Evaluation of tracer labelled methionine load test in vitamin B-12 deficient adolescent women.

BACKGROUND: Methionine loading test (MLT) has been used primarily to identify defects in transsulfuration of homocysteine in cystathionine beta synthase deficiency. It may not be as useful to evaluate remethylation pathway, in vitamin B-12 and folate deficiencies. OBJECTIVE: We used tracer isotope labelled MLT to interrogate transsulfuration and remethylation independently in vitamin B-12 deficiency. DESIGN: We studied vitamin B-12 deficient women with a tracer labelled MLT before and eleven months after treatment with vitamin B-12. The fractional contribution of [13C]homocysteine to breath CO2 was used as a measure of transsulfuration, and difference in the intracellular enrichment of [13C]methionine and that of [C2H3]methionine as a measure of remethylation of homocysteine. Combined pre- and post-treatment results were analyzed to investigate the association between plasma vitamin B-12 concentrations and measures of homocysteine metabolism. RESULTS: The subjects were 17 years old, with a BMI of 19.4 kg/m2. Treatment with vitamin B-12, 2µg/day increased plasma B-12 from 93 (78.7, 106.2) [median (25th, 75th centiles)] to 161.5 (125.5, 226.2) pmol/L; 44% were below <150pmol/L after treatment. Fasting homocysteine concentration was significantly lower and that of cysteine higher in subjects with B-12 levels >150pmol/L. The tracer estimated transsulfuration of homocysteine was lower and remethylation higher with B-12 levels >150pmol/L when compared with those <150pmol/L. CONCLUSIONS: The tracer labelled MLT in combination with fasting parameters is a robust way to estimate parameters of methionine metabolism and can be used in the field where prime-constant rate infusion studies cannot be done efficiently.

Increased mitochondrial arginine metabolism supports bioenergetics in asthma.

High levels of arginine metabolizing enzymes, including inducible nitric oxide synthase (iNOS) and arginase (ARG), are typical in asthmatic airway epithelium; however, little is known about the metabolic effects of enhanced arginine flux in asthma. Here, we demonstrated that increased metabolism sustains arginine availability in asthmatic airway epithelium with consequences for bioenergetics and inflammation. Expression of iNOS, ARG2, arginine synthetic enzymes, and mitochondrial respiratory complexes III and IV was elevated in asthmatic lung samples compared with healthy controls. ARG2 overexpression in a human bronchial epithelial cell line accelerated oxidative bioenergetic pathways and suppressed hypoxia-inducible factors (HIFs) and phosphorylation of the signal transducer for atopic Th2 inflammation STAT6 (pSTAT6), both of which are implicated in asthma etiology. Arg2-deficient mice had lower mitochondrial membrane potential and greater HIF-2α than WT animals. In an allergen-induced asthma model, mice lacking Arg2 had greater Th2 inflammation than WT mice, as indicated by higher levels of pSTAT6, IL-13, IL-17, eotaxin, and eosinophils and more mucus metaplasia. Bone marrow transplants from Arg2-deficient mice did not affect airway inflammation in recipient mice, supporting resident lung cells as the drivers of elevated Th2 inflammation. These data demonstrate that arginine flux preserves cellular respiration and suppresses pathological signaling events that promote inflammation in asthma.

Whole body creatine and protein kinetics in healthy men and women: effects of creatine and amino acid supplementation.

Creatine kinetics were measured in young healthy subjects, eight males and seven females, age 20-30 years, after an overnight fast on creatine-free diet. Whole body turnover of glycine and its appearance in creatine was quantified using [1-(13)C] glycine and the rate of protein turnover was quantified using L-ring [(2)H5] phenylalanine. The creatine pool size was estimated by the dilution of a bolus [C(2)H3] creatine. Studies were repeated following a five days supplement creatine 21 g.day(-1) and following supplement amino acids 14.3 g day(-1). Creatine caused a ten-fold increase in the plasma concentration of creatine and a 50 % decrease in the concentration of guanidinoacetic acid. Plasma amino acids profile showed a significant decrease in glycine, glutamine, and taurine and a significant increase in citrulline, valine, lysine, and cysteine. There was a significant decrease in the rate of appearance of glycine, suggesting a decrease in de-novo synthesis (p = 0.006). The fractional and absolute rate of synthesis of creatine was significantly decreased by supplemental creatine. Amino acid supplement had no impact on any of the parameters. This is the first detailed analysis of creatine kinetics and the effects of creatine supplement in healthy young men and women. These methods can be applied for the analysis of creatine kinetics in different physiological states.

