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Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Zinc Supplementation Reduces Iron Absorption through Age-Dependent Changes in Small Intestine Iron Transporter Expression in Suckling Rat Pups¹

Department of Nutritional Biology, University of California, Davis, CA 95616

² To whom correspondence should be addressed. E-mail: slkelleher{at}ucdavis.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Zinc (Zn) supplementation negatively affects iron (Fe) absorption; however, the molecular mechanisms are not understood. We determined effects of Zn supplementation during mid- and late infancy on intestinal Fe transport mechanisms using a suckling rat model. Suckled rat pups were supplemented with 0 (control), 300 (low), or 750 (high) µg Zn/d until weaning at postnatal day (PN) 20. At mid- (PN10) and late (PN20) infancy, tissue Fe distribution, Fe absorption, intestine DMT1, ferroportin-1 (FPN) and hephaestin expression, and localization and liver hepcidin expression were measured. During early infancy, DMT1 and FPN were localized intracellularly. Negative effects of Zn supplementation on Fe absorption were associated with increased small intestine Fe retention, decreased hephaestin, and increased FPN expression. During late infancy, both DMT1 and FPN were appropriately localized to the apical and basolateral membrane, respectively, and negative effects of Zn supplementation on Fe absorption were absent. Although FPN protein level was lower in Zn-supplemented pups, hephaestin protein level was increased, which may have facilitated enhanced Fe efflux. These results indicate that Zn supplementation reduced Fe absorption during early infancy as a consequence of increased intestinal Fe retention due to reduced hephaestin levels. These effects were age-dependent, further demonstrating that Fe transport regulation is not fully developed until weaning, which may have important implications regarding the safety and efficacy of Zn supplementation programs for infants.

KEY WORDS: • zinc • iron transporters • DMT1 • ferroportin-1 • hephaestin

Zinc requirements are relatively high during infancy due to increased nutritional demand during rapid growth and development. Zinc deficiency in infants is primarily associated with decreased growth, neurological and cognitive dysfunction, and impaired immune function (1,2). Although healthy breast-fed infants consume adequate amounts of Zn through mid-infancy (3), it has been proposed that infants born from women in Zn-deficient populations may be marginally Zn deficient and thus would benefit from Zn supplementation (4). Interestingly, the success of Zn supplementation programs has been mixed (5–7), possibly due to the heterogeneity of Zn status within the supplemented population (as Zn status is very difficult to assess) but also to negative effects of elevated Zn intake on the metabolism of other trace minerals, such as iron (Fe).

The body maintains Fe homeostasis principally by inversely regulating Fe absorption relative to liver Fe stores, which is controlled primarily through systemic regulation of basolateral Fe efflux mediated by ferroportin (FPN)³, in coordination with the oxidation of Fe⁺² to Fe⁺³ by hephaestin, a copper-requiring, basolateral membrane-associated ferroxidase homologous to ceruloplasmin (8). Recently, the communication link between liver Fe stores and intestine Fe absorption has been proposed to be the liver-derived peptide hepcidin, which is supported by the observations that liver hepcidin expression is increased during Fe overload (9,10) and that hepcidin binds to FPN to stimulate its internalization and degradation in mature enterocytes, thereby decreasing Fe efflux (11). Conversely, at the apical membrane, divalent metal transporter-1 (DMT1) imports ferrous Fe into the enterocyte in a proton-coupled manner (12), and its expression is reciprocally regulated by cellular Fe levels in animal models and human enterocytes (13–15) through alterations in iron-regulating protein (IRP) binding to the 3' iron-responsive elements (IRE) (16). The combinatorial control of apical Fe uptake and basolateral Fe efflux finely regulates Fe absorption in adults; however, we have previously determined that this system is under ontogenic control (17) and is not capable of adjusting to Fe deficiency until late infancy (18).

