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The effects of a new amino-acid dipeptide solution on nitrogen balance and humoral growth factors in the postoperative state in man
Clinical Nutrition (1995) 14:97-104 © Pearson Professional Ltd 1995
The effects of a new amino-acid dipeptide solution on nitrogen balance and humoral growth factors in the postoperative state in man M. WIRI~N, T. E.* ADRIAN, F.t HAMMARQVIST, K-E. JOHANSSON, J. PERMERT, B.t PETERSSON, j.t WERNERMAN and J. LARSSON Department of Surgery, University Hospital, Link6ping, #Departments of Surgery and Anaesthesia, St G6rans Hospital, Stockholm. *Department of Biomedical Sciences, Creighton University, Omaha, USA (Correspondence to: MW, Department of Surgery, Faculty of Health Sciences, Link6ping University, S - 581 85 Link6ping, Sweden A B S T R A C T - - 27 patients admitted for elective abdominal surgery were allocated to receive postoperative total parenteral nutrition supplemented with glutamine (glycyl-glutamine) and tyrosine (glycyl-tyrosine) containing dipeptides (DP-GIn 20; 0.16 g glutamine/kg BW/24 h) or isonitrogenous Vamin 18 for 5 days. The aim was to evaluate safety and effects on short-life plasma proteins, nitrogen balance, 3-methylhistidine excretion and alimentary growth factors in plasma. No differences in transthyretin or retinol binding protein levels, nitrogen balance or 3methylhistidine excretion were found in patients receiving DP-GIn 20 compared to Vamin 18. There were higher plasma levels of peptide YY in the dipeptide group 5 days after surgery (p < 0.05). A correlation between insulin levels and nitrogen balance was found only in DP-GIn 20 treated patients day 6 (r = 0.91, p < 0.01). DP-GIn 20 is a glutamine dipeptide (Gly-GIn) containing amino acid solution which is considered safe in the postoperative state in man. No beneficial effects on whole body protein metabolism were found by adding DP-GIn 20 to total parenteral nutrition.
Introduction
glutamine parenterally have demonstrated improvement in nitrogen balance postoperatively (4-6), restoration of intestinal permeability characteristics during TPN-treatment (7), a shortened length of stay at hospital in bone marrow transplant (BMT) patients (8, 9), and reduction of excess total body water during TPN treatment (10). Moreover, a positive effect on the mood of BMT-patients receiving glutarnine has been reported (11). On the other hand, the reduction in infectious complications in bone marrow transplant patients originally described, has not been reproduced (9). In home parenteral nutrition, liver toxicity has been described (12). In severe pancreatitis it has not been possible to demonstrate beneficial effects of glutamine (13). Glutamine added to TPN exerts a positive effect on glutamine and muscle protein metabolism after surgery (4-6). The otherwise characteristic decreases in muscle free-glutamine concentration and protein synthesis were attenuated in parallel with decreased whole body nitrogen losses. The uptake of amino
Specific nutrients and growth factors are attracting increasing interest in nutritional research and clinical practice (1). In general surgery, much attention has been focused on the requirements of the gastrointestinal tract after abdominal operations, during total parenteral nutrition (TPN) and bowel rest periods. Glutamine has been proposed to be a necessary nutritional substrate for the organism in catabolic situations. It is also considered a specific intestinal nutrient, capable of restoring intestinal mucosa exposed to trauma or atrophy (2). The possibility of administering parenteral glutamine has been achieved by adding glutamine-containing dipeptides. Tyrosine has also been considered important in patients exposed to trauma and stress, because it is a precursor for hormone and neurotransmittor synthesis (3). DPGln 20 is a new dipeptide amino acid solution containing glycyl-glutamine and glycyl-tyrosine. Previous studies using comparable amounts of 97
98
NITROGEN BALANCE AND HUMORAL GROWTH FACTORS
acids and subsequent protein synthesis has been shown to be dependent on various factors; nutrients, hormones, cytokines and growth factors being of crucial interest. Growth hormone given to humans has been shown to improve postoperative protein metabolism (14). Growth factors, hormones and gastrointestinal peptides have also been proposed to be of interest in posttraumatic regulation of gastrointestinal function, metabolism and growth. Nutritional support could influence hormonal release and paracrine actions of growth factors, thereby indirectly affecting the utility of nutrients for anabolism. PYY, enteroglucagon, EGF and IGF-I and II are some of the factors being involved in the regulation of growth of the gastrointestinal tract, as shown in animal experiments involving intestinal resection. As glutamine is a primary gut nutrient an improvement in intestinal integrity could be associated with changes in alimentary growth factors. The aim of this study was to examine the effects of glycyl-glutamine and glycyl-tyrosine (DP-Gln 20) supplemented TPN for 5 days after major abdominal surgery on the plasma levels of short-life proteins, alimentary hormones and growth factors, urinary 3methylhistidine excretion and whole body nitrogen balance.
and 13 patients in the Vamin 18-group completing the protocol. The patients were informed about the purpose and the risks of the study in writing and orally and were also informed that they may leave the study at any time, with no deleterious effects concerning treatment. Patients were randomized after giving oral informed consent. Demographic characteristics of patients and underlying diseases are shown in Table 1. The surgical procedures were equally distributed in the two groups (Table 2). The protocol of the study was approved by the Ethical Committee of the Karolinska Institute, Stockholm, Sweden. The two groups received an iso-nitrogenous (0.18 g N/kg/24 h) and iso-volumetric (28.4 ml/kg/24 h) TPN solution for 5 days following surgery. The differences in composition of DP-20 and Vamin 18 are presented in Table 3. The solutions were prepared blind by the pharmacy at the two hospitals in an all-in-one TPN bag according to the patients weight. The dosage of non-protein energy was approximately 109 KJ/kg Table 1 Demographicdata and diagnosesof the study population. Mean (SEM).
