Stable isotope spectrometry to study human protein and amino acid metabolism

O desenvolvimento e uso de técnicas laboratoriais, envolvendo espectrometria de massa, permitiram que os isótopos estáveis pudessem ser utilizados na avaliação do metabolismo aminoacídico e protéico, em seres biológicos. Assim, medidas precisas de enriquecimento isotópico, ao nível de 3-6% além do basal, após infusão de 13C-, 15N- ou de ²H2 -aminoácido, tornaram possível realizar a avaliação das interrelações metabólicas protéicas no organismo, in vivo. O método pode ser considerado pouco invasivo, sendo necessário, geralmente, coleta de ar expirado e sangue periférico, para determinação de substratos na concentração em torno de nanomolar. Desta maneira, períodos de infusão isotópica, de 4-6 horas, de 13C-leucina, permitem estimativa do metabolismo protéico corpóreo total, pela medida direta da oxidação, via determinação do 13CO2, no ar expirado, e da 13C-Leu ou seu metabólito, 13C-KIC, no sangue, assim como estimativa da degradação e síntese protéica. Mais recentemente, o advento de nova técnica, ou seja, a da associação de combustão da amostra à cromatografia e espectrometria, tornou o método mais sensível, sendo possível a determinação de substrato, marcado em amostras diluídas por um fator mínimo de 10. O método também permite o estudo da interrelação do metabolismo protéico com o de hidrato de carbono e lipídeo, quando aplicado a aminoácidos, essenciais ou não, associados a glicídios e a triglicerídeos.The aim of this work is to describe the gas chromatography/mass spectrometry (GC/MS) and its clinical application. GC/MS is becoming a tool for clinical diagnosis and research procedure interested in the molecular pathogenesis of diseases. The method is highly sensitive analytical that can obtain precise information on molecule weight and structure from a trace amount of sample. It has been applied to a wide range of fields from physics and chemistry, to life sciences such as biochemistry, pharmacy and medicine

Cord Blood Glutathione Depletion in Preterm Infants: Correlation with Maternal Cysteine Depletion

Background: Depletion of blood glutathione (GSH), a key antioxidant, is known to occur in preterm infants. Objective: Our aim was to determine: 1) whether GSH depletion is present at the time of birth; and 2) whether it is associated with insufficient availability of cysteine (cys), the limiting GSH precursor, or a decreased capacity to synthesize GSH. Methodology: Sixteen mothers delivering very low birth weight infants (VLBW), and 16 mothers delivering healthy, full term neonates were enrolled. Immediately after birth, erythrocytes from umbilical vein, umbilical artery, and maternal blood were obtained to assess GSH [GSH] and cysteine [cys] concentrations, and the GSH synthesis rate was determined from the incorporation of labeled cysteine into GSH in isolated erythrocytes ex vivo, measured using gas chromatography mass spectrometry. Principal Findings: Compared with mothers delivering at full term, mothers delivering prematurely had markedly lower erythrocyte [GSH] and [cys] and these were significantly depressed in VLBW infants, compared with term neonates. A strong correlation was found between maternal and fetal GSH and cysteine levels. The capacity to synthesize GSH was as high in VLBW as in term infants. Conclusion: The current data demonstrate that: 1) GSH depletion is present at the time of birth in VLBW infants; 2) As VLBW neonates possess a fully active capacity to synthesize glutathione, the depletion may arise from inadequate cysteine availability, potentially due to maternal depletion. Further studies would be needed to determine whether maternal-fetal cysteine transfer is decreased in preterm infants, and, if so, whether cysteine supplementation of mothers at risk of delivering prematurely would strengthen antioxidant defense in preterm neonates

The apparent breastfeeding paradox in very preterm infants: relationship between breast feeding, early weight gain and neurodevelopment based on results from two cohorts, EPIPAGE and LIFT