Adverse effects of Zn supplementation on Fe status have been demonstrated, such as decreased Fe absorption, hemoglobin, and serum ferritin levels in adults (19–22). Although the mechanisms through which these effects are mediated are not understood, Zn treatment increased DMT1 and FPN expression in enterocyte-like Caco-2 cells (23), suggesting specific effects of Zn supplementation on intestine Fe transporters. Although no effect of Zn supplementation on Fe status in infants >6 mo from developing countries has been observed (24), we have previously determined that young infant rhesus monkeys supplemented with Zn (7.5 mg/d) from birth to 4 mo had reduced Fe absorption and impaired Fe status (25); we speculate that this age effect is due to the ontogeny of the Fe transport machinery in the small intestine (17). Therefore, in this study, we hypothesized that Zn supplementation during infancy alters intestine Fe transporter expression and functionally decreases Fe absorption. Whereas we have previously documented ontogenic development of the intestine Fe transport system, we further hypothesize that these effects occur in an age-dependent manner. Thus, our objective was to determine effects of Zn supplementation on ⁵⁹Fe absorption and tissue retention, tissue Fe distribution, DMT1, FPN, and hephaestin expression in the small intestine, and hepcidin expression in the liver during mid- and late infancy using a suckling rat pup model.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals. This study was approved by the Animal Research Services at the University of California, Davis, which is accredited by the American Association for the Accreditation of Laboratory Animal Care. Female virgin Sprague Dawley rats (n = 12) were obtained commercially (Charles River) and housed individually in suspended polycarbonate cages containing wood shavings. They were maintained at a 12 h light/dark cycle with controlled temperature and humidity (20°C, humidity above 60%). Rats were fed commercial rat chow (LabDiet) and deionized water ad libitum throughout the study (10 µg Cu/g, 77 µg Zn/g, and 160 µg Fe/g). The rats were bred and on postnatal day (PN) 2, pups were culled to 14–15 pups per litter and randomized (n = 56 pups/group). Pups were orally supplemented daily with sucrose (25 µL of 10% sucrose, control) containing 300 µg (low Zn) or 750 µg Zn as ZnSO₄ (high Zn) until mid- (PN10) or late (PN20) infancy. The choice of supplemental Zn concentration was based on the following assumptions: breast-fed human infants consume 1 mg Zn/d and are routinely supplemented with 10 mg Zn/d, resulting in 10-fold the normal level. Suckling rat pups consume 50 µg Zn/d and so were supplemented with 10 times that, 500 µg Zn/d. We chose an amount below (300 µg) and above (750 µg) this level to account for intake and body weight differences as a consequence of age. All pups were food deprived for 6 h prior to being killed.

    Effects of Zn supplementation on tissue Fe retention. To determine effects of Zn supplementation on Fe absorption and tissue retention, pups were given an oral dose of ⁵⁹Fe. On PN10 and PN20 pups (n = 12 pups · group^–1 · d^–1) were food-deprived for 6 h, gavaged with 0.5 mL of a solution containing 2.5 µg Fe as FeSO₄, L-ascorbic acid (30-fold molar excess), and 0.1 µCi ⁵⁹FeSO₄ (Amersham Pharmacia Biotech) and killed by CO₂ asphyxiation 6 h later. Small intestine was dissected and perfused with 3 mL of ice-cold 0.9% NaCl. The small intestine, perfusate, liver, spleen, kidneys, and brain were removed, and tissue and carcass radioactivity was measured in a -counter.

    Effects of Zn supplementation on tissue Fe distribution, small intestine Fe transporter, and liver hepcidin expression. To determine effects of Zn supplementation on tissue Fe distribution, pups (n = 8 pups · group^–1 · d^–1) were anesthetized by CO₂ inhalation and blood was collected by cardiac puncture into heparinized tubes on PN10 and PN20. Whole blood hemoglobin level was measured using a commercially available kit (Sigma). Pups were decapitated and tissues (femur, brain, liver, muscle, small intestine, and spleen) were removed and frozen at –20°C until mineral analysis. Samples of small intestine and liver were collected from the remaining pups (n = 8 pups · group^–1 · d^–1) and snap frozen in liquid nitrogen or immediately homogenized in 3 mL TriZOL (Life Technologies) and stored at –80°C prior to protein and RNA extraction, respectively.

    Tissue Fe, Zn, and Cu analysis. Tissue (0.3 g) was wet-ashed in concentrated nitric acid. Fe, Zn, and Cu concentrations were determined by atomic absorption spectrophotometry as previously described (26).

