Schierbeek, H.

Lysine requirement of the enterally fed term infant in the first month of life (Article)

2011-12-01T00:00:00Z

Background: Infant nutrition has a major impact on child growth and functional development. Low and high intakes of protein or amino acids could have a detrimental effect. Objective: The objective of the study was to determine the lysine requirement of enterally fed term neonates by using the indicator amino acid oxidation (IAAO) method. L-[1-13C]phenylalanine was used as an indicator amino acid. Design: Twenty-one neonates were randomly assigned to lysine intakes that ranged from 15 to 240 mg·kg-1·d-1. Breath, urine, and blood samples were collected at baseline and during the plateau. The mean lysine requirement was determined by using biphasic linear regression crossover analysis on the fraction of13CO2recovery from L-[1-13C] phenylalanine oxidation (F13CO2) and phenylalanine oxidation rates calculated from the L-[1-13C]phenylalanine enrichment of urine and plasma. Results: The mean (±SD) phenylalanine flux calculated from urine and plasma L-[1-13C]phenylalanine enrichment data were 88.3 ± 6.9 and 84.5 ± 7.4 μmol·kg-1·h-1, respectively. Graded intakes of lysine had no effect on phenylalanine fluxes. The mean lysine requirement determined by F13CO2was 130 mg·kg-1·d-1(upper and lower CIs: 183.7 and 76.3 mg·kg-1·d-1, respectively). The mean requirement was identical to the requirement determined by using phenylalanine oxidation rates in urine and plasma. Conclusions: The mean lysine requirement of enterally fed term neonates was determined by using F13CO2and phenylalanine oxidation rates calculated from the L-[1-13C] phenylalanine enrichment of urine and plasma. These methods yielded a similar result of 130 mg lysine·kg-1·d-1. This study demonstrates that sampling of13CO2in expired air is sufficient to estimate the lysine requirement by using the IAAO method in infants. This trial was registered at www.trialregister.nl as NTR1610.

Amino acid metabolism in the human fetus at term: Leucine, valine, and methionine kinetics (Article)

2011-12-01T00:00:00Z

Human fetal metabolism is largely unexplored. Understanding how a healthy fetus achieves its fast growth rates could eventually play a pivotal role in improving future nutritional strategies for premature infants. To quantify specific fetal amino acid kinetics, eight healthy pregnant women received before elective cesarean section at term, continuous stable isotope infusions of the essential amino acids [1-13C,15N]leucine, [U-13C5]valine, and [1-13C]methionine. Umbilical blood was collected after birth and analyzed for enrichments and concentrations using mass spectrometry techniques. Fetuses showed considerable leucine, valine, and methionine uptake and high turnover rates. α-Ketoisocaproate, but not α-ketoisovalerate (the leucine and valine ketoacids, respectively), was transported at net rate from the fetus to the placenta. Especially, leucine and valine data suggested high oxidation rates, up to half of net uptake. This was supported by relatively low α-ketoisocaproate reamination rates to leucine. Our data suggest high protein breakdown and synthesis rates, comparable with, or even slightly higher than in premature infants. The relatively large uptakes of total leucine and valine carbon also suggest high fetal oxidation rates of these essential branched chain amino acids.

Reducing glucose infusion safely prevents hyperglycemia in post-surgical children (Article)

2011-12-01T00:00:00Z

Background & aims: To investigate the effects of two different glucose infusions on glucose homeostasis and amino acid metabolism in post-surgical children. Methods: This randomized crossover study evaluated glucose and amino acid metabolism in eight children (age 9.8 ± 1.9 months, weight 9.5 ± 1.1 kg) admitted to a pediatric intensive care unit in a tertiary university hospital after surgical correction for non-syndromal craniosynostosis. Patients were randomized to receive low (LG; 2.5 mg kg-1min-1) and standard (SG; 5.0 mg kg-1min-1) glucose infusion in a crossover setting. After a bolus (4 g kg-1) of deuterium oxide, we conducted a primed, constant, 8 h tracer infusion with [6,6-2H2]Glucose, [1-13C]Leucine, [ring-2H5]Phenylalanine and [3,3-2H2]Tyrosine. Results: SG resulted in hyperglycemia (defined as > 6.1 mmol L-1), while during LG plasma glucose levels were normoglycemic (5.9 ± 0.6 vs. 7.5 ± 1.7 mmol L-1; LG vs. SG respectively, p = 0.02). Hypoglycemia did not occur during LG infusion. Endogenous glucose production was not fully suppressed during the hyperglycemic state under SG and increased with reduced glucose infusion (2.6 ± 1.5 vs. 1.1 ± 1.4 mg kg-1min-1; LG vs. SG; p = 0.05). Whole body protein balance derived from leucine and phenylalanine kinetics was slightly negative but not further affected with a decrease in glucose infusion. Conclusions: The current recommended glucose infusion induces hyperglycemia in post-surgical children. A reduced glucose infusion safely reduced high glucose levels, while children were capable to sustain normoglycemia with increased endogenous glucose production. The reduced glucose infusion did not exacerbate the mild catabolic state in which the patients were.

