Infant iron status affects iron absorption in Peruvian breastfed infants at 2 and 5 mo of age

Infant iron status affects iron absorption in Peruvian breastfed infants at2 and 5 mo of age13

Julia L Finkelstein, Kimberly O OBrien, Steven A Abrams, and Nelly Zavaleta

ABSTRACTBackground: Effects of prenatal iron supplementation on maternalpostpartum iron status and early infant iron homeostasis remainlargely unknown.Objective: We examined iron absorption and growth in exclusivelybreastfed infants in relation to fetal iron exposure and iron statusduring early infancy.Design: Longitudinal, paired iron-absorption (58Fe) studies wereconducted in 59 exclusively breastfed Peruvian infants at 23 moof age (2M) and 56 mo of age (5M). Infants were born to womenwho received $5100 or #1320 mg supplemental prenatal Fe. Ironstatus was assessed in mothers and infants at 2M and 5M.Results: Infant iron absorption from breast milk averaged 7.1%and 13.9% at 2M and 5M. Maternal iron status (at 2M) predictedinfant iron deficiency (ID) at 5M. Although no infants were irondeficient at 2M, 28.6% of infants had depleted iron stores (ferritinconcentration ,12 mg/L) by 5M. Infant serum ferritin decreased(P , 0.0001), serum transferrin receptor (sTfR) increased (P ,0.0001), and serum iron decreased from 2M to 5M (P , 0.01).Higher infant sTfR (P , 0.01) and breast-milk copper (P , 0.01)predicted increased iron absorption at 5M. Prenatal iron supplemen-tation had no effects on infant iron status or breast-milk nutrientconcentrations at 2M or 5M. However, fetal iron exposure predictedincreased infant length at 2M (P , 0.01) and 5M (P , 0.05).Conclusions: Fetal iron exposure affected early infant growth butdid not significantly improve iron status or absorption. Young, ex-clusively breastfed infants upregulated iron absorption when ironstores were depleted at both 2M and 5M. Am J Clin Nutr2013;98:147584.

INTRODUCTION

An estimated 1.6 billion people worldwide are anemic (1, 2)and iron deficiency (ID)4 is the leading cause of anemia (1, 3).Pregnant women are at increased risk of anemia and ID becauseof, in part, increased nutritional requirements of pregnancycoupled with a lack of adequate dietary iron intake and bio-availability to meet these increased demands (3, 4). In resource-limited settings, it has been estimated that 50% of pregnantwomen had anemia (1) compared with 1225% in developedregions (3, 5, 6). When anemia or ID occurs during pregnancy,risk of maternal (7) and infant mortality (8) is increased, butlittle is known about how this affects early infant iron homeo-stasis or growth.

The impact of maternal iron status and iron supplementationduring pregnancy on neonatal iron status has not been fully

characterized. Several studies have shown no significant asso-ciation between maternal and infant iron status (9, 10), whereasother evidence has suggested an association between maternaliron status in pregnancy and infant iron stores postpartum (1114); maternal iron-deficiency anemia (IDA) in pregnancy hasbeen associated with compromised fetal iron reserves (11, 1517). Recent data by Roberfroid et al (18) have indicated that thephysiologic anemia of pregnancy occurs even in women re-ceiving iron supplementation.

Several studies have shown that placental iron transfer can beupregulated when maternal or fetal iron stores are limited (1923). We have previously reported that a significantly largerfraction of maternally ingested iron is transferred to the fetus inwomen with depleted iron stores (23, 24). Recent animal studiesalso suggested that the timing at which iron supplementation isinitiated during pregnancy may also influence subsequent neo-natal outcomes (25). Although acute studies of placental irontransport have highlighted the adaptability of maternal-fetal ironpartitioning, little is known about the long-term impact of thistransfer on early iron homeostasis. In Peru, the estimated pre-valence of anemia in infants 68 mo old is 69.3% (26); reasonsfor this high prevalence in relation to early iron absorption needto be investigated.

To address this issue, we conducted longitudinal, paired iron-absorption studies in exclusively breastfed Peruvian infants at 2and 5 mo of age to 1) characterize longitudinal determinants ofiron absorption from breast milk in young infants as the ironstores of birth are depleted and 2) examine the impact of fetaliron exposure on early infant iron homeostasis and growth.

1 From the Division of Nutritional Sciences, Cornell University, Ithaca,

NY (JLF and KOO); the USDA/Agricultural Research Service Childrens

Nutrition Research Center, Baylor College of Medicine, Houston, TX

(SAA); and the Instituto de Investigacion Nutricional, Lima, Peru (NZ).2 Supported by a grant from the Nestle Foundation.3 Address reprint requests and correspondence and to KO OBrien, Divi-

sion of Nutritional Sciences, Cornell University, 230 Savage Hall, Ithaca,

NY 14853. E-mail: [email protected] Abbreviations used: ID, iron deficiency; IDA, iron-deficiency anemia;

SF, serum ferritin; sTfR, serum transferrin receptor; 2M, 23 mo of age;

5M, 56 mo of age; 2Fe, #1320 mg supplemental prenatal Fe; +Fe, $5100mg supplemental prenatal Fe.

ReceivedDecember 19, 2012. Accepted for publication September 11, 2013.

First published online October 2, 2013; doi: 10.3945/ajcn.112.056945.

