Glutamine and alpha-ketoglutarate prevent the decrease in muscle free glutamine concentration and influence protein synthesis after total hip replacement.

After surgical trauma, protein synthesis, as well as the concentration of free glutamine in muscle, decreases. Total parenteral nutrition (TPN) alone does not prevent the decrease of glutamine in muscle, but TPN supplemented with glutamine or its precursor, alpha-ketoglutarate, maintains amino acid concentration in muscle and preserves protein synthesis. The aim of this study was to characterize a human trauma model using patients undergoing total hip replacement, and furthermore to investigate whether glutamine or alpha-ketoglutarate alone without TPN can prevent the postoperative decrease in muscle free glutamine. Metabolically healthy patients undergoing total hip replacement were randomized into three groups. The control group (n = 13) received glucose 2 g/kg body weight (BW) during surgery and the first 24 postoperative hours. The glutamine group (n = 10) received glucose 2 g/kg BW and glutamine 0.28 g/kg BW, and the alpha-ketoglutarate group (n = 10) received glucose 2 g/kg BW and alpha-ketoglutarate 0.28 g/kg BW. Muscle biopsies were performed before surgery and 24 hours postoperatively. Free glutamine concentration in muscle decreased from 11.62 +/- 0.67 to 9.80 +/- 0.36 mmol/kg wet weight in the control group (P < .01), whereas it remained unchanged in both the glutamine group and alpha-ketoglutarate group. Protein synthesis, as reflected by the concentration of total ribosomes, decreased significantly in the control group, but not in glutamine and alpha-ketoglutarate groups. Polyribosome concentration decreased significantly in both the control and alpha-ketoglutarate groups. Total hip replacement can be used as a reproducible trauma model, with characteristic changes in the muscle amino acid pattern and protein synthesis 24 hours postoperatively.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term effects of postoperative total parenteral nutrition supplemented with glycylglutamine on subjective fatigue and muscle protein synthesis.

Seventeen patients undergoing elective open cholecystectomy were given conventional total parenteral nutrition either with (nine patients) or without (eight) glutamine supplementation of 20 g/day for 3 days after surgery and thereafter ordinary food for the following 27 days. Muscle protein synthesis, as assessed by the total concentration of ribosomes, decreased in control patients on day 3 following surgery and remained low on days 10, 20 and 30 (P < 0.05). In patients who received glutamine the total ribosome concentration was maintained on the third day after operation. Concurrently, the subjective feeling of fatigue increased on days 3 and 10 after surgery and the nitrogen balance was negative after operation in both groups, without any difference related to glutamine supplementation. Intravenous glutamine after surgery counteracts a decline in muscle protein synthesis only for as long as it is provided.

Stress hormone and amino acid infusion in healthy volunteers: short-term effects on protein synthesis and amino acid metabolism in skeletal muscle.

To study the immediate effects of stress hormones and intravenous amino acid support, healthy male volunteers were administered a stress-hormone infusion including epinephrine, cortisol, and glucagon either alone (Triple, n = 8) or combined with a balanced glutamine-free amino acid solution (Triple AA, n = 8) over a period of 6 hours. The amino acid infusion was started 2 hours after the hormone infusion. A third group (AA, n = 8) received the balanced amino acid solution alone. After 6 hours of the stress-hormone infusion, a decrease was observed in skeletal muscle protein synthesis as measured by the size distribution and concentration of ribosomes. The decrease was prevented by an infusion of the balanced amino acid solution. Following the triple-hormone infusion, a decrease was noted in the content of the total free amino acids in both muscle and plasma. After including amino acids in the infusion solution, the significant decrease in muscle glutamine caused by the triple hormones was not seen. Plasma cortisol, insulin, and glucose increased in response to the triple-hormone infusion alone or in combination with amino acids. In summary, the results show that the signs of muscle protein catabolism elicited by administration of stress hormones can be attenuated by simultaneous administration of a conventional amino acid solution, although it does not contain glutamine.

Metabolic effects on growth and muscle of soya-bean protein feeding in cod (Gadus morhua).