    Real time relative RT-PCR. To determine effects of Zn supplementation on intestine Fe transporters and liver hepcidin mRNA levels, total RNA was isolated from homogenized small intestine and liver following the manufacturer's instructions (Life Technologies). RNA integrity was assessed following electrophoresis through agarose (2%) followed by ethidium bromide staining. Relative expression of DMT1, FPN, hephaestin, and hepcidin mRNA was determined by real time relative RT-PCR, using the SYBR Green detection system (Perkin Elmer Applied Biosystems). Gene specific primers were designed using Primer Express software (Perkin Elmer Applied Biosystems) to span exons in order to avoid coamplification of genomic DNA (Qiagen). Primer pairs used were as follows: DMT1, 5'-TTT GTC ATG GAG GGA TTC CT-3' and 5'-CAT TCA TCC CTG TCA GAT GCT-3'; FPN, 5'-GTG CCT CCC AGA TCG CAG-3' and 5'-GGG CTG GTT ATA GTA GGA GAC CC-3'; hephaestin, 5'-TGT CAT CCT GAT CCA CCT GAA G-3' and 5'-AAC ACC GTG AGG GTG AAT GG-3'; hepcidin, 5'-TGC GCT GCT GAT GCT GAA-3' and 5'-AGC ATT TAC AGC AGA AGA GGC AT-3'; GAPDH, 5'-TGC CAA GTA TGA TGA CAT CAA CAA G-3' and 5'-AGC CCA GGA TGC CCT TTA GT-3'). cDNA was generated from 1 µg RNA using a reverse transcription kit (Perkin Elmer Applied Biosystems) following the manufacturer's instruction, and the reaction was performed at 48°C for 30 min followed by 95°C for 5 min. Real time RT-PCR was performed on 4 µL of the cDNA reaction mixture using the ABI 7900 HT real-time thermocycler (Perkin Elmer Applied Biosystems) at the following cycling parameters: 50°C for 2 min; 95°C for 10 min; 40 cycles of 95°C for 15 s; 60°C for 1 min; 95°C for 15 s; 60°C for 15 s; and 95°C for 15 s. The linearity of the dissociation curve was analyzed using the ABI 7900 HT software and the mean cycle time of the linear part of the curve was designated Ct. Each sample was analyzed in duplicate and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the following equation: Ct_GENE = Ct_GENE – Ct_GAPDH. The fold change, relative to control rat pups, was calculated using the following equation: 2^(CtGENE) where Ct_GENE = mean Ct_GENE of the control rats – Ct_GENE of each Zn-supplemented rat. Values are mean fold change ± SD.

    Western blot analysis. To determine effects of Zn supplementation on the relative protein level of intestine Fe transporters, small intestine (0.5 g) was homogenized in Hepes-EDTA buffer containing protease inhibitors, as previously described (27). The homogenate was centrifuged at 5000 g for 10 min at 4°C. The protein concentration of the postnuclear supernatant was determined using the Bradford Assay (Bio-Rad). An equal amount of total protein (100–200 µg) was electrophoresed by SDS-PAGE (4–20%, Bio-Rad) under reducing conditions and transferred to nitrocellulose for 1 h at 350 mA. Membranes were blocked for 1 h in 5% nonfat milk in PBS/0.1% Tween-20 (PBS-T) and washed 3 times in PBS-T, followed by incubation with primary antibody (1:1000) for 45 min, and then washed 3 times in PBS-T. DMT1 and FPN antisera were previously characterized by Leong et al. (15) and production of hephaestin antibody is described below. Proteins were detected following incubation with donkey-anti-rabbit IgG conjugated to horseradish peroxidase (Amersham Pharmacia Biotech), visualized with Super Signal Femto Chemiluminescent Detection System (Pierce), and exposed to autoradiography film. Relative band density was quantified using the Chemi-doc Gel Quantification System (Bio-Rad).

    Generation and characterization of hephaestin antibody. Peptides generated from the predicted protein sequence of hephaestin (IDVHTAFFHGQTLSIRGHRTDVAHI) were synthesized with an additional cysteine residue for conjugation to keyhole limpet hemocyanin at the C-terminal end (Genemed Synthesis). Sequences were verified by amino acid analysis and mass spectroscopy and keyhole limpet hemocyanin–conjugated peptides were injected into New Zealand white rabbits (1 mg peptide/rabbit) for polyclonal antibody production. Antibody specificity was documented by antibody blocking experiments as follows: hephaestin antiserum was diluted 1:1000 in PBS-T or preabsorbed with hephaestin peptide (1 mg) for 2 h prior to being incubated with nitrocellulose membranes of electrophoresed small intestine protein (200 µg), as described above. Illustration of nonspecific bands was conducted with preimmune serum diluted 1:1000.

    Localization of DMT1 and FPN in the small intestine. To determine whether effects of Zn supplementation are associated with alterations in DMT1 and FPN localization, small intestine (duodenum) was perfused and fixed in 4% phosphate-buffered paraformaldehyde, pH 7.4 at 4°C for 24 h, washed extensively with PBS, and sequentially dehydrated in ethanol as previously described (27). Paraffin-embedded intestine was sectioned (4 µm) and immunostained with affinity purified antibody (2 mg/L) for 1 h at room temperature (17). The localization of DMT1 and FPN was detected with 3,3'-diaminobenzidine tetrahydrochloride following incubation with anti-rabbit IgG conjugated to horseradish peroxidase (Vector Labs) and counterstained with hematoxylin.

