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Glutamine improves protein metabolism during liver regeneration
Topic 19 - GLUTAMINE
Those differences could reflect difference in protein turn over rate and protein accretion between adult and infants.
Date: Wednesday, September 9 09.45-l 1.45 Chair: N.N. Abumrad (USA), A. Revhaug (Norway) Hankard, R. (Paris) Study of Glutamine Metabolism using Stable isotope in Infants with Neonatal Short Bowel Syndrome Hijrig, H. (Basel) Glutamine (GLN) ment following Antigen Receptor Activation of Human Lymphocytes
0.50
Glutamine antigen receptor lymphocytes
R. (Maastricht) Decreased Duodenal Mucosa Glutamine Concentration in the Depleted Preoperative Patient H. (Mainz) Glutamine Dipeptide Supplemented TPN maintains Intestinal Function in Critically III
Tremel,
L. (Milano) Nutritional Effects of Ornithine Alpha Ketoglutarate in Burn Patients
Donati,
M. (Phoenix, AZ) Substrate and Hormonal Changes due to Dietary Supplementation with Ornithine Alpha Ketoglutarate (OKG) in Critically III Trauma Victims
Jeevanandam,
B. (Stockholm) Alpha-Ketoglutarate given together with TPN improves the Free Glutamine-Levels in Glutamine-Depleted Intensive Care Patients
Petersson,
Study of glutamine metabolism isotope in infants with neonatal syndrome
using short
R. Hankard, 0. Goulet, M. Rongier, J-F. Desjeux, V. Colomb, C. Ricour and D. Darmaun lnserm tJ290, Hopital St. Lazare and Hopital Necker-Enfants Malades, 149 rue de Sevres, 75015 Paris cedex, Paris, France Glutamine (Gln) can be considered as a major fuel for small bowel (SB). Extensive SB resection could alter whole body Gln metabolism in vivo. Very few data are available on Gln metabolism in infants. Eleven infants with extensive neonatal SB resection leaving them with 35 + 12 cm SB length and 4 controls were studied. During the post-absorptive state infants received primed continuous infusion of 1.5 mg/kg/h L-[2-‘5N] Gln and 0.4 mg/kg/h L-[1 -13C] leucine. From stable isotopes plasma enrichments were calculated: Gln and Leu fluxes (Ra Gln) (Ra Leu). The 2 inflow components of RaGln were calculated: release from protein breakdown (B Gln)O and de novo synthesis (G Gln): pmollkglh Patients
Ra Gin 568+-124 816+149’
D Gin 175*100 359i181
B Gin 396k98 457+07
following of human
In this study we investigated on the GLN requirement in different steps of human peripheral blood mononuclear cells (PBMC) activation induced bya monoclonal antibody (mAb) directed against the CD3-T-cell receptor molecular complex. Methods: Density gradient separated PBMC were cultured in the presence or absence of anti-CD3 mAb and GLN. Phenotypic expression of activation markers was evaluated by flow cytometry, using specific mAb. Total cellular RNA was extracted after 8 hours of culture, reverse transcribed, and cDNA thus generated was assayed, by specific cytokine primers pairs, in 25 cycles polymerase chain reaction (PCR) for the presence of relevant gene transcription. Cell cycle was analyzed by flow-cytometry on 48 hours stimulated PBMC with propidium iodide based reagents. Finally, proliferation was assessed on day three by a standard 3H-thymidine incorporation technique. Results: Anti-CD3 stimulated expression of CD69, an early activation marker, on T cells, was unaffected in the absence of GLN, whereas the expression of p55 low affinity IL-2 receptor and late activation markers such as CD45RO and CD71 transferrin receptor was dose dependently correlated with GLN concentration in the culture medium. Transcription of IL-2, IL-4, IL-5, IL-6 and IFN-gamma genes was found to be inducible even in the absence of GLN by anti-CD3 mAb. In contrast, entering of stimulated PBMC into cell cycling was dependent on the presence of GLN in the culture medium, as also observed for proliferation assays. Conclusion: Early events of antigen receptor triggered lymphocyte activation can take place in the absence of GLN, whereas later stages, including entering into cell cycling and proliferation, depend on the presence of GLN. These data confirm that an adequate GLN supply is a prerequisite for an intact immune response.
