Premature infants are at risk for low iron storage, iron deficiency (ID), and anemia. Erythropoietin (EPO) has been used in preterm infants to prevent and treat anemia, and is usually given 3 times a week as a subcutaneous injection. The iron store is low in preterm infants and rapidly gets depleted during the first 6 to 8 weeks.[2,3] As iron is needed for various tissue functions, iron supplementation is essential for preterm infants. Oral iron therapy is not well tolerated due to gastrointestinal immaturity in preterm infants and hence intravenous iron therapy appears to be an alternative treatment strategy.[4,5] Guidelines on pediatric parenteral nutrition (PN) suggest a dose of 200 μg/kg/d iron,[6–8] but the time of initiation of therapy remains controversial in these infants. In the present study, we aimed to elucidate whether parenteral iron combined erythropoietin (EPO) could improve anemia in preterm infants.
2 Patients and methods
We recruited 96 preterm infants with gestational age between 28 and 34 weeks treated in the neonatal intensive care unit (NICU) of the First People's Hospital in Kunshan, Jiangsu University between February 2014 and June 2014. Exclusion criteria were infants with (1) liver and kidney dysfunction; (2) hemolytic disease of newborns; (3) hemorrhage (gastrointestinal, pulmonary, and intracranial hemorrhage grade III–IV, 24 h decline in Hb> 2 g/L); (4) blood transfusion; (5) major or life-threatening malformations. Approval was obtained from the Ethics Committee of the First People's Hospital in Kunshan, Jiangsu University, and the study was registered with ClinicalTrials.gov (Identifier: NCT02060851). Parents gave informed consent.
The study was a single-blind, randomized, and controlled clinical trial. The patients were randomly assigned to 1 group in the following manner: treatment cards were imprinted with a unique randomization code and placed in sequentially numbered, opaque envelopes. At the study site, the treatment cards were taken out in the sequential order, and the patients were assigned to the corresponding treatment group based on the randomization number. All investigators, physicians, and nurses involved in patient care and parents were blinded to this assignment. The randomization process was made available only to the pharmacist who supervised the quality of iron sucrose (IS) and the PN preparation. Study infants were followed up until discharge from the hospital. Infants were withdrawn from the trial if severe adverse effects developed or parents withdrew the consent.
2.3 Dosing of study drug
Infants were randomized to 3 groups: (1) group 1—a control group who received standard PN; (2) group 2—an iron-supplemented group (IS); (3) group 3—iron-supplemented combined EPO group (IS+EPO). EPO, 400U/kg, was given twice a week (Monday and Friday). IS, 200 μg/kg per day, was given everyday with PN, and the dose was continued until 2 weeks after birth. The patients were treated for 2 weeks, or as long as they received parenteral nutrition. Criteria for discontinuation of drug administration included a systemic adverse reaction to iron such as hypotension, respiratory compromise, rash, or hypertension. All infants received parenteral nutrition from the second day of birth until infants could receive 120 mL/kg per day of enteral feedings.
2.4 Nutrition support
Parenteral nutritional support for the study neonates was provided according to published guidelines. The “all-in-one” solution contained lipids (20% lipfundin; B. Braun), amino acids (6% pediatric amino acid), glucose, minerals, trace elements, and vitamins. The infants in group 2 received iron and group 3 received EPO along with PN. All preterm infants were fed standard preterm formula (SPF), NeoSure or Similac, which contained 0.9 mg of iron per 100 mL. The formula was started with 10 mL/kg per day and increased at 10 mL/kg per day until 150 mL/kg per day. The enteral feeding was stopped when infants developed symptoms of necrotizing enterocolitis (NEC). The amount of enteral nutrition and the symptoms of feeding intolerance were recorded.
2.5 Data collection
The general information, number of ventilator days, length of stays in hospital, number of blood examination in hospital and incidence of NEC and retinopathy of prematurity (ROP) were recorded. The levels of blood sugar and liver and renal function were examined before and after the interventions. Assessment of Hb levels was the primary objective, whereas, the secondary objectives included assessment of RBC count, mean corpuscular volume (MCV), serum iron (SI), transferrin saturation (TS), ferritin, the percentage of reticulocyte count (RET), total iron binding capacity (TIBC), and malondialdehyde (MDA) and superoxide dismutase (SOD) at baseline and 2 weeks after supplementation. RBC count, Hb and MCV levels were measured at corrected ages of 1 month and 3 months. A radio-immunoassay method, chemical method, and an automatic biochemical analyzer was used to test the serum ferritin (DADE BEHRING, Germany), serum iron (automatic biochemical analyzer, 7600, HITACHI), respectively. The extraction method was used to test SOD (SOD Assay Kit, Nanjing built biological science and Technology Co., Ltd) and thiobarbituric acid (TBA) method was used to test MDA (MDA Assay Kit, Nanjing built biological science and Technology Co., Ltd).
2.6 Statistical analyses
The sample size was 21 for each group, which calculated according to 0.05 as type I error of 5%, a power of 80% and 20 g/LHb as clinically significant increase. Considering a dropout 20%, we enrolled 26 patients for each group and 78 patients in total. The data was analyzed using SPSS 21.0 for the Windows software, and the results are expressed as mean ± SD (x ± s) or median and interquartile range. Differences in rates were analyzed by the chi-square test. P<0.05 was considered statistically significant.
3.1 General characteristics
Of 96 premature infants enrolled, 91 infants completed the study (30 belonged to the control group, 31 to the IS group, and 30 to the IS+ EPO group). Five infants could not complete the study for the following reasons: 1 in the IS group died because of respiratory failure, and 4 infants (1 in the control group, 1 in the IS group, and 2 in the IS+PN group) were discharged because treatments were discontinued by their parents. There were no significant differences between the baseline characteristics among 3 groups (Table 1).