DP-Gln 20
Vamin 18
Demography Patients and methods
31 patients admitted for elective abdominal surgery in two surgical centres were randomly allocated to double-blind administration of either a glutaminecontaining dipeptide solution, DP-Gln 20 (Pharmacia, Sweden) or Vamin 18 (Pharmacia, Sweden), for 5 days after operation. The study was conducted at the Departments of Surgery and Anesthesia, St. G6rans Hospital in Stockholm and Department of Surgery, Linktping University Hospital. Male and female adult patients between 25 and 75 years undergoing elective, major, abdominal surgery were included in the study. Patients with inborn errors of amino acid metabolism, reduced liver or kidney function (creatinine > 200 gmol/l, Quick < 65%, ALAT > 2 gkat/1), circulatory instability, diabetes mellitus and female patients with child-bearing potential were excluded from the trial. Excluded were also all patients with a per-operative blood loss exceeding 1500 ml, or receiving transfusion of blood products exceeding > 1000 ml postoperatively, as proper evaluation of protein metabolism otherwize would be hazardous (15). 4 patients were excluded due to premature termination of TPN or withdrawal of consent, resulting in 14 patients in the DP-Gln 20-group
Female/Male Age, years Body weight, kg Body weight, cm
7/7 58.7 (3.7) 73.4 (2.9) 173 (2.4)
6/7 51.7 (3.0) 73.5 (4.0) 171 (2.4)
Diagnoses
Reflux oesophagitis Gastric cancer Colorectalcancer Other malignancies Benign upper GI disease Benign colorectaldisease Vascular disease
6 4 1 2 1 0 0
6 1 2 I 1 1 1
Table 2 Surgicalprocedures and operativetrauma. Mean (SEM). DP-Gln 20 n = 14
Vamin 18 n = 13
Surgical procedures
Fundoplicatio Gastrectomy Colorectalresection Gastric resection/GE Other abdominaloperation
6 2 1 1 4
6 1 3 1 2
Operative trauma
Operatingtime (min) Blood loss (ml) Transfusions (bloodor plasma)
155 (14) 432 (89) n=3
159 (9) 429 (89) n =3
CLINICALNUTRITION 99 Table 3 Compositionof the two amino acid solutions, amino acids (g/l).
Glutamine Glycine Isoleucine Leucine Lysine Methionine Phenylalanine Tyrosine Threonine Tryptophan Valine Arginine Histidine Alanine Aspartatic acid Cysteine Glutamicacid Proline Serine
DP-Gln 20
Vamin 18
16.06" 9.00* 4.49 6.34 7.23 4.49 4.69 1.83* 4.49 1.53 5.86 9.07 5.46 12.85 2.73 0 4.49 5.46 3.61
0 7.90 5.60 7.90 9.00 5.60 7.90 0.23 5.60 1.90 7.30 11.30 6.80 16.00 3.40 0.56 5.60 6.80 4.50
Analytical methods
*componentsof dipeptides: Glycyl-glutamine24.31 g (Gly = 8.23 g, Gln= 16.06 g) and Glycyl-tyrosine2.77 g (Gly = 0.75, Tyr = 1.83 g).
body weight. 45% of the non-protein energy was fat emulsion (Intralipid 20%, Pharmacia, Sweden) and 55% was given as glucose (30%). TPN was administered via a central venous line and started the first day after operation. On the day of surgery patients received glucose solutions and volume substitution. Low molecular heparin, Fragmin (Pharmacia, Sweden), was used as thromboprophylaxis, starting the evening before surgery. Routine antibiotic prophylaxis included tetracyclin and cefuroxim, with or without metronidazole, which was used in all colorectal and vascular procedures. The patients were clinically assessed
Preop.
Op.
and heart rate and blood pressure were recorded twice daily. Blood samples were taken preoperatively after an overnight fast and during the study period according to the flow chart presented in Figure 1. Blood samples for peptide analysis were collected into ice-cold tubes containing aprotinin (Trasylol, Bayer, 400 KIU/ml) and EDTA (5 mg/ml blood). The samples were immediately separated in a refrigerated centrifuge (1500 g for 15 rain), the plasma decanted and frozen at -70°C for subsequent analysis. Urine collection was performed every 2 4 h starting the first day after surgery and continued for 5 days. Completeness of urinary sampling was monitored by daily measurements of creatinine output. Growth factor analyses were performed only on patients included at the surgical department in Link6ping.
Day 1
Routine biochemistry including analysis of hematologic, electrolytes and liver function tests were performed at the departments of chemistry in the two hospitals according to standard procedures. Frozen (-30°C) plasma was used for analysis of transthyretin and retinol-binding-protein by nephelometry at the laboratories of Pfrimmer Kabi, Erlangen, Germany.
Urine analysis Nitrogen excretion was determined according to Kjeldahl and the nitrogen balance was calculated estimating nitrogen losses in stool or skin to 30 mg/kg BW/day. 3-methylhistidine was analyzed with a conventional amino acid analyzer (Beckman System 6300, Beckman Instruments Inc., Palo Alto, CA, USA). Nitrogen, urea and 3-methylhistidine excretion was corrected for varying muscle mass by taking the ratio with creatinine into account.
Day2
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Lab. assessments A 1" ? Lab. assessments B T ? ?$ T? Body temperature 1" $ $ ?$ t? Heart rate, blood pressure 1" $ $ Urine collection ,~ TPN Admixture infusion A B Electrolytes Bilirubin Retinol binding Hb Urea Triglycerides protein WBC Crcatinine Albumin Platelets Liver enzymes Prealbumin Quick
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Fig. 1. Studydesign. Laboratoryassessments,safetycontrol and urinary collectionwas monitored5 days after surgery. TPN was givenduring the day-timeand laboratoryassessmentswere taken in the fasting state.
100 NITROGENBALANCEAND HUMORALGROWTHFACTORS
Measurement of growth factors by radioimmunoassay The samples were acidified in two volumes of acidified ethanol (0.1 M HC1 in 75% ethanol). After vortex mixing and centrifugation, at 1500 g for 10 min at 4°C, the supernatant was decanted. Supernatants were dried in a vacuum centrifuge and reconstituted in assay buffer prior to radioimmunoassay. Insulin, islet amyloid polypeptide (IAPP), glucagon, gastrin, peptide YY (PYY), enteroglucagon, epidermal growth factor (EGF), transforming growth factor a (TGF-~) and insulin-like growth factors I and II (IGF-I and IGF-II) were measured using specific and sensitive radioimmunoassays previously described in detail (16-18).
Statistics All values are given as means + SEM. Student's t-test for unpaired samples was used to compare differences between the two groups and for paired samples to compare observations within the groups, p < 0.05 was considered significant. Linear regression and ANOVA were used to analyse correlations between growth factor results and metabolic data.
Table 4 Transthyretine and retinol-binding protein in plasma, changes from day 1 to day 6. Cumulative nitrogen balance and mine excretion of nitrogen and 3-methyl-histidine in urine. Mean (SEM).