Am J Clin Nutr 2013;98:147584. Printed in USA. 2013 American Society for Nutrition 1475

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SUBJECTS AND METHODS

Study population

Two groups of Peruvian women and their 23-mo-old infantswere recruited from Villa El Salvador, which is a low-incomeperiurban area of Lima, Peru. One group of women (n = 30) hadreceived supplemental iron [60 mg Fe/d for $3 mo duringpregnancy for a total of $5100 mg supplemental prenatal iron(+Fe)], whereas the other group (n = 29) had not consumed ironsupplements or consumed ,1 mo of supplemental iron duringpregnancy [defined as#1320 mg supplemental prenatal Fe (2Fe)across pregnancy]. Maternal supplemental iron intake and ad-herence during pregnancy was evaluated by self-report. Thisstudy was conducted during 1998; at that time, pregnant womenreceiving care at health centers frequently did not ingest ironsupplements either by low adherence to prenatal supplementationor because supplements were not regularly available in healthcenters. Recent survey data on prenatal iron supplementation (60mg/d) in this community reported an average intake of 6360 mgFe (IQR: 48607980 mg Fe) over the course of pregnancy (27).

All infants recruited were receiving care at the well baby clinicat the Cesar Lopez Silva Hospital. Infants were eligible forparticipation if they were 23 mo old, healthy, full-term sin-gletons ($37 to ,42 wk of gestation) with a birth weight$2500 g. Data on infant weight and length at birth were ob-tained from medical records. Infants were exclusively breastfed(ie, none had received any infant formula, herbal infusions, orfoods), had not received any supplemental iron, and lived inLima (at sea level) since birth. The study protocol was explainedin detail to mothers by the field nurse and study doctor, andinformed written consent was obtained before study enrollment.The study protocol was approved by the Committee on HumanResearch at Johns Hopkins Bloomberg School of Public Healthand by the Institutional Review Board at the Instituto de In-vestigacion Nutricional, Lima, Peru.

Study design

Longitudinal, paired iron-absorption (58Fe) studies were un-dertaken in infants at 23 mo of age (2M) and again at 56 mo ofage (5M). All study visits were conducted at the outpatient wellbaby clinic in Cesar Lopez Silva Hospital, Villa El Salvador, Lima,Peru. On the morning of the first iron-absorption study, the infantwas examined by the study pediatrician, weight was measured byusing a digital scale (SECA) (610 g), and recumbent length wasmeasured to the nearest 0.1 cm by using a wooden measuringboard. Each infant then had a 3-mL venous blood sample collectedto assess baseline iron status, including hemoglobin, serumtransferrin receptor (sTfR), serum ferritin (SF), and C-reactiveprotein (evacuated tubes containing EDTA; SARSDET). Breast-milk samples were collected from each mother into containers freefrom trace minerals; samples were obtained from early, middle,and late feed for assessment of iron, zinc, and copper concentra-tions. A maternal 3-mL venous blood sample was obtained on theday the infant was dosed at both 2M and 5M to evaluate the sameiron-status indicators as measured in each infant.

In the initial iron-absorption studies, women expressed breastmilk into a sterile trace mineralfree container, iron tracer (150mg 58Fe) was added to the expressed breast milk, and the mix-ture was allowed to sit overnight in a cold room on a shaker. At

the clinic the next morning, infants were fed the extrinsicallylabeled breast milk (the container with milk was warmed in hotwater) from a bottle ad libidum after a 2-h fast, and the quantityof milk and tracer ingested was determined by preweighing andpostweighing the feed. However, the initial iron-absorption datafrom the first 9 infants were much lower than anticipated on thebasis of existing literature at the time the study was undertaken.As a result, to ensure that all tracer was delivered, the studydesign was modified to administer 150 mg 58Fe (after a 2-h fast)in a flavored syrup by syringe directly into the infants mouth.Then, mothers breastfed their infants ad libidum. Infant weightwas recorded before and after the feeding interval to assess thetotal intake of breast milk. Statistical analyses were conductedboth with and without adjustment for the method of tracer ad-ministration; there were no significant differences in iron ab-sorption in subjects who received tracer that had equilibrated inbreast milk overnight (n = 15; 9 +Fe and 62Fe), compared withthose who received tracer alone before being breastfed (n = 44;21 +Fe and 23 2Fe). Thus, the dosing method was not adjustedfor in subsequent analyses.

Syringes were weighed before and after dosing to determinethe exact quantity of isotope administered. All mother and infantpairs studied remained in the clinic for 1 h postdosing to ensurethat tracer was not regurgitated; no breast milk, other food, orliquid was given for 2 h after the feed that contained the iron dosewas completed. Anthropometric, dietary, and health data werecollected. Fourteen days after dosing, mothers and infantsreturned to the study hospital. A 3-mL venous blood sample wascollected to assess concentrations of stable iron isotopes andbiochemical indicators in infants. Anthropometric, dietary, andhealth data were also collected. After the blood collection,mothers and infants returned home.

Three months after the first iron-absorption study, mother-infant pairs returned to the study hospital, and a second ironabsorption study was conducted by using the same labelingmethod as used at the first study except that a 3-mL venous bloodsample was collected from each infant before dosing to assessbaseline 58Fe enrichment, and the 58Fe dose was increased to200 mg. The baseline enrichment at 5M was used as the newbaseline value for the second absorption-study calculations.Maternal blood samples were also collected for the same anal-yses as those measured in the first study. As in the first study, themother and infant remained in the clinic for the following hourpostdosing to ensure that tracer was not regurgitated and thesame anthropometric, biochemical, dietary, and health data werecollected. Fourteen days after the second dosing study, mothersand infants returned to the study hospital, and a venous bloodsample (3 mL) was obtained from each infant to assess ironenrichment in red blood cells and concentrations of biochemicalindicators. Anthropometric data, dietary, and health data werealso collected, and mothers and infants returned home. At theend of the second study, any mother or infant who was shown tobe anemic was treated for anemia in accordance with standard ofcare guidelines for the WHO and government of Peru (3).

Biochemical indicators

Hemoglobin concentrations were assessed in whole bloodsamples by using the Hemocue method. The packed cell volumewas analyzed by using the microhematocrit method. SF was

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measured by using an ELISA with human antiferritin and anti-ferritin peroxidase antibodies purchased from DAKO. sTfR wasmeasured by using a commercially available ELISA (Quantikine;R&D Systems). Laboratory samples were tested in batches by anexperienced biochemist at Instituto de Investigacion Nutricional,and instruments were calibrated daily by using standardizedprocedures.

