Comparative Study of Preterm Infants Fed New and Existing Human Milk Fortifiers (PDF)

Received: 17 April 2019 | Accepted: 20 August 2019 DOI: 10.1111/apa.14981 REGULAR ARTICLE Comparative study of preterm infants fed new and existing human milk fortifiers showed favourable markers of gastrointestinal status Jacques Rigo1 | Jean‐Michel Hascoët2 | Jean-Charles Picaud3...

Received: 17 April 2019 | Accepted: 20 August 2019 DOI: 10.1111/apa.14981 REGULAR ARTICLE Comparative study of preterm infants fed new and existing human milk fortifiers showed favourable markers of gastrointestinal status Jacques Rigo1 | Jean‐Michel Hascoët2 | Jean-Charles Picaud3 | Fabio Mosca4 | 5 6 7 8 Amandine Rubio | Elie Saliba | Michaël Radkë | Umberto Simeoni | 9 10 11 Bernard Guillois | Nicholas P. Hays | Mickaël Hartweg | Claude Billeaud12 | 13 Johannes Spalinger 1 Department of Neonatology, CHR Citadelle, University of Liège, Liège, Belgium 2 Maternité Régionale Universitaire A. Pinard, CHRU, Nancy, France 3 Service de Neonatologie, Hôpital de la Croix Rousse, Lyon, France 4 Neonatal Intensive Care Unit, Department of Clinical Science and Community Health, Fondazione IRCCS “Ca' Granda” Ospedale Maggiore Policlinico, University of Milan, Milano, Italy 5 Hôpital Couple Enfant, CHU de Grenoble, Grenoble, France 6 Hôpital Clocheville, CHU de Tours, Tours, France 7 Klinikum Westbrandenburg GmbH, Potsdam, Germany 8 Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland 9 Hôpital Clemenceau, CHU de Caen, Caen, France 10 Nestlé Product Technology Center – Nutrition, La Tour‐de‐Peilz, Switzerland 11 Nestlé Clinical Development Unit, Lausanne, Switzerland 12 CIC Pédiatrique 1401 CHU, Bordeaux, France 13 Children's Hospital of Lucerne, Lucerne, Switzerland Correspondence Nicholas P. Hays, Nestlé Product Technology Abstract Center – Nutrition, Rue Entre‐deux‐Villes Aim: This study examined the influence of different human milk fortifiers on bio‐ 10, 1814 La Tour‐de‐Peilz, Switzerland. Email: [email protected] markers of gastrointestinal immaturity and inflammation in preterm infants. Methods: We report secondary outcomes from a controlled, double‐blind, ran‐ Funding information This study was sponsored by Nestlé domised, parallel group study conducted from 2011 to 2014 in neonatal intensive Nutrition. care units at 11 metropolitan hospitals in France, Belgium, Germany, Switzerland and Italy. Preterm infants born at up to 32 weeks or weighing up to 1500 g were ran‐ domised to a new powdered human milk fortifier (n = 77) or a control fortifier (n = 76) for a minimum of 21 days. We analysed faecal markers of gut inflammation, namely alpha‐1 antitrypsin and calprotectin, and maturity, namely elastase‐1. Abbreviations: ANCOVA, analysis of covariance; FS, fortification strength; NEC, necrotising enterocolitis. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2019 Société des Produits Nestlé SA. Acta Pædiatrica Published by John Wiley & Sons Ltd on behalf of Foundation Acta Pædiatrica Acta Paediatrica. 2020;109:527–533.  wileyonlinelibrary.com/journal/apa | 527 528 | RIGO et al. Results: Faecal alpha‐1 antitrypsin was slightly lower in the new than control forti‐ fier group after 21 days of full enteral feeding, with a geometric mean and standard deviation of 1.52 ± 1.32 vs 1.82 ± 1.44 mg/g stools (P =.01). There was no significant difference in faecal calprotectin (median [Q1‐Q3] of 296 [136‐565] μg/g stools in both groups combined at study day 21). Faecal elastase‐1 was lower in the new fortifier than control fortifier group (202.5 ± 1.6 vs 257.7 ± 1.5 μg/g stools, P =.016). Conclusion: Mean values for each parameter were within the ranges in healthy term infants, indicating favourable markers of gastrointestinal status in both groups. In ad‐ dition, for faecal calprotectin, the relatively high concentration observed in preterm infants fed fortified human milk suggests that the threshold level for detecting necro‐ tising enterocolitis should be revised. KEYWORDS alpha‐1 antitrypsin, calprotectin, elastase‐1, low birthweight infant, prematurity 1 | I NTRO D U C TI O N Key notes Preterm infants have immature development at birth1 and are more This study examined the influence of different human likely to need specialised care in the immediate postpartum period milk fortifiers on biomarkers of gastrointestinal immatu‐ than term infants. Immaturity of the gastrointestinal system is a par‐ rity and inflammation in preterm infants. ticular concern, as it has been associated with severe morbidities, in‐ We report secondary outcomes from a controlled, dou‐ cluding necrotising enterocolitis (NEC).2 It also influences the ability of ble‐blind, randomised, parallel group study in neona‐ the infant to digest, absorb and use the nutrients needed to support tal intensive care units at 11 metropolitan hospitals in further growth and development.3 Gut