QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hicks, P. D. Articles by Lönnerdal, B. Search for Related Content PubMed PubMed Citation Articles by Hicks, P. D. Articles by Lönnerdal, B.

---

Community and International Nutrition

Iron Deficiency, but Not Anemia, Upregulates Iron Absorption in Breast-Fed Peruvian Infants

¹ University of California at Davis, Department of Nutrition, Davis, CA 95616; ² Instituto de Investigación Nutricional, Lima, Peru 18-0191; ³ U.S. Department of Agriculture/Agricultural Research Service, Children's Nutrition Research Center, Department of Pediatrics, Texas Children's Hospital, Baylor College of Medicine, Houston, TX 77030

^* To whom correspondence should be addressed. E-mail: pennih{at}bcm.edu.

	ABSTRACT

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
Iron absorption in adults is regulated by homeostatic mechanisms that decrease absorption when iron status is high. There are few data, however, regarding the existence of a similar homeostatic regulation in infants. We studied 2 groups of human milk-fed infants using ⁵⁷Fe (given as ferrous sulfate without any milk) and ⁵⁸Fe (given at the time of a breast-milk feeding) stable isotopes to determine whether healthy infants at risk for iron deficiency would regulate their iron absorption based on their iron status. We studied 20 Peruvian infants at 5–6 mo of age and 18 infants at 9–10 mo of age. We found no effect of infant hemoglobin concentration on iron absorption with 5–6 mo–old infants absorbing 19.2 ± 2.1% and 9- to 10-mo–old infants absorbing 25.8 ± 2.6% of the ⁵⁷Fe dose. For ⁵⁸Fe, 5- to 6-mo–old infants absorbed 42.6 ± 5.0% and 9 to 10-mo–old infants absorbed 51.9 ± 10.3%. Following log transformation, iron absorption from ⁵⁷Fe (r = –0.61, P = < 0.001) and ⁵⁸Fe (r = –0.61, P = < 0.001) were inversely correlated to serum ferritin (S-Ft). For both the ⁵⁷Fe and ⁵⁸Fe doses, infants with S-Ft <12 mg/L (n = 11) had significantly higher iron absorption than those with S-Ft >12 mg/L. We concluded that iron absorption in infants is related to iron status as assessed by serum ferritin but not hemoglobin concentration. Infants with low iron status upregulate iron absorption from breast milk at both 5–6 and 9–10 mo of age.

	Introduction

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

As iron deficiency is of varying prevalence in infants (3), we chose to evaluate iron absorption in a population where iron status is less likely to be adequate. According to a national survey conducted in 2000, Peru has a high prevalence of anemic infants. The survey reported a 59% incidence of anemia among infants at 6–9 mo of age and 72% among 10- to 12-mo–old infants. This can be explained in part by the effect maternal iron status has on the delivery of fetal iron (4) and by the low iron intake of the infants (5). The aim of the present study was to evaluate whether 2 groups of healthy term, breast-fed infants, ages 5–6 and 9–10 mo, at high risk for iron deficiency can attempt to compensate for their poor iron status by increasing their iron absorption.

	Subjects and Methods

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
Subjects

Two groups of infants were recruited from Villa El Salvador, a low-income peri-urban area of Lima, Peru. Recruitment of subjects was done by clinic staff from the local population. One group consisted of 5- to 6-mo–old infants (150–195 d) and the other of infants at 9–10 mo of age (270–315 d). All infants were healthy, full-term (37 wk gestation), singleton infants, had a birth weight >2500 g, and were being breast-fed. The 5–6 mo–old infants (n = 20) were exclusively breast-fed. The 9- to 10-mo–old infants (n = 18) were breast-fed and allowed age-appropriate complementary foods at the mother's discretion. None of these infants were being supplemented with iron.

The Investigational Review Board of the Baylor College of Medicine and Affiliated Hospitals and the Ethics Committee at the Instituto de Investigación Nutricional Lima, Peru approved the protocol. Informed written consent was obtained from the families prior to enrollment.

Five to 6 mo–old infants were recruited based on a screening of their hemoglobin values. We targeted an enrollment of 10 infants with a hemoglobin <105 g/L and 10 with a hemoglobin 105 g/L. Similarly, 9- to 10-mo–old infants were recruited based on a screening of their hemoglobin. We targeted an enrollment of 10 infants with a hemoglobin <100 g/L and 10 with a hemoglobin 100 g/L. Twenty infants were recruited for each of the age groups. Two infants in the 9- to 10-mo–old age group were excluded from the study due to unsuccessful blood draws.