DP Gin-20 Transthyretine, A day 6- day 1 (rag/l) Retinol binding protein, A day 6day 1, (mg/1) Nitrogen excretion, cumulative (g/5 days) Nitrogerdcreatinine excretion cumulative (g/p.mol/5 days) 3-methylhistidine excretion, cumulative, (gmol/5 days) 3-metbylhistidine/creatinine excretion, cumulative (I.tmol/mmol/5 days) Nitrogen balance, cumulative (g/5 days)
Vamin 18
0.43 (0.09) 0.17 (0.02) 59.0 (4.1) 6.0 (0.3) 1224 (121) 121 (5)
0.26 (0.21) 0.09 (0.04) 64.7 (5,5) 5,5 (0,4) 1474 (150) 121 (6)
-3.7 (3.2)
-7,8 (4.2)
nitrogen balance were not statistically significant (Fig. 2). The cumulated 3-methylhistidine excretion corrected for creatinine was identical in the two groups (Fig. 2).
Growth factors Results
Biochemistry There were no adverse effects related to treatment from any of the two amino-acid solutions. The most frequent laboratory changes were increases in liver function tests and urea levels in plasma, but no significant differences in the two groups were found. Effects upon hematologic profile, electrolytes, glucose and kidney function tests of DP-Gln 20 were not different to results in patients treated with Vamin 18. The albumin level was lower initially in the DPGin 20 group (39:5 (1.5) vs 43.3 (0.5), p < 0.05) and during treatment there was a significant increase in plasma albumin concentration from day 3 to day 6 that was not seen in Vamin 18 treated patients [30.8 (0.7)-32.6 (1.0) g/1 DP-Gln 20, p <0.05 vs 34.0 (1.4)-34.5 (1.5) Vamin 18, NS].
There were higher insulin, IAPP, and glucagon levels in both groups. Blood glucose was within normal limits. There were increases in the concentration of circulating PYY in the DP-20 treated patients, and trends in this direction were also seen for enteroglucagon, EGF and TGF-c~. No effects upon gastrin, IGF-I or IGF-II were seen (Table 5).
Growth factors and nitrogen balance Insulin demonstrated the strongest positive correlation with nitrogen balance in the whole study population (r = 0.49, p < 0.07) but did not seem to influence prealbumin levels or 3-methylhistidine excretion. In the Vamin 18 group there was however no correlation between insulin levels and nitrogen excretion or balance (Fig. 3a). In the DP-Gln 20 treated patients on the contrary, there was a strong correlation (r = 0.91, p < 0.01) between insulin levels and nitrogen balance (Fig. 3b).
Protein metabolism There were no significant differences in transthyretin or RBP plasma levels, nitrogen balance or 3methylhisfidine excretion between the two groups from day 1 to day 6 (Table 4). Nitrogen balance was numerically better in the DP-Gln 20 treated patients in 4 days out of 5 but the differences in cumulated
Discussion The interest in specific nutrient support to optimize parenteral nutrition has produced a large body of results in animal experiments, suggesting glutamine to be beneficial posttraumatically in reducing protein
CLINICAL NUTRITION 101 at
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Fig. 2. Cumulative urinary excretion of: a) 3-metylhistidine/creatinine (gmol/mmol/24 h), and b) cumulative nitrogen balance (g/24 h) during 5 days postoperatively. Mean (SEM). DP-Gln 20-group in striped bars and Vamin 18 in filled bars. No significant differences were seen.
Table 5 Hormones/growth factors preoperatively (preop) and day 6 postoperatively (postop) in the Vamin group and the DP-Gln 20 group. Mean (SEM). Statistical significance is indicated within the groups * = p < 0.05, * = p < 0.01. Dp-Gln 20 Hormones/ growth factors Insulin (pmol/1) IAPP (pmol/l) Glucagon (pmol/1) Enteroglucagon (pmol/1) Gastrin (pmol/1) PYY (pmol/1) IGF-I (nmolB) IGF-II (nmol/1) EGF (pmol/1) TGFct (pmol/1)
Vamin 18
Preop
Postop
Preop
Postop
42.1 (3.2) 8.7 (1.5) 5.9 (0.5) 42.0 (6.3) 22.9 (2.8) 11.5 (2.1) 14.6 (2.6) 4.2 (0.4) 9.4 (1.8) 7.9 (1.5)
68.0 * (5.9) 14.4 (3.1) 7.6* (0.4) 66.5 (11.9) 22.4 (2.9) 18.5" (3.1) 15.2 (2.7) 4.2 (0.5) 13.0 (1.3) 10.7 (1.5)
44.9 (4.7) 8.4 (1.2) 6.3 (0.3) 40.4 (8.6) 23.6 (2.4) 12.4 (2.1) 10.6 (2.2) 4.2 (0.6) 9.6 (2.1) 7.6 (1.5)
65.7* (3.7) 12.2 (2.4) 7.7* (0.3) 44.3 (12.9) 21.0 (2.4) 16.5 (2.4) 10.0 (1.8) 3.3 (0.2) 9.4 (2.0) 6.9 (1.5)
IAPP = islet amyloid polypeptide, PYY = peptide YY, IGF = insulin-like growth factor, EGF = epidermal growth factor, TGF-o~ = transforming growth factor alpha.
breakdown and improving intestinal growth as well as immunological function (19). This hypothesis has also been tested in human experiments, partly supporting glutamine as a beneficial supplement to TPN
(4-11, 20-22). In the present study, the effects of postoperative glutamine supplementation in the form of a new amino-acid dipeptide solution, DP-Gln 20, after major abdominal surgery on patients without major blood loss were evaluated. We could find no differences between DP-Gln 20 and Vamin in plasma levels of transthyretin and RBP, nor on nitrogen balance or 3-methylhistidine excretion. An interesting correlation between endogenous insulin levels and nitrogen balance was found only in DP-Gln 20-treated patients. In some aspects these results contradict previously published papers. Improvement in nitrogen balance has been reported in most of the studies. When using elective cholecystectomy, effects on nitrogen balance have been reported both when glutamine or glutamine as a dipeptide, alanyl-glutamine, was given as a part of TPN (4, 6). However, this was not reproduced in a study when glycyl-glutamine was given together with TPN (22). On the other hand, positive effects were shown regarding muscle protein synthesis, in terms of a diminished decrease in polyribosomes, and regarding the level of muscle free glutamine, that showed a profound decrease in the control groups not receiving glutarnine (5, 6, 22). Stehle could show a highly significant improvement already by day 1 and each subsequent day up to day 5 after abdominal surgery with administration of glutamine in dipeptide solution (4). In the work by Ziegler on BMT-patients an improvement in nitrogen balance was seen but the study arms were not strictly isonitrogenous (8). One reason for the lack of positive results could be the fact that the glutamine dose is lower than in
102 NITROGEN BALANCE AND HUMORAL GROWTH FACTORS a.
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Insulin pmol/1 Fig. 3. Correlation between nitrogen balance on day 6 (g/24 h) and plasma levels of insulin (pmolfl) on day 6: a) in Vamin 18 group (y = (0,05) x -2,21), and b) in DP-Gln 20 group (y = (0.11) x -5.34) r = 0.92, p < 0.01.