The aim of the present study was to investigate the effect of soya-bean protein on growth and muscle metabolism in fish. Cod, Gadus morhua, were fed on a fish-feed formula with the high-quality fish-meal protein being replaced by 100, 200 or 300 g soya-bean protein/kg fish-meal protein. The feeding experiment lasted for 43 d at a water temperature of 7-8 degrees and a sea water salinity of 3.5%. At the 200 g/kg level of soya-bean protein, food intake and growth rate were similar to those of the controls. At the 300 g/kg level of soya-bean protein, food intake was diminished by 6% and growth by 67% relative to control levels. In muscle, sarcoplasmic protein (/g wet weight) was significantly decreased by 14%. Myofibrillar protein (/g wet weight) was unchanged. Levels of RNA in the myofibrillar fraction decreased at all three levels of soya-bean protein, and that of the sarcoplasmic fraction decreased at the highest level of legume-protein. With increased levels of soya-bean protein, RNA:DNA declined by 18% from 1.88 to 1.54. The contractile protein myosin heavy chain (/mg protein and /g wet weight) and myosin heavy chain-specific mRNA (/mg RNA) were not significantly affected by dietary conditions. Expressed per g wet weight, the decline by 21% of the specific mRNA depended on the total RNA content which decreased with the increase in soya-bean protein. Acid proteinase activity was lowest at the 200 g/kg level, showing a decrease of 23%.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of the Atlantic salmon hepatic 21 kDa protein and classification as a high mobility group chromatin protein.

The 21 kDa protein of liver from Atlantic salmon (Salmo salar) has been purified. Hepatic nuclei were extracted with 0.75 M HClO4. The extracted proteins were fractionated using reversed phase high performance liquid chromatography. The purity of the protein was analysed by isoelectric focusing in the first, and SDS-polyacrylamide gel electrophoresis in the 2nd dimension. Isoelectric focusing separated the protein into 5 spots. In gel trypsin digestion after isoelectric focusing followed by SDS-polyacrylamide gel electrophoresis resulted in identical migration of the tryptic peptides. The amino acid composition of the 21 kDa protein was similar to that of high mobility group (HMG) proteins C and D from rainbow trout (Oncorhynchus mykiss). The N-terminal sequence of the amino acids 1-19 revealed a conserved region characteristic for HMG 14/17 proteins of mammals and avians, and their equivalents in rainbow trout. Considering the electrophoretic mobility, amino acid composition and N-terminal amino acid sequence it is concluded that the 21 kDa protein of Atlantic salmon is a member of the HMG protein family resembling the HMG D protein of rainbow trout.

The condensing action of Mg2+, hexamminecobalt3+ and spermidine3+ on chromatin with gene regions activated by 17-beta estradiol.

1. Hepatic vitellogenin synthesis was induced by injecting 17-beta estradiol into fish. Liver nuclei were incubated with the endonuclease EcoRI at an increasing concentration of Mg2+ (0.15-1.50 mM), hexamminecobalt3+ or spermidine3+ (0.10-1.00 mM). Chromatin was separated into a 5000 g supernatant, S-fraction, and pellet fraction. 2. The release of chromatin into the S-fraction was higher for the induced than the control chromatin. Hybridization of the vitellogenin gene retained in the pellet fraction of the controls was unaffected by the individual cations. After activation of the vitellogenin gene, Mg2+ at its lowest concentration retained a high amount of the vitellogenin gene in the pellet fraction. The level of hybridization decreased by increasing the Mg2+ concentration. Retention of the gene rose by adding hexamminecobalt3+ and more so by adding spermidine3+. 3. The condensing action of spermidine3+ was extended to the activated vitellogenin gene regions of chromatin.

Biosynthetic human growth hormone preserves both muscle protein synthesis and the decrease in muscle-free glutamine, and improves whole-body nitrogen economy after operation.

As a reproducible human trauma model, patients (n = 17) undergoing elective cholecystectomy were studied for 3 postoperative days. They were randomly allocated to receive either recombinant human growth hormone (hGH; 0.3 U/kg/24 hours) or placebo together with total parenteral nutrition, including 0.2 gN/kg/24 hours and 135 kJ/kg/24 hours. Before operation and on the third postoperative day, percutaneous muscle biopsies were performed to determine the concentration and size distribution of ribosomes and the free amino acid concentrations. The significant postoperative decrease in the total ribosome concentration (15.3 +/- 6.4%) and the polyribosome concentration (20.9 +/- 6.5%) in the control group was impeded in the group receiving synthetic hGH. Muscle free glutamine decreased by 35.6 +/- 4.2% in the control group and to a lesser extent in the group that was given hGH after operation (p less than 0.05). The protein content of skeletal muscle was unchanged. The cumulated nitrogen balance for the study period was negative in the control group (-7.09 +/- 0.71 gN), but was not different from zero in the hGH group (-2.32 +/- 1.66 gN). It is concluded that synthetic hGH administered after operation has beneficial effects on the whole-body nitrogen economy, as indicated by the unchanged capacity for protein synthesis in skeletal muscle, the preserved levels of muscle free glutamine, and improvement in the whole-body nitrogen balance. The effects of hGH on skeletal muscle protein and amino acid metabolism can explain the postoperative nitrogen-sparing effect attributed to hGH.