    Data analysis. Data are expressed as means ± SD. Tissue Zn and Fe concentrations, ⁵⁹Fe retention, and hemoglobin concentrations were analyzed by 2-way ANOVA to test for interaction between age and Zn supplementation. If a significant interaction was determined, 1-way ANOVA was performed to determine the significant effect of Zn supplementation at each age and was posttested using a Tukey test. Bartlett's test for equal variances was performed, and, if significance was demonstrated (P < 0.05), data were logarithmically transformed to equalize the variances prior to analysis by 1-way ANOVA. Messenger RNA and protein levels were analyzed by 1-way ANOVA at each age. Statistical analysis was conducted using GraphPad Prism 3.02 and significance was demonstrated at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Zinc supplementation affects Fe absorption and tissue Fe distribution. Rat pups supplemented with high Zn had a lower (P < 0.05) hemoglobin concentration (115 ± 22 g/L,) than control pups (140 ± 28 g/L) and pups supplemented with low Zn (131 ± 21 g/L) at PN10. The groups did not differ at PN20 (control, 84 ± 11; low Zn, 83 ± 7; and high Zn, 79 ± 9 g/L) and the group x age interaction was not significant. Pups supplemented with high Zn had significantly lower whole body ⁵⁹Fe retention than control pups or pups supplemented with low Zn at PN10, although this difference was not observed at PN20 (Table 1). In addition, pups supplemented with Zn (low or high) had significantly higher small intestine ⁵⁹Fe retention than control pups at PN10, although no significant difference was observed at PN20. These results indicate that Zn supplementation decreases Fe absorption by increasing Fe retention in the small intestine and this occurs in an age-dependent manner.

    Age but not Zn supplementation affects DMT1 and FPN localization in the small intestine. In the small intestine, DMT1 and FPN stained throughout the entire enterocyte with little plasma membrane association during mid-infancy at PN10 (Fig. 1A, B). However, during late infancy at PN20, the staining pattern was remarkably different in that DMT1 was appropriately localized to the apical membrane and FPN to the basolateral membrane (Fig. 1C, D). There was no significant effect of Zn supplementation on DMT1 or FPN localization (data not shown).

View larger version (120K): [in this window] [in a new window]   FIGURE 1  The localization of DMT1 and FPN in the small intestine at PN10 and PN20 in unsupplemented suckling rat pups. DMT1 (A) and FPN (B) stain within the enterocyte at PN10. However, at PN20, DMT1 (C) and FPN (D) are appropriately localized to the apical and basolateral membrane, respectively. Arrows indicate brown staining of DMT1 or FPN; magnification 40x under oil.

View larger version (37K): [in this window] [in a new window]   FIGURE 2  Characterization of specificity of hephaestin antibody by Western analysis in rat pup small intestine at PN20. Representative Western blot of small intestine protein (100 µg) resolved by SDS-PAGE under reducing conditions and incubated with preimmune serum (Pre-I, 1:1000) or antiserum (1:1000). Preabsorption with excess antigen (+peptide) blocked detection of the hephaestin specific band corresponding to a protein at 160 kDa.

View larger version (19K): [in this window] [in a new window]   FIGURE 3  Effects of zinc supplementation on DMT1, FPN, and hephaestin (Heph) mRNA levels in the small intestine and hepcidin mRNA expression in the liver at PN10 (A) and PN20 (B) in suckling rat pups. Values are fold-changes relative to the control. Data were normalized to GAPDH levels. Values are means ± SD, n = 8. Means with different letters differ, P < 0.05.

View larger version (30K): [in this window] [in a new window]   FIGURE 4  Effects of zinc supplementation on DMT1, FPN, and hephaestin (Heph) protein levels in the small intestine atPN10 (A, B) and PN20 (C, D) in suckling rat pups. Representative Western blot (A and C) of small intestine protein isolated from three individual rats at each Zn dose. Each Western blot was conducted 3 separate times to include analysis of all individual rats. Values are protein expressions relative to controls. Values are means ± SD, n = 8 (B, D). Means with different letters differ, P < 0.05; ND, not detected.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
A number of studies indicate that high Zn intake negatively affects Fe absorption and Fe metabolism in adults (28,29). Fortunately, significant negative effects of Zn supplementation on indices of Fe metabolism in infants, specifically from Zn deficient populations, have not been detected thus far (30–32). However, similar to our observations in healthy infant rhesus monkeys (25), Heinig and colleagues (M. J Heinig, K. H. Brown, B. Lonnerdal, and K. G. Dewey, unpublished data) observed lower Fe status in healthy Zn-supplemented breast-fed infants (17.8 µg ferritin/L) in the United States compared with infants receiving placebo (42.7 µg ferritin/L), which suggests that routine Zn supplementation programs in Zn-adequate populations should be approached with caution. Although Zn supplementation is usually not initiated until 4–6 mo, infants born prematurely or with low birth weight may receive early Zn supplementation and, to our knowledge, the consequences on Fe metabolism have not been investigated. Fundamental to this concern is that the regulation of Fe absorption may not be fully developed until weaning (18) due to ontogenic regulation of Fe transport machinery (15).