other studies. We have provided nitrogen at a level of 0.18 gN/kg/24h which is comparable to the other studies (0.20-0.23 gN/kg/24 h). In this study a dose of glutamine (0.16 g/kg/24 h) was given, compared with (0.19-0.57 g/kg/24 h) in the other studies. It has also been suggested that Ala-Gln is more rapidly cleared and results in higher plasma levels of glutamine than Gly-Gln (23). The combination of slightly less nitrogen intake and a low dose of glutamine administered in glycyl-glutamine dipeptide could explain the discrepancy in results. There is also a possibility that the patients in the study group were more affected by disease, as their plasma albumin level was lower preoperatively. In this study it is not possible to differentiate between the effects of glutamine, tyrosine or the amino acid composition. The glutamine supplementation is however the quantitatively most significant difference compared to Vamin 18. Glutamine could affect metabolism in several ways, and has been shown to improve protein synthesis in isolated enterocytes (24). The main aim has been to reduce protein degradation and glutamine export from skeletal muscle, as restoration of intracellular glutamine levels in muscle parallels a stimulated protein synthesis, albeit no causal relationship has been established. In earlier studies involving postoperative patients, the decrease in intracellular glutamine could not be abolished but was significantly reduced compared to control groups (4-6). Surprisingly, this is achieved in spite of slightly reduced levels of plasma glutamine postoperatively. Consequently, there seems to be no
simple correlation between plasma levels of glutamine, intracellular levels in skeletal muscle and protein synthesis. This does not exclude the possibility that supplying glutamine in a catabolic situation could stimulate protein synthesis in separate organs or in a whole body perspective. Enteroglucagon has been considered an enterotrophic factor in humans since the findings of Bloom of a patient with an enteroglucagon-producing turnout and secondary villous hyperplasia. PYY levels in plasma and tissue increases after resection in rats, but PYY administration to normal animals exogenously does not stimulate enterocyte turnover. In humans PYY-levels increase after colorectal surgery, more substantially in short bowel patients, probably reflecting functional adjustments in transit time and uptake capacity (17, 25). In the present study PYY levels were significantly increased only in the ghitamine supplemented group, a finding supported by observations we have done in a resection study on rats using oral glutamine administration (unpublished data). The results in this study are probably not only related to surgery as there was a slightly higher proportion of patients undergoing colorectal surgery in the Vamin 18 treated group. No significant changes in any of the other growth factors examined were seen between ghitamine-supplemented patients and controls. As nutrients could have diverse actions on the pattern of anabolism and catabolism in different organ systems, the beneficial effects of a specific nutrient on whole body protein metabolism will continue to be difficult to prove. The analysis of growth regulating
CLINICALNUTRITION 103 factors in p l a s m a and tissues increases the possibility to evaluate m e d i a t e d anabolic effects o f nutrients. In this study we f o u n d a strong correlation b e t w e e n increasing i n s u l i n levels and i m p r o v e d nitrogen retention in glutamine-treated patients compared to ordinary a m i n o acid composition. G l u t a m i n e does not seem to raise i n s u l i n levels more than V a m i n 18. A synergistic effect b e t w e e n this anabolic h o r m o n e and g l u t a m i n e on protein m e t a b o l i s m m a y exist. It has b e e n shown that i n s u l i n release is stimulated by a m i n o acids and that i n s u l i n stimulates protein synthesis as well as diminishes protein degradation both in skeletal m u s c l e and liver, but also that results from growing animals can not be automatically translated to adult h u m a n s (26, 27). The exact m e c h a n i s m s for i n s u l i n to influence protein m e t a b o l i s m are not k n o w n but could i n v o l v e the e n e r g y - d e m a n d i n g a m i n o acid uptake, the protein synthesis at t r a n s c r p f i o n a l or translational level or degradation. W e could not find any signs of i n s u l i n effects on m u s c l e degradation, evaluated by 3-methylhistidine excretion. The question as to how g l u t a m i n e could potentiate the anabolic effects o f insulin b y increasing a m i n o acid uptake to the m u s c l e cell, b y stimulating protein synthesis or attenuating degradation needs further studies. The possibility of i m p r o v i n g nitrogen accretion using exogenous i n s u l i n to postoperative patients recieving a m i n o acids at comparable a m o u n t s to our study has recently b e e n demonstrated (28). G l u t a m i n e s u p p l e m e n t a t i o n to T P N at a dose of 0 . 1 6 g glutamine/kg B W / 2 4 h . after major surgery did not significantly alter levels of short-lived p l a s m a proteins, nitrogen balance or 3-methylhistidine excretion c o m p a r e d to a c o n v e n t i o n a l a m i n o acid solution. In the search of optimal nutritional support, further investigations on the effects of specific nutrients and c o m b i n a t i o n s with growth factors as well as interactions with e n d o g e n o u s anabolic substances are awaited.
3.
4.
5.
6.
7. 8.
9. 10.
11.
12.
13. 14.
15.
Acknowledgements This study was presented in preliminary form at ESPEN 94, Birmingham, UK, 31 August- 2 September 1994. The study was supported by grants from the Medical Research Council (Proj. No. 6251 and 10402), the County Council of OstergOtland, and Pbarmacia AB.
16. 17. 18.
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104 NITROGENBALANCE AND HUMORALGROWTH FACTORS 20. Mc Anena O J, Moore F A, Moore E E, Jones T N, Parsons P. Selective uptake of glutamine in the gastrointestinal tract: confirmation in a human study. Br J Surg 1991; 78: 480-482. 21. Powell-Tuck J. Glutamine, parenteral feeding and intestinal nutrition. Lancet 1993; 342: 451-452. 22. Peterson B, yon der Decken A, Vinnars E, Wernermann J. Long-term effects of postoperative TPN supplemented with glycyl-glutamine on subjective fatigue and muscle protein synthesis. Br J Surg 1994; in press 23. Lochs H, Htibl W. Metabolic base for selecting glutaminecontaining substrates for parenteral nutrition. JPEN 1990; 14 (4) (Suppl): 114S-117S. 24. Higashiguchi T, Hasselgren P O, Wagner K, Fisher J E. Effect
25.
