Association of Standardized Parenteral Nutrition with Early Neonatal Growth of Moderately Preterm Infants: A Population-Based Cohort Study.

In preterm infants, early nutrient intake during the first week of life often depends on parenteral nutrition. This study aimed to evaluate the influence of standardized parenteral nutrition using three-in-one double-chamber solutions (3-in-1 STD-PN) on early neonatal growth in a cohort of moderately preterm (MP) infants. This population-based, observational cohort study included preterm infants admitted to neonatal centers in the southeast regional perinatal network in France. During the study period, 315 MP infants with gestational ages between 320/7 and 346/7 weeks who required parenteral nutrition from birth until day-of-life 3 (DoL3) were included; 178 received 3-in-1 STD-PN solution (56.5%). Multivariate regression was used to assess the factors associated with the relative body-weight difference between days 1 and 7 (RBWD DoL1-7). Infants receiving 3-in-1 STD-PN lost 36% less body weight during the first week of life, with median RBWD DoL1-7 of -2.5% vs. -3.9% in infants receiving other PN solutions (p < 0.05). They also received higher parenteral energy and protein intakes during the overall first week, with 85% (p < 0.0001) and 27% (p < 0.0001) more energy and protein on DoL 3. After adjusting for confounding factors, RBWD DoL1-7 was significantly lower in the 3-in-1 STD-NP group than in their counterparts, with beta (standard deviation) = 2.08 (0.91), p = 0.02. The use of 3-in-1 STD-PN provided better energy and protein intake and limited early weight loss in MP infants.

Low Vitamin D Levels at Birth and Early Respiratory Outcome in Infants With Gestational Age Less Than 29 Weeks.

BACKGROUND: Vitamin D (VitD) is involved in lung development but its influence on respiratory distress syndrome of extremely preterm (EPT) infants have been little investigated. In this study, we examined the influence of low vitamin D status at birth on early respiratory outcomes of this vulnerable infant population. METHODS: Cord blood 25(OH)D levels ≤ 75 nmol/L were considered as Low vitamin D levels. Stepwise logistic regression and classification regression-tree analyses were used and the primary outcome was the combined outcome of death or mechanical ventilation need by the end of the first week (death or MV DoL7) as a marker od RDS severity. RESULTS: The mean (SD) GA and birth weight were 26 (1.4) weeks and 801 (212) gr, respectively; 81/109 (74%) infants had low 25(OH)D levels. Infants with low VitD levels had 25% higher initial FiO2 levels (p < 0.05) and were more likely to be mechanically ventilated on DoL7 (36 vs. 7%, p < 0.05). Adjusted for gestational age, they had 10-fold higher odds of death or MV DoL7 (p < 0.01). By regression tree analysis, the rate of death or MV DoL7 increased from 18 to 71% in infants with GA < 26 weeks and with cord blood 25(OH)D levels higher and lower than 74 nmol/L, respectively (p < 0.05). CONCLUSION: Low vitamin D levels at birth are associated with early adverse respiratory outcomes in infants with GA less 29 weeks. Further largest studies are needed to confirm this association.

Culture of Methanogenic Archaea from Human Colostrum and Milk.

Archaeal sequences have been detected in human colostrum and milk, but no studies have determined whether living archaea are present in either of these fluids. Methanogenic archaea are neglected since they are not detected by usual molecular and culture methods. By using improved DNA detection protocols and microbial culture techniques associated with antioxidants previously developed in our center, we investigated the presence of methanogenic archaea using culture and specific Methanobrevibacter smithii and Methanobrevibacter oralis real-time PCR in human colostrum and milk. M. smithii was isolated from 3 colostrum and 5 milk (day 10) samples. M. oralis was isolated from 1 milk sample. For 2 strains, the genome was sequenced, and the rhizome was similar to that of strains previously isolated from the human mouth and gut. M. smithii was detected in the colostrum or milk of 5/13 (38%) and 37/127 (29%) mothers by culture and qPCR, respectively. The different distribution of maternal body mass index according to the detection of M. smithii suggested an association with maternal metabolic phenotype. M. oralis was not detected by molecular methods. Our results suggest that breastfeeding may contribute to the vertical transmission of these microorganisms and may be essential to seed the infant's microbiota with these neglected critical commensals from the first hour of life.

Effect of preterm birth and birth weight on eating behavior at 2 y of age.