van der Hulst,
0.49
(GLN) requirement triggered activation
H. Herig, L. Filgueira, G. Spagnoli, R. Babst and M. Heberer Departments of Surgery and Research, University of Basel, Switzerland
Requiretriggered
Skullman, S. (Linkoping) Glutamine improves Protein Metabolism during Liver Regeneration
stable bowel
AND ANALOGUES
0.51 Glutamine improves during liver regeneration
protein
metabolism
S. Skullman, M. Wiren, M. Chu, P. Garlick”, M. McNurlan’ and J. Larsson Department of Medico-Surgical Gastroenterology, University Hospital, Linkoping, Sweden and * Rowett Research lnstitute, Aberdeen, Scotland Glutamine is a preferential nutritional substrate for small bowel enterocytes and other fast replicating cells. Liver regeneration is a model often used to study cell replication and fast growth. We have previously shown a reduced regeneration rate in malnourished rats and the limiting factor seems to be a diminished protein synthesis. The aim of this study was to examine if glutamine could influence regeneration rate and protein metabolism in regenerating liver. Materials and methods: Eighty-eight rats (225 g) were allocated to either shamoperation (n = 40) or 70% liver
Ra Leu 228&60 263k50
‘p < 0.05 vs controls. Mann Whitney tests
Gln fluxes are reduced after SB resection, mainly due to reduced de novo synthesis. In controls, Gln and Leu fluxes are significantly higher than in adult respectively: 344 * 47 and 88 k 12 pmol/kg/h (Metabolism 1991, 49: 42-4). 24
resection (n = 48). Postoperatively they received an isonitrogenous, isocaloric elementary diet (12% of BW/day) containing 0%, 2% or 4% glutamine, (4% is equal to 25% of aminoacid content). The resected part of the liver was weighed and analysed for protein content, After 2 or 7 days protein synthesis was measured by injecting a flooding dose of L-[4-3H]-phenylalanine intravenously and the liver was analysed for RNA and protein content. Results: There were no differences between the different sham groups or between the 2% and 4% glutamine groups. Total protein content was increased in regenerating liver in the 2 and 4% glutamine groups compared to the 0% group (day 2: 2% p < 0.05, 4% p < 0.001; day 7: 2% p < 0.001 and 4% p < 0.01). Regeneration rate based on liver weight measurements was increased in the 2% group (p < 0.05 on day 2 and day 7) but not in the 4% group compared to the 0% group. There was a tendency towards improved protein synthesis after 7 days of regeneration in the 2 and 4% group compared to the 0% group, measured as mg protein/day, mg RNA (p = protein day/100 g BW and mg protein/day/mg 0.06-0.11). Conclusions: 1. Glutamine containing diet improved liver regeneration rate and total protein content in regenerating liver. 2. There was a tendency towards improved protein synthesis during regeneration in glutamine fed animals. 3. An excess of glutamine in the diet did not improve the results.
Our results suggest an alteration of intracellular duodenal mucosa glutamine metabolism in the patients with low PIW. Because GLN is used as a preferential fuel in the small bowel, specific depletion of mucosa GLN could be of significance.