Current recommended parenteral protein intakes do not support protein synthesis in critically ill septic, insulin-resistant adolescents with tight glucose control (Article)

2011-11-01T00:00:00Z

Objective: To investigate the effects of insulin infusion and increased parenteral amino acid intakes on whole body protein balance, glucose kinetics, and lipolysis in critically ill, insulin-resistant, septic adolescents. Design: A single-center, randomized, crossover study. Setting: A medicosurgical intensive care unit in a tertiary university hospital. Patients: Nine critically ill, septic adolescents (age 15.0 ± 1.2 yrs, body mass index 20 ± 4 kg m-2) receiving total parenteral nutrition. Interventions: Patients received total parenteral nutrition with standard (1.5 g•kg-1•day-1) and high (3.0 g•kg-1•day-1) amino acid intakes in a 2-day crossover setting, randomized to the order in which they received it. On both study days, we conducted a primed, constant, 7-hr stable isotope tracer infusion with [1-13C]leucine, [6,6-2H2]glucose, and [1,1,2,3,3-2H5]glycerol, in combination with a hyperinsulinemic euglycemic clamp during the last 3 hrs. Measurements and Main Results: Insulin decreased protein synthesis at standard amino acid and high amino acid intakes (p < .01), while protein breakdown decreased with insulin at standard amino acid intake (p < .05) but not with the high amino acid intake. High amino acid intake improved protein balance (p < .05), but insulin did not have an additive effect. There was significant insulin resistance with an M value of ∼3 (mg•kg-1•min-1)/(mU•mL-1) which was 30% of reported normal values. At high amino acid intake, endogenous glucose production was not suppressed by insulin and lipolysis rates increased. Conclusion: The current recommended parenteral amino acid intakes are insufficient to maintain protein balance in insulin-resistant patients during tight glucose control. During sepsis, insulin decreases protein synthesis and breakdown, and while high amino acid intake improves protein balance, its beneficial effects may be offset by enhanced endogenous glucose production and lipolysis, raising concerns that insulin resistance may have been exacerbated and that gluconeogenesis may have been favored by high amino acid intakes. Dose-response studies on the effect of the level of amino acid intakes (protein) on energy metabolism are needed.

Foreword (Article)

2011-10-30T00:00:00Z

Measuring body composition and energy expenditure in children with severe neurologic impairment and intellectual disability (Article)

2011-09-01T00:00:00Z

Background: Accurate prediction equations for estimating body composition and total energy expenditure (TEE) in children with severe neurologic impairment and intellectual disability are currently lacking. Objective: The objective was to develop group-specific equations to predict body composition by using skinfold-thickness measurements and bioelectrical impedance analysis (BIA) and to predict TEE by using data on mobility, epilepsy, and muscle tone. Design: Measures of body composition with the use of skinfoldthickness measurements (percentage of body fat) and BIA (total body water) were compared with those from isotope dilution (reference method) by using intraclass correlation coefficients (ICCs) and Bland and Altman limits of agreement analyses. With the use of the same methods, the outcomes of cerebral palsy - specific TEE equations were compared with those of the doubly labeled water method (reference method). Group-specific regression equations were developed by using forward-stepwise-multiple-correlationregression analyses. Results: Sixty-one children with a mean (±SD) age of 10.1 ± 4.3 y (32 boys) were studied. A new equation based on the sum of 4 skinfold-thickness measurements did not improve agreement (n = 49; ICC = 0.61), whereas the newly developed BIA equation - which includes tibia length as an alternative for standing height - did improve agreement (n = 61; ICC = 0.96, SEE = 1.7 kg, R2= 0.92). The newly developed TEE equation, which uses body composition, performed better (n = 52; ICC = 0.87, SEE = 180 kcal, R2= 0.77) than did the equation of Schofield (n = 52; ICC = 0.82, SEE = 207 kcal, R2= 0.69). Conclusions: Current cerebral palsy - specific equations for measuring body composition and energy expenditure are inaccurate. BIA is more accurate at assessing nutritional status in this population than is the measurement of skinfold thickness. The newly developed TEE equation, which uses body composition, provides a reasonable estimate of energy expenditure in these children despite its variability.