Hemoglobin cut-offs were selected based on the levels determined by Domellöf et al. (2) and Emond et al. (6) to be more appropriate for children of these ages.

Study procedure

All study visits took place at a community clinic in Villa El Salvador. Each infant had 2 iron absorption measurements made and the study required 4 visits.

    Visit 1. Infants were screened by a finger stick for group assignment, and 120 mL of breast milk was collected from each mother. All milk collections took place 2–3 d prior to the absorption measurement and the milk was kept refrigerated at the clinic until the time of the study.

    Visit 2. In the morning of the absorption experiment, 60 mL of mother's milk mixed with 150 µg of ⁵⁸Fe was given to the infant after a 2-h fast. The milk was heated to 37°C in a water bath prior to being fed to the infant in a preweighed feeding bottle. The bottle was weighed after the feeding and all milk losses (e.g., spitting up, spilling) were determined by collection in preweighed napkins. The bottle was rinsed with an additional 60 mL of mother's milk, and fed to the infant to ensure the entire amount of isotope was consumed. No food or drink was given for 2 h after the feeding. Anthropometric data, dietary, and health records were collected.

    Visit 3. The following day, 2 mg of ⁵⁷Fe as the sulfate was given in juice containing 50 mg ascorbic acid in a preweighed feeding bottle. Again, the bottle was weighed after the feeding, and all losses were determined by collection in preweighed napkins. No food or drink was given for 2 h before and after the feeding.

    Visit 4. Fourteen days after Visit 2, the infants returned to the clinic and a venous blood sample (3 mL) was drawn for isotope ratio measurement and other biochemical indicators. Anthropometric data, dietary, and health records were collected.

Blood samples and laboratory analyses

Each sample was centrifuged at 2000 x g for 10 min at room temperature and separated into serum, plasma, and RBCs. The RBCs were saved for isotope ratio analysis on a thermal ionization mass spectrometer. Serum ferritin (S-Ft) (Coat-A-Count Ferritin IRMA, DPC), C-reactive protein (CRP) (C-Reactive Protein RID, The Binding Site), and plasma folate and vitamin B-12 (Quantaphase II, BioRad) were also measured.

Stable isotope methods

Iron isotopes of ⁵⁷Fe (95.9%) and ⁵⁸Fe enriched (93.1%) were purchased in the elemental form from Trace Sciences International. Iron isotope solutions were prepared as the sulfate by dissolving the metals in 30 µL of 7 mol/L nitric acid and 125 µL of 0.5 mol/L sulfuric acid for every mg of elemental iron. The solution was dried at 120°C, 230°C, and 500°C for 30 min each. The following day the powdered product was reconstituted with 240 µL of 0.2 mol/L sulfuric acid for every mg elemental iron. Deionized water was added to produce a solution of 0.02 g/L for ⁵⁷Fe and 0.005 g/L for ⁵⁸Fe.

Iron isotope ratios were measured from red blood cells 14 d after dosing. Briefly, 0.3–0.5 mL of RBCs were digested with 10 mL 15 mol/L nitric acid until dry. After cooling, the digest was reconstituted with 0.6 mL 6 mol/L hydrochloric acid (HCl). The solution was then put through an anion exchange column for separation. A filter was placed in an 8 cm x 0.4 cm column and loaded with 2 mL of anion exchange resin. The column was prewashed with 4 mL 6 mol/L HCl, 4 mL of deionized water, and 1 mL of 6 mol/L HCl again. The sample was then loaded into the column and followed by 6 mL 6 mol/L HCl and 0.5 mL 0.5 mol/L HCl. The iron was collected from the column (1 mL 0.5 mol/L HCl) into a Teflon vial and dried on a hotplate. The sample was then reconstituted with 40 µL of 3% ultrapure HCl and 10 µL of sample was placed onto a rhenium filament with 2 µL of 0.7 mol/L phosphoric acid and 6 µg silica gel. The iron isotope ratios were measured with a thermal ionization magnetic sector mass spectrometer (MAT 261; Finnigan ThermoQuest). The results were expressed as the ratio of ⁵⁷Fe:⁵⁶Fe and ⁵⁸Fe:⁵⁶Fe.

