0, 30, or 150 µg Fe from day 2 to day 20 after birth. On days 10 and 20 after birth, 59Fe absorption, tissue minerals, and intestinal DMT1, FPN1, and ferritin expression were examined. To assess developmental expression, DMT1 and FPN1 were examined in control rats from days 1 to 50 after birth.
Results: Intestinal DMT1 and FPN1 were significantly affected by age; expression increased dramatically by day 40. On day 10, no significant effect of iron supplementation on DMT1 and FPN1 gene expression or on iron absorption was observed. By day 20, DMT1 and FPN1 expression and iron absorption had decreased significantly with iron supplementation.
Conclusions: During early infancy, rat pups are unable to down-regulate intestinal iron transporters or iron absorption in response to iron supplementation, whereas down-regulation occurs during late infancy. The current findings provide evidence of the developmental regulation of iron absorption, which emphasizes the need for caution when giving iron supplements to infants at an early age.
Key Words: Rat pups • infants • divalent metal transporter 1 • DMT1 • ferroportin 1 • FPN1 • iron transporters • ferritin • iron supplementation • iron absorption
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although these iron intakes are expected to prevent iron deficiency, little is known about the effects of such intakes on the mucosal regulation of iron absorption in infants. In adults, iron homeostasis is maintained primarily by regulating intestinal iron absorption. However, this regulatory process may not be fully developed in infants. The results of a stable-isotope study of human infants at 6 and 9 mo of age strongly suggest developmental changes in the regulation of iron absorption (4). At 6 mo of age, iron absorption from human milk was not different between infants given daily iron supplementation and control infants. In contrast, at 9 mo of age, iron absorption in the iron-supplemented infants was significantly lower than that in the control group. In addition, hemoglobin responses to iron supplementation differed before and after 6 mo of age (5). At 6 mo of age, hemoglobin increased in response to supplementation, regardless of initial iron status. Thus, iron was absorbed and used for hemoglobin synthesis, apparently without any feedback mechanism, regardless of the fact that the infants were iron sufficient. By 9 mo of age, there was no significant effect of iron supplementation on hemoglobin in infants who were iron replete at 6 mo, but a significant increase was observed in infants who had low hemoglobin concentrations at 6 mo. These findings suggest a developmental change in the regulation of iron metabolism. However, little is known about the molecular mechanisms that regulate iron absorption during infancy.
Our knowledge of the mechanisms that regulate iron absorption has recently advanced dramatically. Two iron transporters, divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1), are critical for intestinal iron absorption and are regulated by body iron stores. DMT1 is a transmembrane protein that transports ferrous iron across the apical membrane of intestinal epithelial cells (6–10), whereas FPN1 is an iron exporter located on the basolateral membrane (11–13). To date, all studies that have investigated the molecular mechanisms of the regulation of iron absorption have been conducted either in cell culture or in adult humans or animals. Little is known about the gene and protein expression of DMT1 and FPN1 or their role in the regulation of iron absorption during infancy. On the basis of these observations in human infants, we believe that the regulation of the molecular mechanisms of iron absorption during early life might differ from that during adulthood. The purpose of this study, therefore, was to investigate at a molecular level the regulation of iron absorption during development in a rat pup model. We examined the developmental gene expression of DMT1 and FPN1 and the effects of iron supplementation on iron absorption and utilization and on the expression of DMT1, FPN1, and ferritin.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hemoglobin
Whole-blood hemoglobin was analyzed after conversion to cyanomethemoglobin with the use of a commercially available kit (Sigma, St Louis). Blood samples (20 µL) were mixed with 5 mL Drabkin’s solution (0.1% sodium bicarbonate, 0.005% potassium cyanide, and 0.02% potassium ferricyanide) for hemoglobin measurement.
