PEDIATRICS Vol. 106 No. 4 October 2000, pp. 700-706

Prospective Randomized Trial of Early Versus Late Enteral Iron Supplementation in Infants With a Birth Weight of Less Than 1301 Grams

Axel R. Franz, MD^*, Walter A. Mihatsch, MD^*, Silvia Sander, Martina Kron, PhD, and Frank Pohlandt, MD, MSc^*

From the ^* Department of Pediatrics, Division of Neonatology and Pediatric Critical Care, and the  Department of Biometry and Medical Documentation, University of Ulm, Ulm, Germany.

	ABSTRACT

Top Abstract Methods Results Discussion Conclusion References

Objectives.  To examine whether early enteral iron supplementation (EI) would improve serum ferritin as a measure of nutritional iron status at 2 months of age and would prevent definite iron deficiency (ID) in infants with a birth weight of <1301 g.

Methods.  Infants were randomly assigned to receive enteral iron supplementation of 2 to 6 mg/kg/day as soon as enteral feedings of >100 mL/kg/day were tolerated (EI) or at 61 days of life (late enteral iron supplementation [LI]). Nutritional iron status was assessed: 1) at birth, 2) at 61 days of life, 3) when the infants reached a weight of 1.6 times birth weight, and 4) before blood was transfused at a hematocrit of <.25. ID was defined by any one of the following criteria: ferritin, <12 µg/L; transferrin saturation, <17%; or increase of absolute reticulocyte counts by >50% one week after the onset of enteral iron supplementation. Restrictive red cell transfusion guidelines were followed and all transfusions were documented. Erythropoietin was not administered. The primary outcome variables were: 1) ferritin at 61 days and 2) the number of infants with ID.

Results.  Ferritin at 61 days was not different between the groups. Infants in the LI group were more often iron-deficient (26/65 vs 10/68) and received more blood transfusions after day 14 of life. No adverse effects of EI were noted.

Conclusions.  EI is feasible and probably safe in infants with birth weight <1301 g. EI may reduce the incidence of ID and the number of late blood transfusions. ID may occur in very low birth weight infants despite early supplementation with iron and should be considered in the case of progressive anemia.preterm infant, iron supplementation, iron deficiency, blood transfusion.

The smaller preterm infants are at birth, the more susceptible they are to iron deficiency (ID) owing to their proportionately smaller iron store at birth^1,2 and their higher relative growth in the first months of life. However, little is known about the optimal time and dose for iron supplementation in extremely low birth weight infants. In infants with a mean birth weight of 1650 g who did not receive blood transfusions, 2 mg/kg/day of enteral iron starting at 2 weeks of age was sufficient to prevent ID at 3 months of age,³ whereas multiply transfused extremely low birth weight infants may have high iron stores without iron supplementation up to 16 weeks of age.⁴

In 1985, the Committee on Nutrition of the American Academy of Pediatrics recommended to start iron supplementation at a dose of 2 to 3 mg/kg/day at 2 months of age or at latest once the low birth weight infant reaches ~2000 g in weight and/or goes home.⁵ This recommendation has not been updated despite the fact that much smaller infants survive today than have survived in the 1980s, reflecting the need for further studies.

This is the first randomized trial of early enteral iron supplementation (EI) in infants with birth weight <1301 g who do not receive erythropoietin. We hypothesized that EI: 1) will improve nutritional iron status as measured as serum ferritin at 61 days of age and 2) will reduce the number of infants with definite ID if compared with a standard iron supplementation according to current recommendations.

	METHODS

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The study was approved by the ethics committee of the University of Ulm and written informed parental consent was obtained.

Setting

The study was conducted at a level 3 neonatal referral center and its affiliated children's hospitals caring for a population of ~2 million.

Study Population

All inborn infants with a birth weight of <1301 g admitted between June 1996 and June 1999. Exclusion criteria were major anomalies, hemolytic disease, twin-to-twin transfusion syndrome, and missing parental consent.

Infants were assigned to 1 of 2 strata according to the need for blood transfusion within the first 7 days of life (stratum 1: no transfusion; stratum 2: 1 transfusion within the first 7 days of life). At day 7 of life, the infants were randomly allocated in blocks of 10 within each stratum to 1 of 2 treatment strategies.

Treatment Groups

EI was started at a dose of 2 mg/kg/day³ of ferrous sulfate as soon as 100 mL/kg/day of enteral feedings were tolerated. The dose was increased to 4 mg/kg/day when the hematocrit fell below .30.