Methionine and protein metabolism in non-alcoholic steatohepatitis: evidence for lower rate of transmethylation of methionine.

Hepatic metabolism of methionine is the source of cysteine, the precursor of glutathione, the major intracellular antioxidant in the body. Methionine also is the immediate precursor of SAM (S-adenosylmethionine) the key methyl donor for phosphatidylcholine synthesis required for the export of VLDL (very-low-density lipoprotein) triacylglycerols (triglycerides) from the liver. We have examined the kinetics of methionine, its transmethylation and trans-sulfuration with estimates of whole body rate of protein turnover and urea synthesis in clinically stable biopsy-confirmed subjects with NASH (non-alcoholic steatohepatitis). Subjects with NASH were more insulin-resistant and had significantly higher plasma concentrations of usCRP (ultrasensitive C-reactive protein), TNFα (tumour necrosis factor α) and other inflammatory cytokines. There was no significant effect of insulin resistance and NASH on whole body rate of protein turnover [phenylalanine Ra (rate of appearance)] and on the rate of urea synthesis. The rates of methylation of homocysteine and transmethylation of methionine were significantly lower in NASH compared with controls. There was no difference in the rate of trans-sulfuration of methionine between the two groups. Enteric mixed nutrient load resulted in a significant increase in all the measured parameters of methionine kinetics. Heterozygosity for MTHFR (5,10-methylene-tetrahydrofolate reductase) (677C→T) did not have an impact on methionine metabolism. We speculate that, as a result of oxidant stress possibly due to high fatty acid oxidation, the activity of methionine adenosyltransferase is attenuated resulting in a lower rate of transmethylation of methionine and of SAM synthesis. These results are the first evidence for perturbed metabolism of methionine in NASH in humans and provide a rationale for the development of targeted intervention strategies.

Metabolic and genomic response to dietary isocaloric protein restriction in the rat.

We have examined hepatic, genomic, and metabolic responses to dietary protein restriction in the non-pregnant Sprague-Dawley rat. Animals were pair-fed either a 6 or 24% casein-based diet for 7-10 days. At the end of the dietary period, a microarray analysis of the liver was performed, followed by validation of the genes of interest. The rates of appearance of phenylalanine, methionine, serine, and glucose and the contribution of pyruvate to serine and glucose were quantified using tracer methods. Plasma and tissue amino acid levels, enzyme activities, and metabolic intermediates were measured. Protein restriction resulted in significant differential expression of a number of genes involved in cell cycle, cell differentiation, transport, transcription, and metabolic processes. RT-PCR showed that the expression of genes involved in serine biosynthesis and fatty acid oxidation was higher, and those involved in fatty acid synthesis and urea synthesis were lower in the liver of protein-restricted animals. Free serine and glycine levels were higher and taurine levels lower in all tissues examined. Tracer isotope studies showed an ∼50% increase in serine de novo synthesis. Pyruvate was the primary (∼90%) source of serine in both groups. Transmethylation of methionine was significantly higher in the protein-restricted group. This was associated with a higher S-adenosylmethionine/S-adenosylhomocysteine ratio and lower cystathione ß-synthase and cystathionine γ-lyase activity. Dietary isocaloric protein restriction results in profound changes in hepatic one-carbon metabolism within a short period. These may be related to high methylation demands placed on the organism and caused by possible changes in cellular osmolarity as a result of the efflux of the intracellular taurine.

Methionine metabolism in human pregnancy.

