Effect of enteral bovine lactoferrin on neonatal iron status

Mohammed El Barbary; Nancy A Shady; Hebatallah A Shaaban; Menat A.A Shaaban; Ola Y Ahmed

doi:10.4103/ejh.ejh_30_18

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ORIGINAL ARTICLE

Year : 2018 | Volume : 43 | Issue : 4 | Page : 212-216

Effect of enteral bovine lactoferrin on neonatal iron status

Mohammed El Barbary¹, Nancy A Shady¹, Hebatallah A Shaaban¹, Menat A.A Shaaban², Ola Y Ahmed¹
¹ Department of Pediatrics, Faculty of Medicine, Ain Shams University, Cairo, Egypt
² Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Date of Submission	13-Aug-2018
Date of Acceptance	29-Aug-2018
Date of Web Publication	10-Apr-2019

Correspondence Address:
Menat A.A Shaaban
lecturer in department of Clinical Pathology, Faculty of Medicine, Ain Shams University. Abbassia Square, PO box 11381, Cairo
Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/ejh.ejh_30_18

Abstract

Background All infants experience a decrease in hemoglobin (Hb) soon after birth, which results in varying degrees of anemia. Oral bovine lactoferrin (LF) supplementation, an iron-binding glycoprotein, is a promising therapy for iron-deficiency anemia.
Objectives To evaluate the effect of enteral LF supplementation on the levels of Hb, hematocrit, and serum ferritin in infants admitted in the neonatal intensive care units. This was an interventional double-blind trial conducted on 52 neonates who were randomized into LF group (n=26) and placebo group (n=26). LF was administered at a dose of 100 mg/day once by enteral route starting from birth to 30th day of life. Complete blood count and serum ferritin were assessed in patients at 7th and 30th day of life.
Results There were no significant differences between both the studied groups as regards serum ferritin, Hb, hematocrit, mean corpuscular volume, red cell distribution width, platelet count, and total leukocytic count on day 7. There were statistically significant higher serum ferritin, Hb, hematocrit, and mean corpuscular volume, and lower red cell distribution width and total leukocytic count in the LF group than the placebo group on day 30. The placebo group had significantly higher mortality than LF group (19.2% vs. zero; P=0.051).
Conclusion Bovine LF is an effective and safe therapy to prevent anemia in neonates.

Keywords: ferritin, hematocrit, hemoglobin, lactoferrin, neonates

How to cite this article:
El Barbary M, Shady NA, Shaaban HA, Shaaban MA, Ahmed OY. Effect of enteral bovine lactoferrin on neonatal iron status. Egypt J Haematol 2018;43:212-6

How to cite this URL:
El Barbary M, Shady NA, Shaaban HA, Shaaban MA, Ahmed OY. Effect of enteral bovine lactoferrin on neonatal iron status. Egypt J Haematol [serial online] 2018 [cited 2023 Jun 5];43:212-6. Available from: http://www.ehj.eg.net/text.asp?2018/43/4/212/255876

Introduction

All term and preterm infants experience a decrease in hemoglobin (Hb) during the 8–10 postnatal weeks, which results in varying degrees of anemia. This is due to multiple physiological and nonphysiological factors; a decline in erythropoietin due to the transition from a relatively hypoxic state in utero to a relatively hyperoxic state after birth, nutritional deficiencies of iron, vitamin E, vitamin B₁₂, and folate may exaggerate the degree of anemia and reduce the red cell life span. Among the preterm infants, the expected decline in Hb is more severe than for term infants, and unlike term infants, the more rapid and profound fall in Hb may be associated with clinical signs of anemia [1],[2].

Iron is an essential micronutrient that plays an important role in many cellular functions. It is thought to be particularly important for neonates, especially preterm infants [3]. Thus, iron supplementation is essential for normal growth and development. However, the pro-oxidant role of nonprotein-bound iron and the poorly developed antioxidant measures in preterm infants cautions against aggressive iron supplementation in this population. There are wide variations in iron supplementation practices among neonatal intensive care units (NICU) [4].