Albumin synthesis rates in post-surgical infants and septic adolescents; influence of amino acids, energy, and insulin (Article)

2011-08-01T00:00:00Z

Background & aims: To investigate the effects of glucose, parenteral amino acids, and intravenous insulin on albumin synthesis rates in critically ill children. Methods: Two studies were performed in 8 post-surgical infants (age 9.8 ± 1.9 months; weight 9.5 ± 1.1 kg) and 9 septic adolescents (age 15 ± 1 yr; BMI 23 ± 4 kg m-2), respectively. All received a primed, constant, tracer infusion with [1-13C]Leucine. The infants in study 1 were randomized to receive low (2.5 mg kg-1min-1) and standard (5.0 mg kg-1min-1) glucose intake in a cross-over setting of two periods of 4 h each. The adolescents in study 2 were randomized to receive total parenteral nutrition with standard (1.5 g kg-1day-1) and high (3.0 g kg-1day-1) amino acid intake in a two day cross-over setting. On both study days, during the last 3 h of the tracer study, they received insulin infused at 80 mU m-2min-1. Results: The post-surgical infants and the septic adolescents were mildly hypoalbuminemic (∼2.5 g dL-1) with high synthesis rates, which were not affected by different intakes of glucose, amino acids, or insulin infusion. Conclusions: Albumin synthesis rates in hypoalbuminemic critically ill children are high but were not upregulated through nutrient supply, and in septic adolescents are unaffected by insulin.

Intestinal threonine utilization for protein and mucin synthesis is decreased in formula-fed preterm pigs (Article)

2011-07-01T00:00:00Z

Threonine is an essential amino acid necessary for synthesis of intestinal (glyco)proteins such as mucin MUC2 to maintain adequate gut barrier function. In premature infants, reduced barrier function may contribute to the development of necrotizing enterocolitis (NEC). Human milk protects against NEC compared with infant formula. Therefore, we hypothesized that formula feeding decreases the MUC2 synthesis rate concomitant with a decrease in intestinal first-pass threonine utilization, predisposing the preterm neonate to NEC. Preterm pigs were delivered by caesarian section and received enteral feeding with formula (FORM; n = 13) or bovine colostrum (COL; n = 6) for 2 d following 48 h of total parenteral nutrition. Pigs received a dual stable isotope tracer infusion of threonine to determine intestinal threonine kinetics. NEC developed in 38% of the FORM pigs, whereas none of the COL pigs were affected (P = 0.13). Intestinal fractional first-pass threonine utilization was lower in FORM pigs (49 ± 2%) than in COL pigs (60 ± 4%) (P = 0.02). In FORM pigs compared with COL pigs, protein synthesis (369 ± 31 mg·kg-1·d-1vs. 615 ± 54 mg·kg-1·d-1; P = 0.003) and MUC2 synthesis (121 ± 17%/d vs. 184 ± 15%/d; P = 0.02) were lower in the distal small intestine (SI). Our results suggest that formula feeding compared with colostrum feeding in preterm piglets reduces mucosal growth with a concomitant decrease in first-pass splanchnic threonine utilization, protein synthesis, and MUC2 synthesis in the distal SI. Hence, decreased intestinal threonine metabolism and subsequently impaired gut barrier function may predispose the formula-fed infant to developing NEC.