26. 27. 28.
of glutamine on protein synthesis in isolated intestinal epithelial cells. JPEN 1993; 17: 307-314. Adrian T E, Ballantyne G H, Longo W E et al. Deoxycholate is an important releaser of peptide YY and enteroglucagon from the human colon. Gut 1993; 34: 1219-1224. Long C, Lowry S. Hormonal regulation of protein metabolism. JPEN 1990; 14: 555-562. Millward D J. The hormonal control of protein turnover. Clin Nutr 1990; 9: 115-126. Valarini R, Sonsa M F, Kalil R, Abumrad N N, Riella M C. Anabolic effects of insulin and amino acids in promoting nitrogen accretion in postoperative patients. JPEN 1994; 18: 214-218.
Submission date: 19 October 1994; Accepted after revision: 26 January 1995
The effects of a new amino-acid dipeptide solution on nitrogen balance and humoral growth factors in the postoperative state in man M. WIRI~N, T. E.* ADRIAN, F.t HAMMARQVIST, K-E. JOHANSSON, J. PERMERT, B.t PETERSSON, j.t WERNERMAN and J. LARSSON Department of Surgery, University Hospital, Link6ping, #Departments of Surgery and Anaesthesia, St G6rans Hospital, Stockholm. *Department of Biomedical Sciences, Creighton University, Omaha, USA (Correspondence to: MW, Department of Surgery, Faculty of Health Sciences, Link6ping University, S - 581 85 Link6ping, Sweden A B S T R A C T - - 27 patients admitted for elective abdominal surgery were allocated to receive postoperative total parenteral nutrition supplemented with glutamine (glycyl-glutamine) and tyrosine (glycyl-tyrosine) containing dipeptides (DP-GIn 20; 0.16 g glutamine/kg BW/24 h) or isonitrogenous Vamin 18 for 5 days. The aim was to evaluate safety and effects on short-life plasma proteins, nitrogen balance, 3-methylhistidine excretion and alimentary growth factors in plasma. No differences in transthyretin or retinol binding protein levels, nitrogen balance or 3methylhistidine excretion were found in patients receiving DP-GIn 20 compared to Vamin 18. There were higher plasma levels of peptide YY in the dipeptide group 5 days after surgery (p < 0.05). A correlation between insulin levels and nitrogen balance was found only in DP-GIn 20 treated patients day 6 (r = 0.91, p < 0.01). DP-GIn 20 is a glutamine dipeptide (Gly-GIn) containing amino acid solution which is considered safe in the postoperative state in man. No beneficial effects on whole body protein metabolism were found by adding DP-GIn 20 to total parenteral nutrition.
Introduction
glutamine parenterally have demonstrated improvement in nitrogen balance postoperatively (4-6), restoration of intestinal permeability characteristics during TPN-treatment (7), a shortened length of stay at hospital in bone marrow transplant (BMT) patients (8, 9), and reduction of excess total body water during TPN treatment (10). Moreover, a positive effect on the mood of BMT-patients receiving glutarnine has been reported (11). On the other hand, the reduction in infectious complications in bone marrow transplant patients originally described, has not been reproduced (9). In home parenteral nutrition, liver toxicity has been described (12). In severe pancreatitis it has not been possible to demonstrate beneficial effects of glutamine (13). Glutamine added to TPN exerts a positive effect on glutamine and muscle protein metabolism after surgery (4-6). The otherwise characteristic decreases in muscle free-glutamine concentration and protein synthesis were attenuated in parallel with decreased whole body nitrogen losses. The uptake of amino
Specific nutrients and growth factors are attracting increasing interest in nutritional research and clinical practice (1). In general surgery, much attention has been focused on the requirements of the gastrointestinal tract after abdominal operations, during total parenteral nutrition (TPN) and bowel rest periods. Glutamine has been proposed to be a necessary nutritional substrate for the organism in catabolic situations. It is also considered a specific intestinal nutrient, capable of restoring intestinal mucosa exposed to trauma or atrophy (2). The possibility of administering parenteral glutamine has been achieved by adding glutamine-containing dipeptides. Tyrosine has also been considered important in patients exposed to trauma and stress, because it is a precursor for hormone and neurotransmittor synthesis (3). DPGln 20 is a new dipeptide amino acid solution containing glycyl-glutamine and glycyl-tyrosine. Previous studies using comparable amounts of 97
98
NITROGEN BALANCE AND HUMORAL GROWTH FACTORS
acids and subsequent protein synthesis has been shown to be dependent on various factors; nutrients, hormones, cytokines and growth factors being of crucial interest. Growth hormone given to humans has been shown to improve postoperative protein metabolism (14). Growth factors, hormones and gastrointestinal peptides have also been proposed to be of interest in posttraumatic regulation of gastrointestinal function, metabolism and growth. Nutritional support could influence hormonal release and paracrine actions of growth factors, thereby indirectly affecting the utility of nutrients for anabolism. PYY, enteroglucagon, EGF and IGF-I and II are some of the factors being involved in the regulation of growth of the gastrointestinal tract, as shown in animal experiments involving intestinal resection. As glutamine is a primary gut nutrient an improvement in intestinal integrity could be associated with changes in alimentary growth factors. The aim of this study was to examine the effects of glycyl-glutamine and glycyl-tyrosine (DP-Gln 20) supplemented TPN for 5 days after major abdominal surgery on the plasma levels of short-life proteins, alimentary hormones and growth factors, urinary 3methylhistidine excretion and whole body nitrogen balance.
and 13 patients in the Vamin 18-group completing the protocol. The patients were informed about the purpose and the risks of the study in writing and orally and were also informed that they may leave the study at any time, with no deleterious effects concerning treatment. Patients were randomized after giving oral informed consent. Demographic characteristics of patients and underlying diseases are shown in Table 1. The surgical procedures were equally distributed in the two groups (Table 2). The protocol of the study was approved by the Ethical Committee of the Karolinska Institute, Stockholm, Sweden. The two groups received an iso-nitrogenous (0.18 g N/kg/24 h) and iso-volumetric (28.4 ml/kg/24 h) TPN solution for 5 days following surgery. The differences in composition of DP-20 and Vamin 18 are presented in Table 3. The solutions were prepared blind by the pharmacy at the two hospitals in an all-in-one TPN bag according to the patients weight. The dosage of non-protein energy was approximately 109 KJ/kg Table 1 Demographicdata and diagnosesof the study population. Mean (SEM).