Results from this study indicate that age has an effect on the interference of Zn supplementation on Fe absorption. Our data indicate that decreased Fe absorption during mid-infancy is actually a consequence of increased Fe retention in the small intestine, facilitated through reduced basolateral Fe efflux and enterocytic Fe trapping. Interestingly, by the time of weaning, this effect is resolved, potentially as a result of the "maturation" of Fe absorptive mechanisms (17). For example, DMT1 and FPN are not localized to the apical and basolateral membrane, respectively, until weaning and are instead found primarily in intracellular compartments, similar to their localization in the liver (33). Although this may be considered "immature localization," the possibility exists that this reflects homeostatic control in response to high neonatal Fe stores via DMT1 and FPN internalization (34,35) in the enterocyte.

Enterocytic Fe export is mediated by FPN coordinated with Fe⁺² oxidation to Fe⁺³ via hephaestin, a basolateral membrane–associated multicopper ferroxidase (36). The dependence upon hephaestin for Fe efflux from the enterocyte into circulation for delivery to apo-transferrin is illustrated by the anemic phenotype of sex-linked anemia mice that have a mutant form of hephaestin resulting in enterocytic Fe efflux defects (8). During mid-infancy, Zn supplementation decreased hephaestin protein abundance in a dose-dependent and posttranscriptional manner, providing a mechanistic explanation behind reduced basolateral Fe efflux. Recent studies have determined that hephaestin protein stability is sensitively regulated through polyubiquitination and proteosomal degradation (37), a process that can be specifically induced by Zn exposure at nontoxic concentrations (38). Additionally, secondary effects of Zn supplementation on Cu metabolism have been well documented and our data clearly indicates that Zn supplementation in infant rat pups reduced intestine Cu concentration. Although the precise mechanisms responsible for this observation are currently unknown, studies examining the effects of Zn supplementation on Cu absorption and intestine Cu transporters during infancy are currently underway. Nevertheless, our data suggest that secondary effects of Zn supplementation on Cu metabolism may interfere with hephaestin translation, as decreased hephaestin protein abundance paralleled small intestine Cu concentration, thus providing in vivo evidence that reduced Cu availability for hephaestin incorporation may result in conformational changes that target the apo-hephaestin protein for increased turnover as described by Nittis and Gitlin (37). Thus, the reduction of hephaestin protein abundance, in combination with inability of hephaestin protein to interact at the basolateral membrane due to the intracellular nature of FPN (39,40) during mid-infancy, likely reduces Fe absorption at this age.

During mid-infancy, FPN protein, but not mRNA level, was higher in Zn-supplemented pups, suggesting that Zn supplementation may directly increase FPN mRNA or protein stability. Hepcidin has recently been shown to stimulate FPN internalization and degradation, suggesting that increased FPN levels may result from reduced hepcidin levels in circulation (41). However, we determined that Zn supplementation increased liver hepcidin expression, leading us to speculate that Zn supplementation interferes with hepcidin translation or secretion into circulation; however, this remains to be confirmed empirically. In contrast to the systemic regulation of FPN via hepcidin, DMT1 is inversely regulated through changes in enterocyte Fe levels, as high cellular Fe maintains the 4Fe-4S structure of IRP, preventing IRE binding to a 3' IRE (42) and thereby decreasing DMT1 mRNA levels (43). This suggests that during mid-infancy, when enterocyte Fe is elevated in response to Zn supplementation, DMT1 expression should be decreased. However, the fact that it is not inversely related to intestine Fe concentration, similar to observations in the heart and brain by Ke et al. (44), but is positively associated with intestine Zn concentration, suggests that Zn may play a direct role in regulating DMT1 expression, which is similar to observations in Caco-2 cells (23). Although there is currently no evidence that IRP/IRE binding is sensitive to changes in cellular Zn level, the promoter region of DMT1 contains several metal response elements (43), suggesting that Zn exposure can positively regulate DMT1 mRNA level via metal transcription factor-1 activation (45,46) or perhaps through other Zn-dependent transcription factors, such as peroxisome proliferator-activated receptor-, nuclear factor-B, and activator protein-1 (47).