RNA extraction
Rat pups were deprived of food for 2 h (day 1 rat pups), 4 h (day 10 rat pups), 6 h (day 20 and day 30 rat pups), or 8 h (day 40 and day 50 rat pups) and killed by carbon dioxide asphyxiation. Duodenum and liver from the rat pups were dissected and stored in RNAlater (Ambion, Austin, TX) and kept at -20 °C until extraction. Tissues (0.1 g/L) were then homogenized in TRIzol reagent (Life Technologies, Rockville, MD), and total RNA was extracted according to the TRIzol protocol.
Real-time quantitative reverse transcriptase–polymerase chain reaction
The relative expression levels of DMT1 and FPN1 were determined by real-time quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) with TaqMan EZ RT-PCR Core Reagents (Applied Biosystems, Foster City, CA). Primers and probes were designed by using PRIMER EXPRESS software (Applied Biosystems) to span introns to avoid coamplification of genomic DNA and were purchased from Applied Biosystems. For quantification of DMT1 complementary DNA (cDNA), the following primers were used: 5-GTT TGT CAT GGA GGG ATT CCT-3 and 5-CAT TCA TCC CTG TCA GAT GCT-3, which recognize both the iron-responsive element (IRE) and the non-IRE forms. For quantification of FPN1 cDNA, the following primers were used: 5-GTG CCT CCC AGA TCG CAG-3 and 5-GGG CTG GTT ATA GTA GGA GAC CC-3. The probes for DMT1 (5-AAA ATG GTC GCG CTT TGC CCG A-3) and FPN1 (5-ACC CTT CCG CAC TTT TCG AGA TGG A-3) were 5-labeled with 6-carboxyfluorescein and 3-labeled with 6-carboxytetramethylrhodamine.
The RT-PCR reactions were carried out according to the manufacturer’s protocol on an ABI Prism 7700 Sequence Detector (Applied Biosystems). RT-PCR conditions were 30 min at 60 °C and then 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Data were analyzed with Sequence Detector 1.7 software. All reactions were performed in duplicate, and the change in fluorescence value and the threshold cycle number were calculated from fluorescence activity data collected during PCR. A no-template control was included in every reaction. The housekeeping gene 18 S ribosomal RNA was used for internal normalization.
Membrane and soluble protein preparation
Frozen duodenum (300 mg) samples were homogenized in 5 mL Hepes-EDTA buffer [20 mmol Hepes/L, pH 7.4; 1 mmol EDTA/L; 250 mmol sucrose/L; a protease inhibitor mixture containing 4-(2-aminoethyl)benzenesulfonyl fluoride, trans-epoxysuccinyl-L-leucylsmido(4-guanidino)butane, bestatin, leupeptin, aprotinin, and sodium EDTA (Sigma)]. The homogenate was centrifuged at 500 x g for 5 min at 4 °C. The supernatant fluid was then centrifuged at 100 000 x g for 30 min at 4 °C. The supernatant fluid (soluble protein) was collected for ferritin protein determination, and the crude membrane fraction (pellet) was resuspended in 0.3 mL homogenization buffer for DMT1 and FPN1 protein determination. Samples were stored at -80 °C until analyzed. Protein concentrations of the soluble fractions and the membrane fractions were quantified by the method of Lowry et al (14).
Production of DMT1 and FPN1 antibodies
Peptide fragments of DMT1 (VKPSQSQVLKGMFV) and of FPN1 (KQLTSPKDTEPKPLEGTH) were synthesized with an additional cysteine residue for conjugation to keyhole limpet hemocyanin at the carboxy-terminal end (Genemed Synthesis Inc, South San Francisco). Sequences were verified by amino acid analysis and mass spectroscopy. Keyhole limpet hemocyanin–conjugated peptides were injected into New Zealand White rabbits (1 mg peptide/rabbit) for polyclonal antibody production. DMT1 peptide was synthesized according to the predicted amino acid sequence obtained from the human DMT1 cDNA corresponding to amino acids 235–248 of the protein in the presumed fourth external loop between putative transmembrane regions 5 and 6. Because this region is common to both the IRE and non-IRE forms of DMT1, it will react with both types of DMT1. The amino acid sequence of this region is highly conserved in mammals with little sequence homology to Nramp1. This peptide is in an extracellular region of the protein away from the glycosylation sites and has only 6 amino acids in common with human Nramp1. The FPN1 peptide was synthesized according to the predicted amino acid sequence obtained from the rat FPN1 cDNA corresponding to amino acids 253–270 of the protein in the external loop between putative transmembrane regions 5 and 6. Comparison of both peptide sequences with public sequences databases with the use of BLAST (Basic Local Alignment Search Tool) identified only DMT1 and FPN1 sequences.