DP-Gln 20
Vamin 18
Demography Patients and methods
31 patients admitted for elective abdominal surgery in two surgical centres were randomly allocated to double-blind administration of either a glutaminecontaining dipeptide solution, DP-Gln 20 (Pharmacia, Sweden) or Vamin 18 (Pharmacia, Sweden), for 5 days after operation. The study was conducted at the Departments of Surgery and Anesthesia, St. G6rans Hospital in Stockholm and Department of Surgery, Linktping University Hospital. Male and female adult patients between 25 and 75 years undergoing elective, major, abdominal surgery were included in the study. Patients with inborn errors of amino acid metabolism, reduced liver or kidney function (creatinine > 200 gmol/l, Quick < 65%, ALAT > 2 gkat/1), circulatory instability, diabetes mellitus and female patients with child-bearing potential were excluded from the trial. Excluded were also all patients with a per-operative blood loss exceeding 1500 ml, or receiving transfusion of blood products exceeding > 1000 ml postoperatively, as proper evaluation of protein metabolism otherwize would be hazardous (15). 4 patients were excluded due to premature termination of TPN or withdrawal of consent, resulting in 14 patients in the DP-Gln 20-group
Female/Male Age, years Body weight, kg Body weight, cm
7/7 58.7 (3.7) 73.4 (2.9) 173 (2.4)
6/7 51.7 (3.0) 73.5 (4.0) 171 (2.4)
Diagnoses
Reflux oesophagitis Gastric cancer Colorectalcancer Other malignancies Benign upper GI disease Benign colorectaldisease Vascular disease
6 4 1 2 1 0 0
6 1 2 I 1 1 1
Table 2 Surgicalprocedures and operativetrauma. Mean (SEM). DP-Gln 20 n = 14
Vamin 18 n = 13
Surgical procedures
Fundoplicatio Gastrectomy Colorectalresection Gastric resection/GE Other abdominaloperation
6 2 1 1 4
6 1 3 1 2
Operative trauma
Operatingtime (min) Blood loss (ml) Transfusions (bloodor plasma)
155 (14) 432 (89) n=3
159 (9) 429 (89) n =3
CLINICALNUTRITION 99 Table 3 Compositionof the two amino acid solutions, amino acids (g/l).
Glutamine Glycine Isoleucine Leucine Lysine Methionine Phenylalanine Tyrosine Threonine Tryptophan Valine Arginine Histidine Alanine Aspartatic acid Cysteine Glutamicacid Proline Serine
DP-Gln 20
Vamin 18
16.06" 9.00* 4.49 6.34 7.23 4.49 4.69 1.83* 4.49 1.53 5.86 9.07 5.46 12.85 2.73 0 4.49 5.46 3.61
0 7.90 5.60 7.90 9.00 5.60 7.90 0.23 5.60 1.90 7.30 11.30 6.80 16.00 3.40 0.56 5.60 6.80 4.50
Analytical methods
*componentsof dipeptides: Glycyl-glutamine24.31 g (Gly = 8.23 g, Gln= 16.06 g) and Glycyl-tyrosine2.77 g (Gly = 0.75, Tyr = 1.83 g).
body weight. 45% of the non-protein energy was fat emulsion (Intralipid 20%, Pharmacia, Sweden) and 55% was given as glucose (30%). TPN was administered via a central venous line and started the first day after operation. On the day of surgery patients received glucose solutions and volume substitution. Low molecular heparin, Fragmin (Pharmacia, Sweden), was used as thromboprophylaxis, starting the evening before surgery. Routine antibiotic prophylaxis included tetracyclin and cefuroxim, with or without metronidazole, which was used in all colorectal and vascular procedures. The patients were clinically assessed
Preop.
Op.
and heart rate and blood pressure were recorded twice daily. Blood samples were taken preoperatively after an overnight fast and during the study period according to the flow chart presented in Figure 1. Blood samples for peptide analysis were collected into ice-cold tubes containing aprotinin (Trasylol, Bayer, 400 KIU/ml) and EDTA (5 mg/ml blood). The samples were immediately separated in a refrigerated centrifuge (1500 g for 15 rain), the plasma decanted and frozen at -70°C for subsequent analysis. Urine collection was performed every 2 4 h starting the first day after surgery and continued for 5 days. Completeness of urinary sampling was monitored by daily measurements of creatinine output. Growth factor analyses were performed only on patients included at the surgical department in Link6ping.
Day 1
Routine biochemistry including analysis of hematologic, electrolytes and liver function tests were performed at the departments of chemistry in the two hospitals according to standard procedures. Frozen (-30°C) plasma was used for analysis of transthyretin and retinol-binding-protein by nephelometry at the laboratories of Pfrimmer Kabi, Erlangen, Germany.
Urine analysis Nitrogen excretion was determined according to Kjeldahl and the nitrogen balance was calculated estimating nitrogen losses in stool or skin to 30 mg/kg BW/day. 3-methylhistidine was analyzed with a conventional amino acid analyzer (Beckman System 6300, Beckman Instruments Inc., Palo Alto, CA, USA). Nitrogen, urea and 3-methylhistidine excretion was corrected for varying muscle mass by taking the ratio with creatinine into account.
Day2
Day3 ?
Lab. assessments A 1" ? Lab. assessments B T ? ?$ T? Body temperature 1" $ $ ?$ t? Heart rate, blood pressure 1" $ $ Urine collection ,~ TPN Admixture infusion A B Electrolytes Bilirubin Retinol binding Hb Urea Triglycerides protein WBC Crcatinine Albumin Platelets Liver enzymes Prealbumin Quick
Day4
Day5
$$ $?
?? ?T
Day6 ? ? ? T
Fig. 1. Studydesign. Laboratoryassessments,safetycontrol and urinary collectionwas monitored5 days after surgery. TPN was givenduring the day-timeand laboratoryassessmentswere taken in the fasting state.
100 NITROGENBALANCEAND HUMORALGROWTHFACTORS
Measurement of growth factors by radioimmunoassay The samples were acidified in two volumes of acidified ethanol (0.1 M HC1 in 75% ethanol). After vortex mixing and centrifugation, at 1500 g for 10 min at 4°C, the supernatant was decanted. Supernatants were dried in a vacuum centrifuge and reconstituted in assay buffer prior to radioimmunoassay. Insulin, islet amyloid polypeptide (IAPP), glucagon, gastrin, peptide YY (PYY), enteroglucagon, epidermal growth factor (EGF), transforming growth factor a (TGF-~) and insulin-like growth factors I and II (IGF-I and IGF-II) were measured using specific and sensitive radioimmunoassays previously described in detail (16-18).