0.53 Glutamine dipeptide supplemented maintains intestinal function in critically
H. Tremel, B. Kienle. L.S. Weilemann, R. Abele’, B. FichtF, P. Stehle3 and P. Fur.& Medical Clinic II, University of Maim; ’Fresenius AG, Oberurself Ts; 2 Walther Straub Inst. Pharmacol. & Toxicol., University of Munich; ‘Inst. Biol. Chem. & Nutr., University of Hohenheim, Stuttgart, FRG Long term total parenteral nutrition (TPN) is accompanied with villous atrophy and subsequent malabsorption syndrome. The underlying reasons for the impaired intestinal function are yet to be determined, though current information attest the important role of glutamine (Gln) in maintaining intestinal structure and function. Parenteral provision of synthetic highly soluble Gln-containing dipeptides facilitate Gln nutrition in practical clinical setting. Twelve critically ill intensive care patients were uniformly randomized (controls: 55.3 k 2.3 y; 76.7 + 4.4 kg; peptide group: 64.7 + 4.6 y; 80.7 k 5.6 kg) to receive isonitrogenous (0.26 g N kg-‘d’) and isoenergetic (155 kJ kg-‘.d-‘) TPN over 9 days. Non-protein energy was derived from FGX solution and fat emulsion (Combisteril and Lipovenos, 20% Fresenius). The control group received a conventional amino acid solution (Traumasteril 10%; Fresenius; 1.5 g amino acids kg-‘d’). The peptide group was given a complete solution containing the dipeptide L-alanyl-L-glutamine (20 g I-’ = 0.3 g kg-‘.d-‘). On the morning of day 8 and 9 a modified D-xylose test (Craig et al, 1985) was performed. This test is considered as an integrated assessment of the absorption efficiency of the small intestine. Excretion of D-xylose during the 5 hour test period was 7.4 t 1.1 g vs 3.8 + 0.9 g (p < 0.05, Wilcoxon) in the peptide and control group, respectively. The serum D-xylose concentrations 2 hours after its ingestion were 38.7 +_ 3.0 vs 27.8 k 2.9 mg.dl-’ (p < 0.05). Kinetic evaluation (Yamaoka et al, 1983) revealed higher maximum D-xylose blood concentration (c,,,: 41.2 k 14.8 vs 27.2 & 6.9 mg.dl-‘; p < 0.05) and higher estimated values for the area under the curve (AUC,,,,: 56.4 + 22.5 vs 31.7 + 10.9 mg.h.dl-‘; p < 0.05) with the peptide. The estimated effective duodenal absorption was within the malabsorption range in the controls (22.4% + 1.2%) but normal in the peptide group (30.3 f 3.4%; p < 0.05). TheresultsstronglysuggestthatGIn-dipeptidesupplemented nutritional support may stand for a routinely manageable method to avoid TPN related intestinal atrophic response and thus represents an important adjunct for patients with intestinal mucosal injury secondary to trauma and sepsis.
0.52 Decreased duodenal mucosa glutamine concentration in the depleted preoperative patient R.R.W.J. van der Hulst, N.E.P. Deutz, M.F. van Meyenfeldt, R. W. Stockbrugger’ and P.B. Soeters Departments of Surgery and Gastroenterology’, University of Limburg, Maastricht, The Netherlands The small intestine has been identified as an important site for the metabolism of circulating glutamine (GLN). Utilization of GLN provides an important energy source for the mucosa of the gut. Nutritional depletion may result in a decreased GLN supply for the gut. This study was done to evaluate the intestinal GLN concentrations during nutritional depletion. A gastro-duodenoscopy was performed in patients (n = 12) admitted to our hospital for preoperative total parenteral nutrition (TPN). The indication for preoperative nutrition was bowel rest for inflammatory bowel disease (n = lo), small bowel fistula (n = 1), and gastric entrance stenosis after a Mason procedure (n = 1). Percent ideal body weight was calculated using the Metropolitan height and weight tables 1983. After an overnight fast, before starting TPN, biopsies were taken from the second part of the duodenum, for routine pathological examination and amino acid analysis. Patients (n = 7) with complaints of dyspepsia, without pathological findings, were evaluated as a control group. Venous blood samples were taken for plasma amino acid analysis. Samples were kept at -70” until determination. The patients in the TPN with a percent ideal body weight (PIW) below 95% (n = 7) had an alanine and glutamine concentration (bmol/kg dry weight) 40% (p < 0.01) respectively 20% (p < 0.02) lower than in the patient group with a PIW > 95%. In comparison with the control group, mucosal glutamate was increased in the PIW < 95% group (p < 0.01). No difference in plasma amino acids were found in the patient groups (not shown). Controls (n = 7) PIW 295% (n = 5) PIW <95% (n = 7)
Glutamine 3126k185 3670t277 2608k226’
(‘I = p < 0.02 “ers”s PIW > 95%. I#)
Alanine 4636k558 4286+487 2651 f 244’ #
TPN ill
Nutritional effects of ketoglutarate in burn patients
0.54
ornithine
alpha
L. Donati, and M. Signorini Department of Plastic Surgery and Burn Centre, University Hospital Niguarda. Milan, Italy Severe burn injury is accompanied by protein catabolism and negative nitrogen (N) balance. Conventional nutritional support fails to normalise the nutritional status of the patients, but positive effects of ornithine alpha ketoglutarate (OKG) have been reported (Cynober et al, Nutrition 1987).
