Directly after birth, preterm infants have an impaired gut barrier function, reflected by an increased intestinal permeability(Reference van den Berg, Fetter and Westerbeek1, Reference van Elburg, Fetter and Bunkers2). Due to this increased intestinal permeability, delayed intestinal colonisation and immaturity of the host immune defence system, potentially pathogenic bacteria may translocate from the intestinal lumen and cause systemic infections(Reference Dai and Walker3–Reference Duffy5). Early enteral feeding is associated with decreased intestinal permeability(Reference Shulman, Schanler and Lau6). In a recent study, intestinal permeability was decreased in preterm infants who received breast milk feeding v. preterm infants who received formula feeding(Reference Taylor, Basile and Ebeling7). These beneficial effects of breast milk may partially be attributed to the ‘bifidogenic’ effect of human milk. Increasing the number of ‘bifidogenic’ bacteria may improve gut barrier function and prevent systemic infections from translocation of gut bacteria(Reference Guarner and Malagelada8, Reference Guarner9). Non-human milk oligosaccharides such as short-chain galacto-oligosaccharides (SCGOS) and long-chain fructo-oligosaccharides (LCFOS) have been developed(Reference Boehm and Moro10). Non-human milk acidic oligosaccharides (AOS) can be derived from pectin. In studies with non-human milk oligosaccharides, enteral supplementation of neutral oligosaccharides stimulates the growth of bifidobacteria and lactobacilli, resulting in increased SCFA production(Reference Boehm and Moro10). In an in vitro model, SCFA were able to stimulate mucin-2 production and improve gut barrier function(Reference Burger-van Paassen, Vincent and Puiman11). In an experimental animal study, a diet containing a prebiotic mixture of inulin and oligofructose had a trophic effect on colonic mucosal architecture(Reference Kleessen, Hartmann and Blaut12). In addition, translocation of Salmonella typhimurium after oral inoculation was partly decreased by the prebiotic diet(Reference Kleessen and Blaut13), suggesting a beneficial effect on gut barrier function.
In the initial study, we found a trend towards a decreased incidence of serious endogenous infections after enteral supplementation of a prebiotic mixture consisting of neutral oligosaccharides (SCGOS/LCFOS) and AOS, if given in sufficient amounts(Reference Westerbeek, van den Berg and Lafeber14). We hypothesised that the lower endogenous infection rate in preterm infants receiving SCGOS/LCFOS/AOS may originate from an improved gut barrier function.
Until now, studies into the effect of prebiotics on intestinal permeability in adults and newborn infants showed controversial results(Reference Guarner9, Reference Olguin, Araya and Hirsch15–Reference Stratiki, Costalos and Sevastiadou19). In preterm infants, intestinal permeability was found to decrease spontaneously in the first week of life(Reference van den Berg, Fetter and Westerbeek1, Reference van Elburg, Fetter and Bunkers2, Reference Stratiki, Costalos and Sevastiadou19, Reference van Elburg, van den Berg and Bunkers20). We hypothesise that enteral supplementation of a prebiotic mixture consisting of neutral oligosaccharides (SCGOS/LCFOS) and AOS may improve gut barrier function, especially in the first week of life, as reflected by decreased intestinal permeability. Therefore, the aim of the present study was to determine the effect of enteral supplementation of SCGOS/LCFOS/AOS on intestinal permeability as measured with the sugar absorption test (SAT) in the first week of life in preterm infants. Furthermore, we determined host- and treatment-related factors associated with intestinal permeability in these preterm infants.
Experimental methods
Subjects
Infants with a gestational age (GA) < 32 weeks and/or birth weight (BW) < 1500 g admitted to the level III neonatal intensive care unit (NICU) of the VU University Medical Center, Amsterdam were eligible for participation in the study. Exclusion criteria were small-for-GA infants with a GA >34 weeks, major congenital or chromosomal anomalies, death < 48 h after birth and transfer to another hospital < 48 h after birth. The present study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures involving human patients were approved by the medical ethical review board of our hospital. Written informed consent was obtained from all the parents.