BACKGROUND: Hyperhomocysteinemia during pregnancy, which is a consequence of perturbations in methionine and/or folate metabolism, has been implicated in adverse outcomes such as neural tube defects, preeclampsia, spontaneous abortion, and premature delivery. The adaptive changes in methionine metabolism during pregnancy in humans have not been determined. OBJECTIVE: Our objective was to examine the kinetics of methionine and its rate of transsulfuration and transmethylation in healthy women with advancing gestation. DESIGN: The whole-body rate of appearance (Ra) of methionine and phenylalanine was measured in healthy pregnant women during the first (n = 10), second (n = 5), and third (n = 10) trimesters of pregnancy. These data were compared with those for nonpregnant women (n = 8). Tracers [1-(13)C]methionine, [C(2)H(3)]methionine, and [(2)H(5)]phenylalanine were administered as prime-constant rate infusions. The effect of enteral high-protein, mixed-nutrient load on tracer-determined variables was also examined. RESULTS: In pregnant women, the Ra of phenylalanine was significantly (P < 0.05) lower in the first trimester than in the second and third trimesters and was significantly lower than that in nonpregnant women. A linear positive correlation was evident between gestational age and phenylalanine Ra. The fractional rate and total rate of transsulfuration of methionine was significantly (P < 0.05) higher during the first trimester, whereas the rate of transmethylation was higher during the third trimester. Plasma concentrations of total cysteine and homocysteine were lower during pregnancy. CONCLUSIONS: Uncomplicated pregnancy in humans is associated with a higher rate of transsulfuration early in gestation and a higher rate of transmethylation of methionine in late gestation. These data may have implications for understanding the role of methionine and homocysteine in complications of pregnancy and for the nutritional care of pregnant women.

Simultaneous assay of isotopic enrichment and concentration of guanidinoacetate and creatine by gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometry (GC-MS) method for the simultaneous measurement of isotopic enrichment and concentration of guanidinoacetate (GAA) and creatine in plasma sample for kinetic studies is reported. The method, based on preparation of the bis(trifluoromethyl)pyrimidine methyl ester derivatives of GAA and creatine, is robust and sensitive. The lowest measurable m(1) and m(3) enrichment for GAA and creatine, respectively, was 0.3%. The calibration curves for measurements of concentration were linear over ranges of 0.5 to 250microM GAA and 2 to 500microM for creatine. The method was reliable for inter- and intraassay precision, accuracy, and linearity. The technique was applied in a healthy adult to determine the in vivo fractional synthesis rate of creatine using primed-constant rate infusion of [1-(13)C]glycine. It was found that isotopic enrichment of GAA reached a plateau by 30min of infusion of [1-(13)C]glycine, indicating either a small pool size or a rapid turnover rate (or both) of GAA. In contrast, the tracer appearance in creatine was slow (slope=0.00097), suggesting a large pool size and a slow rate of synthesis of creatine. This method can be used to estimate the rate of synthesis of creatine in vivo in human and animal studies.

Glycine and urea kinetics in nonalcoholic steatohepatitis in human: effect of intralipid infusion.

The rates of oxidation of glycine and ureagenesis were quantified in the basal state and in response to an intravenous infusion of intralipid with heparin (IL) in healthy subjects (n = 8) and in subjects with nonalcoholic steatohepatitis (NASH) (n = 6). During fasting, no significant difference in weight-specific rate of appearance (R(a)) of glycine, glycine oxidation, and urea synthesis was observed. Intralipid infusion resulted in a significant increase in plasma beta-hydroxybutyrate in both groups. The correlation between free fatty acids and beta-hydroxybutyrate concentration in plasma was 0.94 in NASH compared with 0.4 in controls, indicating greater hepatic fatty acid oxidation in NASH. Intralipid infusion resulted in a significant decrease in urea synthesis and glycine R(a) in both groups and did not impact glycine oxidation. The fractional contribution of glycine carbon to serine was lower in subjects with NASH before and after IL infusion. In contrast, the fractional contribution of serine carbon to cystathionine was higher in NASH before and following IL infusion. These results suggest that hepatic fatty acid oxidation is higher in NASH compared with controls and that glycine oxidation and urea synthesis are not altered. An increase in oxidative stress, induced by a higher rate of fatty acid oxidation in NASH, may have caused an increase in the contribution of serine to cystathionine to meet the higher demands for glutathione.

Metabolism of methionine in the newborn infant: response to the parenteral and enteral administration of nutrients.