immaturity, and the associated France, Belgium, Germany, Switzerland and Italy. increased risk in morbidity and feeding intolerance, makes the choice Mean values for each parameter were within the ranges of appropriate enteral nutrition extremely important. Although giving in healthy term infants, indicating favourable markers of a preterm infant human milk provides many them with benefits,4,5 the gastrointestinal status in both groups. nutritional profile of unfortified human milk is insufficient to meet the increased demands for growth in this population.6 As a result, human milk fortification is recommended for all infants born weighing less than 1800 g.7 While feeding fortified human milk has been shown to exocrine function, and differentiating severe from milder pancreatic have no adverse effect on feeding tolerance in preterm infants,8 the insufficiency.12 In preterm infants, these parameters can be used as influence of different human milk fortifiers on markers of gut maturity non‐invasive markers for pancreatic function, intestinal permeability and gastrointestinal inflammation has not been well studied. and intestinal complications such as NEC.12-14 Maturity of gastrointestinal function and inflammation can be The purpose of this study was to assess the effect of two human assessed using faecal biomarkers, which provide a non‐invasive and milk fortifiers with different composition on faecal biomarkers of early method for detecting any increased risk for these conditions.9 gastrointestinal inflammation and maturity of gut function in clini‐ Alpha‐1 antitrypsin is a serum trypsin inhibitor that is highly resis‐ cally stable preterm infants. Faecal biomarkers were the secondary tant to intestinal proteolysis. The extravasation of alpha‐1 antitryp‐ endpoints of a trial that evaluated weight gain velocity, other growth sin into the gut can be measured in the stools and is a classic marker parameters and nutritional biomarkers, including lipid profiles, in clin‐ for protein‐losing enteropathies or conditions that result in loss of ically stable preterm infants The new fortifier was PreNAN Human 10 blood proteins into the intestinal tract. This process happens in Milk Fortifier (Nestlé, Vevey, Switzerland), and the control fortifier intestinal inflammation, as a result of mucosal ulceration or aug‐ was FM85 Human Milk Fortifier (Nestlé, Vevey, Switzerland). The mented permeability. The total protein in stools is another marker primary results of the trial have already been reported.15,16 Briefly, of protein‐losing enteropathy and gut inflammation. Faecal calpro‐ they showed that the unadjusted weight gain rate was 18.3 g/kg/d tectin reflects various pathological processes that occur in the mu‐ for the new fortifier and 16.8 g/kg/d for the control fortifier and cosa of paediatric patients, with elevated concentrations indicating that both fortifiers were well tolerated, with a similar incidence of pathogenesis.11 Faecal elastase‐1 is a highly specific and sensitive adverse gastrointestinal events. The adjusted mean weight gain was test for determining gut functional maturity, specifically pancreatic 2.3 g/d greater for the new fortifier and the lower limit of the 95% RIGO et al. | 529 confidence interval of 0.4 g/d exceeded both the noninferiority and be insufficient, a second collection was made from a later bowel superiority margins. movement on the same or following day. Concentrations of faecal calprotectin and elastase‐1 were assessed by enzyme‐linked immu‐ nosorbent assays, following sample treatment with the Smart Prep 2 | M E TH O DS faecal extraction device (Roche Diagnostics). These were as fol‐ lows: EK‐Cal (Bühlmann Laboratories); ScheBo Pancreatic Elastase This report presents secondary outcomes from a controlled, dou‐ 1 (ScheBo Biotech AG) and Euroimmun Analyzer A1 (Euroimmun). ble‐blind, randomised, parallel group study that was conducted in Faecal alpha‐1 antitrypsin concentration was assessed by immu‐ neonatal intensive care units at 11 metropolitan hospitals in France, nonephelometry, using the IMMAGE 800 (Beckman Coulter) and Belgium, Germany, Switzerland and Italy. Each provided between faecal total protein by near‐infrared spectroscopy with the Fenir 25 and 45 beds. We included clinically stable preterm infants born 8820 (Perten Instruments). All the analyses were completed in a cen‐ from April 2011 to March 2014 at up to 32 weeks of gestation or tral laboratory (Rothen Medizinische Laboratorien AG). with a birthweight of up to 1500 g. The mothers agreed to provide FS day one values were log‐transformed and groups were com‐ expressed or donor breast milk for the 21‐day study duration. The pared using t tests computed with the Satterthwaite method. Day inclusion and exclusion criteria have previously been reported.15,16 21 values were log‐transformed and analysed using analysis of cova‐ The