You Are What You Eat: Mass spectrometry in paediatric kinetic studies using stable isotopes

An overview will be presented about applications of stable isotopes in paediatric research. Mass spectrometry has proven to be an essential tool for unravelling kinetic studies in a large range of different research disciplines related to intestinal diseases, obesities, severe cerebral palsy, oxidative stress and foetal metabolism. Due to the diversity and complexity of the different metabolites involved in these studies, there is a high demand on sophisticated mass spectrometric instruments. Several new methods have been developed for measurement of isotopic labelled compounds in body fluids. 13C isotopic glucose enrichment in human plasma is analysed, using liquid chromatography isotope ratio mass spectrometry (LC/IRMS). Also new methods were developed for measuring the glutathione (GSH) fractional synthesis rate (FSR) in neonates after infusion of [1-13C]glycine as a tracer. For measuring energy expenditure and total body water composition in humans the doubly labelled water method is valuable technique. It usually involves blood or urine sampling, which might be difficult in neonates and children with cerebral palsy or other disabilities. We therefore aimed to validate a method making use of saliva samples analyzed by automated thermal conversion elemental analyzer in combination with isotope ratio mass spectrometry (TC/EA/IRMS). The different types of mass spectrometric instruments will be discussed here as well as several applications in paediatric research utilizing these techniques. The applications cover amino acid metabolism and body composition, energy expenditure, and the synthesis of specific proteins such as glutathione and albumin in different groups of children and even in foetuses. Finally, the aims and outline of this dissertation are covered

Albumin synthesis in preterm infants on the first day of life studied with [1-13C]leucine

Albumin is the major binding protein in the human neonate. Low production of albumin will lower its transport and binding capacity. This is especially important in preterm infants, in whom albumin binds to potentially toxic products such as bilirubin and antibiotics. To study the metabolism of plasma albumin in preterm infants, we administered a 24-h constant infusion of [1-(13)C]leucine to 24 very low birth weight (VLBW) infants (28.4 +/- 0.4 wk, 1,080 +/- 75 g) on the first day of life. The caloric intake consisted of glucose only, and therefore amino acids for albumin synthesis were derived from proteolysis. The fractional synthesis rate (FSR) of plasma albumin was 13.9 +/- 1.5%/day, and the absolute synthesis rate was 148 +/- 17 mg x kg(-1) x day(-1). Synthesis rates were significantly lower (P <0.03) in infants showing intrauterine growth retardation. Albumin synthesis increased with increasing SD scores for gestation and weight (P <0.05). The FSR of albumin tended to increase by 37% after administration of antenatal corticosteroids to improve postnatal lung function (P=0.09). We conclude that liver synthetic capacity is well developed in VLBW infants and that prenatal corticosteroids tend to increase albumin synthesis. Decreased weight gain rates in utero have effects on protein synthesis postnatall

Continuous Enteral Administration Can Enable Normal Amino Acid Absorption in Rats with Methotrexate-Induced Gastrointestinal Mucositis

It is unknown what feeding strategy to use during chemotherapy-induced gastrointestinal mucositis, which causes weight loss and possibly malabsorption. To study the absorptive capacity of amino acids during mucositis, we determined the plasma availability of enterally administered amino acids (AA), their utilization for protein synthesis, and the preferential side of the intestine for AA uptake in rats with and without methotrexate (MTX)-induced mucositis. Four days after injection with MTX (60 mg/kg) or saline (controls), rats received a primed, continuous dual-isotope infusion (intraduodenal and intravenous) of labeled L-leucine, L-lysine, L-phenylalanine, L-threonine, and L-methionine. We collected blood samples, assessed jejunal histology, and determined labeled AA incorporation in proximal and distal small intestinal mucosa, plasma albumin, liver, and thigh muscle. MTX-induced mucositis was confirmed by histology. The median systemic availability of all AA except for leucine was similar in MTX-treated rats and in controls. However, the individual availability of all AA differed substantially within the group of MTX-treated rats, ranging from severely reduced (40% of intake in 5 of 9 rats). More AA originating from basolateral uptake than those originating from apical uptake were used for intestinal protein synthesis in MTX-treated rats (>= 420% more, P <0.05). We conclude that continuous enteral administration can enable normal AA absorption in rats with MTX-induced mucositis. The intestine prefers basolateral AA uptake to meet its need for AA for protein synthesis during mucositis. J. Nutr. 142: 1983-1990, 2012

Methionine transmethylation and transsulfuration in the piglet gastrointestinal tract