Late enteral iron supplementation (LI) was started at 61 days of life at a dose of 2 mg/kg/day.⁵ If ID was diagnosed at any time throughout the study, iron was started at 4 mg/kg/day.

In both groups, iron was administered with the milk feeds.⁶

Dietary Regimen

Infants of both groups received either protein and energy enriched milk of their own mother (not containing supplemental iron) or an iron-fortified preterm infant cow's milk formula (12 mg of iron/L).⁷ It was aimed to achieve a protein intake of 3.5 to 4.0 g/kg/day⁸ and an energy intake of 500 to 550 kJ/kg/day.

Treatment Guidelines

Erythropoietin was not administered. Restrictive red cell transfusion guidelines were followed. Infants who were: 1) on mechanical ventilation with a fraction of inspired oxygen of >.25, or 2) 7 days of age with a birth weight of <1000 g, or suffered from 3) cyanotic heart disease, or 4) septic shock, or 5) necrotizing enterocolitis, were not transfused unless their hematocrit fell below .40. Infants who were: 1) on mechanical ventilation with a fraction of inspired oxygen of <.26 or 2) breathing spontaneously with a fraction of inspired oxygen of >.25, or 3) breathing spontaneously with apnea or desaturations were not transfused unless their hematocrit fell below .30. Healthy growing infants were not transfused unless their hematocrit fell below .21. All transfusions were prospectively recorded.

Hematologic and Biochemical Determinations

Ferritin, transferrin, iron, reticulocytes, and blood count were determined: 1) at birth, 2) when the infants reached a weight of 1.6 times their birth weight, 3) at 61 days of life, and 4) at any time when hematocrit fell below .25 and blood was taken for cross-match before transfusion. Blood count and reticulocyte counts were repeated at day 68 of life. The laboratory personnel handling the blood samples were unaware of the infant's group assignment.

Ferritin, transferrin, and iron were determined from serum or lithium-heparin plasma. Blood samples were centrifuged within 2 hours after sampling.

Ferritin was measured by an enzyme immunoflourescence assay (AIA21, Tosoh/Eurogenetics, Brussels, Belgium). The detection limit was 1.5 µg/L. The assay was calibrated up to 1000 µg/L, and interassay variations were <6% at 18.8, 242, and 658 µg/L. Transferrin was measured by rate nephelometry (Beckman Array 2.0, Beckman Instruments, Munich, Germany). The detection limit was .75 g/L. The assay was calibrated up to 7.5 g/L, and interassay variations were <5% at 1.43, 2.97, and 4.42 g/L. Iron was measured by the Ferrozine method (Dimension XL, Dade Behring, Marburg, Germany). The detection limit was 2 µmol/L. The assay was calibrated up to 180 µmol/L, and interassay variations were <1.4% at 13.0, 16.8, and 34.6 µmol/L. The transferrin saturation (TS) was calculated as TS (%) = 3.98 × iron (µmol/L)/transferrin (g/L).⁹

Ferritin, transferrin, and TS values were only evaluated if C-reactive protein was normal from 48 hours before to 48 hours after blood sampling, and the patient was not treated for infection at that time.

Complete blood counts were performed on a cell counter (Abbott CD3500, Abbott, Wiesbaden, Germany). Reticulocytes were manually counted by 4 experienced technicians.

Definition of ID

The diagnosis of ID was made based on at least one of the following criteria: 1) ferritin: <12 µg/L,^3,10-15 2) TS: < 17%,^11,13,16 or 3) a relative increase of absolute reticulocyte counts by >50% one week after the onset of enteral iron supplementation.^12,15

Statistical Analysis

Primary outcome variables were: 1) ferritin at 61 days of life and 2) the number of infants who fulfilled the criteria of ID at any time throughout the study. The study hypotheses were tested as a-priori-ordered hypotheses; therefore, no Bonferroni-Holm correction for multiple testing was required.

The sample size calculation for a 1-sided test problem was based on an expected difference of natural logarithm ferritin at day 61 of .5³ and an estimated standard deviation of natural logarithm ferritin of .95 (twice that found in term infants according to reference 12). With an  = .05 and a power of 90%, 63 patients were required in each group.