fortifiers were both based on cows' milk and they provided riance (ANCOVA), adjusted for the FS day one value of the relevant similar energy, namely 17 kcal per 100 mL of human milk. For every parameter, sex and centre (random effect). Changes from FS day one 100 mL of HM, the new human milk formula provided 1.4 g par‐ to day 21 were analysed using ANCOVA, adjusted for postmenstrual tially hydrolysed whey protein, 0.7 g lipids, 1.3 g carbohydrate and age and weight at study day one, when full enteral feeding and full a blend of micronutrients. The control human milk fortifier provided fortification had been achieved, the FS day one value of the relevant 1.0 g extensively hydrolysed whey protein, no lipids, 3.3 g carbohy‐ parameter, sex and centre (random effect). As many data were miss‐ drate and a blend of micronutrients. The new fortifier was devel‐ ing for total faecal protein, only summary statistics were calculated oped with a higher protein to energy ratio, with protein provided for this parameter. as partially hydrolysed whey and non‐protein energy from lipids, as The study was reviewed and approved by the institutional review medium‐chain triglycerides and docosahexaenoic acid. It also con‐ board or independent ethics committee at each hospital and the par‐ tained higher electrolyte and vitamin levels, while achieving a lower ent or legal representative of each participant provided written, in‐ osmolality. The aim was to comply with the recommendation from formed consent prior to enrolment. The Clinical Trial Registration for the European Society for Paediatric Gastroenterology Hepatology the study was NCT01771588. and Nutrition17 and the discussions by an expert group reported by Koletzko et al.18 Infants tolerating ≥100 mL/kg/d of human milk for >24 hours 3 | R E S U LT S were randomised to receive either the new or control fortifier for 21 days after they had achieved full enteral feeding. The fortifiers The study flow diagram, baseline subject characteristics and paren‐ that were given to the infants started at half‐strength, and this was tal demographics have previously been reported.15 A total of 153 called fortification strength (increase) day one (FS day one). It was infants were enrolled and randomised to either the new fortifier then increased in line with hospital practice, with the full‐strength (n = 77) or the control fortifier (n = 76). One infant in the new group fortification being reached when infants could maintain intakes of was excluded due to a history of systemic disease, and two in the con‐ 150‐180 mL/kg/d, namely full enteral feeds and full fortification. trol group were small for gestational age, leaving 76 and 74, respec‐ This was called study day one and occurred approximately 3 days tively. The infant characteristics were similar between the new and after fortification began. The measurements then continued for control groups: 50% and 53% of girls, 32% and 27% of vaginal births another 21 days. FS day one occurred at a median (Q1‐Q3) of 13 and 24% and 22% of twins, respectively. The mean birthweights (11‐18) days in the new group and 14 (10‐20) days in the control were 1147 ± 258 g and 1156 ± 289 g, and the mean gestational ages group. The mean days of life for study day one were 16 (13‐20) vs at birth were 28.8 ± 2.1 weeks and 28.7 ± 1.18 weeks. As shown in 17 (13‐23) days, respectively, and 36 (33‐40) vs 37 (33‐43) for study Figure 1, only 56%‐94% of the population provided data for the stool day 21. analyses (depending on time point and parameter), due to the diffi‐ Stool samples were collected on FS day one or the day after culty in obtaining sufficient stool quantities for all subjects. and at 21 ± 1 days (day 21). They were analysed for the following There were no significant differences in any faecal biomarkers parameters: gastrointestinal inflammation, namely faecal alpha‐1 at FS day one (Figure 2). The geometric mean concentrations of fae‐ antitrypsin, calprotectin and total protein, and maturity of gut func‐ cal alpha‐1 antitrypsin (Figure 2A) and faecal elastase‐1 (Figure 2C) tion, namely faecal elastase‐1. Approximately 5‐8 g was collected were significantly lower in the new fortifier group compared to from each infant within 2 hours of a bowel movement. Samples the control fortifier group at study day 21 (P =.010 and.016, re‐ were stored frozen at −20°C and shipped for analysis on dry ice. spectively). In addition, the increase from FS day one to study day If the stool sample quantity was below 5.5 g, which was judged to 21 for both of these parameters was significantly less in the new 530 | RIGO et al. F I G U R E 1 Flow of study participants and sample sizes for stool analyses. FS day 1 = fortification strength day one. Day 21 = study day 21 fortifier group compared to the control fortifier group (P =.007 and human milk19,20 and appears to increase with lactation.19 