Randomisation, blinding and treatment
After assignment to one of three BW groups ( ≤ 799, 800–1199 and ≥ 1200 g), infants were randomly allocated < 48 h after birth to receive either an enteral supplementation of a prebiotic mixture of 80 % SCGOS/LCFOS and 20 % AOS or a placebo mixture of maltodextrin. An independent researcher used a computer-generated randomisation table (provided by Danone Research, Friedrichsdorf, Germany) to assign infants to treatment with SCGOS/LCFOS/AOS or placebo. Investigators, parents, medical and nursing staff were unaware of treatment allocation. The randomisation code was broken after complete data analysis had been performed.
SCGOS/LCFOS/AOS and the placebo (maltodextrin) were prepared and packed sterile (Danone Research). The two powders were indistinguishable by appearance, colour and smell. During the study period, SCGOS/LCFOS/AOS and placebo were monitored for stability and possible microbiological contamination.
The supplementation of SCGOS/LCFOS/AOS or placebo was administered in increasing doses between days 3 and 30 of life to a maximum of 1·5 g/kg per d to breast milk or preterm formula (Nenatal Start®). Due to osmolarity reasons, each infant had an individual feeding scheme depending on BW and daily amount of feeding. If an infant received ≤ 100 ml/kg per d enteral feeding, 1 g SCGOS/LCFOS/AOS or placebo was added per 60 ml enteral feeding. If an infant received >100 ml/kg per d, 1 g SCGOS/LCFOS/AOS or placebo was added per 100 ml enteral feeding. Two members of the nursing staff added the daily supplementation to breast milk or to preterm formula according to the parents' choice. Per 100 ml, the preterm formula provided 350 kJ (80 kcal), 2·4 g protein (casein-to-whey protein ratio 40:60), 4·4 g fat and 7·8 g carbohydrate. The preterm formula did not contain oligosaccharides. When infants were transferred to another hospital before the end of the study, the protocol was continued under supervision of the principal investigator (E. A. M. W.).
Nutritional support
Protocol guidelines for the introduction of parenteral and enteral nutrition followed current practice at our NICU. Nutritional support was administered as previously described(Reference van den Berg, van Elburg and Westerbeek21), except for minimal enteral feeding which was defined as 12–24 ml/kg per d. Enteral nutrition was advanced either from day 2 or from day 4 in case of a BW < 10th percentile, GA < 26 weeks, Apgar score < 6 at 5 min, umbilical artery pH < 7·10 or base deficit >10 mmol/l. For each infant in the study, a feeding schedule was proposed based on BW and the guidelines as mentioned previously. After discharge, all the infants received breast milk or preterm formula (Nenatal Start® without oligosaccharides) until term, and a post-discharge formula (Nenatal 1® without oligosaccharides) until the corrected age of 6 months. The medical staff of our NICU and the responsible paediatricians in the regional hospitals had final responsibility for the administration of parenteral nutrition and advancement of enteral nutrition. Further details on the initial study have previously been published(Reference Westerbeek, van den Berg and Lafeber14, Reference Westerbeek, van Elburg and van den Berg22).
Intestinal permeability
Intestinal permeability was measured by SAT, as previously described(Reference van Elburg, Fetter and Bunkers2), at three time points: before the start of the study (t = 0), at day 4 (t = 1), and at day 7 (t = 2) after birth. In the SAT, urine was collected for 6 h after instillation of 2 ml/kg of the test solution (100 mg mannitol and 250 lactulose/5 ml sterile water) by nasogastric tube. After collection, 0·1 ml chlorhexidine digluconate (20 %) was added to the urine as a preservative, and samples were stored at − 20°C until analysis. Lactulose and mannitol were measured by GC, and the lactulose/mannitol (L/M) ratio was calculated.
Nutrition and clinical characteristics of the infants at the time of SAT were assessed, including administration of type of feeding in the week preceding the SAT, parenteral nutrition, achievement of full enteral feeding and presence of serious infection(s). A serious infection was defined as sepsis, meningitis, pyelonephritis, pneumonia or arthritis as diagnosed by a combination of clinical signs and a positive culture(Reference van der Zwet, Kaiser and van Elburg23) within 48 h preceding the SAT.