BACKGROUND: Preterm infants frequently present eating difficulties in early childhood. Determinants of these difficulties are not known. OBJECTIVE: We assessed the influence of neonatal and maternal characteristics on eating behaviors at 2 y of age. DESIGN: The following 2 cohorts were compared: 234 preterm children born <33 wk of gestational age from the POLYmorphisme génétique, Nutrition et Comportement Alimentaire cohort and 245 term children from the Observation des Préférences ALImentaires du Nourrisson et de l'Enfant cohort. Eating behaviors were assessed by using the validated Children's Eating Difficulties Questionnaire, which assesses the following 2 dimensions: a low drive to eat and narrow food repertoire. Each dimension was graded from 2 to 10 with more severe difficulties reflected by a higher score. Children in the upper quintile were classified as having eating disorders. RESULTS: Compared with term children, preterm children had a worse drive-to-eat score (4.3 ± 1.6 compared with 3.6 ± 1.5, respectively; P = 0.001) and a marginally lower food-repertoire score (5.0 ± 1.5 compared with 4.8 ± 1.6, respectively; P = 0.05). In a multilevel logistic regression model, female sex [adjusted OR (aOR): 1.76; 95% CI: 1.08, 2.88; P = 0.025] and birth weight less than -1 z score (aOR: 2.88; 95% CI: 1.47, 5.67; P = 0.002) but not gestational age were associated with a worse drive to eat. A maternal level of education beyond high school was associated with lower risk of a poor food-repertoire score (aOR: 0.54; 95% CI: 0.32, 0.90; P = 0.02). CONCLUSIONS: Preterm children have more eating difficulties than term children do, but after adjustment for maternal and neonatal characteristics, gestational age is not associated with impaired eating behaviors at the age of 2 y. Female sex, a low maternal level of education, and less than -1 SD intrauterine growth are associated with eating difficulties at 2 y of age. This trial was registered at clinicaltrials.gov as NCT 00663572.

Urinary citrulline in very low birth weight preterm infants receiving intravenous nutrition.

As gut immaturity precludes full enteral feeding, very low birth weight (VLBW) preterm infants receive parenteral nutrition (PN) during the first few weeks of life. Weaning VLBW infants off PN, however, is a top priority since PN is associated with a high risk of complications. The decision making is purely empirical, as there is currently no suitable index of gastrointestinal (GI) maturity. Plasma citrulline concentration is considered an index of GI function in conditions such as short-bowel syndrome and coeliac disease in adults. To identify the factors determining urinary citrulline excretion, and determine whether urinary citrulline excretion could be used as a non-invasive index of GI tolerance to enteral feeding, nutritional intake and urinary citrulline were monitored bi-weekly in forty-seven preterm infants < 1500 g (interquartiles 880-1320 g), during their stay in the Neonatology unit. Median urinary citrulline was 24·7 µmol/mmol creatinine (14·5-38·6 µmol/mmol creatinine). No relationship was observed with the percentage of energy tolerated enterally. In multivariate regression analysis, weak correlations were found with post-conceptional age (P = 0·001), parenteral amino acid supply (P = 0·001) and the daily volume of enteral mixture administered (P = 0·043). A significant correlation was found with urinary nitrite+nitrate excretion (r 0·47; P < 0·001). We conclude that in preterm infants: (1) one of the major determinants of urinary citrulline may be the biosynthesis of citrulline from arginine by NO-synthase; (2) urinary citrulline cannot be used to predict GI tolerance. This is consistent with the observations that, in neonatal gut, citrulline is converted to arginine in situ rather than exported towards the kidneys as observed in adults.

[Cobedding for premature twins]. / Le cobedding pour les jumeaux prématurés.

The cobedding of premature twins consists in lying them together in a single cocoon to maintain the closeness developed in the womb. It is associated with beneficial effects on the quality of sleep and could favour weight gain. Moreover, it does not seem to increase the incidence of infections or the frequency of the accidental removal of medical devices.

Oral supplementation with probiotics in very-low-birth-weight preterm infants: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Although recent reports suggest that supplementation with probiotics may enhance intestinal function in premature infants, the mechanisms are unclear, and questions remain regarding the safety and efficacy of probiotics in extremely low-birth-weight infants. OBJECTIVE: The objective was to evaluate the efficacy of probiotics on the digestive tolerance to enteral feeding in preterm infants born with a very low or extremely low birth weight. DESIGN: In a bicentric, double-blind, randomized controlled clinical trial that was stratified for center and birth weight, 45 infants received enteral probiotics (Bifidobacterium longum BB536 and Lactobacillus rhamnosus GG; BB536-LGG) and 49 received placebo. The primary endpoint was the percentage of infants receiving >50% of their nutritional needs via enteral feeding on the 14th day of life. A triangular test was used to perform sequential analysis. RESULTS: The trial was discontinued after the fourth sequential analysis concluded a lack of effect. The primary endpoint was not significantly different between the probiotic (57.8%) and placebo (57.1%) groups (P = 0.95). However, in infants who weighed >1000 g, probiotic supplementation was associated with a shortening in the time to reach full enteral feeding (P = 0.04). Other than colonization by the probiotic strains, no alteration in the composition of intestinal microbiota or changes in the fecal excretion of calprotectin was observed. No colonization by probiotic strains was detected in infants who weighed < or =1000 g, presumably because of more frequent suspensions of enteral feeding, more courses of antibiotic treatment, or both. CONCLUSIONS: Supplementation with BB536-LGG may not improve the gastrointestinal tolerance to enteral feeding in very-low-birth-weight infants but may improve gastrointestinal tolerance in infants weighing >1000 g. This trial was registered at clinicaltrials.gov as NCT 00290576.