Glutamate 18715+698 23642f 2812 22337+947#
= p c 0.02 YWS”Scontrol
25
Those differences could reflect difference in protein turn over rate and protein accretion between adult and infants.
Date: Wednesday, September 9 09.45-l 1.45 Chair: N.N. Abumrad (USA), A. Revhaug (Norway) Hankard, R. (Paris) Study of Glutamine Metabolism using Stable isotope in Infants with Neonatal Short Bowel Syndrome Hijrig, H. (Basel) Glutamine (GLN) ment following Antigen Receptor Activation of Human Lymphocytes
0.50
Glutamine antigen receptor lymphocytes
R. (Maastricht) Decreased Duodenal Mucosa Glutamine Concentration in the Depleted Preoperative Patient H. (Mainz) Glutamine Dipeptide Supplemented TPN maintains Intestinal Function in Critically III
Tremel,
L. (Milano) Nutritional Effects of Ornithine Alpha Ketoglutarate in Burn Patients
Donati,
M. (Phoenix, AZ) Substrate and Hormonal Changes due to Dietary Supplementation with Ornithine Alpha Ketoglutarate (OKG) in Critically III Trauma Victims
Jeevanandam,
B. (Stockholm) Alpha-Ketoglutarate given together with TPN improves the Free Glutamine-Levels in Glutamine-Depleted Intensive Care Patients
Petersson,
Study of glutamine metabolism isotope in infants with neonatal syndrome
using short
R. Hankard, 0. Goulet, M. Rongier, J-F. Desjeux, V. Colomb, C. Ricour and D. Darmaun lnserm tJ290, Hopital St. Lazare and Hopital Necker-Enfants Malades, 149 rue de Sevres, 75015 Paris cedex, Paris, France Glutamine (Gln) can be considered as a major fuel for small bowel (SB). Extensive SB resection could alter whole body Gln metabolism in vivo. Very few data are available on Gln metabolism in infants. Eleven infants with extensive neonatal SB resection leaving them with 35 + 12 cm SB length and 4 controls were studied. During the post-absorptive state infants received primed continuous infusion of 1.5 mg/kg/h L-[2-‘5N] Gln and 0.4 mg/kg/h L-[1 -13C] leucine. From stable isotopes plasma enrichments were calculated: Gln and Leu fluxes (Ra Gln) (Ra Leu). The 2 inflow components of RaGln were calculated: release from protein breakdown (B Gln)O and de novo synthesis (G Gln): pmollkglh Patients
Ra Gin 568+-124 816+149’
D Gin 175*100 359i181
B Gin 396k98 457+07
following of human
In this study we investigated on the GLN requirement in different steps of human peripheral blood mononuclear cells (PBMC) activation induced bya monoclonal antibody (mAb) directed against the CD3-T-cell receptor molecular complex. Methods: Density gradient separated PBMC were cultured in the presence or absence of anti-CD3 mAb and GLN. Phenotypic expression of activation markers was evaluated by flow cytometry, using specific mAb. Total cellular RNA was extracted after 8 hours of culture, reverse transcribed, and cDNA thus generated was assayed, by specific cytokine primers pairs, in 25 cycles polymerase chain reaction (PCR) for the presence of relevant gene transcription. Cell cycle was analyzed by flow-cytometry on 48 hours stimulated PBMC with propidium iodide based reagents. Finally, proliferation was assessed on day three by a standard 3H-thymidine incorporation technique. Results: Anti-CD3 stimulated expression of CD69, an early activation marker, on T cells, was unaffected in the absence of GLN, whereas the expression of p55 low affinity IL-2 receptor and late activation markers such as CD45RO and CD71 transferrin receptor was dose dependently correlated with GLN concentration in the culture medium. Transcription of IL-2, IL-4, IL-5, IL-6 and IFN-gamma genes was found to be inducible even in the absence of GLN by anti-CD3 mAb. In contrast, entering of stimulated PBMC into cell cycling was dependent on the presence of GLN in the culture medium, as also observed for proliferation assays. Conclusion: Early events of antigen receptor triggered lymphocyte activation can take place in the absence of GLN, whereas later stages, including entering into cell cycling and proliferation, depend on the presence of GLN. These data confirm that an adequate GLN supply is a prerequisite for an intact immune response.