Measuring energy expenditure and body composition in children with neurological impairment and intellectual disability using the doubly labeled water method: Comparing urine and saliva sampling (Article)

2010-10-01T00:00:00Z

Background & Aims: Information is lacking on the comparability of the outcomes of the doubly labeled water method using urine and saliva samples in children with severe neurological impairment and intellectual disability (ID). Our aim is to compare outcomes and feasibility based on both sampling methods. Methods: Total energy expenditure (TEE) and total body water (TBW) were calculated using urine and saliva samples of thirteen children (aged 3-15 y). To correct for age and weight, TEE was expressed as a percentage of recommended daily allowance (%TEE of RDA) and TBW as a percentage of weight (%TBW). Agreement between methods was evaluated using Bland and Altman analyses. Results: 88.5% of urine and 79.5% of saliva samples were successfully analyzed. Limits of agreement between urine and saliva samples were favorable for the outcomes %TEE of RDA (mean difference -1.9% ± 7.5%) and %TBW (mean difference -1.9% ± 3.0%). Conclusions: Both urine and saliva are feasible sample media for the doubly labeled water method in children with severe neurological impairment and ID. Clinical outcomes based on either urine or saliva samples agree well. Thus, choosing either one of the sampling methods is justified if the other fails.

Almost all enteral aspartate is taken up in first-pass metabolism in enterally fed preterm infants (Article)

2010-06-01T00:00:00Z

Background & aims: The intestine is a major site of amino acid metabolism, especially in neonates. Neonatal animals derive energy needed for metabolic processes from dietary glucose and amino acids. Rats were found to oxidize non-essential amino acids such as aspartate, glutamate and glutamine in the intestine at a high rate. We have previously found that glutamate and glucose are important sources of energy for the splanchnic tissues in fully fed preterm infants. However, no data are available on splanchnic aspartate metabolism in human preterm infants. In the present study we studied whole-body and splanchnic aspartate metabolism and determined the metabolic fate of aspartate. Methods: In eight, enterally fed, preterm infants (gestational age 31 weeks (wk)±3 SD, range: 26-34wk) splanchnic and whole-body aspartate kinetics were assessed by dual tracer ([U-13C]aspartate and [D3]aspartate) techniques. Results: Splanchnic first-pass aspartate uptake was almost complete (77±15%). Almost all (80±9%) of the13C administered as [U-13C]aspartate used in first-pass was recovered as CO2in expired breath. Conclusion: The splanchnic tissues extract almost all of the dietary aspartate in preterm infants. The majority of the labeled carbon is recovered in expired breath, making it most likely that the sequestered carbon skeleton of aspartate is utilized for energy generation.

Glutathione synthesis rates in early postnatal life (Article)

2010-04-01T00:00:00Z

Preterm infants have diminished antioxidant defenses. Glutathione (GSH), the main intracellular antioxidant, increases upon amino acid (AA) administration in preterm infants, without an accompanying rise of the fractional synthesis rate of GSH (FSRGSH) This study investigated the mechanism behind this increased GSH concentration by determining GSH synthesis in the first days after birth using stable isotope techniques in very low-birth-weight (VLBW) infants receiving i.v. AAs. Advanced oxidized protein products (AOPPs) were determined to quantify oxidative stress. Eighteen infants (birth weight 989 ± 241 g, gestational age of 27/7 ± 1/7 weeks) were studied either on postnatal day 1 or 2 (7 or 31 h postnatally, respectively). Concentration of GSH increased with postnatal age (1.45 ± 0.48 mM versus 1.99 ± 0.40 mM, p = 0.019). FSRGSH was not significantly different, but the absolute synthesis rate of GSH (ASRGSH) tended to be higher in the infants studied on day 2 [8.1 ± 2.7 mg/(kg • d) versus 10.6 ± 2.4 mg/(kg • d), p = 0.054]. AOPP concentrations were not different between groups. In conclusion, GSH concentration in VLBW infants increases significantly after birth. A concomitant increased synthesis rate was not found, suggesting that GSH consumption decreases upon AA administration.

Majority of dietary glutamine is utilized in first pass in preterm infants (Article)

2010-02-01T00:00:00Z

Glutamine is a conditionally essential amino acid for very low-birth weight infants by virtue of its ability to play an important role in several key metabolic processes of immune cells and enterocytes. Although glutamine is known to be used to a great extend, the exact splanchnic metabolism in enterally fed preterm infants is unknown. We hypothesized that preterm infants show a high splanchnic first-pass glutamine metabolism and the primary metabolic fate of glutamine is oxidation. Five preterm infants (mean ± SD birth weight 1.07 ± 0.22 kg and GA 29 ± 2 wk) were studied by dual tracer ([U-C]glutamine and [N2]glutamine) cross-over techniques on two study days (at postnatal week 3 ± 1 wk). Splanchnic and whole-body glutamine kinetics were assessed by plasma isotopic enrichment of [U-C]glutamine and [N2]glutamine and breath CO2 enrichments. Mean fractional first-pass glutamine uptake was 73 ± 6% and 57 ± 17% on the study days. The splanchnic tissues contributed for a large part (57 ± 6%) to the total amount of labeled carbon from glutamine retrieved in expiratory air. Dietary glutamine is used to a great extent by the splanchnic tissues in preterm infants and its carbon skeleton has an important role as fuel source.