Metabolic effects of different protein intakes after short term undernutrition in artificially reared infant rats.

BACKGROUND: Early postnatal nutrition is involved in metabolic programming. Small for gestational age and premature babies commonly receive insufficient dietary protein during the neonatal period due to nutrition intolerance, whereas high protein formulas are used to achieve catch up growth. Neither the short term, nor the long term effects of such manipulation of protein intake are known. AIM: We hypothesized that high or low protein intake during infancy would induce metabolic alterations both during early-life and in adulthood. METHODS: Gastrostomized neonatal rat pups received either 50% (P50%), 100% (P100%), or 130% (P130%) of the normal protein content in rat milk from the 7th to the 15th day of life (D7 to D15), when they were either sacrificed or placed with mothers for the long term study. Glucose tolerance tests (GTT) were performed at D230. Long term rats were sacrificed at D250. RESULTS: At D15, weight of P50% pups was lower than P100% and P130% pups. Neither liver and kidney mass, nor islet beta-cell areas were altered. Brain weight (adjusted to body weight) was higher in P50% vs. P130% (p<0.05). Insulin/glucose ratio was lower in P50% vs. P130%. Expression of GLUT4 on adipocyte cell membrane and GLUT2 in liver cytosol was significantly enhanced in P50% vs. P130%. Long term, neither GTT results nor body nor organ weights differed between groups. CONCLUSION: In neonatal rats, higher protein intakes via the enteral route led to enhanced short term weight gain, insulin resistance, and modified expression of glucose transporters. However, these differences were not sustained.

Determination of citrulline in human plasma, red blood cells and urine by electron impact (EI) ionization gas chromatography-mass spectrometry.

A method was developed by using gas chromatography-mass spectrometry in the electron impact ionization mode to quantify citrulline in plasma, red blood cells (RBC) and urine. For all three fluids, citrulline was extracted on ion exchange resins, before derivatization to its propyl-heptaflorobutyryl-ester. Assay precision (coefficient of variation, CV) was <5%, recovery% was >90% and the within- and between-day CV were <10% on 200 microL of plasma and RBC, and 400 microL of urine. The current method allows for the detection of 20 pmol of natural citrulline in aqueous standards, and small volumes (<100 microL) of biological fluids.

Neonatal formula feeding leads to immunological alterations in an animal model of type 1 diabetes.

Neonatal diet may influence the development of type 1 diabetes (T1D) in susceptible individuals through an intestinal mucosal inflammatory response, resulting in loss of self-tolerance. We tested the hypothesis that formula feeding during the neonatal period accelerates the development of T1D in diabetes-prone BioBreeding (BBDP) rats through regulation of CD4+CD25+ regulatory T lymphocytes (T(reg)) and anti-inflammatory cytokines. BBDP rat pups fed rat milk substitute (RMS) via a "pup-in-the cup" system were compared with mother-fed (MF) rats. The spleen and thymus were analyzed for Foxp3-expressing CD4+/CD25+ T cells. Multiplex enzyme-linked immunosorbent assays (ELISAs) were performed to measure cytokine-induced neutrophil chemoattractant (CINC), tumor necrosis factor alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin (IL)-4, IL-10, and IL-18. Diabetes-free survival, time of disease onset, and T(reg)/total T lymphocyte ratios were not different. MF pups had higher ileal CINC (p < 0.001) and IL-18 (p = 0.002), but no differences in the liver. There were no differences in ileal cytokine concentrations of 75-d-old rats, but the formula-fed rats had greater liver TNF-alpha (p < 0.001), IFN-gamma, and IL-4 (p < 0.01) and lower IL-10 (p = 0.002) compared with MF animals. Formula versus maternal milk altered the hepatic cytokine profile at 75 d toward an inflammatory pattern but did not result in altered T(reg) cell frequencies or the development of T1D.