Antibody specificities were verified by peptide competition analysis. Briefly, membrane proteins from the intestine were resolved and transferred as described below. After blocking, the blots were incubated with primary antibodies in the presence (100 µg) or absence of the corresponding peptides for 1 h. The blots were visualized by using enhanced chemiluminescence after incubation with secondary antibody (Figure 1
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7 and sodium azide was added.
Western blotting
DMT1, FPN1, and ferritin protein expression in the duodenum of the animals were measured by using Western blots. Protein fractions or the soluble protein fractions (100 µg) in duodenum membrane were solubilized in Laemmli buffer, boiled for 5 min, and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Similar loading and transfer of proteins was verified by staining the blots with Ponceau S (Sigma). Proteins were transferred to nitrocellulose membranes by electroblotting, which was then blocked with 5% nonfat powdered milk in PBS with Tween 20 (PBST) at room temperature for 2 h and at 4 °C overnight. The membranes were then washed with several changes of PBST and incubated for 1 h at room temperature in either affinity-purified DMT1 (1:1000), affinity-purified FPN1 (1:750), or mouse anti-human ferritin antibody (1:2500; Alpha Diagnostic, San Antonio, TX) in PBST. The membranes were washed again with several changes of PBST and then incubated with donkey anti-rabbit IgG, peroxidase-linked species-specific whole antibody (Amersham Biosciences UK Ltd, Little Chalfonte, United Kingdom) for DMT1 and FPN1 or peroxidase-conjugated rabbit anti-mouse IgG (Dako, Copenhagen) for ferritin for 1 h at room temperature. Membranes were washed again with several changes of PBST. The immunologically detected proteins were visualized by using enhanced chemiluminescence (Amersham). Processed blots were exposed to X-ray film for the optimum exposure time and quantified by using the Chemi-doc Gel Quantification System (Bio-Rad).
Preparation of iron-absorption test solutions
The iron-absorption test dose contained 5 µg Fe as ferrous sulfate/mL PBS, with 2.68 mmol ascorbic acid/L. This concentration of iron is similar to the iron concentration of rat milk during days 10–20 of lactation (15). 59Fe as FeCl3 (Amersham) was then added to the test dose to provide 200,000 cpm/mL. This solution was prepared immediately before use and was prewarmed before intubation.
Iron absorption
Animals were fasted for 4 h (day 10) or 6 h (day 20) and intubated with 0.5 mL of the test dose directly into the stomach by using an animal feeding needle. Animals were killed by carbon dioxide asphyxiation 4–5 h after intubation. The small intestines were then perfused with 2–3 mL saline to remove unabsorbed 59Fe from the lumen. Radioactivity in the brain, kidney, liver, small intestine, spleen, carcass, and perfusate were counted in a 
-counter (Gamma 8500;Beckman, Irvine, CA). The results were expressed as a percentage of the total radioactivity received:F
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Tissue mineral analysis
Brain, kidney, liver, small intestine, and spleen were dissected and wet ashed in 16 mol HNO3/L for 7 d (16). Samples were then diluted with appropriate amounts of distilled water and analyzed for iron, zinc, copper, and manganese by atomic absorption spectrophotometry (model Smith-Heifjie 4000; Thermo Jarrell Ash, Franklin, MA).