Lactoferrin (LF) is predominately available in mammalian milk and are secreted by exocrine glands and by neutrophils. Relative high levels of LF are observed in colostrum, human breast milk, and in other body secretions like tear, seminal plasma, and vaginal secretions [5],[6]. LF (also referred as lacto-transferrin) is an iron-binding glycoprotein with anti-inflammatory, immunomodulatory, antimicrobial, anticarcinogenic, antioxidant properties, and is an enhancer of iron absorption [6],[7],[8]. In infants, it binds a major proportion of iron in breast milk, and is not readily digested and thus could enhance the iron absorption in the small intestine. Consequently, exclusive breastfed infants usually have satisfactory iron status by 6 months of age though breast milk is low in iron content [7],[9]. Thus, LF is emerging as an important regulator of systemic iron homeostasis [10].

Human LF possesses high sequence homology with bovine lactoferrin (BLF). Therefore, the majority of the in-vitro and in-vivo studies have been carried out using BLF [5].

The present study was designed to evaluate the effect of enteral BLF supplementation on Hb, hematocrit, and serum levels of ferritin in infants admitted in the NICU.

Patients and methods

The present double-blinded cohort study took place in the NICU, Faculty of Medicine, Ain Shams University. The mothers of the newborns were provided written consent for the protocol, which was approved by the Research Ethics Committee of Ain Shams University Hospitals (ID: FMASU MS 27/2017 and are in accordance with the Helsinki Declaration of 1975).

Fifty-two neonates were simply randomized according to the sequence of enrollment:

The LF cases group consisted of 26 neonates who received LF granules Pravotin (Produced in Egypt by: Medizen Pharmaceutical Industries. For: HYGINT Pharmaceuticals) in a dose of 100 mg/day once by enteral route starting from birth to the 30th day of life
Twenty-six healthy neonates were recruited as the placebo group, who received distilled water once per day by enteral route starting from birth to the 30th day of life.

Neonates with conditions necessitating nil per os, congenital anomalies, suspected inborn error of metabolism, renal and hepatic problems, and any patient with chronic metabolic disease were excluded from the study.

A full history was taken for all the neonates including maternal, obstetric, and perinatal history. Gestational age was calculated based on the date of last menstrual period and confirmed by using the modified Ballard score [11]. Anthropometric parameters were measured at birth and on day 30 of life. All neonates received routine neonatal care according to our NICU protocol.

All neonates were subjected to laboratory investigations which included complete blood count (CBC) analysis and serum ferritin on days 7 and 30 of life.

Blood samples for CBC analysis were collected on potassium-EDTA in sterile vacutainers, and right after collection, the tube was gently inverted several times and placed into a cold box at 4–8°C. The samples were transported and analyzed on the same day. CBC was performed using the automated hematology analyzer; Sysmex XT-1800i (Sysmex, Kobe, Japan). The XT-1800i performs an analysis of white blood cells with an optical detector based on the flow cytometry method. Red blood cells (RBCs) and platelet count analyses were done by the RBC detector using the Hydrodynamic Focusing method.

Samples for serum ferritin analysis were obtained in iron-free polyethylene tubes without the addition of anticoagulant and centrifuged for 15 min for separation of the serum, and then the samples were stored at −20°C till ferritin analysis. Serum ferritin was assayed by cobas e411 (Roche Diagnostics, Indianapolis, Indiana, USA) by an electrochemiluminescence immunoassay. The sandwich principle was used.

The primary outcome was to evaluate the effect of enteral LF supplementation on the Hb, hematocrit, and serum levels of ferritin in infants admitted in the NICU. The secondary outcomes were to determine the frequency of the required packed RBC transfusion and weight gain on the 30th day of life.

Sample size calculation

The required sample size has been calculated using the G*Power Software (Universität Düsseldorf, Germany).

It is estimated that a sample size of 26 neonates in either group would achieve a power of 80% (type II error, 0.2) to detect a statistically significant difference between the two groups for an effect size corresponding to a Cohen d coefficient of 0.8 using a two-sided unpaired t test with a confidence level of 95% (type I error, 0.05). The effect size (d) is calculated as follows:[INLINE 1]

A targeted effect size of d=0.8 has been selected as it could be regarded as a clinically relevant difference to seek in this exploratory study.

Statistical analysis

Data were analyzed using IBM SPSS Statistics, version 23 (IBM Corp., Armonk, New York, USA). Parametric quantitative data were presented as mean, SD, and range, while nonparametric data were presented as median and interquartile ranges. Qualitative variables were presented as number and percentages. Between-group comparisons of normally distributed numerical data were done using the unpaired t test. Paired numerical data were compared using the paired samples t test. Categorical data were compared using Fisher’s exact test (for nominal data) or the χ² test for trend (for ordinal data). Two-sided P value less than 0.05 was considered statistically significant.