The rates of transmethylation and transsulfuration of methionine were quantified using [1-(13)C]methionine and [C2H3]methionine tracers in newborn infants born at term gestation and in prematurely born low birth weight infants. Whole body rate of protein breakdown was also measured using [2H5]phenylalanine. The response to enteral formula feeding and parenteral nutrition was examined in full term and prematurely born babies, respectively. The relative rates of appearance of methionine and phenylalanine were comparable to the amino acid composition of mixed body proteins. Rates of transmethylation were high, both in full term infants (fast 32 +/- 14 micromol kg(-1) x h(-1); fed 21.7 +/- 3.2) and in preterm infants (57.2 +/- 14.8). Significant flux through the transsulfuration pathway was evident (full term: fast 6.0 +/- 4.4, fed 4.1 +/- 2.1; preterm: 24.9 +/- 9.9 micromol kg(-1) x h(-1)). Transsulfuration of methionine is evident in the human newborn in the immediate neonatal period, suggesting that cysteine may not be considered a "conditionally" essential amino acid for the neonate. The high rate of transmethylation may reflect the high methylation demand, whereas high rates of transsulfuration in premature babies may be related to high demands for glutathione and to the amounts of methionine in parenteral amino acid mixtures.

Effect of intravenous amino acids on glutamine and protein kinetics in low-birth-weight preterm infants during the immediate neonatal period.

Glutamine may be a conditionally essential amino acid in low-birth-weight (LBW) preterm neonates. Exogenously administered amino acids, by providing anaplerotic carbon into the tricarboxylic acid cycle, could result in greater cataplerotic efflux and glutamine de novo synthesis. The effect of dose and duration of amino acid infusion on glutamine and nitrogen (N) kinetics was examined in LBW infants in the period immediately after birth. Preterm neonates (<32 weeks gestation, birth weights 809-1,755 g) were randomized to initially receive either 480 or 960 micromol x kg(-1) x h(-1) of an intravenous amino acid solution for 19-24 hours, followed by a higher or lower amino acid load for either 5 h or 24 h. Glutamine de novo synthesis, leucine N, phenylalanine, and urea kinetics were determined using stable isotopic tracers. An increase in amino acid infusion from 480 to 960 micromol x kg(-1) x h(-1) for 5 h resulted in decreased glutamine de novo synthesis in every neonate (384.4 +/- 38.0 to 368.9 +/- 38.2 micromol x kg(-1) x h(-1), P < 0.01) and a lower whole body rate of proteolysis (P < 0.001) and urea synthesis (P < 0.001). However, when the increased amino acid infusion was extended for 24 h, glutamine de novo synthesis increased (369.7 +/- 92.6 to 483.4 +/- 97.5 micromol x kg(-1) x h(-1), P < 0.001), whole body rate of proteolysis did not change, and urea production increased. Decreasing the amino acid load resulted in a decrease in glutamine rate of appearance (R(a)) and leucine N R(a), but had no effect on phenylalanine R(a). Acutely stressed LBW infants responded to an increase in amino acid load by transiently suppressing whole body rate of glutamine synthesis, proteolysis, and oxidation of protein. The mechanisms of this transient effect on whole body protein/nitrogen metabolism remain unknown.

Amino acids, glutamine, and protein metabolism in very low birth weight infants.

Glutamine has been proposed to be conditionally essential for premature infants, and the currently used parenteral nutrient mixtures do not contain glutamine. De novo glutamine synthesis (DGln) is linked to inflow of carbon into and out of the tricarboxylic acid (TCA) cycle. We hypothesized that a higher supply of parenteral amino acids by increasing the influx of amino acid carbon into the TCA cycle will enhance the rate of DGln. Very low birth weight infants were randomized to receive parenteral amino acids either 1.5 g/kg/d for 20 h followed by 3.0 g/kg/d for 5 h (AA1.5) or 3.0 g/kg/d for 20 h followed by 1.5 g/kg/d for 5 h (AA3.0). A third group of babies received amino acids 1.5 g/kg/d for 20 h followed by 3.0 g/kg/d for 20 h (AA-Ext). Glutamine and protein/nitrogen kinetics were examined using [5-(15)N]glutamine, [2H5]phenylalanine, [1-(13)C,15N]leucine, and [15N2]urea tracers. An acute increase in parenteral amino acid infusion for 5 h (AA1.5) resulted in decrease in rate of appearance (Ra) of phenylalanine and urea, but had no effect on glutamine Ra. Infusion of amino acids at 3.0 g/kg/d for 20 h resulted in increase in DGln, leucine transamination, and urea synthesis, but had no effect on Ra phenylalanine (AA-Ext). These data show an acute increase in parenteral amino acid-suppressed proteolysis, however, such an effect was not seen when amino acids were infused for 20 h and resulted in an increase in glutamine synthesis.