Analysis of [U-13C6]glucose in human plasma using liquid chromatography/isotope ratio mass spectrometry compared with two other mass spectrometry techniques (Article)

2009-12-15T00:00:00Z

The use of stable isotope labelled glucose provides insight into glucose metabolism. The13C-isotopic enrichment of glucose is usually measured by gas chromatography/mass spectrometry (GC/MS) or gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). However, in both techniques the samples must be derivatized prior to analysis, which makes sample preparation more labour-intensive and increases the uncertainty of the measured isotopic composition. A novel method for the determination of isotopic enrichment of glucose in human plasma using liquid chromatography/ isotope ratio mass spectrometry (LC/IRMS) has been developed. Using this technique, for which hardly any sample preparation is needed, we showed that both the enrichment and the concentration could be measured with very high precision using only 20μL of plasma. In addition, a comparison with GC/MS and GC/IRMS showed that the best performance was achieved with the LC/IRMS method making it the method of choice for the measurement of13C-isotopic enrichment in plasma samples. Copyright

You Are What You Eat: Mass spectrometry in paediatric kinetic studies using stable isotopes (Doctoral Thesis)

2009-12-10T00:00:00Z

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.

Validation of deuterium and oxygen18 in urine and saliva samples from children using on-line continuous-flow isotope ratio mass spectrometry (Article)

2009-11-01T00:00:00Z

The doubly labelled water method is valuable for measuring energy expenditure in humans. 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 subjects received labelled water orally and urine and saliva samples were collected and analyzed. Deuterium as well as oxygen18was measured in one single run using a peak jump method. Excellent linearity was found for measurement of enrichments of deuterium (R2= 0.9999) and oxygen18(R2= 0.9999). The intra-assay precision and the inter-assay precision of the measurement of two standards were good for both deuterium and oxygen18. The variation between urine and saliva samples was small (4.83% for deuterium and 2.33% for oxygen18n = 40). Saliva sampling is to be preferred, therefore, as it can be easily collected and is non-invasive. Moreover, its time of production is almost exactly known. The TC-EA/IRMS method is a good alternative to the more laborious off-line IRMS measurements. Copyright

High-dose cysteine administration does not increase synthesis of the antioxidant glutathione preterm infants (Article)

2009-11-01T00:00:00Z

OBJECTIVE: Our aim was to evaluate whether administration of additional cysteine is safe and stimulates glutathione synthesis in preterm infants in early life. METHODS: We conducted a prospective, randomized, clinical trial with infants with birth weights of <1500 g (N = 20). The infants were assigned randomly to receive either a standard dose (45 mg/kg per day) or a high dose (81 mg/kg per day) of cysteine. Intakes of other amino acids were similar, providing a total protein intake of 2.4 g/kg per day in both groups. We recorded base requirements in the first 6 days of life. On postnatal day 2, we conducted a stable isotope study to determine glutathione concentrations and synthesis rates in erythrocytes. RESULTS: Base requirements were higher in the high-dose cysteine group on days 3, 4, and 5. Despite an 80% increase in cysteine intake, plasma cystine concentrations did not increase. Glutathione concentrations and synthesis rates did not increase with additional cysteine administration. CONCLUSIONS: Administration of a high dose of cysteine (81 mg/kg per day) to preterm infants seems clinically safe but does not stimulate glutathione synthesis, compared with a lower dose (45 mg/kg per day). Further research is required to determine whether there is significant benefit associated with cysteine supplementation. Copyright

Simultaneous analysis of 13C-glutathione as its dimeric form GSSG and its precursor [l-13c]glycine using liquid chromatography/isotope ratio mass spectrometry (Article)