Preparation of stable iron isotope

A stable iron isotope (58Fe at 93.13% enrichment) was pur-chased in elemental form from Trace Sciences International.The metal was converted into ferrous sulfate solutions (0.5 mg/mL) according to the procedure of Kastenmayer et al (28), ex-cept that no ascorbic acid was added during the tracer prepa-ration to avoid its influence on iron absorption. Isotope solutionswere checked for sterility before administration. The 58Fe en-richment of the final ferrous sulfate tracer solution was de-termined by using magnetic sector thermal ionization massspectrometry (Thermoquest Triton TI), and the total iron contentwas measured by using atomic absorption spectrophotometry(Perkin Elmer 3300;Perkin Elmer).

Isolation of iron from red blood cells

Approximately 1 mL whole blood was digested on a hot platewith Ultrex nitric acid following previously published methods(24, 29). When the digest was clear, it was evaporated to dryness,reconstituted, and iron was extracted from this digest by usinganion-exchange chromatography as previously reported (24, 29).After the iron was eluted from the column, the sample was driedand reconstituted in 30 mL 0.3N ultrapure nitric acid.

Measurement of iron isotope enrichment

Extracted iron (10 mL) was loaded onto rhenium filamentsand isotopic ratios (58Fe:56Fe ratios normalized for isotopicfractionation by using the 54Fe:56Fe ratio) were analyzed byusing a magnetic sector thermal ionization mass spectrometer(Thermoquest TI). The instrumentation achieved a relative SDof 0.16% for the 58Fe isotope used.

Iron-absorption calculations

The amount of circulating iron in each infant was estimatedafter assuming that the blood volume was 80 mL/kg by using thefollowing equation:

Circulating iron mg hemoglobin concentration g=L3 blood volume mL3 concentration of iron in hemoglobin

3:47mg Fe=g hemoglobin3 1L=1000mL 1

The percentage of iron absorption was determined by measuring theincorporation of 58Fe tracer into red blood cells collected 14 d post-dosing by using previously reported equations (29) and assuming that90% of the absorbed iron was incorporated into red blood cells (28).

Statistical analyses

Linear regression was used to examine associations betweeniron-status indicators and iron absorption at 2M and 5M and

assess determinants of iron absorption. Binomial regression wasused to obtain risk ratio estimates (3032) to examine the effectof fetal iron exposure on categorical outcomes. To adjust formultiple hypothesis testing, significance was determined afterapplying the Bonferroni correction. All P values presented areoriginal (unadjusted) P values for interpretation purposes, andthe threshold used to determine statistical significance was

aO n 2

where a is the level of significance (a = 0.05), and n is thenumber of multiple comparison tests conducted. If results weresignificant after the application of the Bonferroni correction, thesignificance is reported in the text.

Conventional cutoffs were used to categorize risk factors,where available; otherwise, medians were used to classify var-iables. Anemia was defined as hemoglobin concentrations,12.0g/dL in postpartum women and ,11.0 g/dL in infants in ac-cordance with WHO criteria and clinical guidelines in Peru (3).ID was defined as SF concentrations ,12 mg/L in both mothersand infants (5). IDA was defined in the presence of both anemiaand depleted ferritin stores. Maternal sTfR concentrations.5.33 mg/L were classified as ID on the basis of recentNHANES data from nonpregnant women (33). BMI (in kg/m2)was defined as the ratio of weight to height squared. Infantponderal index was calculated as the ratio of grams to the lengthcubed (g/cm3 3 100). Ferritin values were log-transformed toaccount for the nonnormal distribution of this variable, butvalues in the text are presented as nontransformed values forinterpretation purposes. In predictor analyses, variables withunivariate P values ,0.20 were included in each of the multi-variate regression models and retained if their P values were,0.05.

We examined potential confounders, and adjusted all gesta-tional iron exposure and growth analyses for infant sex. Themissing indicator method was used to account for missing pre-dictor data (34).

Potential predictors of study outcomes were also examined ascontinuous variables. We explored the potential nonlinearity ofrelations between covariates and outcomes nonparametrically byusing stepwise restricted cubic splines (35, 36). We used tests fornonlinearity by using the likelihood ratio test and comparing themodel with only the linear term to the model with the linear andthe cubic spline terms. If nonlinear associations are not reported,they were not significant. Statistical analyses were performedwith SAS software (version 9.3; SAS Institute Inc).

RESULTS

Baseline characteristics of the 59 women and infant pairsincluded in the analyses are presented in Table 1. The averageage of women in the study was 24.3 y, and the average BMI was25.2. None of the women were underweight (BMI ,18.5), and50.9% of women were overweight (BMI $25.0) at the 2Mstudy. Of the 59 infants initially enrolled, 30 infants were born to+Fe women, and 29 infants were born to 2Fe women (#1320mg Fe supplementation across gestation).

Maternal and infant iron status at 2M and 5M are presented inTable 2. At the 2M postpartum baseline assessment, 64.9% of

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women were anemic, with a mean (6SD) hemoglobin concen-tration of 11.4 6 1.6 g/dL. The prevalence of anemia in infantswas similarly high; 77.2% of infants had hemoglobin concen-trations ,11.0 g/dL (ranging from 8.0 to 14.6 g/dL, with 31.6%and 8.9% of infants with hemoglobin concentrations ,10.0 and,9.0 g/dL, respectively). Despite the high prevalence of infantanemia, maternal and infant ferritin concentrations averaged20.66 20.3 and 164.46 103.8 mg/L, respectively, at 2M. A totalof 49.0% of mothers were iron deficient (ferritin concentration,12 mg/L), and 36.7% of mothers had IDA at 2M comparedwith 31.9% (P = 0.01) and 21.3% (P . 0.05) of mothers at 5Mpostpartum. Although no infants had ID (ferritin ,12 mg/L) orIDA at 2M, 28.6% of infants had ferritin concentrations ,12mg/L, 59.2% of infants had ferritin concentrations ,30 mg/L,and 24.5% of infants had IDA at 5 mo of age. Prenatal ironsupplementation had no significant effects on indicators ofpostpartum maternal or infant iron status measured, except he-matocrit and sTfR (compared with infants born to 2Fe mothers,infants born to +Fe mothers had higher hematocrit and sTfR at2M; P , 0.05). After correction for multiple hypothesis testing,prenatal iron supplementation had no significant effects on in-dicators of postpartum maternal or infant iron status measured(P . 0.0025).