Secondary outcome variables were: TS, hematocrit, reticulocyte count, mean corpuscular volume, and mean corpuscular hemoglobin at day 61, number of infants who required transfusions at days 14 to 68, and blood volume transfused at days 14 to 68.

All continuous measures were compared using 1-sided Mann-Whitney U test for primary outcome variables and 2-sided Mann-Whitney U test for secondary outcome variables. Dichotomous outcome measures were analyzed using Fisher's exact test.

	RESULTS

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From June 1996 through June 1999, 380 inborn infants with a birth weight of <1301 g were admitted, of whom 204 were randomized (Fig 1). Because of death, early discharge or referral to peripheral children's hospitals not participating in the trial, 71 patients were lost to follow-up before day 68 of life. One hundred thirty-three infants completed the protocol.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Trial profile.

The demographic data of patients randomized and patients who completed the protocol are shown in Tables 1 and 2. Patients in both study groups were very similar for gestational age, birth weight, and Clinical Risk Index for Babies score.¹⁷ Markers of nutritional iron status were similar in both groups at birth (Tables 1 and 2). There was a similar incidence of necrotizing enterocolitis and intraventricular hemorrhage in both groups and there was a trend toward more infants with chronic lung disease and severe retinopathy of prematurity in the LI group (Tables 1 and 2). Of a total of 306 red cell transfusions, only 10 (6 in the EI group and 4 in the LI group) were not in agreement with the transfusion guidelines.

Markers of nutritional iron status were not different between groups at 61 days of age (Table 3). However, 10 of 68 infants in the EI group fulfilled criteria of ID, compared with 26/65 in the LI group. Fewer infants with EI received blood transfusions after day 14. All secondary outcome variables are shown in Table 3.

In infants who had not been transfused after day 14 of life, median and range of ferritin at day 61 in the EI group were 33.5 µg/L (9-189) and in the LI group were 23.0 µg/L (9-61; P < .05). The median and range of TS at day 61 in these infants were 29% (13-63) and 22.5% (11-44), respectively (P < .02).

Fifty-three of 65 infants (82%) in the LI group were either transfused after day 14 or fulfilled criteria of ID, compared with 36 of 68 (53%) in the EI group (P < .001).

Ten patients in the EI group were incorrectly started on iron >7 days (9-33 days) after they had reached enteral feedings of >100 mL/kg/day. Seven patients of the LI group were started on iron >1 week before day 61 of life because of ID. All these patients were evaluated within the assigned group on an intent-to-treat basis. A per-protocol analysis resulted only in minor changes (not shown).

Of the 68 infants in the EI group, only 11 did not tolerate enteral feeding after the introduction of enteral iron for a mean duration of 8.9 dayssimilar to the incidence of feeding intolerance observed in the LI group. None of these episodes of feeding intolerance was thought to be related to EI, and feeding intolerance did not recur in these infants after EI was resumed as soon as enteral feedings of >100 mL/kg/day were tolerated.

Taking into account both supplemental iron and iron from milk feeding and assuming that breast milk contained 1 mg iron/L, total enteral iron intake during the first 61 days of life was 181.7 ± 55.7 mg/kg (mean ± standard deviation) in the EI group and 52.4 ± 29.8 mg/kg in the LI group. Iron intake from iron-supplemented formula was similar in both groups.

	DISCUSSION

Top Abstract Methods Results Discussion Conclusion References

This study of 133 infants with a birth weight of <1301 g showed that ID commonly occurs during the first 2 months of life in very premature infants and that EI may not only reduce the incidence of ID but also the need for blood transfusions after the second week of life.

Erythropoiesis depends on adequate iron stores: iron supplementation of 2 mg/kg/day starting at 2 weeks of life in nontransfused preterm infants of >1000 g of birth weight was associated with higher hemoglobin levels at 2 to 6 months of age,³ and iron supplementation may both increase the endogenous erythropoietin production (in mice and rabbits)^18,19 and the response to exogenous erythropoietin in premature human infants.²⁰ In agreement with this data, this study showed that iron supplementation enhanced erythropoiesis in very premature infants even without exogenous erythropoietin: supplemented infants tended to have higher reticulocyte counts at the same hematocrit and achieved similar hemoglobin levels or hematocrits at 61 days of life despite receiving lower numbers and volumes of red blood cell transfusions (Table 3).