Statistical analysis
Values are expressed as means ± SDs. Comparisons among the groups were tested by one-factor analysis of variance (ANOVA) with PRISM GRAPHPAD (version 3.02; GraphPad Software, San Diego). When the P value obtained from ANOVA was significant, differences between groups were assessed with Tukey’s test. Significance was considered to be P < 0.05. When the variances within the group differed significantly according to Bartlett’s test, the data were log transformed before being tested by one-factor ANOVA. A two-factor repeated-measures ANOVA was used to assess the interactions between the dietary treatment groups (control, medium-Fe, and high-Fe groups) and time (days 10 and 20) on body iron uptake, mucosal iron retention, and unabsorbed iron. Because the interactions were significant, subgroup analysis was done by using Student’s unpaired t test to compare data.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). On day 10, the mean hemoglobin concentration in the high-Fe group was significantly greater than that in the other 2 groups, whereas both of the 2 iron-supplemented groups had significantly higher hemoglobin concentrations than did the control group on day 20. By day 10, iron supplementation had resulted in significantly greater mucosal iron retention and decreased body iron uptake, but total iron absorption (total - perfusate) had not changed with iron supplementation (Table 1). Compared with the control and medium-Fe groups, only the high-Fe group had significantly higher mucosal iron retention and significantly lower body iron uptake by day 20, but total iron absorption was significantly lower in both iron-supplemented groups (Table 1). Mucosal iron retention was significantly lower in each group on day 20 than it was on day 10 (Table 1). Conversely, body iron uptake was significantly greater in each group on day 20 than it was on day 10.
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). On day 20, only the high-Fe group had significantly greater iron concentrations in the small intestine than did the control and the medium-Fe groups. Liver iron concentrations were significantly greater in the iron-supplemented groups than in the control group. Also shown in Table 2 are the tissue concentrations of other minerals.
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). Although there was a 7–9-fold increase in DMT1 gene expression from day 1 to the period between days 10 and 30, there was no significant difference in expression on days 10, 20, and 30. By day 40, there was a significant increase in DMT1 gene expression, which was 116 times greater than that on day 1. For intestinal FPN1, similar but more modest changes were seen during development. The values for relative intestinal gene expression on days 1, 10, 20, 30, 40, and 50 were 1, 7, 9, 7, 116, and 35 for DMT1 and were 1, 7, 9, 5, 19, and 11 for FPN1, respectively.
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). In contrast, expression of DMT1 in the liver on day 10 had significantly decreased by 70% in the high-Fe group and by 52% in the medium-Fe group, whereas no significant differences were noted on day 20 (Figure 4).
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). There were no significant differences in liver FPN1 expression between the groups on either day 10 or day 20 (Figure 4).
Effect of iron supplementation on intestinal DMT1, FPN1, and ferritin protein concentrations
DMT1 and FPN1 protein concentrations were measured to determine whether changes in gene expression corresponded with changes in protein concentrations. On day 10, the DMT1 protein concentration was unchanged with iron supplementation, but it decreased by 50% on day 20 with iron supplementation (Figure 1). The change in DMT1 protein concentration paralleled the change in DMT1 gene expression. FPN1 protein concentration was not affected by iron supplementation on days 10 or 20. The 68-kDa FPN1 protein observed on day 20 was not detectable on day 10, whereas the 130-kDa band observed on day 10 was present in smaller amounts on day 20 (Figure 1). The ratio of messenger RNA to protein was calculated for both DMT1 and FPN1 with regard to dietary iron and development; the values did not support a posttranscriptional mechanism. After 10 d of supplementation, ferritin protein concentrations were 2-fold and 4.3-fold higher in the medium-Fe and high-Fe groups than in the control group. After 20 d of supplementation, ferritin protein concentrations were 2.8-fold and 4.2-fold higher in the medium-Fe and high-Fe groups, respectively (Figure 1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Furthermore, we found that the iron supplementation of rat pups leads to an increase in hemoglobin as occurs in human infants. Finally, we showed that rat pups are unable to regulate iron absorption on day 10 but have the capacity to down-regulate iron absorption in response to supplementation on day 20. Together these findings support the use of this model to study mechanisms of iron absorption and regulation during development.
