Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in the neonatal intensive care unit (NICU). The prevalence of NEC is largely related to birth weight and gestational age (GA), with approximately 1 in 10 very-low-birth-weight infants (<1500 g) developing NEC (1). Presently, an excessive inflammatory response against commensal bacteria by the immature intestine following mucosal injury in the postnatal period is the leading hypothesis in the pathophysiology of NEC (2,3). Because there is an estimated 3- to 10-fold risk reduction in infants fed with breast milk compared with those fed formula milk (4–8), it can be hypothesized that breast-feeding is protective against NEC development through improved gut maturation. Furthermore, animal research has shown formula-related mucosal atrophy, dysfunction, and subsequent elevated NEC risk (9).
Intestinal fatty acid–binding protein (I-FABP) is a small (14–15 kDa) cytosolic protein, exclusively present in mature enterocytes of the small intestine and colon (10,11). It is released into the circulation when enterocytes detach from villi at the end of their lifespan (12,13). We have previously shown that I-FABP reflects gut maturation in utero (14). I-FABP passes the glomerular filter and can easily be detected in urine (plasma half-life ±11 minutes).
The effects of breast milk on intestinal growth have principally been studied in vitro and in animal experiments (15). This study aimed to evaluate the effects of breast-feeding compared with formula feeding on human intestinal maturation in prematurely born babies in vivo by evaluating changes in urinary I-FABP levels during the first weeks after birth.
All of the babies admitted to the NICU of the Maastricht University Medical Center between July 2007 and July 2008 were eligible for participation. These patients were enrolled in a prospectively maintained database for the study of factors associated with NEC, with regular collection of urine and stool. Written informed consent was obtained from parents and/or caregivers, and the study was conducted with approval from local medical ethical committees and according to the Declaration of Helsinki. The principles of Good Clinical Practice were followed during this study. Infants were included if they met the following inclusion criteria: <37 weeks of gestation, first enteral feeding within 4 days after birth, and diet consisting of either exclusively breast milk or exclusively formula milk. The only exclusion criterion was development of significant gastrointestinal pathology during the 30-day study period, defined as disease of the gastrointestinal tract necessitating surgery, antibiotic treatment, cardiopulmonary support, or discontinuation or reduction of enteral feeding. Initiation of feeding and advancement of feeding volumes were realized according to local protocol. The standard guidelines consisted of initiation of oral feeding at days 1 to 4 after birth. Depending on the infant's GA and general condition, the feeding volume was increased with 10 to 20 mL · kg−1 · day−1 and discontinued if there were signs of feeding intolerance including bilious gastric retentions, abdominal distention, emesis, or bloody stools. Differences in intestinal inflammation between breast-fed infants and formula-fed infants were assessed by the fecal inflammatory marker calprotectin in the first stool sample available.
Sample Collection and I-FABP Measurements
Only samples collected on the 5th postnatal day and later were included in the analyses because enteral feeding was initiated in all of the infants by that time, as defined in the inclusion criteria. Urine samples collected on the 5th, 12th, 19th, and 26th day after birth were included in the present study. Samples were obtained by placement of a dental cotton roll in the diaper of the neonate. Once saturated with urine, the roll was placed in a sterile 5-mL syringe from which urine was consequently pressed into Micronic tubes. Samples were stored at −20°C until analysis. The first-available stool samples were collected and stored immediately at −20°C until batch analysis.
After patient inclusion was completed, urinary I-FABP concentrations were measured using an inhouse enzyme-linked immunosorbent assay selectively detecting human I-FABP (lower detection limit 12.5 pg/mL). Values are expressed in picograms per milliliter. Uncorrected urinary I-FABP levels correlate well with urinary I-FABP levels corrected for urine concentration (16).
Fecal Calprotectin Measurement
Fecal calprotectin was measured to assess intestinal inflammatory differences between formula feeding and breast-feeding. Calprotectin is a heterodimeric peptide (36 kDa), which is released from the cytosol of neutrophils upon activation. Fecal calprotectin is a specific marker for neutrophil infiltrate in bowel mucosa. In intestinal inflammation, calprotectin is readily detectable in feces and plasma (17).
After thawing of feces, 100 mg was weighed and 4.9 mL extraction buffer (0.1 mol/L Tris, 0.15 mol/L NaCl, 1.0 mol/L urea, 10 mmol/L CaCl2·2H2O, 0.1 mol/L citric acid, 0.5% bovine serum albumin, pH 8.0) was added (18). After shaking for 30 minutes, 1 mL of suspension was centrifuged at 10,000 rpm for 20 minutes at 4°C and supernatant was aliquoted and stored at −20°C. Calprotectin concentration was measured in lysate using the commercially available calprotectin enzyme-linked immunosorbent assay (lower detection limit 625 ng/mL; Hycult Biotechnology, Uden, the Netherlands). Fecal calprotectin concentration is given in micrograms calprotectin per gram feces.