Statistical analysis
The sample size of 113 infants was based on the sample size calculation for the primary outcome of the main trial (serious infectious morbidity). Normally distributed and non-parametric data are presented as mean (sd) and medians (ranges), respectively. Perinatal and nutritional characteristics were analysed by Student's t test, Mann–Whitney U test, χ2 test or Fisher's exact test for continuous normally distributed, non-parametric continuous and dichotomous data, respectively.
As the parameters of intestinal permeability had a skewed distribution, a natural logarithmic transformation was performed before analysis. In the primary analysis, generalised estimating equations for longitudinal analysis were used to compare changes in lactulose, mannitol and L/M ratio over time between the groups(Reference Twisk, Smidt and de Vente24). This method takes into account the dependency of the observations within a patient and the fact that samples may not be available at each time point. Furthermore, the effect of host- and treatment-related factors (chorioamnionitis, administration of antenatal corticosteroids, mode of delivery, GA, BW, Apgar score at 5 min, administration of antibiotics postpartum, serious infectious morbidity, necrotising enterocolitis(Reference Bell, Ternberg and Feigin25), time to full enteral feeding (>120 ml/kg per d), age at finishing parenteral nutrition and type of feeding during the first week of life on intestinal permeability) was determined by generalised estimating equation analysis. All the statistical analysis was performed on an intention-to-treat basis. For all the statistical analyses, a two-sided P value < 0·05 was considered significant. SPSS 15.0 (SPSS, Inc., Chicago, IL, USA) was used for data analysis.
Results
Between May 2007 and November 2008, 113 of 208 eligible preterm infants entered the study. Reasons for not participating in the study were no informed consent (n 45), participation in another trial (n 7), transfer to a regional hospital within 48 h (n 12), death within 48 h (n 5) and severe congenital malformations (n 12). After randomisation, one infant in the placebo group was excluded, because of strong suspicion of a syndrome. Baseline patient and nutritional characteristics were not different in SCGOS/LCFOS/AOS (n 55) and placebo groups (n 58)(Reference Westerbeek, van den Berg and Lafeber14) (Table 1). SAT was performed at 36 (sd 15) h after birth (t = 0), at postnatal day 4·5 (sd 0·7) (t = 1) and at postnatal day 7·1 (sd 0·5) (t = 2). At t = 0, 17/55 (30·9 %) and 10/58 (17·1 %), at t = 1, 20/55 (36·4 %) and 11/58 (19·0 %), and at t = 2, 15/55 (27·3 %) and 15/58 (25·9 %) of the SAT data were missing in the SCGOS/LCFOS/AOS and placebo groups, respectively. Missing SAT data could mainly be attributed to insufficient urine collections. In both groups, the L/M ratio showed a decrease from t = 0 (0·21 (0·03–2·16) v. 0·34 (0·06–1·86) in SCGOS/LCFOS/AOS-supplemented and placebo groups, respectively, to t = 2 (0·06 (0·00–0·68) v. 0·09 (0·00–2·68)) in SCGOS/LCFOS/AOS-supplemented and placebo groups, respectively (effect 0·34 (95 % CI 0·22, 0·51; P < 0·001) (Fig. 1(a)). Analysis by generalised estimating equations showed no effect of enteral supplementation of SCGOS/LCFOS/AOS on the decrease in L/M ratio. Lactulose and mannitol concentrations were not different in the SCGOS/LCFOS/AOS-supplemented and placebo groups (Fig. 1(b) and (c)).
Table 1 Baseline and nutritional characteristics*
(Percentage values, standard deviations and ranges)
PE, pre-eclampsia; E, eclampsia; HELLP, syndrome of haemolysis, elevated liver enzymes and low platelets.
* Baseline and nutritional characteristics were not statistically different (P < 0·05) between the prebiotic mixture and placebo group.
† Student's t test, Mann–Whitney U test and χ2 test or Fisher's exact test are used to analyse continuous normally distributed, non-parametric continuous data, respectively.
‡ According to Usher & McLean(Reference Usher and McLean39).