van der Hulst,
0.49
(GLN) requirement triggered activation
H. Herig, L. Filgueira, G. Spagnoli, R. Babst and M. Heberer Departments of Surgery and Research, University of Basel, Switzerland
Requiretriggered
Skullman, S. (Linkoping) Glutamine improves Protein Metabolism during Liver Regeneration
stable bowel
AND ANALOGUES
0.51 Glutamine improves during liver regeneration
protein
metabolism
S. Skullman, M. Wiren, M. Chu, P. Garlick”, M. McNurlan’ and J. Larsson Department of Medico-Surgical Gastroenterology, University Hospital, Linkoping, Sweden and * Rowett Research lnstitute, Aberdeen, Scotland Glutamine is a preferential nutritional substrate for small bowel enterocytes and other fast replicating cells. Liver regeneration is a model often used to study cell replication and fast growth. We have previously shown a reduced regeneration rate in malnourished rats and the limiting factor seems to be a diminished protein synthesis. The aim of this study was to examine if glutamine could influence regeneration rate and protein metabolism in regenerating liver. Materials and methods: Eighty-eight rats (225 g) were allocated to either shamoperation (n = 40) or 70% liver
Ra Leu 228&60 263k50
‘p < 0.05 vs controls. Mann Whitney tests
Gln fluxes are reduced after SB resection, mainly due to reduced de novo synthesis. In controls, Gln and Leu fluxes are significantly higher than in adult respectively: 344 * 47 and 88 k 12 pmol/kg/h (Metabolism 1991, 49: 42-4). 24
resection (n = 48). Postoperatively they received an isonitrogenous, isocaloric elementary diet (12% of BW/day) containing 0%, 2% or 4% glutamine, (4% is equal to 25% of aminoacid content). The resected part of the liver was weighed and analysed for protein content, After 2 or 7 days protein synthesis was measured by injecting a flooding dose of L-[4-3H]-phenylalanine intravenously and the liver was analysed for RNA and protein content. Results: There were no differences between the different sham groups or between the 2% and 4% glutamine groups. Total protein content was increased in regenerating liver in the 2 and 4% glutamine groups compared to the 0% group (day 2: 2% p < 0.05, 4% p < 0.001; day 7: 2% p < 0.001 and 4% p < 0.01). Regeneration rate based on liver weight measurements was increased in the 2% group (p < 0.05 on day 2 and day 7) but not in the 4% group compared to the 0% group. There was a tendency towards improved protein synthesis after 7 days of regeneration in the 2 and 4% group compared to the 0% group, measured as mg protein/day, mg RNA (p = protein day/100 g BW and mg protein/day/mg 0.06-0.11). Conclusions: 1. Glutamine containing diet improved liver regeneration rate and total protein content in regenerating liver. 2. There was a tendency towards improved protein synthesis during regeneration in glutamine fed animals. 3. An excess of glutamine in the diet did not improve the results.
Our results suggest an alteration of intracellular duodenal mucosa glutamine metabolism in the patients with low PIW. Because GLN is used as a preferential fuel in the small bowel, specific depletion of mucosa GLN could be of significance.