By the time of weaning, we observed that the majority of effects caused by Zn supplementation had resolved. For example, there was no longer a significant effect of Zn supplementation on Fe absorption, which was likely a result of increased Fe efflux due to increased hephaestin levels in Zn-supplemented pups. Once again, changes in hephaestin protein abundance did not parallel changes in mRNA levels, illustrating the dependence upon Cu incorporation into apo-hephaestin for protein stabilization. Additionally, FPN abundance was decreased, which may have resulted from increased circulating hepcidin levels. However, liver hepcidin expression was decreased by Zn supplementation at this age, again suggesting a potential interaction between Zn supplementation and hepcidin translation or secretion into circulation. Ontogenic control of intestine Fe transport (17) clearly helps to explain the age-specific effects of Zn supplementation on intestine Fe transport during infancy. Although our results indicate that early Zn supplementation has a negative effect on Fe absorption through specific changes in the intestine Fe transport machinery, by late infancy this effect is abrogated, suggesting that Zn supplementation in young infants should be approached with caution.

	ACKNOWLEDGMENTS

 
The authors gratefully acknowledge the excellent technical expertise of Melanie Hupe, Mohammad Bakhtiar Hossain, and Maggie Chiu.

	FOOTNOTES

 
¹ Supported in part by NIH DK 35747 and faculty research grants to S.L.K. and B.L.

³ Abbreviations used: DMT1, divalent metal transporter-1; FPN, ferroportin-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IRE, iron-responsive elements; IRP, iron-regulating proteins; PN, postnatal day.

Manuscript received 12 September 2005. Initial review completed 31 October 2005. Revision accepted 9 February 2006.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Shah D, Sachdev HP. Effect of gestational zinc deficiency on pregnancy outcomes: summary of observation studies and zinc supplementation trials. Br J Nutr. 2001;85: Suppl 2:S101–8.[Medline]

2. Shah D, Sachdev HP. Maternal micronutrients and fetal outcome. Indian J Pediatr. 2004;71:985–90.[Medline]

3. Krebs N, Reidinger CJ, Miller LV, Hambige KM. Zinc homeostasis in breast-fed infants. Pediatr Res. 1996;39:661–5.[Medline]

4. Dijkhuizen MA, Wieringa FT, West CE, Muherdiyantiningsih, Muhilal. Concurrent micronutrient deficiencies in lactating mothers and their infants in Indonesia. Am J Clin Nutr. 2001;73:786–91.[Abstract/Free Full Text]

5. Hamadani JD, Fuchs GJ, Osendarp SJ, Khatun F, Huda SN, Grantham-McGregor SM. Randomized controlled trial of the effect of zinc supplementation on the mental development of Bangladeshi infants. Am J Clin Nutr. 2001;74:381–6.[Abstract/Free Full Text]

6. Sazawal S, Black RE, Menon VP, Dinghra P, Caulfield LE, Dhingra U, Bagati A. Zinc supplementation in infants born small for gestational age reduces mortality: a prospective, randomized, controlled trial. Pediatrics. 2001;108:1280–6.[Abstract/Free Full Text]

7. Bhutta ZA, Black RE, Brown KH, Gardner JM, Gore S, Hidayat A, Khatun F, Matorell R, Nihn NX, et al. Prevention of diarrhea and pneumonia by zinc supplementation in children in developing countries: pooled analysis of randomized controlled trials. Zinc Investigators' Collaborative Group. J Pediatr. 1999;135:689–97.[Medline]

8. Vulpe CD, Kuo YM, Murphy TL, Cowley L, Askwith C, Libina N, Gitschier J, Anderson GJ. Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse. Nat Genet. 1999;21:195–9.[Medline]

9. Frazer DM, Wilkins SJ, Becker EM, Vulpe CD, McKie AT, Trinder D, Anderson GJ. Hepcidin expression inversely correlates with the expression of duodenal iron transporters and iron absorption in rats. Gastroenterology. 2002;123:835–44.[Medline]

10. Yeh KY, Yeh M, Glass J. Hepcidin regulation of ferroportin 1 expression in the liver and intestine of the rat. Am J Physiol Gastrointest Liver Physiol. 2004;286:G385–94.[Abstract/Free Full Text]

11. Leong W-I, Lönnerdal B. Hepcidin, the recently identified peptide that appears to regulate iron absorption. J Nutr. 2004;134:1–4.[Abstract/Free Full Text]