Fig. 1 Results of sugar absorption tests. Results are expressed as median. Time points: before the start of the study (t = 0), at day 4 (t = 1) and at day 7 (t = 2) after birth. (a) Urinary lactulose/mannitol (L/M) ratio. (b) Urinary lactulose concentrations. (c) Urinary mannitol concentrations. ■, Prebiotic mixture; □, placebo. There were no significant differences between both the groups (generalised estimated equations).
As there were no significant differences between the SCGOS/LCFOS/AOS-supplemented and placebo groups, both the groups were analysed together to determine the influence of different host- and treatment-related factors on intestinal permeability (Table 2). Increased BW was related to decreased intestinal permeability (effect 0·54 (95 % CI 0·36, 0·81; P = 0·002)). Both exclusively breast milk feeding and mixed breast milk/formula feeding in the first week of life decreased the L/M ratio compared with exclusively formula feeding (effect 0·49 (95 % CI 0·34, 0·73; P < 0·001) and 0·53 (95 % CI 0·30, 0·93; P < 0·05), respectively). At t = 2, 22/113 (20 %) of the infants had a serious infection. SAT was performed in 16/22 (73 %) of the infants with a serious infection. The median L/M ratio was not different in infants with a serious infection and infants without a serious infection. In total, sixteen infants developed necrotising enterocolitis. The median age at which infants developed necrotising enterocolitis was 21 d (6–60). The median L/M ratio was not different in infants who later developed necrotising enterocolitis compared to infants who did not develop necrotising enterocolitis (Table 2).
Table 2 Factors influencing intestinal permeability (lactulose/mannitol (L/M) ratio) in preterm infants
(Effects and 95 % confidence intervals)
The factor significantly influenced intestinal permeability: *P < 0·05, **P < 0·01, ***P < 0·001.
† Data indicate the effect of a factor on the L/M ratio at all time points (generalised estimated equations). The effect can be interpreted as follows: in case of chorioamnionitis, the L/M ratio is 0·93 (95 % CI) times as high as without chorioamnionitis.
‡ Sepsis, meningitis, pyelonephritis, pneumonia or arthritis as diagnosed by a combination of clinical signs and a positive culture < 48 h preceding sugar absorption test.
§ Compared with exclusive formula feeding.
Discussion
In preterm infants, we found that enteral supplementation of a prebiotic mixture consisting of neutral oligosaccharides (SCGOS/LCFOS) and acidic (AOS) oligosaccharides does not decrease intestinal permeability in the first week of life, as measured by the SAT. The present results are in line with a study in healthy newborns(Reference Colomé, Sierra and Blasco18). Also, in adult burn patients, prebiotic supplementation did not decrease intestinal permeability(Reference Olguin, Araya and Hirsch15).
The lack of effect of prebiotics on intestinal permeability may be explained by high doses of antibiotics used in patients, including preterm infants, admitted at a intensive care unit, which interferes with the growth of bifidobacteria and lactobacilli(Reference Olguin, Araya and Hirsch15). In two recent studies in preterm infants, we found that antibiotics severely delayed the intestinal colonisation(Reference Westerbeek, van den Berg and Lafeber26, Reference van den Berg, van Elburg and Westerbeek27). Delayed intestinal colonisation decreases the production of SCFA which in turn impairs gut barrier function(Reference Boehm and Moro10, Reference Burger-van Paassen, Vincent and Puiman11). In the present study, 75 % of all infants received antibiotics immediately after birth. Furthermore, the prebiotic supplementation dose in the present study may have been insufficient to reach an (maximal) effect on intestinal permeability. This is in accordance with the trend towards a lower incidence of endogenous infections if preterm infants received prebiotics in a sufficient amount and numbers of days(Reference Westerbeek, van den Berg and Lafeber14). In the literature, few data are available about the optimal type, combination and amount of prebiotic supplementation(Reference Manzanares and Hardy28). Olguin et al. (Reference Olguin, Araya and Hirsch15) used one type of prebiotics (oligofructose), and Colomé et al. (Reference Colomé, Sierra and Blasco18) did not state which type and amount of prebiotics were given. Furthermore, probiotics and, especially, the combination of pre- and probiotics could be used, because a synergistic or additional effect may exist(Reference Manzanares and Hardy28). Stratiki et al. (Reference Stratiki, Costalos and Sevastiadou19) found that administration of a probiotic (Bifidobacterium lactis)-supplemented formula decreased intestinal permeability of preterm infants at day 30. In adult trauma patients, synbiotic, but not prebiotic (fermentable fibres), supplementation decreased intestinal permeability(Reference Spindler-Vesel, Bengmark and Vovk16).