Despite 6 weeks of iron supplementation in the EI group, measures of iron stores were similar at 61 days of life. Considering the increased number of infants with ID in the LI group, similar ferritin levels in both groups can only be explained by the increased number and volume of blood transfusions administered to infants in the LI group and not by ineffective iron supplementation to the EI group. In fact, in infants who had not been transfused after day 14, ferritin and TS were reduced in the LI group. Furthermore, 82% of infants in the LI group were either iron-deficient or received blood transfusions after day 14, compared with 53% in the EI group.

Transfusion requirements are primarily determined by iatrogenic blood losses^21-25 and closely correlate to disease severity.²⁶ In agreement with these data, both the number of transfusions and the volume transfused were similar in both groups during the first 2 weeks of life, ie, when iatrogenic blood losses were highest. However, EI resulted in a reduced need for late blood transfusions after day 14, ie, when diagnostic blood sampling became minimal and was at least in part compensated by erythropoiesis.

Planning this study, we expected that a difference in ID between the groups could only be a consequence of different iron stores. Therefore, the study was designed to test the 2 hypotheses that EI: 1) would increase ferritin at day 61, and 2) would decrease the incidence of ID as a-priori-ordered hypotheses, thereby increasing the power of the study. However, because of this design, the second hypothesis could not be tested, because we were unable to show a difference in ferritin levels. Ideally, this study should have been conducted as double-blinded trial. However, blinding for enteral iron supplementation is impossible because of the effect of enteral iron (given as single daily dose) on the stool color. As in any open-label study, there may be bias in favor of the intervention group (ie, in favor of EI). Despite our effort to strictly implement guidelines, bias may in part account for a difference in late transfusions between the groups. However, the primary outcome variables of this study were objective, predefined laboratory criteria and the laboratory personnel were unaware of patient assignment.

A further shortcoming of this study is that although in the LI group reticulocyte counts were measured on day 61 and day 68 to detect an increase in reticulocytes after initiation of iron supplementation on day 61, in the EI group reticulocytes were measured on the same days but under unchanged iron supplementation. Preferably, iron supplementation should have also been increased by 2 mg/kg/day on day 61 in the EI group to match the iron supplementation started on that day in the LI group. If only the first 2 criteria for ID (ie, ferritin: <12 µg/L and TS: <17%) are considered to exclude potential bias from the third criterion, there is still a trend toward more ID in the LI group (10/68 vs 86/65; P = .11), and there would have been significantly more ID in the LI group (12/68 vs 84/65; P = .01) if the threshold for ferritin had been set to 15 µg/L (a threshold previously suggested by other investigators²⁷).

The gold standard for the definition of ID is the lack of iron stores in bone marrow and liver biopsies, but low levels of serum ferritin and TS in combination with a microcytic anemia^16,28 or an increase of hemoglobin after the onset of iron supplementation¹⁵ are also widely accepted as signs of ID. In contrast to adult patients, red cell indices (such as mean corpuscular volume and mean corpuscular hemoglobin) are not useful for the diagnosis of ID anemia in newborn infants, especially if born prematurely and multiply transfused, and were not evaluated in this study. Ferritin and transferrin are acute phase proteins, permitting interpretation only in the absence of inflammation.¹⁰ To overcome this limitation only values of ferritin, transferrin and iron taken at times when the C-reactive protein was normal and infants did not receive antibiotic therapy for suspected infection were evaluated.

There is controversy in the literature about which cutoff of ferritin or TS truly represents ID, but levels of 10 µg/L for ferritin¹² and 16% for TS^11,16 seem to be widely accepted. The significance of these levels in growing preterm infants has not been examined. Considering that ferritin levels as high as 60 µg/L represent the fifth percentile in healthy term infants at birth,^12,29 ferritin levels of 20 to 30 µg/L may seem to be far too low to ensure proper development, especially because up to 80% of infants with hematologic signs of ID anemia may have ferritin levels above 11 µg/L.¹² In contrast, it is well known that ferritin levels decline rapidly after birth in healthy term infants and values as low as 8 µg/L were reported in apparently healthy populations.¹⁰

Similarly, although TS of down to 10% have been repeatedly reported in healthy infants,^30-32 authors of erythropoietin trials carefully kept TS above 30% to allow maximal erythropoiesis.²³