Tissue-specific sensitivity of chromatin and the vitellogenin gene to micrococcal nuclease after continuous exposure of salmon (Salmo salar) to 17 beta-estradiol.

Smoltified Atlantic salmon (Salmo salar), 2 years old and weighing 217 +/- 13 g, were treated for 2 weeks with 17 beta-estradiol containing silastic capsules implanted intraperitoneally. Control fish received empty capsules. Vitellogenin, present in the blood of both groups of fish, was enhanced by estradiol treatment. Nuclei were isolated from liver, blood cells, and brain and incubated with increasing concentrations of micrococcal nuclease (EC 3.1.31.1). In liver there were more mononucleosomes as a percentage of total chromatin in hormone-treated than in control fish. Using vitellogenin cDNA as a probe the highest hybridization signals were seen when 2 to 4% of the chromatin was digested to mononucleosomes. In blood cell and brain nuclei independent of the extent of the chromatin released the hybridization signals remained low. The expression of the vitellogenin gene in immature females was potentiated by exogenous estradiol to give increased micrococcal nuclease sensitivity of the chromatin without enhancement of the hybridization level. Micrococcal nuclease digestion and hybridization of the vitellogenin gene demonstrated that the hepatic specificity of vitellogenin synthesis is manifested as structural modulations of the chromatin containing the vitellogenin gene.

Physiological properties of liver, gonads and muscle during maturation of female Atlantic salmon, Salmo salar. Comparison between a control and xenobiotics containing fish feed.

1. Atlantic salmon, Salmo salar, in their 4th year were maintained in a sea-based farm in the Baltic Sea, salinity 0.2-0.4%. The fish were fed a control diet or a diet containing, among the lipid-soluble xenobiotics, 60 parts of dioxin per 10(12) parts of fish oil. 2. In the fish that attained sexual maturity the liver and gonadal wet weight increased, but decreased after stripping of the roe. Vitellogenesis was also apparent as an elevated level of plasma vitellogenin which was higher after than 2 months prior to removal of the roe. 3. In muscle protein content was highest prior to the removal of the roe. RNA content decreased with time. Following the taking of the roe glycogen content and acid proteinase activity were elevated. 4. Comparison between the feeding groups showed, in the fish fed the experimental diet, a higher gonadal wet weight and plasma vitellogenin content, and in muscle, after stripping of the roe, a lower glycogen content and an elevated level of acid proteinase activity.

Dietary protein levels affect growth and protein metabolism in trunk muscle of cod, Gadus morhua.

Cod (Gadus morhua) of 50 g body weight were kept at 14 degrees C. The fish were fed ad libitum during 80 days a diet containing protein levels which in terms of total energy corresponded to 25%, 45% or 65%. Growth increased in accordance with protein-energy levels. The protein content per gram of wet weight of white trunk muscle was unchanged, as was the myofibrillar protein myosin heavy chain determined by the antigen-antibody reaction of the enzyme-linked immunosorbent assay. The amount of messenger ribonucleic acid (mRNA) coding for myosin heavy chain was lower at 25% than at 45% or 65% protein-energy intake, the differences being significant per gram of wet weight of muscle. Acid proteinase activity was highest at the lowest protein-energy intake. Glycogen content in muscle increased with the protein-energy levels. It is concluded that the metabolic response of white trunk muscle to graded protein-energy intake included a change in the capacity to synthesize myosin heavy chain as judged by its mRNA content. The protein content per gram of wet weight was unaffected by dietary protein-energy levels of 25%, 45% and 65%, but protein accretion and thus growth of the animals increased with the protein intake. Dietary protein-energy restriction caused a rise in acid proteinase activity and a decrease in content of mRNA for myosin heavy chain, resulting in a diminished growth rate at an unchanged protein content per gram of wet weight of muscle.