Context: Supplementation of breast milk is difficult once infants suckle the breast and is often discontinued at end of hospitalisation and after discharge. Thus, breastfed preterm infants are exposed to an increased risk of nutritional deficit with a possible consequence on neurodevelopmental outcome. Objective: To assess the relationship between breast feeding at time of discharge, weight gain during hospitalisation and neurodevelopmental outcome. Design: Observational cohort study. Setting: Two large, independent population-based cohorts of very preterm infants: the Loire Infant Follow-up Team (LIFT) and the EPIPAGE cohorts. Patients: 2925 very preterm infants alive at discharge. Main outcome measure: Suboptimal neurodevelopmental outcome, defined as a score in the lower tercile, using Age and Stages Questionnaire at 2 years in LIFT and Kaufman Assessment Battery for Children Test at 5 years in EPIPAGE. Two propensity scores for breast feeding at discharge, one for each cohort, were used to reduce bias. Results: Breast feeding at time of discharge concerned only 278/1733 (16%) infants in LIFT and 409/2163 (19%) infants in EPIPAGE cohort. Breast feeding is significantly associated with an increased risk of losing one weight Z-score during hospitalisation (LIFT: n=1463, adjusted odd ratio (aOR)=2.51 (95% CI 1.87 to 3.36); EPIPAGE: n=1417, aOR=1.55 (95% CI 1.14 to 2.12)) and with a decreased risk for a suboptimal neurodevelopmental assessment (LIFT: n=1463, aOR=0.63 (95% CI 0.45 to 0.87); EPIPAGE: n=1441, aOR=0.65 (95% CI 0.47 to 0.89) and an increased chance of having a head circumference Z-score higher than 0.5 at 2 years in LIFT cohort (n=1276, aOR=1.43 (95% CI 1.02 to 2.02)) and at 5 years in EPIPAGE cohort (n=1412, aOR=1.47 (95% CI 1.10 to 1.95)). Conclusions: The observed better neurodevelopment in spite of suboptimal initial weight gain could be termed the 'apparent breastfeeding paradox' in very preterm infants. Regardless of the mechanisms involved, the current data provide encouragement for the use of breast feeding in preterm infants

1H-NMR-Based Metabolic Profiling of Maternal and Umbilical Cord Blood Indicates Altered Materno-Foetal Nutrient Exchange in Preterm Infants

Background: Adequate foetal growth is primarily determined by nutrient availability, which is dependent on placental nutrient transport and foetal metabolism. We have used 1H nuclear magnetic resonance (NMR) spectroscopy to probe the metabolic adaptations associated with premature birth. Methodology: The metabolic profile in 1H NMR spectra of plasma taken immediately after birth from umbilical vein, umbilical artery and maternal blood were recorded for mothers delivering very-low-birth-weight (VLBW) or normo-ponderal full-term (FT) neonates. Principal Findings: Clear distinctions between maternal and cord plasma of all samples were observed by principal component analysis (PCA). Levels of amino acids, glucose, and albumin-lysyl in cord plasma exceeded those in maternal plasma, whereas lipoproteins (notably low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) and lipid levels were lower in cord plasma from both VLBW and FT neonates. The metabolic signature of mothers delivering VLBW infants included decreased levels of acetate and increased levels of lipids, pyruvate, glutamine, valine and threonine. Decreased levels of lipoproteins glucose, pyruvate and albumin-lysyl and increased levels of glutamine were characteristic of cord blood (both arterial and venous) from VLBW infants, along with a decrease in levels of several amino acids in arterial cord blood. Conclusion: These results show that, because of its characteristics and simple non-invasive mode of collection, cord plasma is particularly suited for metabolomic analysis even in VLBW infants and provides new insights into the materno-foetal nutrient exchange in preterm infants

Non-Invasive Exploration of Neonatal Gastric Epithelium by Using Exfoliated Epithelial Cells

Background & Aims: In preterm infants, exfoliated gastric epithelial cells can be retrieved from aspirates sampled through the naso-gastric feeding tube. Our aims were to determine (1) whether the recovery of exfoliated cells is feasible at any time from birth through the removal of the nasogastric tube, (2) whether they can be grown in culture in vitro, and (3) whether the physiological state of exfoliated cells expressing H+/K+-ATPases reflects that of their counterparts remaining in situ at the surface of the gastric epithelium in neonatal rat pups. Methods: In infants, gastric fluid aspirates were collected weekly after birth or every 3 hours over 24-h periods, and related to clinical parameters (Biocollection PROG/09/18). In rat pups submitted to a single fasting/refeeding cycle, we explored circadian exfoliation with the cellular counter-parts in the gland. All samples were analyzed by confocal imaging and Enzyme-Linked Immunosorbent Assay. Results: Epithelial cells were identified by microscopy using membrane-bound anti-H+/K+ ATPases antibody, assessed for nucleus integrity, and the expression of selected proteins (autophagy, circadian clock). On 34 infants, the H+/K+-ATPasepositive cells were consistently found quiescent, regardless of gestational age and feeding schedule from day-5 of life to the day of removal of the naso-gastric tube. By logistic regression analysis, we did find a positive correlation between the intensity of exfoliation (cellular loss per sample) and the postnatal age (p,0.001). The H+/K+ ATPase-positive cell

Fecal Calprotectin Excretion in Preterm Infants during the Neonatal Period

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.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.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. (p = 0.047).During the first weeks of life, the high f-calprotectin values observed in preterm infants could be linked to the gut bacterial establishment

Besoins nutritionnels de la femme enceinte et du fœtus

National audienc

Etude cinetique du metabolisme de la glutamine par les isotopes stables in vivo chez l'homme

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 78628 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

L’empreinte nutritionnelle, ou comment la nutrition au début de la vie détermine la santé du futur adulte

National audienc

Interventions nutritionnelles chez le prématuré après l’hospitalisation: bénéfices, risques

National audienc