Iron absorption was calculated as iron incorporated into the erythrocytes at 14 d based on the assumption that 90% of the absorbed iron was incorporated into RBCs (7). There is evidence that <80% of absorbed iron is promptly incorporated into erythrocytes of infants (8). Fomon et al. (9) reported that erythrocyte incorporation of ⁵⁸Fe was significantly greater in older infants (168 d) at 52% than in younger infants (56 d) at 23%. All of our subjects were >56 d old, and a large proportion were older than 168 d; thus, the 90% value may be more suitable for our subjects, however, we realized that using Fomon's 52% value would increase our results. Most previous studies have used the 90% (10,11) value, so there is consistency among studies.

Sample size

We hypothesized that we would find a 50% greater absorption in anemic children relative to non-anemic children. Using previous iron absorption data (2), a group size of 8 infants in each group would give a power of 80% to detect a 50% difference in iron absorption between groups when testing at an alpha level of 0.05. We chose a group size of 10 infants for each group (40 infants total) to account for a 20% attrition rate.

Statistical analysis

The difference in iron absorption between each age group was compared by a 2-sample t test, by ANCOVA, and by linear regression. Data were analyzed by ANCOVA with hemoglobin, serum ferritin, age, gender, and anemia each as a covariant. Data are presented as means ± SEM.

	Results

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
    Baseline characteristics. Body weights and lengths did not differ between the anemic and non-anemic infants in each age group (Table 1). No subject had a serum CRP concentration >11.0 mg/L, indicating no evidence of acute infection.

View larger version (15K): [in this window] [in a new window]   Figure 1  Absorption of 58Fe from human milk (A) and 57Fe when given with ascorbic acid (B) by anemic (A) and non-anemic (NA) 5- to 6-mo–old and 9- to 10-mo–old infants. Values are mean ± SEM, n = 10 (5–6 mo A), n = 10 (5–6 mo NA), n = 9 (9–10 mo A), n = 9 (9–10 mo NA).

    Correlation between serum ferritin and iron absorption. Following log-transformation, iron absorption from both the labeled breast milk (⁵⁷Fe: r = –0.61, P = < 0.001) and from the reference dose (⁵⁸Fe: r = –0.44, P < 0.01) were each inversely correlated with serum ferritin (Fig. 2). For both the non-milk dose (⁵⁷Fe) and breast-milk dose (⁵⁸Fe), infants with S-Ft <12 µg/L (n = 11) had significantly higher iron absorption than those whose S-Ft was 12.0 µg/L (n = 27). ⁵⁷Fe absorption was 36.4 ± 4.5% for infants with S-Ft <12µg/L and 16.7 ± 1.6% for infants with S-Ft 12 µg/L, (P < 0.001). ⁵⁸Fe absorption was 56.4 ± 4.7% for infants with S-Ft <12 µg/L and ⁵⁸Fe absorption was 38.0 ± 5.3% for S-Ft 12 µg/L, (P = 0.04). The Hb concentration did not differ between the groups with S-Ft > or <12 µg/L, (P = 0.45).

View larger version (13K): [in this window] [in a new window]   Figure 2  Correlations between absorption of 58Fe (A) and 57Fe (B) and serum ferritin in 5- to 6-mo–old and 9- to 10-mo–old infants.

	Discussion

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
We found that iron absorption was not affected by Hb concentration in this at-risk population of Peruvian infants. There were no differences in iron absorption between anemic and non-anemic groups at either 6 or 9 mo of age. This finding was unexpected based on previous data referred to as the "iron stores regulator theory." This theory is based on the inverse relation between iron stores and iron absorption in adults (12), but has not been validated in infants. The mechanisms regulating iron absorption may be immature at this age, which has been supported by Domellöf et al. (2) who studied iron absorption in 6 and 9-mo–old breast-fed infants who were given iron supplements (iron sufficient) or a placebo. At 6 mo of age, iron-supplemented infants were not able to downregulate iron absorption (as a consequence of higher iron stores). However, at 9 mo of age, a significant downregulation of iron absorption occurred in the supplemented infants. In our study, infants with lower iron status, as measured by serum ferritin, exhibited an upregulation of iron absorption at both 6 and 9 mo. Results from the latter study suggest that infants with low iron status are able to compensate for their impaired status by increasing iron absorption, whereas infants with adequate iron status and who are given additional iron are not able to decrease iron absorption to protect against fortification or supplementation strategies that may lead to iron intakes above recommendations or that might have unexpected negative implications (13). Our data also suggest that Hb may not be a reliable marker of iron status in healthy infants (14,15). Iron absorption from both labeled breast milk and from the reference dose were inversely correlated with serum ferritin, indicating that, in terms of regulation of absorption, ferritin may be more indicative of iron needs of infants than Hb.