Polyamines in liver and their influence on chromatin condensation after 17-beta estradiol treatment of Atlantic salmon.

Atlantic salmon (Salmo salar) were treated with 17-beta estradiol to induce vitellogenin synthesis in liver. This led to an increase in liver wet weight and total DNA. After incubation with micrococcal nuclease (EC 3.1.31.1) less soluble chromatin was obtained from nuclei of the estradiol treated than the control fish, but active gene regions were solubilized by the nuclease. Thus, in the estradiol treated fish soluble mononucleosomes contained hybridizable vitellogenin gene sequences. As a result of estradiol treatment the content in total liver of putrescine rose 3-fold, that of spermidine 2-fold, while spermine was unchanged. In muscle no significant changes were observed. The regulatory functions of polyamines during gene expression were investigated by binding (14C)spermine to isolated liver nuclei depleted of endogenous polyamines. The number of binding sites was higher in nuclei of estradiol treated than control fish. (14C)spermine associated preferentially with micrococcal nuclease insensitive chromatin. Thus, the high content of putrescine and spermidine in liver supported the view of polyamine accumulation in proliferating tissues. The preferential binding to condensed chromatin indicated a stabilizing effect of polyamines on the organization of inactive chromatin structures.

Ribosome and free amino acid content in muscle during hemodialysis.

Patients (N = 8) with chronic renal failure and uremia treated with hospital hemodialysis were in a pilot study investigated before and after a single hemodialysis session. The extracorporeal dialysis circuit was flushed regularly with saline to avoid clotting and the use of heparin. Percutaneous skeletal muscle biopsies were taken before and after the dialysis to determine the content of free amino acids together with the concentration and size distribution of ribosomes before and after dialysis. After dialysis the alanine concentration in muscle decreased by 20% (P less than 0.05), while all other amino acids were unaffected. The total ribosome concentration per mg of DNA decreased by 31% (P less than 0.01) and the relative proportion of polyribosomes by 7% (P less than 0.05) after the dialysis compared to predialytic values. All individual plasma amino acids decreased during the dialysis procedure except for threonine and arginine, which were unaltered, and leucine and isoleucine, which increased. The decline in ribosome and polyribosome content together with the changes in amino acid levels indicate a low capacity for protein synthesis and increased catabolism in muscle of hemodialyzed patients.

Growth hormone improves muscle protein metabolism and whole body nitrogen economy in man during a hyponitrogenous diet.

Healthy male volunteers (n = 12) were given a normocaloric hyponitrogenous diet for a conditioning period of 7 days. Thereafter they were blindly randomized to receive daily injections of methionyl recombinant human growth hormone (met-hGH) 0.06 IU/kg or saline during a second week of hyponitrogenous nutrition. The met-hGH group showed a lower urinary urea excretion and a lower serum concentration of urea as compared with the control group. In skeletal muscle, the polyribosome concentration, indicative of muscle protein synthesis, as well as the concentrations of glutamine, alanine, aspartate, serine, and threonine, decreased in the control group, whereas no such changes were seen in the met-hGH-treated group. Since provision of met-hGH prevented protein catabolism in muscle and improved whole body nitrogen economy, investigations of the possible beneficial effects of met-hGH to prevent skeletal muscle vast after surgical trauma are advocated.

Alpha-ketoglutarate preserves protein synthesis and free glutamine in skeletal muscle after surgery.

Serving as a reproducible human trauma model, patients (n = 21) undergoing elective cholecystectomy received postoperative total parenteral nutrition with (n = 9) or without (n = 12) alpha-ketoglutarate (AKG) supplementation. Skeletal muscle biopsy specimens were taken before surgery and on the third postoperative day. The postoperative decreases in the concentrations of free glutamine and basic amino acids seen in the control group were counteracted in the AKG group (p less than 0.05). Muscle protein synthesis was estimated by ribosome analysis. On the third postoperative day the control group showed a decline in the polyribosome concentration (25.8% +/- 4.5%; p less than 0.001). No significant change was observed in the AKG group. On each postoperative day the nitrogen balance was negative in the control group but not in the AKG group. In the control group the cumulative nitrogen balance amounted to -9.9 +/- 1.8 gm of nitrogen and in the AKG group -2.6 +/- 2.6 gm of nitrogen, which was significantly different (p less than 0.05). Administration of AKG, the carbon skeleton corresponding to glutamine, produced results similar to those seen when glutamine is added to postoperative total parental nutrition. The results suggest that the availability of precursors for glutamine synthesis in skeletal muscle is crucial for the degree of muscle protein catabolism after surgical trauma.

