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PubMed1996

Structure and function of the glutamine phosphoribosylpyrophosphate amidotransferase glutamine site and communication with the phosphoribosylpyrophosphate site.

Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase from Escherichia coli exhibits a basal PRPP-independent glutaminase activity having a kcat/Km that is 0.3% of fully active enzyme. Binding of PRPP activates the enzyme by a structural change that lowers the Km for glutamine 100-fold and couples glutamine hydrolysis to synthesis of 5-phosphoribosylamine. By analysis of the x-ray structure of the glutamine site containing bound 6-diazo-5-oxonorleucine, a glutamine affinity analog, and by site-directed mutagenesis we have identified residues important for glutamine binding, catalysis, and coupling with PRPP. Tyr74 is a key residue in the coupling between the sites for glutamine in the NH2-terminal domain and PRPP in the COOH-terminal domain. Arg73 and Asp127 have roles in glutamine binding. The x-ray structure indicates that there are no amino acid side chains sufficiently close to Cys1 to participate as a proton acceptor in formation of the thiolate needed for nucleophilic attack on the carboxamide of glutamine, nor as a general acid for amide nitrogen transfer. Based on the x-ray model of the glutamine site and analysis of a mutant enzyme we propose that the free NH2 terminus of Cys1 functions as the proton acceptor and donor. The results indicate that the side chain of Asn101 and the backbone nitrogen of Gly102 function to stabilize a tetrahedral oxyanion resulting from attack of Cys1 on the glutamine carboxamide. Cys1, Arg73, Asn101, Gly102, and Asp127 are conserved in the NH2-terminal domain of a subfamily of amidotransferases that includes asparagine synthetase, glucosamine 6-phosphate synthase, and glutamate synthase, implying a common function in the four enzymes. Tyr74, on the other hand, is conserved only in glutamine PRPP amidotransferase sequences consistent with a specific role in interdomain coupling. The catalytic framework of key glutamine site residues supports the assignment of glutamine PRPP amidotransferase to a recently described Ntn (NH2-terminal nucleophile) hydrolase family of enzymes.

Adenosine Monophosphate↗
PubMed2006

Caspase and bid involvement in Clostridium difficile toxin A-induced apoptosis and modulation of toxin A effects by glutamine and alanyl-glutamine in vivo and in vitro.

Clostridium difficile is the leading cause of nosocomial bacterial diarrhea. Glutamine and its stable and highly soluble derivative alanyl-glutamine, have been beneficial in models of intestinal injury. In this study, we extend our work on the mechanisms of Clostridium difficile toxin A (TxA)-induced apoptosis in human intestinal epithelial T84 cells and evaluate the effects of glutamine and alanyl-glutamine on TxA-induced apoptosis in vitro and disruption of ileal mucosa in vivo. T84 cells were incubated with TxA (100 ng/ml) in medium with or without glutamine or alanyl-glutamine (3 to 100 mM). Apoptosis was evaluated by DNA fragmentation in vitro and the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling method in vivo. Caspase and Bid involvement were investigated by Western blotting. Ligated rabbit ileal loops were used for the evaluation of intestinal secretion, mucosal disruption, and apoptosis. TxA induced caspases 6, 8, and 9 prior to caspase 3 activation in T84 cells and induced Bid cleavage by a caspase-independent mechanism. Glutamine or alanyl-glutamine significantly reduced TxA-induced apoptosis of T84 cells by 47% and inhibited activation of caspase 8. Both glutamine and alanyl-glutamine reduced TxA-induced ileal mucosal disruption and secretion. Altogether, we further delineated the apoptosis-signaling cascade induced by TxA in T84 cells and demonstrated the protective effects of glutamine and alanyl-glutamine. Glutamine and alanyl-glutamine inhibited the apoptosis of T84 cells by preventing caspase 8 activation and reduced TxA-induced intestinal secretion and disruption.

Animals↗
PubMed1987

Pathways of glutamine and glutamate metabolism in resting and proliferating rat thymocytes: comparison between free and peptide-bound glutamine.