The closer correlation between iron status and absorption from the dose given separately from breast milk implies that absorption of nonbreast-milk iron (such as that given with a supplement) may more readily respond to iron status changes. A major part of iron in breast milk is bound to lactoferrin (16). The presence of lactoferrin receptors in the small intestine of infants (17) and the fact that a large proportion of breast-milk lactoferrin can survive proteolysis in the gut and is found intact in the stool of breast-fed infants (18), strongly suggest that part of breast-milk iron is taken up by lactoferrin receptors (19). Because there is no evidence of upregulation of the lactoferrin receptor during iron deficiency, breast-milk iron may not be regulated as tightly as iron from supplements.

Studies have reported that iron supplementation before 6 mo of age prevents the physiological fall in Hb that occurs from 1 to 6 mo (20,21) and that this may have beneficial hematologic and developmental outcomes for some infants (22). Thus, early introduction of highly bioavailable iron sources, before 6 mo of age, may be beneficial in this high-risk population. However, some studies have reported detrimental effects such as decreased growth and increased morbidity (23), decreased zinc absorption (24), and altered vitamin A metabolism (25) when iron supplements are provided to infants who do not need them. As described above, it is possible that young infants may lack the capacity to downregulate iron absorption, either due to immaturity or to other micronutrient deficiencies, and that iron given to such infants may cause adverse effects. Population-based consideration of iron status on individual outcomes should be considered when deciding to give an early iron supplement.

We conclude that the regulation of iron absorption is mature by 6 mo but with a relatively modest responsiveness to iron deficiency from iron absorption in breast milk and with a greater response of iron absorption from nonbreast-milk sources. The use of Hb to assess iron status and to thereby identify likely responders to iron supplementation may not be effective in this population.

	ACKNOWLEDGMENTS

 
We would like to thank the study team, Norma Valencia, MD and Juana Callalli, RN, for care of the children and Ian Griffin for helpful discussions about these data.

Manuscript received 4 May 2006. Initial review completed 5 June 2006. Revision accepted 26 June 2006.

	LITERATURE CITED

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 

1. Hallberg L. Bioavailability of dietary iron in man. Annu Rev Nutr. 1981;1:123–41.[Medline]

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

3. International Nutrition Anemia Consultative Group. Guidelines for the use of iron supplements to prevent and treat iron deficiency anemia. Washington, DC: International Nutrition Anemia Consultative Group; 1988.

4. O'Brien KO, Zavaleta N, Abrams SA, Caulfield LE. Maternal iron status influences iron transfer to the fetus during the third trimester of pregnancy. J Clin Nutr. 2003;924–30.

5. Reyes J, Ochoa LH. Encuesta demográfica y de salud familiar. República del Perú: Instituto Nacional de Estadística e Informática, Macro International Inc; 2000. p. 182–3

6. Emond AM, Hawkins N, Pennock C, Golding J. Haemoglobin and ferritin concentrations in infants at 8 months of age. Arch Dis Child. 1996;74:36–9.[Abstract/Free Full Text]

7. Kastenmayer P, Davidsson L, Galan P, Cherouvrier F, Hercberg S, Hurrell R. A double stable isotope technique for measuring iron absorption in infants. Br J Nutr. 1994;71:411–24.[Medline]

8. Fomon SJ, Ziegler EE, Serfass RE, Nelson SE, Rogers RR, Frantz JA. Less than 80% of absorbed iron is promptly incorporated into erythrocytes of infants. J Nutr. 2000;130:45–52.[Abstract/Free Full Text]

9. Fomon SJ, Serfass RE, Nelson SE, Rogers RR, Frantz JA. Time course of and effect of dietary iron level on iron incorporation into erythrocytes by infants. J Nutr. 2000;130:541–5.[Abstract/Free Full Text]

10. Davidsson L, Kastenmayer P, Yuen M, Lönnerdal B, Hurrell R. Influence of Lactoferrin on iron absorption from human milk fed infants. Pediatr Res. 1994;35:117–24.[Medline]

11. Abrams SA, Wen J, Stuff JE. Absorption of calcium, zinc, and iron from breast milk by five-to-seven-month-old infants. Pediatr Res. 1997;41:384–90.[Medline]