Statistics All values are given as means + SEM. Student's t-test for unpaired samples was used to compare differences between the two groups and for paired samples to compare observations within the groups, p < 0.05 was considered significant. Linear regression and ANOVA were used to analyse correlations between growth factor results and metabolic data.
Table 4 Transthyretine and retinol-binding protein in plasma, changes from day 1 to day 6. Cumulative nitrogen balance and mine excretion of nitrogen and 3-methyl-histidine in urine. Mean (SEM).
DP Gin-20 Transthyretine, A day 6- day 1 (rag/l) Retinol binding protein, A day 6day 1, (mg/1) Nitrogen excretion, cumulative (g/5 days) Nitrogerdcreatinine excretion cumulative (g/p.mol/5 days) 3-methylhistidine excretion, cumulative, (gmol/5 days) 3-metbylhistidine/creatinine excretion, cumulative (I.tmol/mmol/5 days) Nitrogen balance, cumulative (g/5 days)
Vamin 18
0.43 (0.09) 0.17 (0.02) 59.0 (4.1) 6.0 (0.3) 1224 (121) 121 (5)
0.26 (0.21) 0.09 (0.04) 64.7 (5,5) 5,5 (0,4) 1474 (150) 121 (6)
-3.7 (3.2)
-7,8 (4.2)
nitrogen balance were not statistically significant (Fig. 2). The cumulated 3-methylhistidine excretion corrected for creatinine was identical in the two groups (Fig. 2).
Growth factors Results
Biochemistry There were no adverse effects related to treatment from any of the two amino-acid solutions. The most frequent laboratory changes were increases in liver function tests and urea levels in plasma, but no significant differences in the two groups were found. Effects upon hematologic profile, electrolytes, glucose and kidney function tests of DP-Gln 20 were not different to results in patients treated with Vamin 18. The albumin level was lower initially in the DPGin 20 group (39:5 (1.5) vs 43.3 (0.5), p < 0.05) and during treatment there was a significant increase in plasma albumin concentration from day 3 to day 6 that was not seen in Vamin 18 treated patients [30.8 (0.7)-32.6 (1.0) g/1 DP-Gln 20, p <0.05 vs 34.0 (1.4)-34.5 (1.5) Vamin 18, NS].
There were higher insulin, IAPP, and glucagon levels in both groups. Blood glucose was within normal limits. There were increases in the concentration of circulating PYY in the DP-20 treated patients, and trends in this direction were also seen for enteroglucagon, EGF and TGF-c~. No effects upon gastrin, IGF-I or IGF-II were seen (Table 5).
Growth factors and nitrogen balance Insulin demonstrated the strongest positive correlation with nitrogen balance in the whole study population (r = 0.49, p < 0.07) but did not seem to influence prealbumin levels or 3-methylhistidine excretion. In the Vamin 18 group there was however no correlation between insulin levels and nitrogen excretion or balance (Fig. 3a). In the DP-Gln 20 treated patients on the contrary, there was a strong correlation (r = 0.91, p < 0.01) between insulin levels and nitrogen balance (Fig. 3b).
Protein metabolism There were no significant differences in transthyretin or RBP plasma levels, nitrogen balance or 3methylhisfidine excretion between the two groups from day 1 to day 6 (Table 4). Nitrogen balance was numerically better in the DP-Gln 20 treated patients in 4 days out of 5 but the differences in cumulated
Discussion The interest in specific nutrient support to optimize parenteral nutrition has produced a large body of results in animal experiments, suggesting glutamine to be beneficial posttraumatically in reducing protein
CLINICAL NUTRITION 101 at
Day 2
Day I 0
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Fig. 2. Cumulative urinary excretion of: a) 3-metylhistidine/creatinine (gmol/mmol/24 h), and b) cumulative nitrogen balance (g/24 h) during 5 days postoperatively. Mean (SEM). DP-Gln 20-group in striped bars and Vamin 18 in filled bars. No significant differences were seen.
Table 5 Hormones/growth factors preoperatively (preop) and day 6 postoperatively (postop) in the Vamin group and the DP-Gln 20 group. Mean (SEM). Statistical significance is indicated within the groups * = p < 0.05, * = p < 0.01. Dp-Gln 20 Hormones/ growth factors Insulin (pmol/1) IAPP (pmol/l) Glucagon (pmol/1) Enteroglucagon (pmol/1) Gastrin (pmol/1) PYY (pmol/1) IGF-I (nmolB) IGF-II (nmol/1) EGF (pmol/1) TGFct (pmol/1)
Vamin 18
Preop
Postop
Preop
Postop
42.1 (3.2) 8.7 (1.5) 5.9 (0.5) 42.0 (6.3) 22.9 (2.8) 11.5 (2.1) 14.6 (2.6) 4.2 (0.4) 9.4 (1.8) 7.9 (1.5)
68.0 * (5.9) 14.4 (3.1) 7.6* (0.4) 66.5 (11.9) 22.4 (2.9) 18.5" (3.1) 15.2 (2.7) 4.2 (0.5) 13.0 (1.3) 10.7 (1.5)
44.9 (4.7) 8.4 (1.2) 6.3 (0.3) 40.4 (8.6) 23.6 (2.4) 12.4 (2.1) 10.6 (2.2) 4.2 (0.6) 9.6 (2.1) 7.6 (1.5)
65.7* (3.7) 12.2 (2.4) 7.7* (0.3) 44.3 (12.9) 21.0 (2.4) 16.5 (2.4) 10.0 (1.8) 3.3 (0.2) 9.4 (2.0) 6.9 (1.5)
IAPP = islet amyloid polypeptide, PYY = peptide YY, IGF = insulin-like growth factor, EGF = epidermal growth factor, TGF-o~ = transforming growth factor alpha.