Pathways of glutamine metabolism in resting and proliferating rat thymocytes were evaluated by in vitro incubations of freshly prepared or 60-h cultured cells for 1-2 h with [U14C]glutamine. Complete recovery of glutamine carbons utilized in products allowed quantification of the pathways of glutamine metabolism under the experimental conditions. Partial oxidation of glutamine via 2-oxoglutarate in a truncated citric acid cycle to CO2 and oxaloacetate, which then was converted to aspartate, accounted for 76 and 69%, respectively, of the glutamine metabolized beyond the stage of glutamate by resting and proliferating thymocytes. Complete oxidation to CO2 in the citric acid cycle via 2-oxoglutarate dehydrogenase and isocitrate dehydrogenase accounted for 25 and 7%, respectively. In proliferating cells a substantial amount of glutamine carbons was also recovered in pyruvate, alanine, and especially lactate. The main route of glutamine and glutamate entrance into the citric acid cycle via 2-oxoglutarate in both cells is transamination by aspartate aminotransferase rather than oxidative deamination by glutamate dehydrogenase. In the presence of glucose as second substrate, glutamine utilization and aspartate formation markedly decreased, but complete oxidation of glutamine carbons to CO2 increased to 37 and 23%, respectively, in resting and proliferating cells. The dipeptide, glycyl-L-glutamine, which is more stable than free glutamine, can substitute for glutamine in thymocyte cultures at higher concentrations.

Animals↗
PubMed1996

The effect of glutamine administration on intestinal glutamine content.

The small intestine has been identified as an important site of metabolism of glutamine. The impairment of gut mucosa integrity during total parenteral nutrition (TPN) has been ascribed to a diminished supply of glutamine, due to the lack of glutamine in standard TPN. In addition, nutritional depletion diminishes glutamine availability in the intestine. The uptake of glutamine in the gut is dependent on the plasma glutamine concentration. This study was performed to observe changes in plasma and mucosa amino acid content during infusion of standard TPN and during infusion of TPN supplemented with glutamine. Twenty patients were randomly allocated to an experimental group (GTPN, TPN containing 0.23 g glutamine/kg.day) and a control group (CTPN, standard TPN). Patients received the iso-nitrogenous and isocaloric TPN during 10-14 days. After an overnight fast, before TPN was started, biopsies were taken from the duodenum. Amino acid concentrations were determined in the biopsies. Blood was taken for routine laboratory measurements and amino acid determination. This procedure was repeated on the last day of TPN infusion. Plasma glutamine in the GTPN group rose about 25% whereas plasma glutamine concentrations in the CTPN group remained unchanged. Mucosa glutamine rose with about 75% in the GTPN group whereas in the CTPN group no increase was observed. The rise of total plasma and mucosa amino acids was equal in both groups. The rise in plasma and mucosa glutamine was limited to nutritionally depleted patients. In conclusion, parenteral glutamine administration increases plasma and subsequently mucosa glutamine, specifically in depleted patients.

Adult↗
PubMed2000

Neither glutamine nor arginine supplementation of diets increase glutamine body stores in healthy growing rats.

The aim of the work was to resolve whether glutamine and arginine supplemented diets affect plasma and tissue (muscle, liver and intestinal mucosa) glutamine concentrations, as well as glutaminase and glutamine synthetase specific activities. The trial was performed in growing rats fed 10% protein diets for 3 weeks. Protein sources were: whey proteins (W); whey proteins+free glutamine (WG); whey proteins+arginine (WA); and casein+wheat protein hydrolysate+acid whey (39:39:22), as source containing protein-bound glutamine (CGW). Rats fed the control diet (6.4% glutamine) (W) showed comparable glutamine body stores to those of rats fed the WG diet. In fact, glutamine sup- plementation down-regulated the hepatic glutamine synthetic capacity of growing rats (W/WG: 6.8+/-0.3 vs 6.0+/-0.2 nmol/min/mg protein). Arginine supplementation of the diet (up to 9% of the protein content) resulted in a decrease in plasma and tissue glutamine concentrations (W/WA: plasma, 1218+/-51 vs 1031+/-48 micromol/L; liver 7.5+/-0.4 vs 6.5+/-0.2 micromol/g; muscle: 5.7+/-0.2 vs 4.0+/-0.2 micromol/g). These data suggest that glutamine supplementation of the diet does not increase plasma and tissue glutamine concentrations in healthy growing rats, while the addition of arginine to the diet decreases glutamine body stores.