12. Gunshin H, Mackenzie B, Berger UV. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature. 1997;388:482–7.[Medline]

13. Tallkvist J, Bowlus CL, Lönnerdal B. Functional and molecular responses of human intestinal Caco-2 cells to iron treatment. Am J Clin Nutr. 2000;72:770–5.[Abstract/Free Full Text]

14. Tallkvist J, Bowlus CL, Lönnerdal B. DMT1 gene expression and cadmium absorption in human absorptive enterocytes. Toxicol Lett. 2001;122:171–7.[Medline]

15. Leong W-I, Bowlus CL, Tallkvist J, Lönnerdal B. Iron supplementation during infancy-effects on expression of iron transporters, iron absorption and iron utilization in rat pups. Am J Clin Nutr. 2003;78:1203–11.[Abstract/Free Full Text]

16. Zoller H, Theurl I, Koch R, Kaser A, Weiss G. Mechanisms of iron mediated regulation of the duodenal iron transporters divalent metal transporter 1 and ferroportin 1. Blood Cells Mol Dis. 2002;29:488–97.[Medline]

17. Leong W-I, Bowlus CL, Tallkvist J, Lönnerdal B. DMT1 and FPN1 expression during infancy: developmental regulation of iron absorption. Am J Physiol Gastrointest Liver Physiol. 2003;285:G1153–61.[Abstract/Free Full Text]

18. Domellöf M, Lönnerdal B, Abrams AA, Hernell O. Iron-absorption in breast-fed infants: effects of age, iron status, iron supplements and complementary foods. Am J Clin Nutr. 2002;76:198–204.[Abstract/Free Full Text]

19. Donangelo CM, Woodhouse LR, King SM, Viteri FE, King JC. Supplemental zinc lowers measures of iron status in young women with low iron reserves. J Nutr. 2002;132:1860–4.[Abstract/Free Full Text]

20. Herman S, Griffin IJ, Suwarti S, Ernawati F, Permaesih D, Pambudi D, Abrams SA. Co-fortification of iron-fortified flour with zinc sulfate, but not zinc oxide, decreases iron absorption in Indonesian children. Am J Clin Nutr. 2002;76:813–7.[Abstract/Free Full Text]

21. Lind T, Lönnerdal B, Stenlund H, Ismail D, Seswandhana R, Ekström E-C, Persson L-Å. A community-based randomized controlled trial of iron and zinc supplementation in Indonesian infants: interactions between iron and zinc. Am J Clin Nutr. 2003;77:883–90.[Abstract/Free Full Text]

22. Yadrick MK, Kenney MA, Winterfeldt EA. Iron, copper, and zinc status: response to supplementation with zinc or zinc and iron in adult females. Am J Clin Nutr. 1989;49:145–50.[Abstract/Free Full Text]

23. Yamaji S, Tennant J, Tandy S, Williams M, Srai SKS, Sharp P. Zinc regulates the function and expression of the iron transporters DMT1 and IREG1 in human intestinal Caco-2 cells. FEBS Lett. 2001;507:137–41.[Medline]

24. Fischer Walker C, Kordas K, Stoltzfus RJ, Black RE. Interactive effects of iron and zinc on biochemical and functional outcomes in supplementation trials. Am J Clin Nutr. 2005;82:5–12.[Abstract/Free Full Text]

25. Kelleher SL, Casas I, Carbajal N, Lönnerdal B. Supplementation of infant formula with the probiotic Lactobacillus reuteri and zinc: impact on enteric infection and nutrition in infant rhesus monkeys. J Pediatr Gastroenterol Nutr. 2002;35:162–8.[Medline]

26. Clegg MS, Keen CL, Lönnerdal B, Hurley LS. Influence of ashing techniques on the concentration of trace elements in animals tissues. I: Wet ashing. Biol Trace Elem Res. 1981;3:107–15.