Recovery of exfoliated cells from the gastrointestinal tract of premature infants: a new tool to perform "noninvasive biopsies?".

To gain insight into specific gene expression in the gastrointestinal (GI) tract of preterm infants, we adapted a method to isolate exfoliated epithelial cells. Gastric residual fluid aspirates (n = 89) or stool samples (n = 10) were collected from 96 neonates (gestational age, 24-36 wk). Cells were characterized by microscopic observation, cytokeratin-18 immunodetection, and expression of transcripts. The human origin of cellular DNA was confirmed by amplification of specific X and Y chromosome sequences. Isolation yielded 100-500 cells per sample for gastric aspirates (n = 8) and 10-20 cells for fecal samples (n = 5). Epithelial origin was confirmed by immunodetection of cytokeratin 18. Analyses of reverse transcribed products, using two independent methods, from 15 gastric fluid and two stool samples showed that 18S-rRNA and transcripts of beta-actin, glyceraldehyde-3-phosphate dehydrogenase (gapdh), and period1 were in quantities corresponding to at least 10 cells. On 59 aspirates, we found beta-actin transcripts (all but one), cytokeratin 18 (eight positive of eight samples), SLC26-A7-1 (13 positive of 19 samples), period2 (17 positive of 17 samples), and clock (25 positive of 26 samples). Exfoliated cells can be recovered from gastric aspirates and fecal samples and serve as a tool to investigate the impact of therapeutic and nutritional regimens on the maturation of GI functions.

Manipulation of citrulline availability in humans.

To determine whether circulating citrulline can be manipulated in vivo in humans, and, if so, whether citrulline availability affects the levels of related amino acids, nitric oxide, urinary citrulline, and urea nitrogen, 10 healthy volunteers were studied on 3 separate days: 1) under baseline conditions; 2) after a 24-h treatment with phenylbutyrate (0.36 g.kg(-1).day(-1)), a glutamine "trapping" agent; and 3) during oral L-citrulline supplementation (0.18 g.kg(-1).day(-1)), in randomized order. Plasma, erythrocyte (RBC), and urinary citrulline concentrations were determined by gas chromatography-mass spectrometry at 3-h intervals between 1100 and 2000 on each study day. Regardless of treatment, RBC citrulline was lower than plasma citrulline, with an RBC-to-plasma ratio of 0.60 +/- 0.04, and urinary citrulline excretion accounted for <1% of the citrulline load filtered by kidney. Phenylbutyrate induced an approximately 7% drop in plasma glutamine (P = 0.013), and 18 +/- 14% (P < 0.0001) and 19 +/- 17% (P < 0.01) declines in plasma and urine citrulline, respectively, with no alteration in RBC citrulline. Oral L-citrulline administration was associated with 1) a rise in plasma, urine, and RBC citrulline (39 +/- 4 vs. 225 +/- 44 micromol/l, 0.9 +/- 0.3 vs. 6.2 +/- 3.8 micromol/mmol creatinine, and 23 +/- 1 vs. 52 +/- 9 micromol/l, respectively); and 2) a doubling in plasma arginine level, without altering blood urea or urinary urea nitrogen excretion, and thus enhanced nitrogen balance. We conclude that 1) depletion of glutamine, the main precursor of citrulline, depletes plasma citrulline; 2) oral citrulline can be used to enhance systemic citrulline and arginine availability, because citrulline is bioavailable and very little citrulline is lost in urine; and 3) further studies are warranted to determine the mechanisms by which citrulline may enhance nitrogen balance in vivo in humans.

Lactobacillus rhamnosus GG decreases lipopolysaccharide-induced systemic inflammation in a gastrostomy-fed infant rat model.