Amino Acids↗
PubMed1990

Alanyl-glutamine counteracts the depletion of free glutamine and the postoperative decline in protein synthesis in skeletal muscle.

Skeletal muscle protein and amino acid metabolism change after surgical trauma during a period characterized by skeletal muscle protein catabolism. Available total parenteral nutrition (TPN) not containing glutamine does not prevent these changes, while TPN enriched with glutamine has been shown to have beneficial effects on postoperative skeletal muscle protein metabolism. Glutamine, in the form of a dipeptide, alanyl-glutamine, was added to TPN. Patients undergoing elective cholecystectomy were given postoperative TPN. Two groups received isocaloric and isonitrogenous conventional TPN, one group with (n = 8) and the other without an addition of alanyl-glutamine (n = 8). Skeletal muscle protein metabolism was studied in muscle biopsy specimens from which the muscle free amino acid pattern and the concentration and size distribution of ribosomes, serving as a measure of protein synthesis, were determined. In the control group, muscle free glutamine decreased by 38.8% +/- 6.6% and the polyribosome concentration per mg of DNA decreased by 21% +/- 5.2% after operation. In the group given TPN supplemented with alanyl-glutamine, these two parameters of muscle protein and amino acid metabolism did not change significantly. Compared to the control group, whole-body nitrogen balance was improved after operation by the addition of alanyl-glutamine to TPN (p less than 0.01). Muscle free glutamine and muscle protein synthesis were preserved after operation and the whole-body nitrogen balance was improved by adding glutamine in the form of alanyl-glutamine to TPN. The dipeptide alanyl-glutamine seems to be a suitable means of providing glutamine in a stable form.

Cholecystectomy↗
PubMed1975

Specificity of the glutamine-binding site involved in the reguation of glutamine-synthetase activity in hepatoma tissue-culture cells.

Glutamine accelerates the degradation of glutamine synthetase in hepatoma tissue culture cells. Compounds structurally related to glutamine were tested for their ability to mimic or antagonize this effect of glutamine. 6-Diazo-5-oxo-L-norleucine, like glutamine depressed the activity of glutamine synthetase in hepatoma tissue culture cells. L-Methionine sulfone, albizzine, L-methionine sulfoxide, L-gamma-glutamyl hydrazide and gamma-N-methyl-L-glutamine (listed in order of decreasing potency) were antagonists which prevented the effect of glutamine on glutamine synthetase activity. These antagonists had little effect on glutamine transport or protein synthesis of hepatoma tissue culture cells and their effects were reversible. The effects of compounds on gluatmine synthetase activity in cell-free extracts of the cells were examined. Diazo-oxonorleucine and albizzine inhibited neither the transferase nor the synthetase activity of glutamine synthetase. This observation is interpreted to mean that the glutamine-binding site involved in the regulation of glutamine synthetase activity of hepatoma tissue culture cells is not the active site of the enzyme.

Biological Transport, Active↗
PubMed1982

Derepression of the glutamine synthetase in neuroblastoma cells at low concentrations of glutamine.

Regulation of the biosynthesis of glutamine synthetase was studied in neuroblastoma cells (Neuro-2A) by use of a recently developed, sensitive radioisotopic assay. The removal of glutamine from the culture medium of these cells for 24 h resulted in a 10-fold increase in glutamine synthetase specific activity (15-fold after 2 weeks) compared with the basal level found in cells grown in the presence of 2 mM glutamine. Following the growth of these cells for 2 weeks in the presence of various concentrations of glutamine, a negative linear correlation was observed between the specific activity of glutamine synthetase (from 1.7 to 0.14 unit/mg) and the concentration of glutamine in the growth medium (from 0.5 to 2 mM). Cycloheximide or actinomycin D blocked the increase in glutamine synthetase activity observed in the absence of glutamine. These results suggest that the removal of glutamine led to the induction of glutamine synthetase by stimulating new enzyme synthesis. The enzyme was not degraded, but only diluted, by growth upon readdition of glutamine to the medium. The influence of glutamine depletion is also reported for C-6 glioma cells and glial cells in primary cultures.

Animals↗
PubMed1987

Feedback inhibition of ammonium (methylammonium) ion transport in Escherichia coli by glutamine and glutamine analogs.