We showed that both DMT1 and FPN1 are expressed in the duodenum of 1-d-old rat pups and that they are developmentally regulated. Expression of both genes increased dramatically on day 40 after birth. This change is far greater than the changes observed for dietary iron supplementation. What induces this dramatic increase is not clear, but it is not likely to be caused by low iron status because iron status improved by day 40 (Table 2). The pronounced increase in the expression of the transporters on day 40 may be regulated by mechanisms that have been observed in other developmentally regulated intestinal genes such as sucrase-isomaltase (17–19), lactase (20), and adenosine deaminase (21, 22). The response of intestinal DMT1 and FPN1 to dietary iron is also developmentally dependent. DMT1 and FPN1 gene expression significantly decreased with iron supplementation by day 20 of birth. This was expected because high iron stores are thought to decrease intestinal iron absorption by decreasing the expression of intestinal iron transporters (23). Our findings agree with those of others who have shown DMT1 mRNA to be lower in iron-loaded weanling rats (9) and in iron-supplemented Caco-2 cells (24) than in their respective unsupplemented controls. However, we found that intestinal DMT1 and FPN1 were not affected by dietary iron supplementation on day 10 after birth, even though the intestinal iron concentration was strongly affected by dietary iron. This difference between days 10 and 20 indicates a developmental change in the regulation of iron absorption, which may explain the different responses to iron supplementation in human infants at 6 mo compared with 9 mo of age. Because iron supplementation is commonly recommended during infancy, it may be prudent not to supplement infants with too much iron at a young age if the intestinal iron transporters are not down-regulated with high dietary iron or iron supplementation.
Unlike intestinal DMT1, liver DMT1 gene expression by in situ hybridization is reported to be unaffected by dietary iron in adult rats (9). During late infancy (day 20), we also found that liver DMT1 gene expression is not changed by iron supplementation. However, we found that liver DMT1 decreased after dietary iron supplementation on day 10. It is possible that the liver plays an important role in regulating iron metabolism during early infancy because, at that age, intestinal iron transporters do not respond well to iron status or dietary iron.
In summary, our results show that in early infancy, rat pups are unable to down-regulate intestinal iron transporters and iron absorption in response to iron supplementation. Unregulated iron absorption results in iron accumulation in many tissues and may result in increased oxidative damage during important developmental stages. Iron supplementation of human infants, in whom iron deficiency anemia is uncommon, has been shown to have potentially harmful effects, particularly if started before 6 mo of age (25) because length gain was lower and diarrhea was more common in supplemented than in unsupplemented infants. Neonatal iron exposure has also been shown to result in neurobehavioral dysfunction in mice (26). Regulation of intestinal iron absorption does not occur until late infancy. At that time, infant rats are able to down-regulate intestinal iron transporters and intestinal iron absorption. The regulatory mechanisms involved in the developmental expression and regulation of these transporters should be identified in future studies. However, the current findings provide evidence of the developmental regulation of iron absorption, which supports the recommendation that careful consideration be given to the potential negative effect of iron supplementation during early infancy.
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ACKNOWLEDGMENTS |
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REFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and in rats supplemented with 30 µg Fe (medium-Fe group, 
) or 150 µg Fe (high-Fe group, •) on days 2–20. The horizontal lines represent the mean, and each point represents an individual rat pup. Groups with different lowercase letters are significantly different (one-factor ANOVA, Tukey’s test): P = 0.001 (DMT1) and P = 0.007 (FPN1); the comparison was within the same time point only. n = 10 ± 1 per group.