Results

The demographic data of the two studied groups are presented in [Table 1].

Table 1 Demographic data of the two studied groups

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There was no significant difference between the two studied groups as regards the type of milk used (breast milk, artificial milk, or both). Also, there was no significant difference between serum ferritin and the type of milk used.

There were no significant differences between both studied groups as regards serum ferritin, Hb, hematocrit, mean corpuscular volume (MCV), red cell distribution width (RDW), platelet count, total leukocytic count (TLC), and C-reactive protein (CRP) on day 7 ([Table 2]). However, there were statistically significant higher serum ferritin, Hb, hematocrit, and MCV, and lower RDW, TLC, and CRP in the LF group than the placebo group on day 30 ([Table 2]).

Table 2 Comparison between the two studied groups as regards laboratory data on days 7 and 30

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None of the LF group patients received packed RBCs transfusion, while in the placebo group nine (34%) required packed RBCs transfusion (P=0.002); two received once and seven received twice. Moreover, the LF group neonates significantly reached more rapid to full enteral feeding, more weight gain at 1 month of age and shorter length of stay in the NICU than the placebo group (P=0.001, 0.011, and 0.029, respectively). Furthermore, the placebo group had significantly higher mortality than the LF group (19.2% vs. zero; P=0.051).

In the LF group both serum ferritin and platelet count were significantly increased (382.3±71.8 vs. 327.1±45.5 ng/ml and 249.8±68.5 vs. 173.1±80.9×1000/mm³, respectively; P<0.001) and RDW significantly decreased (16.1±1.8 vs 16.9±1.7%; P=0.004), on day 30 compared with day 7. However, in the placebo group serum ferritin, Hb, hematocrit, and MCV significantly decreased (294.2±72.7 vs. 341.2±60.9 ng/ml; P=0.015, 11.8±2.5 vs. 14.9±2 g/dl, 35.8±7.3 vs. 42.7±5.8; P<0.001, and 92.7±8.8 vs. 96.8±9.7 fl; P=0.005, respectively), while TLC significantly increased (17±6.8 vs. 12.3±5.6×1000/mm³; P=0.003) on day 30 compared with day 7.

Discussion

Iron deficiency at critical periods during the brain development can result in long-term neurocognitive problems [12]. Moreover, the presence of excess iron during the perinatal period can also be detrimental to the developing organs, especially preterm neonates with immature antioxidant. Maintaining iron homeostasis that avoids both iron deficiency and overload is important for optimal development and function [13]. LF, a high-affinity cationic iron-binding glycoprotein, is emerging as an important regulator of systemic iron homeostasis. BLF represents an attractive and promising alternative to oral iron supplementation [10].

In the present study, there were no significant difference between both the studied groups as regards serum ferritin, Hb, hematocrit, MCV, and RDW on day 7; however, there were significantly higher serum ferritin, Hb, hematocrit, and MCV, and lower RDW in the LF group than the placebo group on day 30. Also, serum ferritin significantly increased in the LF group and significantly decreased in the placebo group on day 30 compared with day 7. We found that none in the LF group patients received packed RBCs transfusion, while in the placebo group nine (34%) required packed RBCs transfusion of which seven received twice. Similarly, Paesano et al. [10] concluded that pregnant women suffering from iron deficiency and iron-deficiency anemia, oral administration of BLF significantly increases the number of RBCs, Hb, total serum iron, and serum ferritin after 30 days of the treatment and that BLF is a more effective and safer alternative than ferrous sulfate in treating pregnant women suffering from iron deficiency and iron-deficiency anemia.

In their study Chierici et al. [14] described that at day 150 of life, infants fed the formula enriched with higher quantities of LF (100 mg/100 ml) had serum ferritin levels significantly higher than infants receiving unsupplemented formula or formula enriched with LF (10 mg/100 ml), and that during the first 3 months of life, Hb, hematocrit, and serum iron levels were not substantially different in the different feeding groups and explained that those parameters are not influenced by the type of feeding supplied to the infants in the first 3 months of life.

Furthermore, a similar efficacy was found for oral LF and for intravenous iron, combined with recombinant human erythropoietin, for the treatment of anemia in advanced cancer patients undergoing chemotherapy [15].

In contrast, other studies [16],[17],[18] declared that the addition of BLF to infant formula with iron did not result in any significant advantages with regard to hematologic indexes or iron status.