Statistical analyses were performed using Prism 5.0 for Windows (GraphPad Software, San Diego, CA) and SPSS 20.0 for Windows (SPSS Inc, Chicago, IL). The Kolmogorov-Smirnov test was used to assess for normal distribution; the Mann-Whitney U test was used for between-group comparisons of continuous data; and the Friedman test was used for repeated measures. Dichotomous variables were compared using the Fisher exact test. Receiver operating curve analysis was performed to differentiate formula-fed infants from breast-fed infants. All data are presented as median and interquartile range. Significance was defined a priori as P < 0.05 for all tests. The a priori sample size calculation was based on the intestinal permeability difference between formula-fed and breast-fed babies at day 7 (median lactulose-to-mannitol ratio 0.205 vs 0.076, respectively) (19). Calculation with α = 0.05 and 1 − β = 0.80 resulted in 20 subjects per group.
A total of 40 patients were enrolled in the study, of which 21 were exclusively breast-fed and 19 were exclusively formula fed. Figure 1 shows the flow diagram of enrollment. Median GA was 30+1 (interquartile range 28+2–33+0) days, and median birth weight was 1400 (1080–1740) g. Both groups contained 10 boys as subjects. Human milk–based breast milk fortifier was administered in 11 of 21 breast-fed infants, on median day 16, that is, between the second and third study time points. There were no significant differences in GA, birth weight, or sex between the 2 groups; however, there was a trend of lower median GA in the breast-fed group (Table 1). Median NICU length of admittance was 20 days, with no significant difference between the 2 groups. Prematurity was the primary reason for admittance to the NICU in all of the babies. Comorbid conditions are given in Table 2. Some subjects were lost to follow-up at 19 days after birth (n = 14, of which 10 were transferred to the referring hospital and 4 to the medium care unit, all because of clinical improvement) and at 26 days after birth (n = 12, of which 7 were transferred to the referring hospital and 5 to the medium care unit, all because of clinical improvement). Fecal calprotectin levels in first-available stool samples were not significantly different between breast-fed infants (42.3 [31.2–195.1] μg/g feces) and formula-fed infants (87.6 [31.2–466.4] μg/g feces, P = 0.23), signifying no difference in intestinal inflammation between groups.
Urinary I-FABP Concentrations in Breast-Fed Versus Formula-Fed Babies
Median I-FABP levels for the breast-fed group were 104 (78–340) pg/mL, 408 (173–1028) pg/mL, 374 (153–1404) pg/mL, and 634 (243–2350) pg/mL at 5, 12, 19, and 26 days after birth, respectively. Median I-FABP levels in formula-fed babies were 152 (79–348) pg/mL, 105 (44–557) pg/mL, 723 (103–1670) pg/mL, and 659 (241–3451) pg/mL at 5, 12, 19, and 26 days after birth, respectively. Both groups showed a significant increase in urinary I-FABP concentrations in the first weeks after birth. For the breast-fed infants this increase in I-FABP levels was observed between 5 and 12 days after birth (104 [78–340] pg/mL to 408 [173–1028] pg/mL, P = 0.002), whereas I-FABP levels in formula-fed infants increased significantly between 12 and 19 days after birth (105 [44–557] pg/mL to 723 [103–1670] pg/mL, P = 0.004). As a consequence, infants who were exclusively formula fed had significantly lower median I-FABP levels at postnatal day 12 (P = 0.01). Receiver operating curve plotting produced an optimal cutoff point of 77.6 pg/mL to differentiate formula-fed from breast-fed infants at day 12, with sensitivity 42% and specificity 100%, and area under the curve 0.74 (95% confidence interval 0.58–0.90). There were no significant differences in median I-FABP levels between the 2 groups at postnatal days 5 and 19. Group sizes at day 26 were too low for statistical comparison because 26 of 40 patients had dropped out of the study at this time point. I-FABP levels at different time points are shown in Fig. 2.
Total volume of enteral feeding was recorded every day to investigate whether the type of feeding correlated with feeding tolerance. Volume of enteral intake (milliliter per kilogram body weight) did not differ significantly between the 2 groups during the studied period (P = 0.93, Fig. 3). There were no differences in other signs of feeding intolerance, defined as episodes of discontinuation of enteral feeding or frequency and cumulative amount of gastric retentions (Table 3).
The results of this study show a significant delay in the postnatal increase of I-FABP levels in formula-fed premature babies compared with breast-fed premature babies, suggesting a delayed physiological development of intestinal mucosa in formula-fed premature infants. I-FABP enters the circulation when enterocytes detach from their villi at the end of their lifespan and upon intestinal cell damage (12,13). Therefore, infants with possible intestinal disease were excluded from the study because high I-FABP levels in these children may obscure the I-FABP release from detaching villi during normal maturation. This was confirmed by low levels of fecal calprotectin in the first stool sample available. We have shown before that I-FABP correlates with gut maturation in utero (14). I-FABP levels within normal ranges represent the continuous release of I-FABP from mature enterocytes detaching into the gut lumen (10). The delay in the increase towards normal values of I-FABP in formula-fed premature babies may therefore indicate decreased maturation of enterocytes, as normal values were measured at postnatal day 19 compared with day 12 in breast-fed babies. We have previously reported mean normal values of I-FABP ranging from 100 to 200 pg/mL in healthy children and adults (20–23) and 434 pg/mL in premature babies (16).