2009-09-30T00:00:00Z

Determination of glutathione kinetics using stable isotopes requires accurate measurement of the tracers and tracees. Previously, the precursor and synthesized product were measured with two separate techniques, liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). In order to reduce sample volume and minimize analytical effort we developed a method to simultaneously determine13C-glutathione as its dimeric form (GSSG) and its precursor [1-13C]glycine in a small volume of erythrocytes in one single analysis. After having transformed13C-glutathione into its dimeric form GSSG, we determined both the intra-erythrocytic concentrations and the13C-isotopic enrichment of GSSG and glycine in 150 μL of whole blood using liquid chromatography coupled to LC/IRMS. The results show that the concentration (range of μmol/mL) was reliably measured using cycloleucine as internal standard, i.e. with a precision better than 0.1 μmol/mL. The13C-isotopic enrichment of GSSG and glycine measured in the same run gave reliable values with excellent precision (standard deviation (sd) <0.3%o) and accuracy (measured between 0 and 5 APE). This novel method opens up a variety of kinetic studies with relatively low dose administration of tracers, reducing the total cost of the study design. In addition, only a minimal sample volume is required, enabling studies even in very small subjects, such as preterm infants. Copyright

Threonine metabolism in the intestine of mice: Loss of mucin 2 induces the threonine catabolic pathway (Article)

2009-07-01T00:00:00Z

Objectives: Previous studies have shown that the intestine uses a major part of the dietary threonine intake for the synthesis of the structural component of the protective intestinal mucus layer, the secretory mucin Muc2. In this context, the high intestinal demand for dietary threonine probably results from its incorporation into secretory mucins rich in threonine residues. Therefore, we compared threonine utilization in the colon of Muc2 knockout (Muc2-/-) and wild-type (Muc2+/+) mice to investigate the intestinal dietary threonine metabolism in the absence of Muc2, which results in inflammation of the colon. Materials and Methods: Concentrations and isotopic enrichment of threonine were measured by gas chromatographyisotope ratio mass spectrometry in the serum, colon, and colonic content of mice given a bolus [U-13C]threonine enterally. Results: We retrieved 37.8% and 40.9% of dietary threonine in Muc2+/+and Muc2-/-mice, respectively, either as free or incorporated threonine. There were no major differences in the availability and concentration of free or incorporated threonine recovered in both serum and colon in both types of mice. However, the Muc2-/-mice did show overall significantly higher threonine oxidation rates compared with Muc2+/+mice. Conclusions: In the absence of Muc2, dietary threonine is mainly used for constitutive protein synthesis or becomes a substrate for metabolic oxidation. This indicates that inflammation also requires high threonine amounts. JPGN 49:99-107, 2009.

Small intestinal MUC2 synthesis in human preterm infants (Article)

2009-05-01T00:00:00Z

Mucin 2 (MUC2) is the structural component of the intestinal protective mucus layer, which contains high amounts of threonine in its peptide backbone. MUC2 synthesis rate might be a potential parameter for intestinal barrier function. In this study, we aimed to determine whether systemic threonine was used for small intestinal MUC2 synthesis and to calculate the MUC2 fractional synthetic rate (FSR) in human preterm infants. Seven preterm infants with an enterostomy following bowel resection for necrotizing enterocolitis received intravenous infusion of [U-13C]threonine to determine incorporation of systemic threonine into secreted MUC2 in intestinal outflow fluid. Small intestinal MUC2 was isolated using cesium chloride gradient ultracentrifugation and gravity gel filtration chromatography. MUC2-containing fractions were identified by SDS-PAGE/ periodic acid-Schiff staining and Western blot analysis and were subsequently pooled. Isotopic enrichment of threonine, measured in MUC2 using gas chromatography isotopic ratio mass spectrometry, was used to calculate the FSR of MUC2. Systemically derived threonine was indeed incorporated into small intestinal MUC2. Median FSR of small intestinal MUC2 was 67.2 (44.3-103.9)% per day. Systemic threonine is rapidly incorporated into MUC2 in the small intestine of preterm infants, and thereby MUC2 has a very high synthesis rate. Copyright

Human fetal amino acid metabolism at term gestation (Article)