When cultured with glutamate or glutamine as the nitrogen source, Escherichia coli expresses a specific ammonium (methylammonium) transport system. Over 95% of the methylammonium transport activity in washed cells was blocked by incubation with 100 microM L-glutamine in the presence of chloramphenicol (100 micrograms/ml). The time course for the onset of this glutamine inhibition followed a first-order rate expression with a t1/2 of 2.8 min. The inhibition of transport by L-glutamine was noncompetitive (Ki = 18 microM) with respect to the [14C]methylammonium substrate. D-Glutamine had no significant effect. The glutamine analogs gamma-L-glutamyl hydroxamate (Ki = 360 microM) and gamma-L-glutamyl hydrazide (Ki = 800 microM) were also noncompetitive inhibitors of methylammonium transport, suggesting that glutamine metabolism is not required. The role of the intracellular glutamine pool in the regulation of ammonium transport was investigated by using mutants carrying defects in the operon of glnP, the gene for the glutamine transporter. The glnP mutants had normal rates of methylammonium transport but were refractory to glutamine inhibition. Glycylglycine, a noncompetitive inhibitor of methylammonium uptake in wild-type cells (Ki = 43 microM), was equipotent in blocking transport in glnP mutants. Although ammonium transport is also subject to repression by growth of E. coli in the presence of ammonia, this phenomenon is unrelated to glutamine inhibition. A GlnL RegC mutant which constitutively expressed ammonium transport activity exhibited a sensitivity to glutamine inhibition similar to that of wild-type cells. These findings indicate that ammonium transport in E. coli is regulated by the internal glutamine pool via feedback inhibition.

Biological Transport, Active↗
PubMed1995

[Glutamine supplemented nutrition in clinical practice--use of glutamine-containing dipeptides].

OBJECTIVE: In this review, the central role of glutamine in a large number of pathways is elucidated and the consequences of bodily glutamine deprivation observed during trauma, hypercatabolism and malnutrition on vital functions of organs and tissues are discussed. Subsequently, the use of glutamine and glutamine-containing dipeptides in the frame of clinical nutrition is critically evaluated. Finally, the prospective importance of glutamine nutrition in routine clinical settings is discussed. DESIGN: For this compilation basic work from human physiology and nutrition science as well as all actual research work dealing with the use of glutamine and glutamine dipeptide supplements in clinical nutrition have been considered. RESULTS: In various diseased states (like trauma, hypercatabolism, intestinal dysfunction, immunedeficiency) glutamine has to he considered a conditionally indispensable substrate and thus should be essential part of all nutritional efforts. Only adequate glutamine supply (about 10-15 g/day) can prevent bodily glutamine deprivation which may beneficially influence the clinical situation. The use of highly soluble and stable glutamine-containing dipeptides facilitates for the first time a safe and efficient glutamine supply within parenteral nutrition. CONCLUSIONS: Enteral/parenteral glutamine therapy may contribute to improve nitrogen economy, to diminish the incidence of bacterial infections, to reduce the risk for bacterial translocation, and presumably to reduce hospital stay in various patient groups.

Animals↗
PubMed1988

Glutamine transport and the role of the glutamine translocator in chloroplasts.

The transport of l-[(14)C]glutamine in oat (Avena sativa L.) and spinach (Spinacia oleracea L.) chloroplasts was studied by a conventional single-layer and a newly developed stable double-layer silicone oil filtering system. [(14)C]Glutamine was actively transported into oat chloroplasts against a concentration gradient. Metabolite uptake was greatly affected by the endogenous dicarboxylate pools, which could be easily changed by preloading the chloroplast with specific exogenous substrate. Glutamine uptake was decreased by 44 to 75% in oat chloroplasts preloaded with malate, 2-oxoglutarate (2-OG), and aspartate, but increased by 52% in chloroplasts preloaded with l-glutamate. On the other hand, the uptake of the other four dicarboxylates was decreased by 47 to 79% in chloroplasts preloaded with glutamine. In glutamine-preloaded chloroplasts the uptake of glutamine was inhibited only by l-glutamate. The observed inhibition by l-glutamate was competitive with an apparent K(i) value of 32.1 millimolar in oat and 6.7 millimolar in spinach chloroplasts. This study indicates that there are two components involved in glutamine transport in chloroplasts. The major component was mediated via a specific glutamine translocator. It was specific for glutamine and did not transport other dicarboxylates except l-glutamate. A K(0.5) value of 1.25 millimolar and V(max) of 45.5 micromoles per milligram of chlorophyll per hour were determined for the glutamine translocator in oat chloroplasts. The respective values were 1.0 millimolar and 16.7 micromoles per milligram of chlorophyll per hour in spinach chloroplasts. A three translocator model, involving the glutamine, dicarboxylate, and 2-OG translocators, is proposed for the reassimilation of photorespiratory NH(3) in chloroplasts of C(3) species. In this three-translocator model the additional transport of glutamine into the chloroplast is coupled to the export of glutamate via the glutamine translocator. This is an extension of the two-translocator model, involving the dicarboxylate and 2-OG translocators, proposed for spinach chloroplasts, (KC Woo, UI Flügge, HW Heldt 1987 Plant Physiol 84: 624-632).