This is the first study to assess I-FABP levels in breast-fed versus formula-fed premature infants. In other studies, intestinal permeability measured by a lactulose-to-mannitol ratio has been used as a functional parameter of gut maturation in neonates. These studies consistently show higher intestinal permeability in formula-fed babies when compared with breast-fed babies at 7 to 14 days postnatally, both in premature and term infants (19,24,25). Taylor et al (19) found significantly higher intestinal permeability in formula-fed preterm babies (≤32 weeks) at postnatal days 7 and 14, but not at postnatal day 30. Catassi et al (24) reported similar results in term neonates, with higher permeability at day 7 of life but similar gastrointestinal permeability 30 days after birth. These data suggest differences in the time course of functional gut maturity for formula-fed compared with breast-fed babies. The present finding of delayed gut maturation in formula-fed babies on postnatal day 12, but not on postnatal day 19, is in line with the above-mentioned studies.
Few markers are available for evaluating gut maturation and/or trophicity. This study indicates that I-FABP may provide such a tool, next to being an accurate marker of mesenteric ischemia, villous atrophy in celiac disease, and NEC (26–28). Citrulline has been proposed as a marker of gut maturity before; however, urinary citrulline in preterm infants did not correlate with gut maturity in a study by Bourdon et al (29). Because accurate measurement of gut maturity may be of clinical importance, for example, in predicting feeding tolerance, further study of I-FABP as a marker of gut maturity is desirable.
Delayed intestinal maturation associated with increased intestinal permeability can play a role in the increased incidence of NEC in formula-fed premature infants. Immaturity of the intestinal barrier can lead to easy passage of bacteria, viral particles, and toxins across the mucosal membrane with excessive inflammation as a result. In line with this hypothesis, classical NEC typically presents at 10 to 12 days after birth (30), the period in which intestinal maturation is still significantly lower in formula-fed infants compared with breast-fed infants. Moreover, these findings may provide an explanation for breast-feeding as protective strategy in the treatment of NEC. The design of the study and the manner in which samples were collected did not enable us to analyze factors associated with impaired gut maturity, for example, growth factors, of which most are quickly metabolized or not excreted in urine. Several growth factors, including insulin-like growth factor (31), epidermal growth factor (32), and transforming growth factor-β2 (33), have been linked to gut development. Moreover, colostrum induces higher enterocyte proliferation compared with mature milk (34), and epidermal growth factor levels are higher in preterm milk compared with term milk, as the preterm infant's need for trophic factors is high owing to the lack of exposure to amniotic fluid (35). Future studies investigating the role of human milk–related growth factors can use urinary I-FABP as a read-out for gut maturation, which is easily obtainable in a noninvasive manner.
Alterations of the gut microbiome by breast milk may be another explanation for the observed difference between formula and breast milk, as early-life microbial colonization is crucial for maturation of the immune system (36,37). Several studies have considered specific human milk oligosaccharides to be the most efficient breast milk substitutes in modulating microbial composition and NEC prevention owing to their prebiotic function, particularly on beneficial Bifidobacterium species (37–39).
Another leading hypothesis for the beneficial effects of breast milk is its immunomodulatory and anti-inflammatory effect, largely orchestrated by concentrations of secretory immunoglobulin A, CD14, transforming growth factor-β, erythropoietin, and interleukin-10 (40,41). It is, however, unlikely that inflammatory differences explain the differences in enterocyte maturation, because fecal calprotectin levels shortly after first enteral feeding were comparable between breast-fed babies and formula-fed babies, excluding the possibility of a low-grade inflammatory state in formula-fed infants.
The present study has some limitations. Owing to discharge to the referring hospitals or medium care wards, several patients were lost to follow-up at day 19. On day 26, >50% of patients had even dropped out. Therefore, representative statistical analysis on day 26 was not possible. On day 19, I-FABP values were almost twice as high in formula-fed infants, and lack of statistical significance may have been caused by a lack of power at this time point. A hypertrophic effect of formula-feeding on the gut in this phase may cause such a difference (42).
Further research should aim at unraveling the mechanisms by which breast-feeding improves intestinal maturation to design novel human milk substitutes, for example, analogues of growth factors or human milk oligosaccharides. The preterm infant's high need for trophic factors should be taken into account. Furthermore, this study underlines the importance of breast milk use in preterm infants. Although evidence is limited, donor breast milk seems to have the same beneficial effects as maternal breast milk when compared with formula feeding (7). Therefore, efforts should be taken to stimulate human milk banking and routine use in preterm infants.
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Keywords:© 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,
breast-feeding; formula feeding; intestinal fatty acid binding protein; mucosal damage; necrotizing enterocolitis