breakdown and improving intestinal growth as well as immunological function (19). This hypothesis has also been tested in human experiments, partly supporting glutamine as a beneficial supplement to TPN
(4-11, 20-22). In the present study, the effects of postoperative glutamine supplementation in the form of a new amino-acid dipeptide solution, DP-Gln 20, after major abdominal surgery on patients without major blood loss were evaluated. We could find no differences between DP-Gln 20 and Vamin in plasma levels of transthyretin and RBP, nor on nitrogen balance or 3-methylhistidine excretion. An interesting correlation between endogenous insulin levels and nitrogen balance was found only in DP-Gln 20-treated patients. In some aspects these results contradict previously published papers. Improvement in nitrogen balance has been reported in most of the studies. When using elective cholecystectomy, effects on nitrogen balance have been reported both when glutamine or glutamine as a dipeptide, alanyl-glutamine, was given as a part of TPN (4, 6). However, this was not reproduced in a study when glycyl-glutamine was given together with TPN (22). On the other hand, positive effects were shown regarding muscle protein synthesis, in terms of a diminished decrease in polyribosomes, and regarding the level of muscle free glutamine, that showed a profound decrease in the control groups not receiving glutarnine (5, 6, 22). Stehle could show a highly significant improvement already by day 1 and each subsequent day up to day 5 after abdominal surgery with administration of glutamine in dipeptide solution (4). In the work by Ziegler on BMT-patients an improvement in nitrogen balance was seen but the study arms were not strictly isonitrogenous (8). One reason for the lack of positive results could be the fact that the glutamine dose is lower than in
102 NITROGEN BALANCE AND HUMORAL GROWTH FACTORS a.
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Insulin pmol/1 Fig. 3. Correlation between nitrogen balance on day 6 (g/24 h) and plasma levels of insulin (pmolfl) on day 6: a) in Vamin 18 group (y = (0,05) x -2,21), and b) in DP-Gln 20 group (y = (0.11) x -5.34) r = 0.92, p < 0.01.
other studies. We have provided nitrogen at a level of 0.18 gN/kg/24h which is comparable to the other studies (0.20-0.23 gN/kg/24 h). In this study a dose of glutamine (0.16 g/kg/24 h) was given, compared with (0.19-0.57 g/kg/24 h) in the other studies. It has also been suggested that Ala-Gln is more rapidly cleared and results in higher plasma levels of glutamine than Gly-Gln (23). The combination of slightly less nitrogen intake and a low dose of glutamine administered in glycyl-glutamine dipeptide could explain the discrepancy in results. There is also a possibility that the patients in the study group were more affected by disease, as their plasma albumin level was lower preoperatively. In this study it is not possible to differentiate between the effects of glutamine, tyrosine or the amino acid composition. The glutamine supplementation is however the quantitatively most significant difference compared to Vamin 18. Glutamine could affect metabolism in several ways, and has been shown to improve protein synthesis in isolated enterocytes (24). The main aim has been to reduce protein degradation and glutamine export from skeletal muscle, as restoration of intracellular glutamine levels in muscle parallels a stimulated protein synthesis, albeit no causal relationship has been established. In earlier studies involving postoperative patients, the decrease in intracellular glutamine could not be abolished but was significantly reduced compared to control groups (4-6). Surprisingly, this is achieved in spite of slightly reduced levels of plasma glutamine postoperatively. Consequently, there seems to be no
simple correlation between plasma levels of glutamine, intracellular levels in skeletal muscle and protein synthesis. This does not exclude the possibility that supplying glutamine in a catabolic situation could stimulate protein synthesis in separate organs or in a whole body perspective. Enteroglucagon has been considered an enterotrophic factor in humans since the findings of Bloom of a patient with an enteroglucagon-producing turnout and secondary villous hyperplasia. PYY levels in plasma and tissue increases after resection in rats, but PYY administration to normal animals exogenously does not stimulate enterocyte turnover. In humans PYY-levels increase after colorectal surgery, more substantially in short bowel patients, probably reflecting functional adjustments in transit time and uptake capacity (17, 25). In the present study PYY levels were significantly increased only in the ghitamine supplemented group, a finding supported by observations we have done in a resection study on rats using oral glutamine administration (unpublished data). The results in this study are probably not only related to surgery as there was a slightly higher proportion of patients undergoing colorectal surgery in the Vamin 18 treated group. No significant changes in any of the other growth factors examined were seen between ghitamine-supplemented patients and controls. As nutrients could have diverse actions on the pattern of anabolism and catabolism in different organ systems, the beneficial effects of a specific nutrient on whole body protein metabolism will continue to be difficult to prove. The analysis of growth regulating
CLINICALNUTRITION 103 factors in p l a s m a and tissues increases the possibility to evaluate m e d i a t e d anabolic effects o f nutrients. In this study we f o u n d a strong correlation b e t w e e n increasing i n s u l i n levels and i m p r o v e d nitrogen retention in glutamine-treated patients compared to ordinary a m i n o acid composition. G l u t a m i n e does not seem to raise i n s u l i n levels more than V a m i n 18. A synergistic effect b e t w e e n this anabolic h o r m o n e and g l u t a m i n e on protein m e t a b o l i s m m a y exist. It has b e e n shown that i n s u l i n release is stimulated by a m i n o acids and that i n s u l i n stimulates protein synthesis as well as diminishes protein degradation both in skeletal m u s c l e and liver, but also that results from growing animals can not be automatically translated to adult h u m a n s (26, 27). The exact m e c h a n i s m s for i n s u l i n to influence protein m e t a b o l i s m are not k n o w n but could i n v o l v e the e n e r g y - d e m a n d i n g a m i n o acid uptake, the protein synthesis at t r a n s c r p f i o n a l or translational level or degradation. W e could not find any signs of i n s u l i n effects on m u s c l e degradation, evaluated by 3-methylhistidine excretion. The question as to how g l u t a m i n e could potentiate the anabolic effects o f insulin b y increasing a m i n o acid uptake to the m u s c l e cell, b y stimulating protein synthesis or attenuating degradation needs further studies. The possibility of i m p r o v i n g nitrogen accretion using exogenous i n s u l i n to postoperative patients recieving a m i n o acids at comparable a m o u n t s to our study has recently b e e n demonstrated (28). G l u t a m i n e s u p p l e m e n t a t i o n to T P N at a dose of 0 . 1 6 g glutamine/kg B W / 2 4 h . after major surgery did not significantly alter levels of short-lived p l a s m a proteins, nitrogen balance or 3-methylhistidine excretion c o m p a r e d to a c o n v e n t i o n a l a m i n o acid solution. In the search of optimal nutritional support, further investigations on the effects of specific nutrients and c o m b i n a t i o n s with growth factors as well as interactions with e n d o g e n o u s anabolic substances are awaited.
3.
4.
5.
6.
7. 8.
9. 10.
11.
12.
13. 14.
15.
Acknowledgements This study was presented in preliminary form at ESPEN 94, Birmingham, UK, 31 August- 2 September 1994. The study was supported by grants from the Medical Research Council (Proj. No. 6251 and 10402), the County Council of OstergOtland, and Pbarmacia AB.
16. 17. 18.
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Submission date: 19 October 1994; Accepted after revision: 26 January 1995