Metabolism of threonine in newborn infants.

Threonine kinetics, threonine oxidative pathway, and the relationship between threonine and whole body protein turnover were quantified in 10 healthy term infants during the first 48 h after birth. The kinetic data were obtained 6 h after the last feed (fasting) and in response to formula feeding, using [U-(13)C(4),(15)N]threonine, [(2)H(5)]phenylalanine, and [(15)N]glycine tracers. The rate of carbon dioxide production (Vco(2)) and (13)C enrichment of the expired CO(2) were measured to quantify the rate of oxidation of threonine. The rate of appearance (R(a)) of threonine (136 +/- 37 micromol.kg(-1).h(-1)) was higher in newborn infants than that reported in adults. Formula feeding resulted in a significant decrease in threonine R(a) (P < 0.05). A significant positive correlation was seen between phenylalanine R(a) and threonine R(a), both during fasting and after formula feeding (r(2) = 0.65). In contrast to a 1:1 ratio of threonine and phenylalanine in mixed muscle protein, threonine R(a) relative to phenylalanine R(a) was 2.2 +/- 0.4. The fractional rate of threonine flux oxidized was 20% during fasting and 26% (P < 0.05) in response to nutrient administration. There was a significant correlation between plasma threonine concentration and threonine oxidation (r(2) = 0.75). No measurable incorporation of threonine in plasma glycine was seen. These data suggest that threonine is exclusively degraded by the glycine-independent serine/threonine dehydratase pathway. A higher flux of threonine relative to phenylalanine indicates higher turnover of threonine enriched proteins.

Glutamine supplement with parenteral nutrition decreases whole body proteolysis in low birth weight infants.

OBJECTIVES: To examine the effect of supplemental glutamine (0.6 g.kg -1 .d -1 ) on whole body protein/nitrogen and glutamine kinetics in low birth weight (LBW) infants receiving parenteral nutrition in the immediate neonatal period. STUDY DESIGN: Premature infants < or =32 weeks gestation with a birth weight from 694 to 1590 g were randomly assigned to either a glutamine-supplemented group (n = 10) or to a control group (n = 10). Tracer isotope studies were performed when the infants were 6 to 7 days old and had been receiving an amino acid intake of approximately 3.0 g.kg -1 .d -1 for at least 3 days. Whole body glutamine and nitrogen kinetics were measured with [5-15N]glutamine, [2H5]phenylalanine, [1-13C, 15 N]leucine, [15N2]urea, and GC-mass spectrometry. RESULTS: Supplemental glutamine was associated with a lower rate of appearance of glutamine ( P = .003), phenylalanine ( P = .001), and leucine C ( P = .003). There was no significant difference in leucine N turnover, urea turnover and plasma cortisol, and C-reactive protein levels in the 2 groups. CONCLUSION: Parenteral glutamine supplement in LBW infants was associated with lower whole-body protein breakdown. Because the decrease in whole body proteolysis is associated with protein accretion, parenteral glutamine supplement may be beneficial in selected populations of LBW infants.

Effect of enteral glutamine or glycine on whole-body nitrogen kinetics in very-low-birth-weight infants.