In analyses of correlations between maternal and infant ironstatus at both 2M and 5M time points, maternal hemoglobinconcentrations were associated with infant hemoglobin at bothtime points; low maternal hemoglobin concentrations were sig-nificantly associated with low infant hemoglobin concentrationsat both 2M (R = 0.38, P = 0.007) and 5M (R = 0.31, P = 0.02),

although the magnitude of association decreased at 5M. Maternaland infant hematocrit were significantly correlated at 5M (R = 0.42,P = 0.01). Infants born to iron-deficient mothers (at 2M) weresignificantly more likely to be iron deficient at 5M (R = 0.33, P =0.008), irrespective of iron group.

In analyses of longitudinal changes in maternal and infant ironstatus between 2M and 5M, maternal sTfR significantly de-creased between 2M and 5M (P = 0.02); and maternal serum ironsignificantly increased during the follow-up period (P = 0.01).There were no significant changes in maternal hemoglobin, SF,or hematocrit between 2M and 5M. Although no infants hadferritin concentrations ,12 mg/L at 2M, infant SF significantlydecreased (P , 0.0001), infant sTfR significantly increased(P , 0.0001), and infant serum iron significantly decreasedbetween 2M and 5M (P , 0.01). Although significant changesin infant iron stores were apparent, there were no significantchanges in infant hemoglobin, hematocrit, or CRP between 2Mand 5M, nor were there any significant differences in changes inany of the iron status indicators previously mentioned by ges-tational iron exposure (Table 2). As expected, infant weight(P , 0.0001) and length (P , 0.0001) significantly increased,and the ponderal index (P , 0.001) significantly decreased be-tween 2M and 5M (Table 1). Findings from these analyses re-mained significant after correction for multiple hypothesis testing,except for changes in maternal sTfR (,0.05 but .0.0125).

Breast-milk micronutrient concentrations and infant iron ab-sorption at 2M and 5M and average changes between time pointsare presented in Table 3. Breast-milk iron concentrations de-creased between 2M and 5M, although this change was not

TABLE 1

Characteristics of the mothers and infants1

Variables Total (n = 59) +Fe (n = 30) 2Fe (n = 29)

Maternal

Age (y) 24.3 6 4.82 24.9 6 5.6 23.7 6 3.8Height (m) 1.5 6 0.1 1.5 6 0.1 1.5 6 0.1Supplemental iron intake (mg) 3050.1 6 3276.3 6118.6 6 2114.3 299.1 6 358.5Weight (2M) (kg) 56.9 6 7.1 57.6 6 6.3 56.2 6 7.8BMI (2M) (kg/m2) 25.2 6 3.2 25.0 6 3.1 25.4 6 3.4Overweight (2M) [n (%)]3 30 (50.9) 14 (46.7) 16 (55.2)

Weight (5M) (kg) 55.7 6 8.1 55.4 6 7.1 56.0 6 9.1BMI (5M) (kg/m2) 24.7 6 3.9 24.1 6 3.4 25.3 6 4.3Overweight (5M) [n (%)]3 26 (47.3) 13 (44.8) 13 (50.0)

Infant

Sex (F) [n (%)] 28 (47.5) 13 (43.3) 15 (51.7)

Birth weight (kg) 3.47 6 0.45 3.57 6 0.44 3.37 6 0.45Birth length (cm) 50.6 6 1.7 50.9 6 1.9 50.2 6 1.4Ponderal index at birth (g/cm3) 2.7 6 0.3 2.7 6 0.3 2.6 6 0.3Age at 2M study (d) 69.2 6 9.1 70.3 6 9.7 68.0 6 8.6Weight (2M) (kg) 5.89 6 0.80 6.0 6 0.7 5.76 6 0.85Weight (5M) (kg) 7.72 6 0.97 7.9 6 0.9 7.58 6 1.01Length (2M) (cm)** 59.1 6 2.7 59.9 6 2.3 58.1 6 2.8Length (5M) (cm) 65.8 6 2.5 66.4 6 2.5 65.1 6 2.3Ponderal index (2M) (g/cm3) 2.9 6 0.3 2.8 6 0.2 2.9 6 0.3Ponderal index (5M) (g/cm3) 2.7 6 0.3 2.7 6 0.3 2.7 6 0.2

1 For statistical analyses, linear regression and binomial regression were used to examine differences in characteristics

between iron groups. **No significant differences between the 2 groups after correction for multiple hypothesis testing,

except for infant length at 2M, P, 0.01. 2M, 23 mo of age; 5M, 56 mo of age;2Fe, infants born to women who received#1320 mg supplemental prenatal Fe; +Fe, infants born to women who received $5100 mg supplemental prenatal Fe.

2Mean 6 SD (all such values).3Overweight was defined as BMI (in kg/m2) $25.




