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.

Fermented infant formula (with Bifidobacterium breve C50 and Streptococcus thermophilus O65) with prebiotic oligosaccharides is safe and modulates the gut microbiota towards a microbiota closer to that of breastfed infants.

BACKGROUND & AIMS: Microbiome-modulators can help positively steer early-life microbiota development but their effects on microbiome functionality and associated safety and tolerance need to be demonstrated. We investigated the microbiome impact of a new combination of bioactive compounds, produced by the food-grade microorganisms Bifidobacterium breve C50 and Streptococcus thermophilus ST065 during a fermentation process, and prebiotics in an infant formula. Tolerance and safety were also assessed. METHODS: An exploratory prospective, randomized, double-blind, controlled, multi-centre study was designed to investigate the effect of bioactive compounds and prebiotics (short-chain galacto-oligosaccharides (scGOS)/long-chain fructo-oligosaccharides (lcFOS) 9:1). Experimental formulas containing these bioactive compounds and prebiotics (FERM/scGOS/lcFOS), prebiotics (scGOS/lcFOS), or bioactive compounds (FERM), were compared to a standard cow's milk-based control formula (Control). Exclusively breastfed infants were included as a reference arm since exclusive breastfeeding is considered as the optimal feeding for infants. The study lasted six months and included visits to health care professionals at baseline, two, four and six months of age. Stool SIgA concentration was the primary study outcome parameter. RESULTS: There were 280 infants randomized over the experimental arms and 70 infants entered the breastfed-reference arm. Demographics were balanced, growth and tolerance parameters were according to expectation and adverse events were limited. At four months of age the median SIgA concentration in the FERM/scGOS/lcFOS group was significantly higher compared to the Control group (p = 0.03) and was more similar to the concentrations found in the breastfed-reference group. Bifidobacterium increased over time in all groups. The FERM/scGOS/lcFOS combination resulted in a microbiota composition and metabolic activity closer to the breastfed infants' microbiome. CONCLUSION: The FERM/scGOS/lcFOS combination showed a significant positive effect on SIgA levels. All formulas tested were associated with normal growth and were well-tolerated. Additionally, at four months of age the FERM/scGOS/lcFOS formula brought the microbiome composition and metabolic activity closer towards that of breastfed infants. CLINICAL TRIAL REGISTRY: Registration number NTR2726 (Netherlands Trial Register; www.trialregister.nl/).

Investigation of the intestinal microbiota in preterm infants using different methods.

Modifications in microbial colonization of the human gut are believed to affect intestinal homeostasis and increase the risk of gastrointestinal diseases. The present study examined different methods for investigating the dynamic characterization of the intestinal microbiota in preterm infants. Fecal samples were collected weekly from ten preterm infants during their stay in a neonatal intensive care unit. The infants had a mean gestational age of 29 weeks (range: 28-32 weeks) and a mean birth weight of 1233g (range: 935-1450g). Bacterial colonization was assessed using conventional culture techniques and molecular biological methods. More specifically, the recently developed denaturing high performance liquid chromatography (dHPLC) technique was compared to established methods such as temporal temperature gradient gel electrophoresis (TTGE) and rRNA gene library sequencing. Our results indicate that the gastrointestinal tract of preterm infants, born at a gestational age of less than 33 weeks, has a low biodiversity of mainly, culturable bacteria. Finally, dHPLC was evaluated in terms of speed, labor and sensitivity for its use as a tool to analyze microbial colonization in preterm infants. We found that this technique provided major improvements over gel-based fingerprinting methods, such as TTGE, that are commonly used for studying microbial ecology. As such, it may become a common analytical tool for this purpose.

Genomic markers for ovarian cancer at chromosomes 1, 8 and 17 revealed by array CGH analysis.