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.

Nuclear proteins and chromatin structure in liver and intestinal epithelial cells of young growing and adult rats.

Nuclei from liver and intestinal epithelial cells of young growing rats (39 days old) and adult rats (98 days old) were isolated. After addition of DNase I, the chromatin was separated by centrifugation (1100 g) into two fractions; the pellet (P) and the supernatant (S). The amount of chromatin released into the S-fraction was the same for the two age groups. The intestinal epithelial cell nuclei underwent self-digestion (in the absence of added DNase I) which was significantly higher in the young rats than in the adults. Subsequent examination using immunotechniques established the presence of non-sarcomeric myosin heavy-chain indicating that active genes were present for that protein. Hybridization of DNA with cDNA specific for myosin heavy-chain revealed that, relative to total DNA, the DNA retained in the P-fraction of both tissues and age groups contained the same amount of hybridizable sequences. In liver, nuclear proteins decreased significantly with age per g wet weight of tissue. In the enterocyte tissue, total DNA and protein increased with age. SDS-polyacrylamide gel or acetic acid-urea gel electrophoresis gave no age-related differences in the pattern of the proteins within each tissue. The results show that both liver nuclear DNA and protein decrease with age per g wet weight but increase per total tissue. In intestinal epithelial cells changes in chromatin structure with age were inherent within the nucleus.

Role of glutamine and its analogs in posttraumatic muscle protein and amino acid metabolism.

Skeletal muscle protein catabolism following trauma has until recently not been possible to counteract by intravenous nutritional means. The obligatory loss of nitrogen with concomitant reduction of skeletal muscle protein synthesis is also accompanied by a decrease of muscle free glutamine, the extent of which is proportional to the muscle protein catabolism. Serving as a human model of surgical trauma, patients undergoing elective cholecystectomy were given total parenteral nutrition including additions of either glutamine or its analogs (ornithine-alpha-ketoglutarate, alpha-ketoglutarate, or alanylglutamine) during 3 postoperative days. The polyribosome concentration and the intracellular glutamine concentration in skeletal muscle, as well as nitrogen balance, showed a less pronounced skeletal muscle catabolism in these groups than when conventional total parenteral nutrition was given. It is concluded that a support of either glutamine or its carbon skeleton, alpha-ketoglutarate, counteracts the postoperative fall of muscle free glutamine and of muscle protein synthesis. Furthermore, statistical correlations could be shown between the changes of muscle glutamine and muscle protein synthesis and the postoperative nitrogen losses.

Elective abdominal surgery depresses muscle protein synthesis and increases subjective fatigue: effects lasting more than 30 days.

Ten patients without metabolic disease undergoing elective cholecystectomy were studied before surgery and on days 3, 10, 20 and 30 after operation. Percutaneous muscle biopsies were taken and protein synthesis was determined from the total concentration and size distribution of ribosomes. The subjective feeling of fatigue was estimated using a visual analogue scale. The nitrogen balance was calculated at 20 days following surgery. The mean (s.e.m.) total concentration of ribosomes per milligram of DNA decreased by 27.5(6.6) per cent (P less than 0.01), 44.5(6.5) per cent (P less than 0.001), 48.3(8.9) per cent (P less than 0.001) and 45.0(8.2) per cent (P less than 0.01) on days 3, 10, 20 and 30, respectively. By 30 days after surgery no sign of restoration of normality was seen. The relative proportion of polyribosomes had decreased by 20.4(6.4) per cent (P less than 0.05) on the third postoperative day and by 20.4(3.9) per cent (P less than 0.01) on the tenth postoperative day and was restored to the preoperative level by day 20. The subjective fatigue score increased after operation and five of nine patients had not regained their preoperative scores 30 days after surgery. The daily nitrogen balance was negative for 5 days. The cumulated nitrogen losses were not restored until after 18 days following surgery. Elective abdominal surgery caused a sustained depression of protein synthesis for over 30 days, a longer period than previously presumed. These results show that long-term follow-up is required when the effect of different postoperative nutritional regimens are to be evaluated.