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TABLE 2

Maternal and infant iron status by prenatal iron supplementation1

Variables Time point Total (n = 59) +Fe (n = 30) 2Fe (n = 29)

Maternal

Hemoglobin (g/dL) 2M 11.4 6 1.62 11.2 6 1.8 11.8 6 1.25M 11.7 6 1.3 11.6 6 1.3 11.8 6 1.3

D2M to 5M 0.2 6 2.0 0.4 6 2.3 0.0 6 1.6Anemic [n (%)]3 2M 37 (64.9) 20 (66.7) 17 (63.0)

5M 26 (47.3) 13 (46.4) 13 (48.2)

Serum ferritin (mg/L) 2M 20.6 6 20.3 21.3 6 22.3 20.0 6 18.45M 22.8 6 18.5 24.6 6 21.4 18.8 6 14.7

D2M to 5M 0.6 6 16.6 3.0 6 18.9 21.8 6 13.8Serum ferritin concentration ,12 mg/L [n (%)] 2M 24 (49.0) 11 (44.0) 13 (54.2)

5M 15 (31.9) 7 (29.2) 8 (34.8)

IDA [n (%)]3 2M 18 (36.7) 10 (40.0) 8 (33.3)

5M 10 (21.3) 4 (16.7) 6 (26.1)

Hematocrit 2M 34.5 6 3.8 33.4 6 4.3 35.8 6 2.75M 35.5 6 3.6 35.3 6 3.9 35.8 6 3.3

D2M to 5M 0.8 6 5.1 1.5 6 6.2 0.0 6 3.6Total iron binding capacity (mg/dL) 2M 391.8 6 62.4 382.8 6 59.3 400.9 6 66.3

5M 370.2 6 70.4 347.2 6 52.4 393.3 6 80.3D2M to 5M 221.6 6 87.0 235.6 6 60.4 27.5 6 108.1

sTfR (mg/L) 2M 6.9 6 3.1 6.5 6 2.8 7.3 6 3.45M 6.3 6 3.0 6.1 6 2.2 6.4 6 3.7

D2M to 5M 20.7 6 1.7 20.4 6 1.6 20.9 6 1.8sTfR concentration .5.33 mg/L (%) 2M 67.6 6 25 50.0 6 9 84.2 6 16

5M 56.8 6 21 44.4 6 8 68.4 6 13Serum iron (mg/dL) 2M 57.6 6 20.8 63.9 6 19.6 49.6 6 21.0

5M 74.3 6 29.7 85.6 6 29.3 59.9 6 25.1D2M to 5M 16.7 6 23.8 21.7 6 25.5 10.3 6 21.5

Infant

Hemoglobin (g/dL) 2M 10.4 6 1.2 10.5 6 1.0 10.4 6 1.55M 10.4 6 1.0 10.2 6 1.0 10.7 6 0.9

D2M to 5M 20.0 6 1.4 20.3 6 1.0 0.3 6 1.8Anemic [n (%)]3 2M 44 (77.2) 24 (80.0) 20 (74.1)

5M 41 (78.9) 24 (88.9) 17 (68.0)

Serum ferritin (mg/L) 2M 164.4 6 103.8 151.1 6 79.5 178.3 6 124.55M 34.5 6 31.4 31.5 6 26.7 37.7 6 35.9

D2M to 5M 2129.9 6 91.3 2119.6 6 61.9 2140.6 6 114.8Serum ferritin concentration ,12 mg/L [n (%)] 2M 0.0 6 0 0.0 6 0 0.0 6 0

5M 28.6 6 14 32.0 6 8 25.0 6 6IDA [n (%)]4 2M 0.0 6 0 0.0 6 0 0.0 6 0

5M 24.5 6 12 32.0 6 8 16.7 6 4sTfR (mg/L) 2M 2.3 6 1.9 2.6 6 1.8** 2.1 6 1.9**

5M 4.7 6 2.0 4.7 6 1.9 4.7 6 2.0D2M to 5M 2.4 6 2.3 2.2 6 2.4 2.7 6 2.2

Hematocrit 2M 30.2 6 5.3 31.0 6 2.0** 29.4 6 7.2**5M 31.3 6 3.1 31.1 6 2.9 31.6 6 3.4

D2M to 5M 1.2 6 5.0 20.2 6 2.7 2.4 6 6.3Serum iron (mg/dL) 2M 70.9 6 25.1 72.2 6 31.0 69.4 6 17.0

5M 60.5 6 26.3 62.8 6 23.8 57.9 6 23.5D2M to 5M 220.7 6 28.0 218.8 6 31.2 223.9 6 23.5

CRP (mg/L) 2M 4.5 6 6.7 3.6 6 3.1 5.5 6 9.15M 4.1 6 3.3 4.3 6 4.4 3.9 6 1.8

D2M to 5M 20.5 6 6.9 0.7 6 5.9 21.6 6 7.7CRP concentration .5 mg/L (%) 2M 15.4 6 4 7.7 6 1 23.1 6 3

5M 15.4 6 4 23.1 6 3 7.7 6 1

1 For statistical analyses, linear and binomial regression analyses were conducted to examine associations between iron supplementation and iron-status

indicators. **No significant differences between the 2 groups after correction for multiple hypothesis testing, P . 0.0025. CRP, C-reactive protein; IDA, iron-deficiency anemia; sTfR, serum transferrin receptor; 2M, 23 mo of age; 5M, 56 mo of age; 2Fe, infants born to women who received #1320 mgsupplemental prenatal Fe; +Fe, infants born to women who received $5100 mg supplemental prenatal Fe.

2Mean 6 SD (all such values).3Anemia if hemoglobin concentrations ,12.0 g/dL for women and ,11.0 g/dL for infants.4 IDA if hemoglobin concentrations ,12.0 g/dL and serum ferritin concentrations ,12 mg/L for women and hemoglobin concentrations ,11.0 g/dL and

serum ferritin concentrations ,12 mg/L for infants.




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statistically significant (P . 0.05). In contrast, breast-milkcopper (P = 0.001) and zinc (P , 0.001) concentrations bothsignificantly decreased across the study period after adjustmentfor multiple hypothesis testing.