3.2 Hematological evaluation
The percentages of RET in the IS and IS+EPO groups (2.1 ± 0.4 and 2.5 ± 0.3, respectively) were significantly higher than those in the control group (1.7 ± 0.3) (Table 2). There were no significant differences in the other indicators of anemia, including RBC count, Hb, hematocrit, and MCV among 3 groups.
3.3 Iron storage evaluation
TIBC in the IS and IS+EPO groups (36.7 ± 4.6 and 36.0 ± 4.7, respectively) were significantly lower than those in the control group (41.6 ± 5.2) (Table 2). There were no significant differences in the indicators of iron storage, including SI, SF, and TS, among the 3 groups.
3.4 Comparison of oxidative stress
The levels of MDA and activity of SOD were not significantly different between preintervention and postintervention among the 3 groups (Table 2).
3.5 Follow-up results
RBC, Hb levels, MCV levels, body weight, body length, and head circumference at a corrected age of 1 month did not differ among 3 groups. Infants in the control group were significantly more likely to have abnormal Hb and MCV levels at a corrected age of 3 months, whereas RBC, body weight, body length, and head circumference among 3 groups were not significantly different (Table 3). None of the patients developed NEC and ROP during the study period.
The present study has demonstrated that the percentages of reticulocyte in the iron-supplemented and iron-supplemented with EPO groups of preterm infants were significantly higher after 2 weeks of therapy. TIBC in the iron-supplemented and iron-supplemented with EPO groups were significantly lower than the TIBC in the control group. At 3 months of age, Hb in the iron-supplemented and iron-supplemented with EPO groups was significantly higher than those in the control group, and MCV in iron-supplemented groups were greater than those in the control group. Early parenteral iron supplementation, mostly from day 2 of life, combined with erythropoietin in preterm infants improved reticulocyte counts and decreased total iron binding capacity without influence on other indices, and improved the Hb and MCV levels at 3 months of age. Therefore, early parenteral iron supplementations with EPO were found to be beneficial for the preterm infants.
The study infants tolerated iron well, there was no rash, fever, or other allergic reactions, and the safety of iron dextran has been confirmed.[8,10] Statistical comparison between groups showed that iron did not increase oxidative stress (P> 0.05). However, the number of cases and the study time was limited, and long-term follow-ups could not be performed.
The percentages of RET in the iron-supplemented and iron-supplemented with EPO groups was significantly higher than those in the control group. Robin et al, demonstrated that preterm infants responded to weekly EPO by increasing the percentages of RET and maintaining hematocrit. There were no significant differences in the other indicators of anemia including RBC, Hb, hematorit, and MCV between the 3 groups. Surico et al showed that after 6 days of intravenous iron sucrose therapy in preterm infants, hemoglobin significantly improved. A study on neonatal dairy calves showed parenteral supply of iron can improve mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and iron level. Two studies had shown that intravenous iron therapy can reduce the number of blood transfusions, and the amount of EPO needed.[14,15] There were no differences in the indicators of anemia in the iron-supplemented and iron-supplemented with EPO groups, as compared with the control group. Infants in the control group were significantly more likely to have abnormal Hb and MCV levels at a corrected age of 3 months. This observation could have resulted from use of lower dosages of iron than the dose of 200 μg/kg per day, which is the recommended amount. Another reason could be the short duration of iron intervention and the early application of EPO.
The indicators of iron storage include SI, SF, TIBC, and TS. TIBC in the iron-supplemented and iron-supplemented with EPO groups was significantly lower compared to the control group. There were no significant differences in the other indicators of iron storage, including SI, SF, and TS, among the 3 groups. Previous studies have shown that 120 μg/kg/d of intravenous iron can maintain positive iron balance in preterm infants; however, in the present study, the duration of intervention was short. Serum ferritin is the body's most sensitive indicator of iron deficiency. Pollak and co-workers demonstrated significantly increased serum ferritin levels after 11 days of intravenous iron and EPO in preterm neonates with anemia compared with the control group.
MDA, SOD, and glutathione peroxidase (GSH-Px) in blood or urine are the main indicators of oxidative stress. Pollak et al found no difference in oxidative stress in the intravenous iron treated preterm infants compared with the control group, and iron sucrose caused only a transient increase of MDA in 4 patients. Our study results showed that the indicators of oxidative stress (MDA and SOD) were not significantly changed (P>0.05), indicating 200 μg/kg of iron per day in preterm PN did not increase oxidative injury or decrease antioxidative capacity. Whether intravenous iron is directly involved in oxidative stress in preterm infants needs to be explored.
In summary, preterm infants at risk of iron deficiency should be identified and treated with iron supplementation. Early parenteral iron supplementations with EPO were beneficial for the preterm infants. The present study results agree with European Society of Parenteral and Enteral Nutrition (ESPEN) recommendation that early intravenous iron added to PN, and EPO given for 2 weeks could significantly improve TIBC and reticulocyte counts, and improve the Hb and MCV levels at 3 months of age. The potential effect of Epo as a neuroprotective agent for preterm brain needs a longer follow-up period. The effects of longer duration and larger dose of intravenous iron supplementation and the potential beneficial effects of iron on long-term neurocognitive and psychomotor development warrant future study.
The colleagues of the department of laboratory in the First People’ Hospital of Kunshan are gratefully acknowledged, for assisting in laboratory work. The authors also thank Rongzhu Lu (PhD scholar of Jiangsu University) and Cuihua Huang for helping in statistical analysis.
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