2009-01-01T00:00:00Z

Background: Knowledge on human fetal amino acid (AA) metabolism, largely lacking thus far, is pivotal in improving nutritional strategies for prematurely born infants. Phenylalanine kinetics is of special interest as is debate as to whether neonates will adequately hydroxylate phenylalanine to the semiessential AA tyrosine. Objective: Our aim was to quantify human fetal phenylalanine and tyrosine metabolism. Design: Eight fasted, healthy, pregnant women undergoing elective cesarean delivery at term received primed continuous stable-isotope infusions of [1-13C]phenylalanine and [ring-D4]tyrosine starting before surgery. Umbilical blood flow was measured by ultrasound. Maternal and umbilical cord blood was collected and analyzed by gas chromatography-mass spectrometry for phenylalanine and tyrosine enrichments and concentrations. Data are expressed as medians (25th-75th percentile). Results: Women were in a catabolic state for which net fetal AA uptake was responsible for ≥25%. Maternal and fetal hydroxylation rates were 2.6 (2.2-2.9) and 7.5 (6.2-15.5) μmol phenylalanine/(kg·h), respectively. Fetal protein synthesis rates were higher than breakdown rates: 92 (84-116) and 73 (68-87) μmol phenylalanine/(kg·h), respectively, which indicated an anabolic state. The median metabolized fraction of available phenylalanine and tyrosine in the fetus was <20% for both AAs. Conclusions: At term gestation, fetuses still show considerable net AA uptake and AA accretion [converted to tissue ≈12 g/(kg·d)]. The low metabolic uptake (AA usage) implies a very large nutritional reserve capacity of nutrients delivered through the umbilical cord. Fetuses at term are quite capable of hydroxylating phenylalanine to tyrosine.

Liquid and gas chromatography coupled to isotope ratio mass spectrometry for the determination of 13C-valine isotopic ratios in complex biological samples (Article)

2008-10-01T00:00:00Z

On-line gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is commonly used to measure isotopic ratios at natural abundance as well as for tracer studies in nutritional and medical research. However, high-precision13C isotopic enrichment can also be measured by liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). Indeed, LC-IRMS can be used, as shown by the new method reported here, to obtain a baseline separation and to measure13C isotopic enrichment of underivatised amino acids (Asp, Thr-Ser, Glu, Pro, Gly, Ala, Cys and Val). In case of Val, at natural abundance, the SD(δ13C) reported with this method was found to be below 1‰. Another key feature of the new LC-IRMS method reported in this paper is the comparison of the LC-IRMS approach with the conventional GC-C-IRMS determination. To perform this comparative study, isotopic enrichments were measured from underivatised Val and its N(O, S)-ethoxycarbonyl ethyl ester derivative. Between 0.0 and 1.0 molar percent excess (MPE) (δ13C = -12.3 to 150.8‰), the calculated root-mean-square (rms) of SD was 0.38 and 0.46‰ and the calculated rms of accuracy was 0.023 and 0.005 MPE, respectively, for GC-C-IRMS and LC-IRMS. Both systems measured accurately low isotopic enrichments (0.002 atom percent excess (APE)) with an SD (APE) of 0.0004. To correlate the relative (δ13C) and absolute (atom%, APE and MPE) isotopic enrichment of Val measured by the GC-C-IRMS and LC-IRMS devices, mathematical equations showing the slope and intercept of the curves were established and validated with experimental data between 0.0 to 2.3 MPE. Finally, both GC-C-IRMS and LC-IRMS instruments were also used to assess isotopic enrichment of protein-bound13C-Val in tibial epiphysis in a tracer study performed in rats. Isotopic enrichments measured by LC-IRMS and GC-C-IRMS were not statistically different (p > 0.05). The results of this work indicate that the LC-IRMS was successful for high-precision13C isotopic measurements in tracer studies giving13C isotopic enrichment similar to the GC-C-IRMS but without the step of GC derivatisation. Therefore, for clinical studies requiring high-precision isotopic measurement, the LC-IRMS is the method of choice to measure the isotopic ratio. Copyright

Novel method for measurement of glutathione kinetics in neonates using liquid chromatography coupled to isotope ratio mass spectrometry (Article)