Iron absorption from breast milk averaged 7.1 6 6.6% and13.9 6 12.1% at 2M and 5M, respectively (Table 3). Iron ab-sorption tended to increase between 2M and 5M (P = 0.09),which was consistent with the response to the depletion of ironstores. Although the percentage iron absorption tended to in-crease, because of the low iron content of breast milk, this in-crease would not lead to a marked increase in net ironabsorption. There were no significant associations of fetal ironexposure and subsequent iron absorption at 2M and 5M or onchanges between time points. There were no significant differ-ences in iron absorption in subjects who received tracer that hadequilibrated in breast milk overnight (n = 15) compared with insubjects who received the tracer alone before being breastfed(n = 44) [median (IQR): 4.0% (2.15.8%) equilibrated comparedwith 5.1% (3.49.5%) nonequilibrated at 2M; 7.7% (3.98.8%)equilibrated compared with 10.4% (6.519.4%) nonequilibratedat 5M]. In analyses of the method of dose administration(equilibrated compared with nonequilibrated with breast milk)and iron absorption, there were no significant differences be-tween iron absorption at 2M and 5M or paired changes betweentime points after adjustment for multiple hypothesis testing.

The value of a 90% red blood cell incorporation rate (28) wasused to be consistent with other data reported in healthy infants(3739) and, in particular, to be consistent with values used inhealthy infants from the same Peruvian community (37). Otherpublished literature has used lower red blood cell incorporationvalues that ranged from 23% to 80% (40, 41). If the middlerange of these estimates were used (60%), the average iron-absorption value obtained would have been 10.6 6 9.9% and20.9 6 18.1% at 2M and 5M, respectively (90%: 7.1 6 6.6%at 2M and 13.9 6 12.1% at 5M; 80%: 8.0 6 7.4% at 2M and15.76 13.6% at 5M). Fomon et al (41) reported that erythrocyte

incorporation of an orally administered 58Fe tracer was 52% inolder infants (168 d old) compared with 23% in younger infants(56 d old); the use of Fomon et al (41) values of 23% and/or52% would have increased our absolute absorption results fur-ther (23%: 27.8 6 25.8% at 2M; 52%: 24.1 6 20.9% at 5M; or23%: 54.5 6 47.2% at 5M) but would not have changed therelative associations we reported for other observations.

Associations of breast-milk nutrient concentrations and infantiron absorption are presented in Table 4. Breast-milk nutrientconcentrations at 2M were not significantly associated with in-fant iron absorption at 2M (P . 0.05). Breast-milk iron con-centrations at 5M (P = 0.07) and changes in iron concentrationsbetween 2M and 5M (P = 0.04) were associated with increasedinfant iron absorption at 5M. Higher breast-milk copperconcentrations (5M) (P = 0.03) were associated with signifi-cantly increased infant iron absorption at 5M. After adjust-ment for multiple hypothesis testing, these associations wereno longer significant (P , 0.05 but .0.0167). There were nosignificant differences in associations between breast-milk

TABLE 3

Breast-milk nutrient concentrations and infant iron absorption by iron supplementation1

Variable Total (n = 59) +Fe (n = 30) 2Fe (n = 29)

Iron absorption (%)

2M 7.1 6 6.6 8.0 6 8.9 6.3 6 3.65M 13.9 6 12.1 13.0 6 13.3 14.8 6 11.1D2M to 5M 5.2 6 13.3 2.8 6 15.9 7.1 6 11.1

Breast-milk nutrient concentrations

Iron (mmol/L)

2M 8.0 6 3.3 8.1 6 4.0 7.9 6 2.35M 7.4 6 2.9 7.2 6 3.0 7.6 6 2.9D2M to 5M 20.6 6 3.7 20.9 6 4.2 20.2 6 3.0

Copper (mmol/L)

2M 5.1 6 1.5 4.9 6 1.4 5.3 6 1.65M 4.3 6 1.3 4.1 6 1.5 4.4 6 1.2D2M to 5M 20.8 6 1.5*** 20.7 6 1.4 20.8 6 1.7

Zinc (mmol/L)

2M 27.1 6 11.2 26.1 6 10.3 28.3 6 12.15M 17.7 6 7.9 16.1 6 6.2 19.3 6 9.4D2M to 5M 29.5 6 8.3*** 210.0 6 9.1 28.9 6 7.5

1All values are means 6 SDs. For statistical analyses, linear regression was used to examine differences in ironabsorption and breast-milk nutrient concentrations by iron supplementation. ***Remained significant after correction for

multiple hypothesis testing (P , 0.01). 2M, 23 mo of age; 5M, 56 mo of age; 2Fe, infants born to women who received#1320 mg supplemental prenatal Fe; +Fe, infants born to women who received $5100 mg supplemental prenatal Fe.

TABLE 4

Breast-milk nutrient concentrations and infant iron absorption1

Iron absorption (5M) Time point Value P

Breast-milk iron (mmol/L) 2M 20.40 6 0.61 0.525M 1.36 6 0.72 0.07

D2M to 5M 1.12 6 0.53 0.04*Breast-milk copper (mmol/L) 2M 2.89 6 1.46 0.06

5M 3.71 6 1.67 0.03*D2M to 5M 20.14 6 1.49 0.93

Breast-milk zinc (mmol/L) 2M 0.12 6 0.18 0.515M 20.02 6 0.25 0.92

D2M to 5M 20.26 6 0.25 0.30

1All values are means 6 SDs. For statistical analyses, linear regressionwas used to examine associations between breast-milk nutrient concentra-

tions and iron absorption. *NS after correction for multiple hypothesis test-

ing (P , 0.05 but .0.0167). 2M, 23 mo of age; 5M, 56 mo of age.




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nutrient concentrations and iron absorption by gestationaliron exposure.