Human.004, respectively). Faecal calprotectin concentrations (Figure 2B) milk alpha‐1 antitrypsin is also resistant to pasteurisation21 and tended to increase in both groups during the study, according to the proteolysis in young infants, 22 meaning that faecal concentra‐ Satterthwaite test. The P‐values associated with the test of mean tions may be high, or change over time, even in the absence of changes from FS day one to study day 21 in the new fortifier and enteropathy. It is likely that this accounted for the higher levels of control fortifier groups were P =.051 and P =.112, respectively, with this protein in the two study groups when they were compared to no significant difference between groups demonstrated at study day infants who were predominantly fed preterm formula.19,20,23 For 21 or in the adjusted change over time. The evaluation of the total example, Sivan et al23 reported concentrations of faecal alpha‐1 protein concentration in stools was limited due to inadequate stool antitrypsin in a sample of preterm infants who were predominantly material in 89% of cases at FS day one and 85% of cases on study day fed formula. These ranged from 0.34 to 1.05 mg/g stools, which 21. Nevertheless, no significant difference was observed between was lower than the geometric mean values observed in our study the groups on FS day one when we looked at the medians and inter‐ (Figure 2). However, it is unlikely that the alpha‐1 antitrypsin con‐ quartile ranges: these were 1.70 (1.45‐1.80) for the new fortifier vs tent of human milk contributed to the small, but significant, dif‐ 1.40 (1.30‐1.53) for the control fortifier (P =.100). The values were ference between the groups on study day 21, since each group also similar in both groups at study day 21, namely 1.60 (1.50‐1.63) consumed a very similar volume of fortified milk during the study for the new fortifier vs 1.60 (1.50‐1.70) for the control fortifier. No period. This was 153 mL/kg/d in both groups, with 49% of the vol‐ inferential statistics were performed at study day 21 because 85% ume from donor milk in the new fortifier group and 51% in the of the values were missing. control fortifier group.15 Alternatively, it can be speculated that these results indicate a slight, but significant, increase in gastro‐ intestinal protein loss and, or, inflammation among infants in the 4 | D I S CU S S I O N control than new fortifier groups. However, the geometric mean concentrations in both groups were substantially lower than the This report presents faecal biomarkers of gastrointestinal inflamma‐ cut‐off value previously associated with various gastrointestinal tion and gut maturity in clinically stable, preterm infants fed human diseases in children (2.6 mg/g stools)24 and the difference between milk fortified with either a new or control multinutrient, powdered the groups was quite small (0.30 mg/g stools). fortifier. Our results indicate that the different human milk fortifiers In a 2016 systematic review, Pergialiotis et al25 concluded that led to limited differences in faecal biomarkers of gastrointestinal sta‐ faecal calprotectin was elevated in newborns with NEC. However, tus in this population. faecal calprotectin has also been shown to be higher in healthy, The faecal alpha‐1 antitrypsin results observed here may have exclusively breastfed infants than in infants fed formula or a mix‐ been due to several factors. First, alpha‐1 antitrypsin is present in ture of breast milk and formula, 26,27 suggesting that higher levels RIGO et al. | 531 Furthermore, no infants in our study experienced NEC, despite the fact that many had calprotectin levels (ie median [Q1‐Q3] of 296 [136‐565] μg/g stools in both groups combined at study day 21) that greatly exceeded those reported to be indicative of this condition. 25,29 In our study, the slight increase in faecal calpro‐ tectin over time in both the fortifier groups was likely to have re‐ flected both feeding with human milk fortifier plus mixed sources of human milk and normal gastrointestinal maturation and devel‐ opment. The lack of a significant difference between the groups was consistent with a previous study that showed no differences in this biomarker prior to commencing fortification or 14 days after fortification with either a human milk or bovine milk‐based fortifier. 