Journal of the American College of Nutrition, Vol. 19, No. 3, 370-373 (2000)
Published by the American College of Nutrition
Lactoferrin Levels in Term and Preterm Milk
Patricia A. Ronayne de Ferrer, PhD,
Andrea Baroni, BS,
María E. Sambucetti, PhD,
Nora E. López, MD and
José M. Ceriani Cernadas, MD
Department of Nutrition and Food Science, School of Pharmacy and Biochemistry, University of Buenos Aires (P.A.R.d.F., A.B., M.E.S.), Buenos Aires, Argentina
Division of Neonatology, Department of Pediatrics, Italian Hospital (N.E.L., J.M.C.C.), Buenos Aires, Argentina
Address reprint requests to: Patricia A. Ronayne de Ferrer, PhD, Department of Nutrition and Food Science, School of Pharmacy and Biochemistry, Junín 956 Piso 2, 1113 Buenos Aires, Argentina.
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ABSTRACT
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Objective: Even though there is no doubt that human milk is the best nourishment for the neonate, there is still controversy regarding its suitability for preterm infants. The aim of this study was to contribute to the knowledge of the anti-infective properties of preterm milk, measuring lactoferrin levels, which are a non-specific protective factor.
Methods: Samples from 26 preterm and 20 term mothers (mean gestational age±standard deviation, 30.9±2.6 and 39.5±1.1 weeks, respectively) were collected during the first month post-partum. Milk samples were obtained by total expression of one breast between 10 a.m. and noon. An aliquot was kept at -20°C until analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylarnide gel electrophoresis). Each sample was run in duplicate.
Results: Lactoferrin levels (X±SD) in colostrum and mature milk varied from 575.0±218.2 mg/dL to 459.4±190.7 mg/dL in preterm samples and from 970.6±288.6 mg/dL to 292.0±167.4 mg/dL in term samples. No significant differences were observed between preterm and term groups, in spite of the trend observed in colostrum, where term milk tended to show higher levels than preterm milk. Decreasing values were observed in both groups along time (ANOVA, p<0.05). However, in the preterm group, lactoferrin levels seemed to maintain rather constant values from the eighth post-partum day onwards.
Conclusions: The trend to higher levels of lactoferrin in preterm mature milk would allow maintenance of the protective effect of human milk in preterm infants in spite of the small volumes ingested by these neonates. These findings support the practice of feeding premature infants with their own mothers’ milk at a time when their immune systems have not completely developed.
Key words: human milk, lactoferrin, prematurity, anti-infective factors
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INTRODUCTION
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It is widely recognized that human milk is the best nourishment for the neonate and that breast-fed infants are protected against infectious diseases of both the digestive and respiratory tract. This protection is afforded by several antimicrobial factors, such as secretory immunoglobulin A, lactoferrin, lysozyme, among others [1,2]. Lactoferrin is an iron-binding protein which is present in a predominantly unsaturated form; for this reason, it competes with microorganisms requiring iron for growth, thus exerting an important bacteriostatic activity in the gastrointestinal tract [1–3]. Recently, a giardicidal activity of lactoferrin and its N-terminal peptides has been reported [4], as well as some antiviral properties against respiratory syncytial virus [5]. Lactoferrin also possesses antiinflammatory properties and seems to be involved in phagocytic killing and immune responses [6]. Besides, human lactoferrin has been shown to stimulate thymidine incorporation into DNA of enterocytes of rat small intestine, acting as a possible growth factor [7]. Regarding lactoferrin effect on iron absorption, further research is needed to define its precise role [8,9]. Finally, recent reports [10] have shown that lactoferrin specifically binds to DNA sequences in the nucleus, suggesting that it may act as a transcription factor.
On the other hand, there is still controversy regarding the suitability of breast milk for preterm infants, but in recent years the use of their own mothers’ milk to feed these neonates is increasing. In a previous paper [11], we found significantly higher IgA levels in preterm milk from the 10th day postpartum onwards. The aim of this study was to contribute to the knowledge of the anti-infective properties of preterm milk, measuring lactoferrin concentrations.
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MATERIALS AND METHODS
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Milk Donors
Milk samples were obtained from 26 preterm and 20 term mothers (mean gestational age±standard deviation, 30.9±2.6 and 39.5±1.1 weeks, ranges 27 to 35 weeks and 38 to 41 weeks, respectively). All were healthy middle-class women. Gestational age was determined from menstrual data and confirmed by physical examination according to Capurro et al. [12]. Birth weights were 1416±332g and 3303±499g for preterm and term infants, respectively.
Sample Collection
Milk samples were collected during the 1st month postpartum in the morning, by complete manual or mechanical emptying of one breast. An aliquot was taken and kept in plastic containers at -20°C until analyzed.
Analytical Methods
Total (TN) and nonprotein nitrogen (NPN) were determined by the Kjeldahl method [13]; NPN was obtained by protein precipitation with 24% TCA acid [14]. Protein content was calculated as (TN-NPN) x 6.25.