Results from analyses of predictors of infant iron absorption at2M and 5M are presented in Table 5. Maternal anemia (2M) (P =0.02) and higher infant sTfR concentrations (2M) (P = 0.02)were associated with increased infant iron absorption at 2M. Inanalyses of predictors of iron absorption at 5M, breast-milkcopper concentrations (5M) (P , 0.01) and infant sTfR con-centrations (5M) (P , 0.01) were associated with increasedinfant iron absorption (5M).

Associations of fetal iron exposure and infant length, weight,and ponderal index are presented in Table 6. Fetal iron exposurewas not associated with differences in birth length but was as-sociated with significantly greater infant length at 2M (P ,0.01) and 5M (P , 0.05), indicative of a change in the earlytempo of growth between these 2 groups. There were no sig-nificant effects of fetal iron exposure on infant weight at anytime point (birth, 2M, or 5M). Fetal iron exposure was associ-ated with a significantly reduced ponderal index at 2M (P ,0.05). Results in analyses of fetal iron exposure on infant growthremained similar (and more significant) after adjustment forinfant sex (Table 6), and the effects of iron exposure on infantlength at 2M and 5M remained significant (P , 0.01) afteradjustment for multiple hypothesis testing.

DISCUSSION

In this study, anemia and ID were common in lactating womenat 2 and 5 mo postpartum and in their exclusively breastfedinfants. Although there are limited data on the iron status ofpostpartum women and young infants, the prevalence of anemiaand ID in this study were similar to previous data from resource-limited settings (1, 2) and higher than in studies in pregnantwomen (4245) and older infants (37, 46, 47). In an analysis ofdata from 6 studies in exclusively breastfed infants at 6 mo ofage, the prevalence of ID (SF ,12 mg/L) was 6% in Sweden,17% in Mexico, 1325% in Honduras, and 1237% in Ghana(46). In a previous study in 612-mo-old Peruvian infants, 67%of infants were anemic and 60% were iron deficient (SF con-centration ,20 mg/L) (47). National surveys in Peru have re-ported an anemia prevalence of 69% (68 mo of age) to 72%

(911 mo of age) in older infants (26, 37). However, little dataare available from infants ,6 mo of age, and most data relied oncross-sectional assessments.

The lack of normative iron status data in young, healthy infantsmay have been attributable to challenges in obtaining venoussamples and the belief that, in most instances, the iron endow-ment of birth should be sufficient to maintain early iron statusover the first 6 mo of life. In this study, iron stores of birth weresignificantly depleted between 2 and 5 mo of age; nearly one-third of infants had depleted iron stores (SF concentration ,12mg/L), and 78.9% of infants were anemic by 5M. These resultssuggested that these full-term, exclusively breastfed Peruvianinfants were not endowed with sufficient body iron at birth tomeet requirements for growth and development over the first 46mo of life (48, 49). It is known that delayed cord clamping hasa considerable impact on infant iron status (50, 51) and canincrease neonatal blood volume by 60% (52) and red blood cellsby.30% (53). Although we did not have data on cord-clampingtimes in these infants, delayed cord clamping is routinely un-dertaken in the Villa El Salvador community. Young, exclusivelybreastfed infants may be vulnerable to ID and IDA unless in-testinal iron absorption can be upregulated in response to irondemands.

Infant iron absorption from breast milk in this study (2M: 7.16 6.6%; 5M: 13.96 12.1%) was similar to data from a previousSwedish study in which iron absorption from tracer given witha breast-milk feed averaged 16.46 11.4% in 56-mo-old infantsand 25.7 6 17.2% in 59-mo-old infants (54). However, thesedata were lower than the 36.8 6 9.3% (anemic) and 41.8 67.9% (nonanemic) iron absorption reported in 56-mo-oldnonexclusively breastfed Peruvian infants when tracer was ad-ministered along with breast milk (37).

TABLE 5

Predictors of infant iron absorption1

Variable

Univariate

(P , 0.20)Multivariate

(P , 0.05)

Value P Value P

Iron absorption (2M)

Maternal anemia (2M)2 4.05 6 2.32 0.09 5.44 6 2.24 0.02Infant sTfR (2M) (mg/L) 1.13 6 0.63 0.08 1.47 6 0.59 0.02

Iron absorption (5M)

Breast-milk copper (5M)

(mmol/L)

3.71 6 1.67 0.03 4.06 6 1.46 ,0.01

Infant sTfR (5M) (mg/L) 2.78 6 1.00 ,0.01 2.96 6 0.92 ,0.01

1All values are parameters 6 SEs. For statistical analyses, linear re-gression was used to assess determinants of iron absorption. sTfR, serum

transferrin receptor; 2M, 23 mo of age; 5M, 56 mo of age.2Anemia was defined as hemoglobin concentrations ,12.0 g/dL for

women.

TABLE 6

Maternal prenatal iron supplementation and infant length, weight, and

ponderal index1

Variable 2Fe (n = 29)+Fe (n = 30)

P P2Difference

Length (cm)

Birth 50.2 6 0.3 0.7 6 0.4 0.12 0.162M 58.1 6 0.5 1.9 6 0.7 ,0.01** ,0.01**5M 65.1 6 0.5 1.3 6 0.7 ,0.05 0.01**

Weight (kg)

Birth 3.4 6 0.1 0.2 6 0.1 0.10 0.132M 5.8 6 0.2 0.2 6 0.2 0.24 0.335M 7.6 6 0.2 0.3 6 0.3 0.29 0.29

Ponderal index (g/cm3)

Birth 2.7 6 0.1 0.1 6 0.1 0.51 0.482M 2.9 6 0.1 20.2 6 0.1 ,0.05 0.045M 2.7 6 0.1 20.1 6 0.1 0.46 0.49

1All values are parameters 6 SEs. Infant length, weight, and ponderalindex are presented for the iron-unsupplemented group; the difference

(6SE) is the average difference of infant length, weight, and ponderal indexbetween iron-supplemented and -unsupplemented groups. For statistical

analyses, binomial regression was used to obtain risk-ratio estimates to

examine the effect of fetal iron exposure on categorical outcomes. **Re-

mained significant after correcting for multiple hypothesis testing, P ,0.0167. 2M, 23 mo of age; 5M, 56 mo of age;2Fe, infants born to womenwho received #1320 mg supplemental prenatal Fe; +Fe, infants born to

women who received $5100 mg supplemental prenatal Fe.2After adjustment for infant sex.