27. Kelleher SL, Lönnerdal B. Zn transporter levels and localization change throughout lactation in rat mammary gland and are regulated by Zn in mammary cells. J Nutr. 2003;133:3378–85.[Abstract/Free Full Text]

28. Rossander-Hulten L, Brune M, Sandström B, Lönnerdal B, Hallberg L. Competitive inhibition of iron absorption by manganese and zinc in humans. Am J Clin Nutr. 1991;54:152–6.[Abstract/Free Full Text]

29. Crofton RW, Gvozdanovic D, Gvozdanovic S, Khin CC, Brunt PW, Mowat NA, Aggett PJ. Inorganic zinc and the intestinal absorption of ferrous iron. Am J Clin Nutr. 1989;50:141–4.[Abstract/Free Full Text]

30. Dijkhuizen MA, Wieringa FT, West CE, Martuti S, Muhilal. Effects of iron and zinc supplementation in Indonesian infants on micronutrient status and growth. J Nutr. 2001;131:2860–5.[Abstract/Free Full Text]

31. Baqui AH, Zaman K, Persson LÅ, Arifeen SE, Yunus M, Begum N, Black RE. Simultaneous weekly supplementation of iron and zinc is associated with lower morbidity due to diarrhea and acute lower respiratory infection in Bangladeshi infants. J Nutr. 2003;133:4150–7.[Abstract/Free Full Text]

32. Lind T, Lönnerdal B, Stenlund H, Ismail D, Seswandhana R, Ekström E-C, Persson L-Å. A community-based randomized controlled trial of iron and zinc supplementation in Indonesian infants: interactions between iron and zinc. Am J Clin Nutr. 2003;77:883–90.[Abstract/Free Full Text]

33. Trinder D, Oates PS, Thomas C, Sadleir J, Morgan EH. Localization of divalent metal transporter 1 (DMT1) to the microvillus membrane of rat duodenal enterocytes in iron deficiency, but to hepatocytes in iron overload. Gut. 2000;46:270–6.[Abstract/Free Full Text]

34. Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, Ganz T, Kaplan J. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science. 2004;306:2090–3.[Abstract/Free Full Text]

35. Yeh KY, Yeh M, Watkins A, Rodriguez-Paris J, Glass J. Dietary iron induces rapid changes in rat intestinal divalent metal transporter expression. Am J Physiol Gastrointest Liver Physiol. 2000;279:G1070–9.[Abstract/Free Full Text]

36. Kuo YM, Su T, Chen H, Attieh Z, Syed BA, McKie AT, Anderson GJ, Gitschier J, Vulpe CD. Mislocalisation of hephaestin, a multicopper ferroxidase involved in basolateral intestinal iron transport, in the sex linked anaemia mouse. Gut. 2004;53:201–6.[Abstract/Free Full Text]

37. Nittis T, Gitlin JD. Role of copper in the proteosome-mediated degradation of the multicopper oxidase hephaestin. J Biol Chem. 2004;279:25696–702.[Abstract/Free Full Text]

38. Wu W, Wang X, Zhang W, Reed W, Samet JM, Whang YE, Ghio AJ. Zinc-induced PTEN protein degradation through the proteasome pathway in human airway epithelial cells. J Biol Chem. 2003;278:28258–63.[Abstract/Free Full Text]

39. Chen H, Su T, Attieh ZK, Fox TC, McKie AT, Anderson GJ, Vulpe CD. Systemic regulation of Hephaestin and Ireg1 revealed in studies of genetic and nutritional iron deficiency. Blood. 2003;102:1893–9.[Abstract/Free Full Text]

40. Frazer DM, Anderson GJ. The orchestration of body iron intake: how and where do enterocytes receive their cues? Blood Cells Mol Dis. 2003;30:288–97.[Medline]

41. Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, Ganz T, Kaplan J. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science. 2004;306:2090–3.[Abstract/Free Full Text]

42. Haile D, Rouault T, Harford J, Kennedy M, Blondin G, Beinert H, Klausner R. Cellular regulation of the iron-responsive element binding protein: disassembly of the cubane iron-sulfur cluster results in high-affinity RNA binding. Proc Natl Acad Sci USA. 1992;89:11735–9.[Abstract/Free Full Text]

43. Lee PL, Gelbart T, West C, Halloran C, Beutler E. The human Nramp2 gene: characterization of the gene structure, alternative splicing, promoter region and polymorphisms. Blood Cells Mol Dis. 1998;24:199–215.[Medline]

44. Ke Y, Chen YY, Chang YZ, Duan XL, Ho KP, Jiang H, Wang K, Qian ZM. Post-transcriptional expression of DMT1 in the heart of rat. J Cell Physiol. 2003;196:124–30.[Medline]

45. Andrews GK, Kage K, Palmiter-Thomas P, Sarras MP. Metal ions induce expression of metallothionein in pancreatic exocrine and endocrine cells. Pancreas. 1990;5:548–54.[Medline]

46. Andrews GK. Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem Pharmacol. 2000;59:95–104.[Medline]

47. Meerarani P, Reiterer G, Toborek M, Hennig B. Zinc modulates PPAR signaling and activation of porcine endothelial cells. J Nutr. 2003;133:3058–64.[Abstract/Free Full Text]

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