Methionine is an indispensable sulfur amino acid that functions as a key precursor for the synthesis of homocysteine and cysteine. Studies in adult humans suggest that splanchnic tissues convert dietary methionine to homocysteine and cysteine by means of transmethylation and transsulfuration, respectively. Studies in piglets show that significant metabolism of dietary indispensable amino acids occurs in the gastrointestinal tissues (GIT), yet the metabolic fate of methionine in GIT is unknown. We show here that 20% of the dietary methionine intake is metabolized by the GIT in piglets implanted with portal and arterial catheters and fed milk formula. Based on analyses from intraduodenal and intravenous infusions of [1-13C]methionine and [2H3]methionine, we found that the whole-body methionine transmethylation and remethylation rates were significantly higher during duodenal than intravenous tracer infusion. First-pass splanchnic metabolism accounted for 18% and 43% of the whole-body transmethylation and remethylation, respectively. Significant transmethylation and transsulfuration was demonstrated in the GIT, representing \xe2\x89\x8827% and \xe2\x89\x8823% of whole-body fluxes, respectively. The methionine used by the GIT was metabolized into homocysteine (31%), CO2(40%), or tissue protein (29%). Cystathionine \xce\xb2-synthase mRNA and activity was present in multiple GITs, including intestinal epithelial cells, but was significantly lower than liver. We conclude that the GIT consumes 20% of the dietary methionine and is a significant site of net homocysteine production. Moreover, the GITs represent a significant site of whole-body transmethylation and transsulfuration, and these two pathways account for a majority of methionine used by the GITs

Albumin synthesis in very low birth weight infants is enhanced by early parenteral lipid and high-dose amino acid administration

Albumin is one of the most important plasma proteins and plays a key role in many physiologic processes, such as preserving colloid osmotic pressure, scavenging radicals, and binding and transporting bilirubin, hormones, and drugs. However, albumin concentrations are often low in preterm infants during the first days of life. We hypothesized that early parenteral lipid and high-dose amino acid (AA) administration to very low birth weight (VLBW) infants from birth onwards increases hepatic albumin synthesis rates. Inborn VLBW infants were randomized to receive from birth onwards either 2.4 g amino acids/(kg(.)d) (control group), 2.4 g amino acids/(kg(.)d) plus 2 g lipids/(kg(.)d) (AA + lipid group), or 3.6 g amino acids/(kg(.)d) plus 2 g lipids/(kg(.)d) (high AA + lipid group). On postnatal day 2, infants received a primed continuous infusion of [U-(13)C6,(15)N]leucine. Mass spectrometry was used to determine the fractional and absolute albumin synthesis rates (FSR and ASR, respectively). In total, 28 infants (median gestational age 27 weeks (IQR 25-28), median birth weight 810 g (IQR 679-998) were studied. The median FSR was 6.5%/d in the control group, 10.6%/d in the AA group, and 12.3%/d in the high AA + lipid group, while the median was 84 mg/(kg(.)d) in the control group, 138 mg/(kg(.)d) in the AA group, and 160 mg/(kg(.)d) in the high AA + lipid group. A group of VLBW infants given parenteral nutrition containing lipids and high-dose amino acids showed a higher rate of albumin synthesis compared to infants receiving no lipids and standard amounts of amino acids during the first two days of lif

Human fetal albumin synthesis rates during different periods of gestation

Despite nutritional intervention, albumin concentrations are often low in critically ill premature neonates. Our aim was to quantify albumin synthesis rates during early life under physiologic circumstances. Human fetuses thereby reflect the developmentally related optimal condition. Pregnant women undergoing elective cesarean delivery received 3 different labeled amino acid infusions starting at different times before surgery. With the use of mass spectrometry techniques, this novel model enabled us to quantify fetal albumin synthesis from a single blood sample taken from the umbilical cord after cesarean delivery. The fractional synthesis rate reflects the fraction of the albumin pool that is daily renewed. The absolute synthesis rate is the absolute amount of albumin that is daily synthesized. Results are expressed as medians (25th-75th percentile). We studied 8 fetuses at 29.9 (28.4-35.4) weeks of gestation and 8 fetuses around term. Fractional synthesis rates in premature fetuses [17.5 (12.1-24.4) %/d] were higher (P = 0.02) than in mature fetuses [10.4 (9.1-13.7) %/d]. Absolute synthesis rates were also higher (P = 0.02) in premature than in mature fetuses: 280 (227-365) versus 205 (184-238) mg . kg(-1) . d(-1). On a weight basis, albumin synthesis rates in premature fetuses were higher than in fetuses at term and were higher than the rates previously found in neonates after preterm birth. Considering that the premature fetal liver can synthesize albumin at a high rate, the observed hypoalbuminemia in premature infants therefore seems to suggest that current (nutritional) therapies fail to meet requirements necessary to sustain optimum albumin synthesis rate