LF modulates the gastrointestinal tract of infants in the very early stages of life, through interfering with its permeability and providing a mucosal trophic effect which are the key factors for the prevention of infections and necrotizing enterocolitis (NEC). Thus by promoting fast proliferation of the enterocytes in a growing intestine, LF creates less gut wall leaks, and the gap junctions become tighter, resulting in fewer colonizing pathogens translocation via a leaky gut wall. Moreover, LF promotes a bifidogenic microflora in the gut in neonates and preterms. LF is considered an important factor in the initiation, development, and/or composition of the neonatal gut microbiota [19]. In the present study, the LF group reached full enteral feeding more rapidly than the placebo group, with less needs to withhold feeding and with more rapid rate of feeding increments and better tolerance of enteral feeding. A trend toward a protective effect was observed but our study was not powered for the NEC outcome. Similarly, Akin et al. [20] in their study documented that infants exposed to LF (either alone or in combination with probiotic) had significantly less NEC than controls. Also, in Cochrane Database Review 2017, the authors concluded that LF supplementation to enteral feeds with or without probiotics decreases late-onset sepsis and NEC stage II or III in preterm infants without adverse effects.

In our study, LF group neonates had significantly more weight gain than in the placebo group. This result is similar to that of a previous study where height and weight were significantly greater in the iron+LF formula group than in the iron+nucleotide formula group at 6 months. BLF has been shown to have a growth-promoting action on the intestinal mucosa and on cells in culture. A surprising finding was that the only effect observed in mice whose LF gene was knocked out was impaired growth [18].

In our study, we found that there were no significant differences between LF and placebo groups as regards TLC and CRP on day 7; however, on day 30 of life, TLC was significantly lower in the LF than placebo groups and there were 12/26 (46.2%) in the placebo group had positive CRP, while all of the LF group neonates had negative CRP. Also, platelet counts were significantly increased on day 30 than day 7 in the LF group. This was in agreement with many studies [21],[22],[23]. Bacterial and fungal late-onset sepsis were reduced by two-thirds, regardless of the pathogen, in LF and LF+probiotic groups [9/153 (5.9%) and 7/151 (4.6%)] versus placebo [29/168 (17.3%)], with no adverse effects [21].The patients that received LF had significantly shorter length of stay in the NICU than those who did not receive LF’; this can be attributed to the rapid increment with no withholding periods for feeding, more rapid weight gain, and less signs of sepsis.

LF used is purified from bovine milk, being potential for adverse events, particularly allergic reactions, has been excluded. Allergy to cow’s milk protein is due mainly to casein, alpha-lactalbumin and beta-lactalbumin, but not to LF [24]. We did not report any side effects to BLF in the LF-treated group like allergy, feeding intolerance, abdominal distension, vomiting, diarrhea, or eczema. This was proved by many studies [19],[23],[25]. Nevertheless, long-term consequences remain to be examined; studies performing long-term evaluations for neurodevelopment must be addressed.