12. Finch C. Regulators of iron balance in humans. Blood. 1994;84:1697–702.[Free Full Text]

13. Dewey KG, Domellöf M, Cohen RJ, Landa Rivera L, Hernell O, Lönnerdal B. Iron supplementation affects growth and morbidity of breast-fed infants: results of a randomized trial in Sweden and Honduras. J Nutr. 2002;132:3249–55.[Abstract/Free Full Text]

14. White KC. Anemia is a poor predictor of iron deficiency among toddlers in the United States: for heme the bell tolls. Pediatrics. 2005;115:315–20.[Abstract/Free Full Text]

15. Bogen DL, Krause JP, Serwint JR. Outcome of children identified as anemic by routine screening in an inner-city clinic. Arch Pediatr Adolesc Med. 2001;155:366–71.[Abstract/Free Full Text]

16. Suzuki YA, Shin K, Lönnerdal B. Molecular cloning and functional expression of a human intestinal lactoferrin receptor. Biochemistry. 2001;40:15771–9.[Medline]

17. Davidson LA, Lönnerdal B. Persistence of human milk proteins in the breast-fed infant. Acta Paediatr Scand. 1987;76:733–40.[Medline]

18. Lönnerdal B. Lactoferrin receptors in intestinal brush border membranes. Adv Exp Med Biol. 1994;357:171–5.[Medline]

19. Davidson LA, Lönnerdal B. Specific binding of lactoferrin to brush-border membrane: ontogeny and effect of glycan chain. Am J Physiol. 1988;254:G580–5.[Medline]

20. Friel JK, Azis K, Andrews WL, Harding SV, Courage ML, Adams RJ. A double-masked, randomized control trial of iron supplementation in early infancy in healthy term breast-fed infants. J Pediatr. 2003;143:582–6.[Medline]

21. Domellöf M, Cohen RJ, Dewey KG, Hernell O, Landa Rivera L, Lönnerdal B. Iron supplementation of breast-fed Honduran and Swedish infants from 4 to 9 months of age. J Pediatr. 2001;138:679–87.[Medline]

22. American Academy of Pediatrics Committee on Nutrition. Pediatric nutrition handbook. 5th ed. Elk Grove Village, IL: American Academy of Pediatrics; 2004. p. 299–312.

23. Dewey KG, Domellöf M, Cohen RJ, Landa Rivera L, Hernell O, Lönnerdal B. Iron supplementation effects growth and morbidity of breast-fed infants: results of a randomized trial in Sweden and Honduras. J Nutr. 2002;132:3249–55.[Abstract/Free Full Text]

24. Lind T, Lönnerdal B, Stenlund H, Gamayanti IL, Ismail D, Seswandhana R, Persson L. A community-based randomized controlled trial of iron and zinc supplementation in Indonesian infants: effects on growth and development. Am J Clin Nutr. 2004;80:729–36.[Abstract/Free Full Text]

25. Wieringa FT, Dijkhuizen MA, West CE, Thurnham DI, Muhilal, Van der Meer JW. Redistribution of vitamin A after iron supplementation in Indonesian infants. Am J Clin Nutr. 2003;77:651–7.[Abstract/Free Full Text]

This article has been cited by other articles:

				E. E Ziegler, S. E Nelson, and J. M Jeter Iron status of breastfed infants is improved equally by medicinal iron and iron-fortified cereal Am. J. Clinical Nutrition, July 1, 2009; 90(1): 76 - 87. [Abstract] [Full Text] [PDF]

				E. E Ziegler, S. E Nelson, and J. M Jeter Iron supplementation of breastfed infants from an early age Am. J. Clinical Nutrition, February 1, 2009; 89(2): 525 - 532. [Abstract] [Full Text] [PDF]

				C. M. Chaparro Setting the Stage for Child Health and Development: Prevention of Iron Deficiency in Early Infancy J. Nutr., December 1, 2008; 138(12): 2529 - 2533. [Abstract] [Full Text] [PDF]

				M. F. Lynch, I. J. Griffin, K. M. Hawthorne, Z. Chen, M. G. Hamzo, and S. A. Abrams Iron Absorption Is More Closely Related to Iron Status Than to Daily Iron Intake in 12- to 48-Mo-Old Children J. Nutr., January 1, 2007; 137(1): 88 - 92. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hicks, P. D. Articles by Lönnerdal, B. Search for Related Content PubMed PubMed Citation Articles by Hicks, P. D. Articles by Lönnerdal, B.