Journal Article↗
PubMed1983

Glutamine- and N-acetyl-L-glutamate-dependent carbamoyl phosphate synthetase from Micropterus salmoides. Purification, properties, and inhibition by glutamine analogs.

The glutamine- and N-acetyl-L-glutamate-dependent carbamoyl phosphate synthetase III present in liver of largemouth bass (Micropterus salmoides) has been highly purified. The properties of the enzyme are generally similar to the properties of the carbamoyl phosphate synthetase III from spiny dogfish (Squalus acanthias) previously described (Anderson, P. M. (1981) J. Biol. Chem. 256, 12228-12238). However, the bass enzyme is not subject to self-association, and the effects of urea and, particularly, trimethylamine-N-oxide, on catalytic activity are considerably reduced. Ammonia can substitute for glutamine as the nitrogen-donating substrate, but the maximum rate is lower. Carbamoyl phosphate synthetase III, like other carbamoyl phosphate synthetases, catalyzes two partial reactions, ATP synthesis from carbamoyl phosphate and ADP, and bicarbonate-dependent hydrolysis of ATP; both reactions are greatly stimulated by the presence of N-acetyl-L-glutamate. Carbamoyl phosphate synthetase III gave no detectable immunological cross-reaction with antibody to the ammonia- and N-acetyl-L-glutamate-dependent carbamoyl phosphate synthetase from rat liver mitochondria. The apparent Km value for N-acetyl-L-glutamate decreases significantly as the concentration of L-glutamine increases in the glutamine-dependent reaction, and vice versa. This effect is glutamine-specific. The apparent Km for N-acetyl-L-glutamate in the ammonia-dependent reaction is not affected by changes in ammonia concentration and the apparent Km for ammonia (8 mM) is also not affected by changes in N-acetyl-L-glutamate concentration. Studies involving inhibition of carbamoyl phosphate synthetase III by the glutamine analogs acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid), DON (6-diazo-5-oxo-L-norleucine), and chloroketone (L-2-amino-4-oxo-5-chloropentanoic acid), provided additional evidence for significant interaction between the L-glutamine- and N-acetyl-L-glutamate-binding sites. Glutamine-dependent but not ammonia-dependent activity is inhibited by preincubating the enzyme with these analogs. This inhibition requires the presence of both MgATP and N-acetyl-L-glutamate, and is prevented by the additional presence of L-glutamine. Inhibition of the glutamine-dependent reaction by DON or chloroketone is accompanied by a decrease in the apparent Km for N-acetyl-L-glutamate in the ammonia-dependent reaction from 0.3 mM to a value which is nearly the same as that observed in the glutamine-dependent reaction when glutamine is saturating (0.015 mM).

Adenosine Triphosphate↗
PubMed1990

Glutamine regulates glutamine synthetase expression in skeletal muscle cells in culture.

Metabolite control of glutamine synthetase expression (by glutamine) was studied in L6 skeletal muscle cells. Depletion of glutamine from the culture medium for 24 hours resulted in a 3-4-fold increase in glutamine synthetase activity. This effect was blocked by cycloheximide but not by actinomycin D. Addition of glutamine to L6 cells maintained in glutamine-free medium caused a rapid return of glutamine synthetase activity to the control level. As reported, dexamethasone caused a striking increase in the glutamine synthetase mRNA level. In contrast, neither depletion nor addition of glutamine caused a change in the glutamine synthetase mRNA level. Therefore, regulation of glutamine synthetase by glutamine is exerted at a post-transcriptional level.