2007-08-30T00:00:00Z

A novel analytical method using liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS) was developed for measuring the fractional synthesis rate (FSR) of glutathione (GSH) in neonates after infusion of [1-13C]-glycine as a tracer. After transformation of GSH into GSSG, its dimeric form, the intra-erythrocytic concentration and13C-isotopic enrichment of GSH were deter-mined using 200 μL of blood. The results showed that, using LC/IRMS, the concentration (range of μmol/mL) was reliably measured using norvaline as internal standard with precision better than 0.1 μmol/mL. In addition, the13C-isotopic enrichment measured in the same run gave reliable values with excellent precision (with standard deviation (sd) lower than 0.3‰) and accuracy (measured between 0 and 2 Atom % Excess (APE)). The inter-assay repeatability of δ13C of norvaline used as internal standard with in vivo samples was assessed at -26.07 ± 0.28‰ with coefficient of variance (CV) at 1.1%. The FSR calculated either with GSH or GSSG showed similar results with slightly higher values for GSSG (41.6 ± 4.7 and 46.5 ± 4.4, respectively). The slightly lower FSR of GSH is probably due to interfering compounds in the biological matrix. Successfully used in a clinical study, this rapid and reliable method opens up a variety of kinetic studies with relatively low administration of tracer infusates, reducing the total cost of the study design. The small volume of blood needed enables studies even in extremely small subjects, such as premature infants, as reported in this study. Copyright

Albumin and whole-body protein synthesis respond differently to intraperitoneal and oral amino acids (Article)

2007-08-01T00:00:00Z

Patients with peritoneal dialysis are at risk for malnutrition and hypoalbuminemia, which are indicators of poor outcome. Recently, it was shown that dialysis solutions containing amino acids (AAs) and glucose improve protein anabolism in peritoneal dialysis patients. We determined if the same solutions could increase the fractional synthesis rate of albumin along with whole-body protein synthesis. Changes in the fractional albumin synthetic rate reflect acute change in hepatic albumin synthesis. A random-order cross-over study compared the effects of Nutrineal® (AA source) plus Physioneal® (glucose) dialysate with Physioneal® alone dialysate. Eight patients in the overnight fasting state were compared to 12 patients in the daytime-fed state. Fractional albumin synthetic rate and whole-body protein synthesis were determined simultaneously using a primed-continuous infusion of L-[1-13C]-leucine. Fractional albumin synthesis on AAs plus glucose dialysis did not differ significantly from that on glucose alone in the fasting or the fed state. Protein intake by itself (fed versus fasting) failed to induce a significant increase in the fractional synthetic rate of albumin. Conversely, the oral protein brought about a significant stimulation of whole-body protein synthesis. Our findings show that the supply of AAs has different effects on whole-body protein synthesis and the fractional synthetic rate of albumin.

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

2007-04-01T00:00:00Z

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 [1-13C]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·kg-1·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 postnatally. Copyright

Methionine transmethylation and transsulfuration in the piglet gastrointestinal tract (Article)

2007-02-27T00:00:00Z

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 ≈27% and ≈23% of whole-body fluxes, respectively. The methionine used by the GIT was metabolized into homocysteine (31%), CO2(40%), or tissue protein (29%). Cystathionine β-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.

Threonine utilization is high in the intestine of piglets (Article)

2005-01-01T00:00:00Z

The whole-body threonine requirement in parenterally fed piglets is substantially lower than that in enterally fed piglets, indicating that enteral nutrition induces intestinal processes in demand of threonine. We hypothesized that the percentage of threonine utilization for oxidation and intestinal protein synthesis by the portal-drained viscera (PDV) increases when dietary protein intake is reduced. Piglets (n = 18) received isocaloric normal or protein-restricted diets. After 7 h of enteral feeding, total threonine utilization, incorporation into intestinal tissue, and oxidation by the PDV, were determined with stable isotope methodology [U-(13)C threonine infusion]. Although the absolute amount of systemic and dietary threonine utilized by the PDV was reduced in protein-restricted piglets, the percentage of dietary threonine intake utilized by the PDV did not differ between groups (normal protein 91% vs. low protein 85%). The incorporation of dietary threonine into the proximal jejunum was significantly different compared with the other intestinal segments. Dietary, rather than systemic threonine was preferentially utilized for protein synthesis in the small intestinal mucosa in piglets that consumed the normal protein diet (P < 0.05). Threonine oxidation by the PDV was limited during normal protein feeding. In protein-restricted pigs, half of the total whole-body oxidation occurred in the PDV. We conclude that, in vivo, the PDV have a high obligatory visceral requirement for threonine. The high rate of intestinal threonine utilization is due mainly to incorporation into mucosal proteins