Lactoferrin separation was carried out by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) according to Laemmli [15], as described previously [16]. Briefly, protein separation was carried out on 10% acrylamide gels and, after migration, the staining was performed with Coomassie brilliant blue R 250; gels were scanned at 550 nm. Lactoferrin peak areas were calculated as peak height times width at half-height [17] and lactoferrin amounts calculated from a calibration curve obtained loading varying amounts of a standard (lactoferrin from human milk, lyophilized powder containing approximately 90% protein, Sigma Chemical Co.). A typical gel of the analysis is shown in Fig. 1.
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Fig. 1. SDS-PAGE of increasing amounts of a lactoferrin standard (lanes B to E) and some milk samples (lanes A and F to L). The lactoferrin band is the one shown as LF.
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Statistical Analysis
Data were compared using ANOVA. Significance was declared at p<0 05.
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RESULTS
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Total protein decreased over time and did not differ between groups (Table 1). No significant differences were observed between preterm and term lactoferrin values, in spite of the trend observed in colostrum samples, where term milk tended to show higher levels than preterm milk (Table 2). Lactoferrin values decreased in both groups over time (p<0.05). However, in the preterm group, lactoferrin levels remained rather constant from the eighth postpartum day onwards. When expressed as a proportion of total protein (Table 3), the profile observed for term values was similar to that observed when expressed in mg/dL. In the case of preterm milk, lactoferrin as a proportion of total protein showed an increasing trend, from about 28% to 34%, while it decreased from 54% to 28% in term samples.
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DISCUSSION
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The findings relative to total protein agreed with our previous data [11,18], which showed no differences between term and preterm milk. Previous reports on lactoferrin levels in preterm milk have shown either significantly higher values than in term milk [19] or no differences between them [20]. In this study, no significant differences in lactoferrin levels were found; besides, in colostrum term values tended to be higher than preterm ones. This is in agreement with the results from a very recent paper [21]. As reported by McClelland et al. [22], in early lactation, a few hours can make a large difference in milk composition; lactoferrin, in particular, decreases substantially on the third day of lactation. This could partly explain the results observed in colostrum values, since some term samples were collected on day two while those from preterm mothers were available only from the third day onwards. For this reason, we recalculated the mean for colostrum values, excluding those term milks from day two. In this case, the mean for the term group was 841.9±261.1 mg/dL instead of 970.6±288.6 mg/dL. It showed the same trend as before, leading to the same conclusions.
It is well known that immaturity of the immune system is more critical in preterm than in term infants, so the antiinfective factors afforded by maternal milk are even more important in premature neonates. The presence of intact milk proteins, like lactoferrin, secretory immunoglobulin A and lysozyme, in stools of breast-fed infants suggests that these proteins play an active role in the infant gut [23]. Lactoferrin is remarkably resistant to proteolytic degradation; this fact, together with the rather high gastric pH of breast-fed infants and low activity of gastric and pancreatic proteases, may explain the ability of lactoferrin partially to escape digestion [9]. It has been reported [24] that a higher proportion of lactoferrin is found in the feces of premature babies. It is interesting to remark that some investigators have also reported higher levels of lactoferrin in the urine of breast-fed than in formula-fed infants [25,26]. Hutchens et al. [27] demonstrated that most of the urinary lactoferrin in premature infants was of maternal origin, showing that lactoferrin was absorbed by the gut and excreted intact in the urine of these infants. Our results, which show, from the eighth day onwards, rather constant lactoferrin levels and higher proportions relative to total protein, in preterm milk, compared to the decreasing trend observed in the term group, suggest that this protein might exert a protective effect in preterm infants, in spite of the lower levels observed in colostrum. This pattern could represent a mechanism that would compensate for the smaller milk volumes ingested by preterm neonates, even though it is presumed that additional factors, other than milk volume, influence the concentration of the different proteins present in human milk [2,20]. According to Atkinson [28], a number of events related to preterm birth could affect lactation performance and/or milk composition. Among them may be one or a combination of altered maternal hormonal profile, immaturity of the mammary gland or differences in perinatal management (such as variations in breast stimulation). Further studies are needed to evaluate these issues.
As pointed out by Goldman et al. [2], the definition of the immune system includes not only direct-acting antimicrobial agents, but also antinflamatory factors and immunomodulators; lactoferrin seems to participate, at least, in the two first categories. Benefits of human milk for preterm infants cannot be explained on the basis of lactoferrin alone; however, the findings of this study, together with our previous report on immunoglobulin A levels [11], support the importance of feeding premature infants with their own mothers’ milk, even if an appropriate supplementation is required. It should be taken into account that the delayed development of the immune system in preterm neonates makes them particularly susceptible to infection.
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ACKNOWLEDGMENTS
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Supported by the University of Buenos Aires, grants FA-044 and AB11.
Received April 1, 1999.
Revised April 1, 2000.
Accepted April 1, 2000.
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ABSTRACT
INTRODUCTION