0.53 Glutamine dipeptide supplemented maintains intestinal function in critically
H. Tremel, B. Kienle. L.S. Weilemann, R. Abele’, B. FichtF, P. Stehle3 and P. Fur.& Medical Clinic II, University of Maim; ’Fresenius AG, Oberurself Ts; 2 Walther Straub Inst. Pharmacol. & Toxicol., University of Munich; ‘Inst. Biol. Chem. & Nutr., University of Hohenheim, Stuttgart, FRG Long term total parenteral nutrition (TPN) is accompanied with villous atrophy and subsequent malabsorption syndrome. The underlying reasons for the impaired intestinal function are yet to be determined, though current information attest the important role of glutamine (Gln) in maintaining intestinal structure and function. Parenteral provision of synthetic highly soluble Gln-containing dipeptides facilitate Gln nutrition in practical clinical setting. Twelve critically ill intensive care patients were uniformly randomized (controls: 55.3 k 2.3 y; 76.7 + 4.4 kg; peptide group: 64.7 + 4.6 y; 80.7 k 5.6 kg) to receive isonitrogenous (0.26 g N kg-‘d’) and isoenergetic (155 kJ kg-‘.d-‘) TPN over 9 days. Non-protein energy was derived from FGX solution and fat emulsion (Combisteril and Lipovenos, 20% Fresenius). The control group received a conventional amino acid solution (Traumasteril 10%; Fresenius; 1.5 g amino acids kg-‘d’). The peptide group was given a complete solution containing the dipeptide L-alanyl-L-glutamine (20 g I-’ = 0.3 g kg-‘.d-‘). On the morning of day 8 and 9 a modified D-xylose test (Craig et al, 1985) was performed. This test is considered as an integrated assessment of the absorption efficiency of the small intestine. Excretion of D-xylose during the 5 hour test period was 7.4 t 1.1 g vs 3.8 + 0.9 g (p < 0.05, Wilcoxon) in the peptide and control group, respectively. The serum D-xylose concentrations 2 hours after its ingestion were 38.7 +_ 3.0 vs 27.8 k 2.9 mg.dl-’ (p < 0.05). Kinetic evaluation (Yamaoka et al, 1983) revealed higher maximum D-xylose blood concentration (c,,,: 41.2 k 14.8 vs 27.2 & 6.9 mg.dl-‘; p < 0.05) and higher estimated values for the area under the curve (AUC,,,,: 56.4 + 22.5 vs 31.7 + 10.9 mg.h.dl-‘; p < 0.05) with the peptide. The estimated effective duodenal absorption was within the malabsorption range in the controls (22.4% + 1.2%) but normal in the peptide group (30.3 f 3.4%; p < 0.05). TheresultsstronglysuggestthatGIn-dipeptidesupplemented nutritional support may stand for a routinely manageable method to avoid TPN related intestinal atrophic response and thus represents an important adjunct for patients with intestinal mucosal injury secondary to trauma and sepsis.
0.52 Decreased duodenal mucosa glutamine concentration in the depleted preoperative patient R.R.W.J. van der Hulst, N.E.P. Deutz, M.F. van Meyenfeldt, R. W. Stockbrugger’ and P.B. Soeters Departments of Surgery and Gastroenterology’, University of Limburg, Maastricht, The Netherlands The small intestine has been identified as an important site for the metabolism of circulating glutamine (GLN). Utilization of GLN provides an important energy source for the mucosa of the gut. Nutritional depletion may result in a decreased GLN supply for the gut. This study was done to evaluate the intestinal GLN concentrations during nutritional depletion. A gastro-duodenoscopy was performed in patients (n = 12) admitted to our hospital for preoperative total parenteral nutrition (TPN). The indication for preoperative nutrition was bowel rest for inflammatory bowel disease (n = lo), small bowel fistula (n = 1), and gastric entrance stenosis after a Mason procedure (n = 1). Percent ideal body weight was calculated using the Metropolitan height and weight tables 1983. After an overnight fast, before starting TPN, biopsies were taken from the second part of the duodenum, for routine pathological examination and amino acid analysis. Patients (n = 7) with complaints of dyspepsia, without pathological findings, were evaluated as a control group. Venous blood samples were taken for plasma amino acid analysis. Samples were kept at -70” until determination. The patients in the TPN with a percent ideal body weight (PIW) below 95% (n = 7) had an alanine and glutamine concentration (bmol/kg dry weight) 40% (p < 0.01) respectively 20% (p < 0.02) lower than in the patient group with a PIW > 95%. In comparison with the control group, mucosal glutamate was increased in the PIW < 95% group (p < 0.01). No difference in plasma amino acids were found in the patient groups (not shown). Controls (n = 7) PIW 295% (n = 5) PIW <95% (n = 7)
Glutamine 3126k185 3670t277 2608k226’
(‘I = p < 0.02 “ers”s PIW > 95%. I#)
Alanine 4636k558 4286+487 2651 f 244’ #
TPN ill
Nutritional effects of ketoglutarate in burn patients
0.54
ornithine
alpha
L. Donati, and M. Signorini Department of Plastic Surgery and Burn Centre, University Hospital Niguarda. Milan, Italy Severe burn injury is accompanied by protein catabolism and negative nitrogen (N) balance. Conventional nutritional support fails to normalise the nutritional status of the patients, but positive effects of ornithine alpha ketoglutarate (OKG) have been reported (Cynober et al, Nutrition 1987).
Glutamate 18715+698 23642f 2812 22337+947#
= p c 0.02 YWS”Scontrol
25