One of the concerns with EI may be that free ferrous iron is thought to fuel the production of free radicals and, thereby, to increase the oxidative stress especially in the premature infant who has a limited capacity to assimilate free iron and to degrade free radicals³³ (also reviewed in references 34 and 35). Unfortunately, we have not been able to measure free iron in our patients. Although free iron could theoretically be increased by EI, the reduction of blood transfusions in the EI group may balance this theoretical risk. So far, an increased incidence of diseases thought to be related to oxidative stress (eg, retinopathy of prematurity and chronic lung disease) has only been reported with frequent blood transfusions.^33,36-38 In contrast, feeding an iron-fortified infant formula (8 mg/L) to preterm infants did not increase markers of oxidative stress.³⁹

In this study, an increased incidence of severe retinopathy was found in the LI group, ie, in infants who received more late blood transfusions but who did not receive enteral iron. However, this is most likely a chance finding and not a true protective effect of EI.

Although EI may theoretically increase the formation of free radicals to some degree, it is unclear what in the end would be more harmful to the infant: more stress caused by reactive oxygen species, which theoretically may be involved in diseases of prematurity, or ID, which has been proven to cause neurodevelopmental impairment,^40,41 or increased need for blood transfusions with the risk of viral infections and of retinopathy of prematurity³⁶ and bronchopulmonary dysplasia.^37,38

Enteral iron supplementation is thought to have few side effects in infancy,^42-44 and despite high doses of enteral iron supplementation (up to 36 mg/kg/day⁴⁵) during numerous trials of erythropoietin for the anemia of prematurity, no side effects of such iron supplementation have been reported. In agreement with these reports, EI was well-tolerated in this study. A similar incidence of necrotizing enterocolitis (6/105 in the EI group and 8/99 in the LI group) and late-onset bacterial infections (76 episodes in 88 EI infants vs 84 episodes in 93 LI infants) was found in both study groups, and hemolysis was not observed in our study population under routine vitamin E administration of ~2 mg/kg/day.

Despite increasing iron supplementation up to 4 mg/kg/day (in formula-fed infants up to 6 mg/kg/day) as soon as the hematocrit fell below .30, a considerable number of cases of ID occurred in the EI group, suggesting that enteral iron of up to 6 mg/kg/day may not be sufficient to meet the needs of growing preterm infants. Considering that only ~8% (range: 1.8%-13.7%) of enteral iron are absorbed when iron is given with the formula and only ~10% (range: 4.1%-16.3%) are absorbed when iron is given in between the feeds,⁶ some infants may require supplementation with up to 12 to 16 mg/kg/day of enteral iron to achieve only approximately one half of the fetal accretion rate (ie, an iron retention of ~500 µg/kg/day⁴⁶)even if iron losses were negligible. In addition to individual differences in iron absorption, iatrogenic iron losses also vary between individuals. Therefore, far higher doses than 4 to 6 mg/kg/day of enteral iron may be necessary in some infants to prevent ID.

As an alternative to oral iron administration, intravenous administration of iron to preterm infants has been shown to be effective and seemed to be safe^46-48 and should be considered if oral iron supplementation is ineffective or impossible.

	CONCLUSION

Top Abstract Methods Results Discussion Conclusion References

We conclude that enteral iron supplementation as soon as enteral feeding (100 mL/kg/day) is tolerated is feasible in preterm infants with birth weight <1301 g. This EI in combination with sufficient protein intake may reduce the incidence of ID and the need for blood transfusions. Nevertheless, ID should be considered in extremely premature infants who become progressively anemic, despite enteral iron administration of up to 6 mg/kg/day.

	ACKNOWLEDGMENTS

We acknowledge the support by the staff at the affiliated Children's Hospitals at Aalen, Esslingen, Friedrichshafen, Göppingen, Ravensburg, Schwäbisch Gmünd, and Stuttgart.

We also thank Sabine Schmid for maintaining the study database.

	FOOTNOTES

Received for publication Sep 28, 1999; accepted Jan 20, 2000.

This work was presented in part at the Annual Meeting of the European Society for Pediatric Research; June 27-29, 1999; Copenhagen, Denmark.

Reprint requests to (A.R.F.) Division of Neonatology and Pediatric Critical Care, University Children's Hospital, Prittwitzstr 43, 89075, Ulm, Germany. E-mail: axel.franz{at}medizin.uni-ulm.de

	ABBREVIATIONS

ID, iron deficiency; EI, early enteral iron supplementation; LI, late enteral iron supplementation; TS, transferrin saturation.

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Top Abstract Methods Results Discussion Conclusion References

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