Acknowledgements

Mohammed El Barbary and Nancy A. Shady conceptualized and designed the study. Hebatallah A. Shaaban contributed to conceptualization and drafted the initial manuscript. Menat A.A. Shaaban and Ola Y. Ahmed supervised data collection, laboratory investigations, and analyzed and interpreted the data. All authors contributed to data interpretation and manuscript writing and have read and approved the final submission.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1.	Widness JA. Pathophysiology of anemia during the neonatal period, including anemia of prematurity. Neoreviews 2008; 9:e520.
2.	Cassady G, Potter CF, Southgate WM: Anemia of prematurity, 2016. [Online]. Avaliable: https://emedicine.medscape.com/article/978238-overview. [Accessed 20 October 2017].
3.	Long H, Yi J, Hu P, Li Z, Qiu W, Wang F, Zhu S. Benefits of iron supplementation for low birth weight infants: a systematic review. BMC Pediatrics 2012; 12:99.
4.	Rao R, Georgieff MK. Iron therapy for preterm infants. Clin Perinatol 2009; 36:27–42.
5.	Rosa L, Cutone A, Lepanto MS, Paesano R, Valenti P. Lactoferrin: a natural glycoprotein involved in iron and inflammatory homeostasis. Int J Mol Sci 2017; 18:1985.
6.	Pandita A, Sharma D, Kumar C. Lactoferrin and its role in neonatology: a review article. J Pediatr Neonatal Care 2015; 2:00062.
7.	Lönnerdal B, Bryant A. Absorption of iron from recombinant human lactoferrin in young US women. Am J Clin Nutr 2006; 83:305–309.
8.	Turin CG, Zea-Vera A, Pezo A, Cruz K, Zegarra J, Bellomo S et al., NEOLACTO Research Group. Lactoferrin for prevention of neonatal sepsis. Biometals 2014; 27:1007–1016.
9.	Lönnerdal B, Hernell O. Iron, zinc, copper and selenium status of breast-fed infants and infants fed trace element fortified milk-based infant formula. Acta Paediatr 1994; 83:367–373.
10.	Paesano R, Berlutti F, Pietropaoli M, Pantanella F, Pacifici E, Goolsbee W, Valenti P. Lactoferrin efficacy versus ferrous sulfate in curing iron deficiency and iron deficiency anemia in pregnant women. Biometals 2010; 23:411–417.
11.	Ballard JL, Khoury JC, Wedig K, Wang L, Eilerswalsman BL, Lipp R. New Ballard Score, expanded to include extremely premature-infants. J Pediatr 1991; 119:417–423.
12.	MacQueen B, Christensen R, Ward D, Bennett S, O’Brien E, Sheffield M et al. The iron status at birth of neonates with risk factors for developing iron deficiency: a pilot study. J Perinatol 2017; 37:436–440.
13.	Rao R, Georgieff MK. Iron in fetal and neonatal nutrition. Semin Fetal Neonatal Med 2007; 12:54–63.
14.	Chierici R, Sawatzki G, Tamisari L, Volpato S, Vigi V. Supplementation of an adapted formula with bovine lactoferrin. 2. Effects on serum iron, ferritin and zinc levels. Acta Paediatr 1992; 81:475–479.
15.	Macciò A, Madeddu C, Gramignano G, Mulas C, Sanna E, Mantovani G. Efficacy and safety of oral lactoferrin supplementation in combination with rHuEPO-β for the treatment of anemia in advanced cancer patients undergoing chemotherapy: open-label, randomized controlled study. Oncologist 2010; 15:894–902.
16.	Fairweather-Tait SJ, Balmer SE, Scott PH, Minski MJ. Lactoferrin and iron absorption in newborn infants. Pediatr Res 1987; 22:651–654.
17.	Schulz-Lell G, Dörner K, Oldigs HD, Sievers E, Schaub J. Iron availability from an infant formula supplemented with bovine lactoferrin. Acta Paediatr Scand 1991; 80:155–158.
18.	Hernell O, Lönnerdal B. Iron status of infants fed low-iron formula: no effect of added bovine lactoferrin or nucleotides. Am J Clin Nutr 2002; 76:858–864.
19.	Manzoni P. Clinical benefits of lactoferrin for infants and children. J Pediatr 2016; 173(Suppl):S43–S52.
20.	Akin IM, Atasay B, Dogu F, Okulu E, Arsan S, Karatas HD et al. Oral lactoferrin to prevent nosocomial sepsis and necrotizing enterocolitis of premature neonates and effect on T-regulatory cells. Am J Perinatol 2014; 31:1111–1120.
21.	Manzoni P, Rinaldi M, Cattani S, Pugni L, Romeo MG, Messner H et al. Italian task force for the study and prevention of neonatal fungal infections, Italian Society of Neonatology. Bovine lactoferrin supplementation for prevention of late-onset sepsis in very low-birth-weight neonates: a randomized trial. JAMA 2009; 302:1421–1428.
22.	Ochoa TJ, Zegarra J, Cam L, Llanos R, Pezo A, Cruz K et al. NEOLACTO Research Group. Randomized controlled trial of lactoferrin for prevention of sepsis in peruvian neonates less than 2500 g. Pediatr Infect Dis J 2015; 34:571–576.
23.	Pammi M, Suresh G. Enteral lactoferrin supplementation for prevention of sepsis and necrotizing enterocolitis in preterm infants. Cochrane Database Syst Rev 2017; 6:CD007137.
24.	Sharma S, Kumar P, Betzel C, Singh TP. Structure and function of proteins involved in milk allergies. J Chromatogr B Biomed Sci Appl 2001; 756:183–187.
25.	Ochoa TJ, Pezo A, Cruz K, Chea-Woo E, Cleary TG. Clinical studies of lactoferrin in children. Biochem Cell Biol 2012; 90:457–467.

Tables

[Table 1], [Table 2]

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