The timing of the prebiotic supplementation may also explain the varying effects on intestinal permeability. The mean age of infants in the study of Colomé et al. (Reference Colomé, Sierra and Blasco18) was 74·4 (sd 30·3) d which could explain the lack of effect on intestinal permeability of the prebiotic supplementation, as previous studies show that intestinal permeability decreases rapidly in the first week of life(Reference van den Berg, Fetter and Westerbeek1, Reference van Elburg, Fetter and Bunkers2). Stratiki et al. (Reference Stratiki, Costalos and Sevastiadou19) found that probiotics decreased intestinal permeability at day 30 of life, but not at day 7. This finding suggests that supplementation of prebiotics, probiotics or synbiotics may affect intestinal permeability, if given for a sufficient number of days.
Postnatal factors may play an important role in the rapid adaptation of the small intestine to the extrauterine circumstances(Reference van Elburg, van den Berg and Bunkers20). In the present study, we found that both exclusively breast milk feeding and mixed breast milk/formula feeding during the first week of life decreased intestinal permeability at day 7. This is in line with a recent study done by Taylor et al. (Reference Taylor, Basile and Ebeling7) who found a decreased intestinal permeability in preterm breast-fed infants during the first 30 d of life. A decreased intestinal permeability after breast milk feeding was also found in term infants(Reference Shulman, Schanler and Lau6, Reference Weaver, Laker and Nelson29). This positive effect of breast milk feeding may be attributed to the ‘bifidogenic’ effect of breast milk and supports the hypothesis that the intestinal microbiota plays an important role in gut barrier function. The intestinal microbiota communicates with the underlying epithelium, which may lead to metabolic and immunologic reactions by the epithelial cells and its underlying lymphoid cells. This process is called bacterial–epithelial ‘crosstalk’(Reference Forchielli and Walker30, Reference Forchielli and Walker31). Preterm infants have an inadequate maturation of the host immune defence system, and due to an inappropriate bacterial–epithelial ‘crosstalk’(Reference Forchielli and Walker31), they have an increased risk to develop serious infections. However, the decreased incidence of endogenous infections(Reference Westerbeek, van den Berg and Lafeber14) could not be explained by improved gut barrier function, as reflected by intestinal permeability.
Besides a high risk for serious infectious morbidity, preterm infants are at high risk for developing necrotising enterocolitis. The pathogenesis of necrotising enterocolitis is not completely understood, but it has been proposed that impaired gut barrier function plays a crucial role(Reference Petrosyan, Guner and Williams32). In a rat gavage model, Zani et al. (Reference Zani, Ghionzoli and Lauriti33) found that rats with experimentally induced necrotising enterocolitis have increased intestinal permeability and develop systemic symptoms such as cardiac damage and renal failure. This suggests bacterial translocation and transfer of endotoxin and other inflammatory mediators causing multi-organ failure(Reference Zani, Ghionzoli and Lauriti33). In the present study, we did not find an increased intestinal permeability in infants who developed necrotising enterocolitis. However, in most cases, the SAT was performed before the infants developed necrotising enterocolitis.