AIMS AND BACKGROUND: The literature data show that the most frequently affected chromosomes in ovarian carcinogenesis are 1, 8 and 17. In the present study we aimed to define more precisely at a high resolution the genomic imbalances of these chromosomes in ovarian cancer and to determine genomic markers separating tumors of different histological types and stages. METHODS: Array comparative genomic hybridization (CGH) with a resolution of approximately 0.8 Mb was applied in 28 primary ovarian tumors. We identified regions of highly frequent gains or losses (affecting more than 40% of ovarian cancers) and determined sites showing alterations of elevated amplitude (amplifications or homozygous deletions). Doing this we also identified at least two adjacent changed clones. RESULTS: We determined anomalies strongly associated with the disease such as deletions at 8p21-23, 17p12-13, 1p35-36 or amplifications at 1q23, 17q12, 17q23.2, 8q13.2, 8q24. We defined more precisely the gains in 17q12-q24, finding as strong candidates for ovarian tumorigenesis the genes LASP1 (17q12), TGF11 (17q21.32), MUL (17q23.2), TBX2 (17q23.2), AXIN2 (17q24.3) and GRB2 (17q25.1). Of particular note was gain of 8q13.2, which occurred at a high frequency in ovarian cancer, especially in serous and late-stage tumors. We found that gains of 1q32-1q43, 8p11-p12, 8q11.23, 8q13.2, and 8q24.21-8q24.22 and losses of 1p36.21, 8p23.1-8p21.1 and 8q21.2 were associated with serous histology, whereas losses of 1q23 and 1q32-43 and gains of 17q11.2-12 and 17q25 were associated with mucinous histology. Gains of 1q23, 8q24, 17q23.2, 17q24.2 and losses of 1p35-36, 8p, 17p, and 17q were specific for late-stage ovarian cancers. CONCLUSIONS: Our study has identified potential genomic markers of interest on chromosomes 1, 8 and 17 in ovarian cancer. Tumors showed a wide variety in the patterns of alteration, suggesting that alternative mechanisms of genomic instability may play a role in this tumor type.

Impact of Maternal Nutrition and Perinatal Factors on Breast Milk Composition after Premature Delivery.

(1) Background: Premature infants require mothers' milk fortification to meet nutrition needs, but breast milk composition may be variable, leading to the risk of inadequate nutrition. We aimed at determining the factors influencing mothers' milk macronutrients. (2) Methods: Milk samples were analyzed for the first five weeks after premature delivery by infrared spectroscopy. Mothers' nutritional intake data were obtained during standardized interviews with dieticians, and then analyzed with reference software. (3) Results: The composition of 367 milk samples from 81 mothers was (median (range) g/100 mL): carbohydrates 6.8 (4.4⁻7.3), lipids 3.4 (1.3⁻6.4), proteins 1.3 (0.1⁻3.1). There was a relationship between milk composition and mothers' carbohydrates intake only (r = 0.164; p < 0.01). Postnatal age was correlated with milk proteins (r = -0.505; p < 0.001) and carbohydrates (r = +0.202, p < 0.001). Multiple linear regression analyses showed (coefficient) a relationship between milk proteins r = 0.547 and postnatal age (-0.028), carbohydrate intake (+0.449), and the absence of maturation (-0.066); associations were also found among milk lipids r = 0.295, carbohydrate intake (+1.279), and smoking (-0.557). Finally, there was a relationship among the concentration of milk carbohydrates r = 0.266, postnatal age (+0.012), and smoking (-0.167). (4) Conclusions: The variability of mothers' milk composition is differentially associated for each macronutrient with maternal carbohydrate intake, antenatal steroids, smoking, and postnatal age. Improvement in milk composition could be achieved by the modification of these related factors.

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.

Comprehensive profiling of 8p11-12 amplification in breast cancer.