Animals↗
PubMed1992

Glutamine and glutamate metabolism in normal and heat shock conditions in Drosophila Kc cells: conditions supporting glutamine synthesis maximize heat shock polypeptide expression.

We have previously reported that Drosophila Kc cells require glutamine for maximal expression of heat shock proteins in stressed conditions (Sanders and Kon: J. Cell. Physiol. 146:180-190, 1991). The mechanism of this effect has been investigated by comparing the metabolic utilization of glutamine in conditions which support hsp expression with that of glutamate in conditions where up to 100-fold less hsp is synthesized. This comparison showed that free ammonia was generated by cells incubated in the presence of glutamine in 37 degrees C (heat shock) conditions, but not at 25 degrees C, and not in the presence of glutamate in either normal or heat shock conditions. There was no difference in the amount of [14C]O2 generated from either [14C]-labeled amino acid in the tricarboxylic acid cycle, but three- to four-fold more alanine was synthesized in cells incubated in glutamine than in glutamate. Treating the cells with aminotransferase inhibitors to artificially increase NH3 release raised hsp expression in the presence of glutamate to maximal levels characteristic of glutamine. This potentiation correlated with inhibition of alanine aminotransferase. Since only NH3 production correlated with hsp expression in heat shock conditions in the presence of glutamine, and NH3 addition to glutamate also resulted in maximal hsp expression, we measured glutamine production in glutamate plus NH3 and observed net glutamine synthesis. The supposition that glutamine itself is responsible for the regulatory changes supporting maximal hsp expression was supported by the finding that the glutamine analog, 6-diazo-5-oxo-L-norleucine (DON), mimicked the effects of glutamine. We conclude that glutamine imposes regulatory changes which alter nitrogen metabolism and support hsp expression in Kc cells.

Amino Acids↗
PubMed1998

Regulation of nitrate reductase transcript levels by glutamine accumulating in the leaves of a ferredoxin-dependent glutamate synthase-deficient gluS mutant of Arabidopsis thaliana, and by glutamine provided via the roots.

The regulation by glutamine of the leaf transcript level corresponding to the Arabidopsis thaliana (L.) Heynh. nitrate reductase gene nia2 was examined using a novel approach: we took advantage of the ability of a ferredoxin-dependent glutamate synthase-deficient gluS mutant of A. thaliana to accumulate glutamine in the leaves when illuminated under conditions that favour photorespiration. The accumulation of glutamine in gluS mutant leaves and the concomitant decline in the leaf glutamate pool were not correlated with a reduction in the foliar nia2 transcript level. This result indicates that glutamine may not exert a negative control of the leaf nia2 transcript pool. The pattern of diurnal nia2 mRNA oscillation did not change upon illumination of the gluS mutant in air, although the leaf glutamine level remained high during the diurnal cycle. The amplitude of the diurnal fluctuation in nia2 transcript abundance, therefore, does not seem to depend on the size of the leaf glutamine pool (which normally fluctuates in opposite phase). This result also appears to argue against a role of glutamine as an effective repressor of nia2 transcript accumulation. The application of a solution containing 100 mM glutamine to the roots of A. thaliana resulted in an increase in the leaf glutamine level and in a decrease in the leaf nia2 transcript level. Net CO2 uptake and chlorophyll fluorescence quenching by attached leaves of A. thaliana were determined as a control of the physiological status of the plants and remained unaffected by the glutamine treatment. However, there was a decrease in the foliar nitrate level. The negative effect on the nia2 transcript pool exerted by exogeneous glutamine may, therefore, be explained as a result of the down-regulation of nitrate-uptake permeases in the roots by glutamine.

Amino Acid Oxidoreductases↗
PubMed2005

Fragmentation pathway for glutamine identification: loss of 73 Da from dimethylformamidine glutamine isobutyl ester.