30 Our results also provide further evidence that more work is needed to identify an appropriate diagnostic cut‐off value for NEC. This finding was consistent with the conclusions reached by Pergialiotis et al. 25 Reduced elastase‐1 secretion in stools is considered to be a marker of pancreatic insufficiency, with values of less than 200 μg/g stools indicating impaired exocrine pancreatic function.31 Values have been shown to normalise for most infants, including preterm infants, within the first few weeks after birth.12,32 Preterm infants have been shown to exhibit pancreatic immaturity and high sensitiv‐ ity to environmental stress, including inappropriate nutrient uptake related to defects in the gut barrier function.3 Given that nutrients within the duodenal lumen are the most important stimulators of the pancreatic exocrine response,33 it can be hypothesised that such in‐ appropriate uptake might lead to excessive elastase production and secretion into the gut lumen. Excessive elastase activity in the gut lumen may contribute to maintenance or exacerbation of inflamma‐ tory status,34 leading to metabolic stress and an increased risk of feeding intolerance and NEC.35 In the present study, preterm infants fed with the new fortifier presented with slightly lower levels of fae‐ cal elastase at study day 21 than the group who received the control fortifier. This finding suggests that the new fortifier may have had a modest impact on gut homeostasis. F I G U R E 2 Geometric mean */÷ geometric SD for faecal To our knowledge, this was the first study to assess multiple alpha‐1 antitrypsin (panel A), faecal calprotectin (panel B) and faecal elastase (panel C). Black circles/solid line represent new faecal markers of gastrointestinal inflammation and maturity of gut fortifier and open squares/dashed line represent control fortifier. function in response to different forms of human milk fortification FS day one = fortification strength day one and day 21 = study in preterm infants. A limitation was that the number of samples of day 21. *P =.017 vs FS day one value (by t test computed using sufficient quantity for all analyses ranged from 10% to 85% of the Satterthwaite method); †P =.010 vs control fortifier (by analysis full sample size. of covariance, adjusting for value at FS day one, sex and centre); ‡ P =.016 vs control fortifier (by analysis of covariance, adjusting for value at FS day one, sex and centre) 5 | CO N C LU S I O N may not always indicate pathology. Consistent with this hypothe‐ These results indicate that different human milk fortifiers can lead sis, Groer et al28 showed that infants who received their mother's to modest differences in markers of gastrointestinal status in clini‐ own milk had increasing faecal calprotectin levels over time, while cally stable, preterm infants. The mean values of each marker in infants who received mixed feeding or pasteurised donor milk did both fortifier groups were within the ranges observed in healthy not show this increase over time. Together, these previous find‐ term infants. In addition, these results suggest that the faecal cal‐ ings suggest that faecal calprotectin may not always represent protectin threshold value for early detection of NEC should be pathological inflammation in preterm infants, but rather feed‐ revised in light of the relatively high concentrations observed in ing pattern and, or, normal maturation of the gut immune axis. 28 our subjects. When they are combined with previously reported 532 | RIGO et al. anthropometry and clinical biochemistry data, these results fur‐ 8. Moody GJ, Schanler RJ, Lau C, Shulman RJ. Feeding tolerance in ther illustrate that the new fortifier was safe and well tolerated premature infants fed fortified human milk. J Pediatr Gastroenterol Nutr. 2000;30(4):408‐412. by our cohort. 9. Siddiqui I, Majid H, Abid S. Update on clinical and research appli‐ cation of fecal biomarkers for gastrointestinal diseases. World J Gastrointest Pharmacol Ther. 2017;8(1):39‐46. AC K N OW L E D G E M E N T S 10. Kosek M, Haque R, Lima A, et al. Fecal markers of intestinal in‐ flammation and permeability associated with the subsequent ac‐ The authors thank Virginie de Halleux (University of Liége) and quisition of linear growth deficits in infants. Am J Trop Med Hyg. Jonathan Jaeger, Laurent Ameye, Philippe Steenhout, Christelle 2013;88(2):390‐396. Perdrieu, Samir Dahbane and Jalil Benyacoub for their important 11. Aydemir G, Cekmez F, Tanju IA, et al. Increased fecal calprotec‐ contributions to this study. They were all affiliated to Nestlé when tin in preterm infants with necrotizing enterocolitis. Clin Lab. 2012;58(7‐8):841‐844. the study was completed. 12. Kori M, Maayan‐Metzger A, Shamir R, Sirota L, Dinari G. Faecal elastase 1 levels in premature and full term infants. Arch Dis Child Fetal Neonatal Ed. 2003;88(2):F106‐F108. C O N FL I C T O F I N T E R E S T 13. Campeotto F, Baldassarre M, Butel MJ, et al. Fecal calprotectin: cutoff values for identifying intestinal distress in preterm infants. J NPH and MH are employed by Nestlé SA and JR, J‐CP and CB are Pediatr Gastroenterol Nutr. 2009;48(4):507‐510. consultants for Nestlé Nutrition. JR, J‐MH, J‐CP, FM, AR, ES, MR, US, 14. Keller KM, Knobel R, Ewe K. Fecal alpha 1‐antitrypsin in newborn BG, CB and JS have received research funding from Nestlé Nutrition. infants. 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