OBJECTIVES: A gastrostomy-fed rat infant "pup-in-a-cup" model was used to test the hypothesis that enterally administered Lactobacillus rhamnosus GG (LGG) decreases the proinflammatory response induced by Escherichia coli lipopolysaccharide (LPS) in the developing infant rat small intestine, plasma, lung and liver. METHODS: Two groups of 6- to 7-day-old pups were fed a rat milk substitute with LPS added via the gastrostomy tube for 6 days. One of the rat milk substitute-fed groups received supplemental LGG; another group received LPS without LGG. Age-matched mother-fed rat pups were used as controls. RESULTS: LPS treatment blunted body growth, but LGG supplementation had no effect on weight increments. LGG decreased LPS-induced inflammation in intestinal tissue; CINC-1 (rodent IL-8 equivalent) production in plasma, liver, lung and distal small intestine; and tumor necrosis factor alpha (TNF-alpha) production in plasma and lung. Cytokine multiplex assay showed lung interleukin (IL)-1beta, IL-6, IL-10, IL-18, growth-related oncogene (GRO)/KC (rat CINC-1) and TNF-alpha were significantly higher in gastrostomy-fed, LPS-treated pups than in mother-reared pups, and LGG significantly blunted the LPS-induced elevation of IL-1beta, IL-10, IL-18, GRO/KC and TNF-alpha; liver GRO/KC was significantly higher in gastrostomy-fed, LPS-treated pups than in mother-reared pups, and LGG significantly blunted the LPS-induced elevation of GRO/KC. CONCLUSIONS: LGG provided by the enteral route is able to downregulate LPS-induced proinflammatory mediators. This effect is not only present in the splanchnic organs, that is, the intestine and the liver, but extends to the plasma and a distal organ, the lung.

Early intestinal bacterial colonization and necrotizing enterocolitis in premature infants: the putative role of Clostridium.

Necrotizing enterocolitis (NEC) is among the most severe conditions that can affect preterm infants. Although the etiology of NEC remains unknown, initial bacterial colonization could play a pivotal role in the development of NEC. To further explore the putative relationship between pathogen microorganisms and NEC, we conducted a prospective case-control study in 12 preterm infants with a new approach based on molecular techniques. Over an inclusion period of 24 mo, 12 neonates of <34 wk gestational age admitted to the neonatal unit were enrolled. The group included three cases of NEC, and nine control infants without evidence of NEC who were matched for gestational age and birth weight. Stool samples were collected at weekly intervals from all infants. PCR and temporal temperature gradient gel electrophoresis of 16S ribosomal DNA were used to detect the establishment of bacterial communities in the digestive tract. A salient feature of the bacteriological pattern was observed only in the three infants who later developed NEC: A band corresponding to the Clostridium perfringens subgroup could be detected in early samples, before diagnosis. There was no evidence for this specific band in any of the nine controls. To our knowledge, the current report is the first to demonstrate that the use of molecular techniques based on the study of bacterial 16S rRNA genes allowed the recognition of C. perfringens species in the first 2 wk of life of three infants who later displayed symptoms of NEC. A significant temporal relationship was thus established between early colonization by Clostridium and the later development of NEC. Compared with conventional bacteriological culturing methods, the use of this new molecular approach to analyze the gastrointestinal ecosystem should therefore allow a more complete and rapid assessment of intestinal flora. Although the current data do not constitute definitive proof that the identified bacterial species was a causative agent in the development of NEC, they outline the promise of this new technique based on molecular biology, and suggest that large-scale studies on a much wider population at high risk for NEC may be warranted.

Acute effects of intravenous glutamine supplementation on protein metabolism in very low birth weight infants: a stable isotope study.

Although very low birth weight infants are subjected to severe stress and glutamine is now considered a conditionally essential amino acid that may attenuate stress-induced protein wasting in adults, current amino acid solutions designed for neonatal parenteral nutrition do not contain glutamine. To determine whether a short-term supplementation with i.v. glutamine would affect protein metabolism in very low birth weight infants, 13 preterm neonates (gestational age, 28-30 wk; birth weight, 820-1610 g) receiving parenteral nutrition supplying 1.5 g x kg(-1) x d(-1) amino acids and approximately 60 nonprotein kcal x kg(-1) x d(-1) were randomized to receive an i.v. supplement made of either 1) natural L-glutamine (0.5 g x kg(-1) x d(-1); glutamine group), or 2) an isonitrogenous glutamine-free amino acid mixture (control group), for 24 h starting on the third day of life. On the fourth day of life, they received a 2-h infusion of NaH(13)CO(3) to assess the recovery of (13)C in breath, immediately followed by a 3-h L-[1-(13)C]leucine infusion. Plasma ammonia did not differ between the groups. Glutamine supplementation was associated with 1) higher plasma glutamine (629 +/- 94 versus 503 +/- 83 microM, mean +/- SD; p < 0.05, one-tailed unpaired t test), 2) lower rates of leucine release from protein breakdown (-16%, p < 0.05) and leucine oxidation (-35%, p < 0.05), 3) a lower rate of nonoxidative leucine disposal, an index of protein synthesis (-20%, p < 0.05), and 4) no change in protein balance (nonoxidative leucine disposal - leucine release from protein breakdown, NS). We conclude that although parenteral glutamine failed to enhance rates of protein synthesis, glutamine may have an acute protein-sparing effect, as it suppressed leucine oxidation and protein breakdown, in parenterally fed very low birth weight infants.