Some remarks may be formulated with regard to the methodology of the present study. First, we only measured intestinal permeability in the first week of life. Study supplementation started at a median postnatal age of 2 d. At postnatal day 7, 86/113 (76 %) of the infants had not yet reached a supplementation dose of 1·5 g/kg per d. The mean supplementation dose during the first week of life was 0·73 (sd 0·43) g/kg per d. Therefore, infants may not have received a sufficient dose of SCGOS/LCFOS/AOS supplementation to reach a significant effect at postnatal day 4 or 7. However, in an additional analysis, we did not find a relationship between supplementation dose during the first week of life and intestinal permeability. Furthermore, in a previous study, in the first week of life, a significant decrease of the intestinal permeability was found, and during the next 3 weeks, the intestinal permeability remained stable(Reference van den Berg, van Elburg and Westerbeek21). Therefore, we hypothesised that the maximum effect of prebiotic supplementation on intestinal permeability would be in the first week of life. In the present study, intestinal permeability decreased in both the groups from day 1 to day 7. This is in line with previous studies(Reference van den Berg, Fetter and Westerbeek1, Reference van Elburg, Fetter and Bunkers2, Reference Stratiki, Costalos and Sevastiadou19, Reference van Elburg, van den Berg and Bunkers20). Secondly, breast milk itself contains neutral and AOS and, as shown in the present study, breast milk feeding has a positive effect on intestinal permeability. Therefore, the effect of enteral supplementation of SCGOS/LCFOS/AOS may be less pronounced in preterm infants who exclusively received breast milk. As breast milk is strongly promoted at our NICU, most infants received breast milk feeding (>60 %), and relatively few received exclusively formula feeding (20 %).
There were no serious adverse events reported after prebiotic supplementation in two recent reviews on prebiotic supplementation, including preterm infants(Reference Sherman, Cabana and Gibson34, Reference Srinivasjois, Rao and Patole35). However, Barrat et al. (Reference Barrat, Michel and Poupeau36) found an increased bacterial translocation in the intestine of immature rats (‘pup in the cup model’) fed a milk formula containing GOS and inulin, without an effect on intestinal permeability. However, intestinal permeability was measured in vitro with Ussing chambers(Reference Barrat, Michel and Poupeau36). The mechanisms underlying this increased bacterial translocation are unclear and should be investigated further. Bacterial translocation may originate from a combination of increased intestinal permeability, delayed intestinal colonisation and immaturity of the host immune defence(Reference Dai and Walker3, Reference Duffy5). It has been speculated that the increased bacterial translocation may not necessarily be harmful, but may be involved in the postnatal maturation of the immune system(Reference Urao, Teitelbaum and Drongowski37, Reference Gebbers and Laissue38). Urao et al. (Reference Urao, Teitelbaum and Drongowski37) and Gebbers & Laissue(Reference Gebbers and Laissue38) speculate that bacterial translocation may be instrumental for tolerance induction against the endogenous microbiota and for the stimulation and normal development of the gut-associated lymphoid tissue.
In conclusion, the present study in preterm infants shows that enteral supplementation of a prebiotic mixture consisting of neutral (SCGOS/LCFOS) and acidic (AOS) oligosaccharides does not enhance the decrease in intestinal permeability in the first week of life, as measured by the SAT. Breast milk feeding during the first week of life decreased the L/M ratio. The trend towards a lower incidence of endogenous infection rate in preterm infants receiving SCGOS/LCFOS/AOS cannot be explained by improved gut barrier function, as reflected by intestinal permeability in the first week of life. A beneficial effect of SCGOS/LCFOS/AOS may involve other aspects of gut barrier function; for example, modulation of the intestinal microbiota and the intestinal inflammatory response.
Acknowledgements
Study supplementation (SCGOS/LCFOS/AOS and maltodextrin) and preterm formula (Nenatal Start®) and post-discharge formula (Nenatal 1®) for the present study were provided by Danone Research. We acknowledge the parents for allowing their infants to participate in the study. Furthermore, we also thank the medical and nursing staff of the NICU of the VU University Medical Center and all participating hospitals and Henk Breukelman (Laboratory Center for Special Analysis, University Medical Center Groningen, The Netherlands) for analysing the urinary samples. The authors have no conflict of interest. The authors' contributions were as follows: E. A. M. W., H. N. L., W. P. F. F. and R. M. v. E. formulated the research questions and participated in the study design. E. A. M. W. and R. M. v. E. coordinated the study. E. A. M. W. and A. v. d. B. analysed the data. E. A. M. W. wrote the draft for the manuscript, and all the authors critically reviewed and revised the manuscript. The funding source had no involvement in the analysis of the data or the interpretation of the results. All the authors approved the final version of the manuscript. The study is registered at isrctn.org as ISRCTN16211826.