In human carcinomas, especially breast cancer, chromosome arm 8p is frequently involved in complex chromosomal rearrangements that combine amplification at 8p11-12, break in the 8p12-21 region, and loss of 8p21-ter. Several studies have identified putative oncogenes in the 8p11-12 amplicon. However, discrepancies and the lack of knowledge on the structure of this amplification lead us to think that the actual identity of the oncogenes is not definitively established. We present here a comprehensive study combining genomic, expression, and chromosome break analyses of the 8p11-12 region in breast cell lines and primary breast tumors. We show the existence of four amplicons at 8p11-12 using array comparative genomic hybridization. Gene expression analysis of 123 samples using DNA microarrays identified 14 genes significantly overexpressed in relation to amplification. Using fluorescence in situ hybridization analysis on tissue microarrays, we show the existence of a cluster of breakpoints spanning a region just telomeric to and associated with the amplification. Finally, we show that 8p11-12 amplification has a pejorative effect on survival in breast cancer.

Enumeration of bacteria from the Clostridium leptum subgroup in human faecal microbiota using Clep1156 16S rRNA probe in combination with helper and competitor oligonucleotides.

Target site inaccessibility represents a significant problem for fluorescent in situ hybridisation (FISH) of 16S rRNA oligonucleotide probes. For this reason, the Clep1156 probe targeting 16S rRNA of the Clostridium leptum phylogenetic subgroup used for dot blot experiments could not be used until now for FISH. Considering that bacteria from the C. leptum subgroup are very abundant in the human faecal microbiota and may play a significant role in host health, we have used unlabelled helper and competitor oligonucleotides to improve the 16S rRNA in situ accessibility and specificity of the Clep1156 probe and applied this approach to enumerate C. leptum bacteria in this ecosystem. Nine C. leptum target strains and five non-target strains were selected to develop and validate the helper-competitor strategy. Depending on the target strains, the use of helpers enhanced the fluorescence intensity signal of Clep1156 from 0.4-fold to 8.4-fold with a mean value of 3.6-fold, switching this probe from the brightness class V-VI (masked sites) to III-IV (accessible sites). The simultaneous use of helper and competitor oligonucleotides with Clep1156 probe allowed the expected specificity without disturbing in situ accessibility. Quantified by FISH combined with flow cytometry, C. leptum bacteria in human faecal samples (n=22) represented 19 +/- 7% of bacteria on average [4.9-37.5]. We conclude that helper oligonucleotides are very useful to circumvent the problem of target site in situ accessibility, especially when probe design is limited to only one 16S rRNA area and that helpers and competitors may be efficiently combined.

Fecal calprotectin excretion in preterm infants during the neonatal period.

BACKGROUND: Fecal calprotectin has been proposed as a non-invasive marker of intestinal inflammation in inflammatory bowel disease in adults and children. Fecal calprotectin levels have been reported to be much higher in both healthy full-term and preterm infants than in children and adults. OBJECTIVE: To determine the time course of fecal calprotectin (f-calprotectin) excretion in preterm infants from birth until hospital discharge and to identify factors influencing f-calprotectin levels in the first weeks of life, including bacterial establishment in the gut. METHODOLOGY: F-calprotectin was determined using an ELISA assay in 147 samples obtained prospectively from 47 preterm infants (gestational age, and birth-weight interquartiles 27-29 weeks, and 880-1320 g, respectively) at birth, and at 2-week intervals until hospital discharge. PRINCIPAL FINDINGS: Although median f-calprotectin excretion was 138 microg/g, a wide range of inter- and intra-individual variation in f-calprotectin values (from day 3 to day 78) was observed (86% and 67%, respectively). In multivariate regression analysis, f-calprotectin correlated negatively with ante and per natal antibiotic treatment (p = 0.001), and correlated positively with the volume of enteral feeding (mL/kg/d) (p = 0.009), the need to interrupt enteral feeding (p = 0.001), and prominent gastrointestinal colonization by Clostridium sp (p = 0.019) and Staphylococcus sp (p = 0.047). CONCLUSION: During the first weeks of life, the high f-calprotectin values observed in preterm infants could be linked to the gut bacterial establishment.

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.

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.