A fragmentation mechanism for the neutral loss of 73 Da from dimethylformamidine glutamine isobutyl ester is investigated. Understanding this mechanism will allow to improve the identification and quantification of 15N-labeled and unlabeled glutamine and the distinguishing of glutamine and glutamic acid by electrospray ionization (ESI)-tandem mass spectrometry. Before mass spectrometry analysis, glutamine and glutamic acid are derivatized with dimethylformamide dimethyl acetal and isobutanol to form dimethylformamidine isobutyl ester. Derivatization conditions are modified based on an existing method to ensure complete derivatization of glutamic acid and to prevent the hydrolysis of glutamine. The fragmentation mechanism of dimethylformamidine glutamine isobutyl ester is studied and possible fragmentation pathways are proposed. Based on the fragmentation mechanism, a quantification method is developed to quantify both 15N-labeled and unlabeled glutamine and glutamic acid at a series of different neutral losses by performing multiple-reaction monitoring (MRM) scans in a triple-quadrupole mass spectrometer. Labeled glutamine includes 15N-amide labeled, 15N-amine labeled glutamine and glutamine 15N-labeled at both amide and amine positions. Deuterium labeled glutamine and glutamic acid are used as internal standards. Isotope effects are characterized for 15N labeled and deuterium labeled glutamine. It is found that the same method can be used to distinguish aspartic acid from asparagine. This study will improve the application of MS/MS for amino acid quantification and stable isotope labeling metabolism studies.

Butanols↗
PubMed2000

Effects of parenteral nutrition supplemented with glutamine or glutamine dipeptides on liver antioxidant and detoxication systems in rats.

Our aim was to determine the effects of glutamine or alanyl glutamine parenteral supplementation on the liver oxidant/antioxidant balance and on cytochrome-P450-mediated detoxication in rats. Animals were infused for 5 d with standard total parenteral nutrition (TPN), glutamine-enriched TPN, or alanyl glutamine-enriched TPN. The hepatic concentration of glutathione was reduced, and the levels of thiobarbituric-acid-reactive substances (TBARS) were increased in animals receiving standard TPN. Both glutamine and alanyl glutamine supplementation normalized glutathione, but thiobarbituric-acid-reactive substance concentration was only decreased by ananyl glutamine. This effect was parallel to a partial recovery of the activity of antioxidant enzymes. Cytochrome-P450 liver content, cytochrome-P450-dependent monooxygenases, and antipyrine clearance were not modified by glutamine or alanyl glutamine. Our data suggest a better protection against free radicals by alanyl glutamine supplementation and an absence of effects of both glutamine and alanyl glutamine on liver oxidative metabolism.

Animals↗
PubMed2001

Glutamine metabolism to glucosamine is necessary for glutamine inhibition of endothelial nitric oxide synthesis.

L-Glutamine is a physiological inhibitor of endothelial NO synthesis. The present study was conducted to test the hypothesis that metabolism of glutamine to glucosamine is necessary for glutamine inhibition of endothelial NO generation. Bovine venular endothelial cells were cultured for 24 h in the presence of 0, 0.1, 0.5 or 2 mM D-glucosamine, or of 0.2 or 2 mM L-glutamine with or without 20 microM 6-diazo-5-oxo-L-norleucine (DON) or with 100 microM azaserine. Both DON and azaserine are inhibitors of L-glutamine:D-fructose-6-phosphate transaminase (isomerizing) (EC 2.6.1.16), the first and rate controlling enzyme in glucosamine synthesis. Glucosamine at 0.1, 0.5 and 2 mM decreased NO production by 34, 45 and 56% respectively compared with controls where glucosamine was lacking. DON (20 microM) and azaserine (100 microM) blocked glucosamine synthesis and prevented the inhibition of NO generation by glutamine. Neither glutamine nor glucosamine had an effect on NO synthase (NOS) activity, arginine transport or cellular tetrahydrobiopterin and Ca(2+) levels. However, both glutamine and glucosamine inhibited pentose cycle activity and decreased cellular NADPH concentrations; these effects of glutamine were abolished by DON or azaserine. Restoration of cellular NADPH levels by the addition of 1 mM citrate also prevented the inhibiting effect of glutamine or glucosamine on NO synthesis. A further increase in cellular NADPH levels by the addition of 5 mM citrate resulted in greater production of NO. Collectively, our results demonstrate that the metabolism of glutamine to glucosamine is necessary for the inhibition of endothelial NO generation by glutamine. Glucosamine reduces the cellular availability of NADPH (an essential cofactor for NOS) by inhibiting pentose cycle activity, and this may be a metabolic basis for the inhibition of endothelial NO synthesis by glucosamine.

Animals↗