BACKGROUND: Glutamine is a critical amino acid for the metabolism of enterocytes, lymphocytes, and other proliferating cells. Although supplementation with glutamine has been suggested for growing infants, its effect on protein metabolism has not been examined. OBJECTIVE: The objective was to examine the effect of enteral glutamine or glycine on whole-body kinetics of glutamine, phenylalanine, leucine, and urea in preterm infants. DESIGN: Infants at <32 wk of gestation were given formula supplemented with either glutamine (0.6 g. kg(-1). d(-1); n = 9) or isonitrogenous amounts of glycine (n = 9) for 5 d. Eight infants fed unsupplemented formula served as control subjects. Glutamine, phenylalanine, leucine nitrogen flux, leucine carbon flux, and urea kinetics were quantified during a basal fasting period and in response to nutrient intake. RESULTS: Growing preterm infants had a high weight-specific rate of appearance of glutamine, phenylalanine, and leucine nitrogen flux. When compared with the control treatment, enteral glutamine resulted in a high rate of urea synthesis, no change in the plasma glutamine concentration, and no change in the rate of appearance of glutamine. Glycine supplementation resulted in similar changes in nitrogen metabolism, but the magnitude of change was less than that in the glutamine group. In the nonsupplemented infants, the rate of appearance of leucine nitrogen flux was negatively correlated (rho = -0.72) with urea synthesis. In contrast, the correlation (rho = 0.75) was positive in the glutamine group. CONCLUSION: Enterally administered glutamine in growing preterm infants is entirely metabolized in the gut and does not have a discernable effect on whole-body protein and nitrogen kinetics.

Serine metabolism in human pregnancy.

Serine plays an important role in intermediary metabolism as a source of one carbon pool for nucleotide biosynthesis, as a precursor for glycine and glucose, and as a contributor to cysteine biosynthesis. A unique serine-glycine cycling between the liver and the placenta has been demonstrated in the sheep fetus. We hypothesized that, because of serine's role in growth and development, significant changes in serine metabolism will occur in pregnancy with advancing gestation. The rate of appearance (R(a)) of serine and its metabolism were quantified in healthy women longitudinally through pregnancy with a [2-(15)N(13)C]serine tracer. The contribution of serine N to urea and the rate of oxidation of serine were measured using the precursor-product relation. Plasma serine concentrations and serine R(a) were lower in pregnant (P) women, in both early and late gestation, compared with nonpregnant (NP) women [plasma serine: NP, 113 +/- 24.5; P early, 71.9 +/- 6.2; P late, 68.5 +/- 9.6 micromol/l; serine R(a): NP (n = 7), 152.9 +/- 42.8; P early (n = 12), 123.7 +/- 21.5; P late (n = 8), 102.8 +/- 18.2 micromol x kg(-1) x h(-1)]. Serine contributed approximately 6% to urea N and 15-20% to the plasma glycine pool, and oxidation of serine represented approximately 8% of R(a). There was no significant difference between P and NP subjects. Glucose infusion, at 3 mg x kg(-1) x min(-1) in P subjects, resulted in a decrease in serine R(a) and an increase in oxidation. The decrease in serine turnover in pregnancy may represent a decrease in alpha-amino nitrogen turnover related to a decreased rate of branched-chain amino acid transamination and caused by pregnancy-related hormones aimed at nitrogen conservation and accretion.

Glutamine and leucine nitrogen kinetics and their relation to urea nitrogen in newborn infants.

Glutamine kinetics and its relation to transamination of leucine and urea synthesis were quantified in 16 appropriate-for-gestational-age infants, four small-for-gestational-age infants, and seven infants of diabetic mothers. Kinetics were measured between 4 and 5 h after the last feed (fasting) and in response to formula feeding using [5-(15)N]glutamine, [1-(13)C,(15)N]leucine, [(2)H(5)]phenylalanine, and [(15)N(2)]urea tracers. Leucine nitrogen and glutamine kinetics during fasting were significantly higher than those reported in adults. De novo synthesis accounted for approximately 85% of glutamine turnover. In response to formula feeding, a significant increase (P = 0.04) in leucine nitrogen turnover was observed, whereas a significant decrease (P = 0.002) in glutamine and urea rate of appearance was seen. The rate of appearance of leucine nitrogen was positively correlated (r(2) = 0.59, P = 0.001) with glutamine turnover. Glutamine flux was negatively correlated (r(2) = 0.39, P = 0.02) with the rate of urea synthesis. These data suggest that, in the human newborn, glutamine turnover is related to a high anaplerotic flux into the tricarboxylic acid cycle as a consequence of a high rate of protein turnover. The negative relationship between glutamine turnover and the irreversible oxidation of protein (urea synthesis) suggests an important role of glutamine as a nitrogen source for other synthetic processes and accretion of body proteins.