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In adults, absorption of nonheme iron is regulated in responseto iron stores (55), but the degree to which this occurs over earlyinfancy is uncertain. Data from Honduras and Sweden suggestedthere is a developmental delay in the regulation of iron ab-sorption, and healthy neonates do not develop the ability toregulate iron absorption in response to iron stores until 69 mo ofage (54, 56). However, a previous study from Peru showed thatinfants with low iron stores (SF concentration ,12 mg/L) up-regulated iron absorption from breast milk at 56 and 910 moof age (37). The current study provides evidence that this effectmay occur even earlier, as early as 25 mo of age.

The effects of maternal iron supplementation on iron statushave been equivocal to date. In the current study, supplementalgestational iron intake had no significant effects on maternal orinfant iron status, breast-milk iron concentrations, or infant ironabsorption in early infancy, although iron intake and dietaryvariability were low in this population (57). Despite a lack ofan impact on iron status, in utero iron exposure significantlyinfluenced early growth potential, as evidenced by increasedinfant length at 2M and 5M. Findings from studies of the effectsof prenatal iron supplementation on early infant growth havebeen inconsistent. In a randomized placebo-controlled trial ofiron supplementation in Niger in pregnant women, prenatal ironsupplementation was associated with significantly increasedinfant length at birth (10). Iron was also associated with increasedinfant length at 3 and 6 mo of age compared with a placebo,although this difference was not statistically significant (10). Ina randomized placebo-controlled trial of iron supplementation inpregnant women in Cleveland, OH, birth length was slightlyhigher in the Fe-supplemented group, although this result was notstatistically significant (58). However, in a meta-analysis, iron-only interventions (n = 27) had no effect on linear growth,whereas multiple micronutrients (n = 20) significantly improvedlinear growth (59). A recent randomized trial in Burkina Fasonoted a significant cumulative effect of both multiple micro-nutrients and iron folate on fetal growth (60). This result wasconsistent with the growing appreciation of the impact of inutero iron status on developmental programming (6163).

In the current study,we examined breast-milk iron, zinc, and copperconcentrations and their associations with infant iron absorption andstatus prospectively. Iron and copper concentrations at 5M andchanges in iron concentrations between 2M and 5Mwere associatedwith significantly increased iron absorption at 5M. Breast-milk iron,zinc, and copper molar concentrations were similar to a previousstudy in Lima, Peru, in breastfeeding mothers at delivery to 6 mopostpartum (64). Breast-milk iron, zinc, and copper concentrationsdeclined between 2M and 5M (P , 0.001).

To date, there has been a limited assessment of iron absorptionin exclusively breastfed, young infants. Few studies haveassessed the impact of fetal iron exposure on early infant ironhomeostasis (65), particularly in ID populations. Our analysiswas distinct from previous studies because of the young age ofinfants studied, assessment of both maternal and infant ironstatus, longitudinal paired iron-absorption measures, selection ofinfants on the basis of gestational iron exposure, and role ofbreast-milk micronutrient ratios in iron absorption. To ourknowledge, this was the first such longitudinal iron absorptionstudy of 25-mo-old infants.

One limitation of this analysis was the lack of data on ma-ternal iron status during pregnancy, delivery practices (eg, cord

clamping), and neonatal iron status at birth. These factorswould have provided additional information on determinants ofearly iron homeostasis. Similarly, maternal supplemental ironintake was based on self-report. Because this study was nota randomized trial of iron supplementation, the study design didnot rule out the possible influence of other potential con-founders, which may have explained the observed associationsbetween iron intake and infant growth. Additional biomarkers,such as hepcidin, or additional measures of inflammation (eg,alpha-1-acid glycoprotein) would have strengthened thisanalysis.

In conclusion, fetal iron exposure did not significantly affectiron status or absorption in young, exclusively breastfed infants at25 mo of age. However, fetal iron exposure across gestationwas associated with significantly improved infant growth, whichwas a finding that warrants additional investigation. Full-term,exclusively breastfed infants from this community may not beendowed with sufficient body iron to meet the iron requirementsneeded for the first 6 mo of life. Young infants are able to up-regulate iron absorption when iron stores are depleted, as earlyas 25 mo of age, but this may not be adequate to meet irondemands. Thus, young exclusively breastfed infants may bevulnerable to ID and IDA and represent an important risk group.Additional attention needs to be focused on optimizing the ironendowment at birth and characterizing sources of variability iniron absorption and determinants of ID across early infancy.Screening and targeted preventive interventions are needed forID and anemia in young infants, particularly in settings withhigh ID burden.

We are grateful to the mothers, children, and field teams, including phy-

sicians, nurses, field staff, laboratory staff, and the administrative staff, who

made this study possible and the Cesar Lopez Silva Hospital Micro Red Villa

El Salvador-Lurin Pachacamac Pucusana and Direccion de Salud Integral

Lima Sur for institutional support.

The authors responsibilities were as followsKOO, SAA, and NZ: de-

signed and conducted the study; JLF: performed statistical analyses and

wrote the initial draft of the manuscript; and all authors: assisted in the

interpretation of data and writing of the manuscript. The funders of the study

had no role in the study design, implementation of data collection, data

analysis, data interpretation, or writing of the manuscript. None of the au-

thors had a conflict of interest.

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Infant iron status affects iron absorption in Peruvian breastfed infants at 2 and 5 mo of age