Br. J. Surg. 1992, Vol. 79, March, 217-220
S. Sandstedt, L. Jorfeldt* and J. Larsson-f Departments of Anaesthesiology, *Clinical Physiology and ?Surgery, University Hospital, S-581 85 Linkoping, Sweden Correspondence to: Associate Professor J. Larsson
Randomized, controlled study evaluating effects of branched chain amino acids and alpha-ketoisocaproate on protein metabolism after surgery The efSect of nutritional supplementation with branched chain amino acids or the ketoacid a-ketoisocaproate on protein metabolism after surgery was studied in 24 patients with gastrointestinal cancer. They were randomized to receive one of three nutritional regimens. Allpatients received a balanced amino acid solution and group 1 ( n = 8 ) received no further supplements, group 2 ( n = 8 ) received supplementation with a-ketoisocaproate (17 g day-') andgroup 3 (n = 8 ) receiveda branched chain solution including leucine, isoleucine and valine, corresponding to 3.3 g nitrogen day- I . Plasma albumin, prealbumin, fibronectin and serum urea concentrations, nitrogen balance and 3-methylhistidine release from the leg and its excretion in the urine were measured. Albumin and prealbumin concentrations fell after surgery in all groups, andJibronectin levels fell in group 2 ( P < 0.001). In group 2 there was also a signiJicant increase in serum urea concentration after operation ( P < 0.05). Cumulative nitrogen balance after 3 days was -5.6 g (group I ) , -3.8 g .(group 2 ) and - 1.7 g (group 3). The release of 3-methylhistidine (nmol100 g - min- ') from the leg after operation, following an overnight fast, was -0.42 (group I ) , -0-51 (group 2 ) and -0.66 (group 3). During infusion the release was -0.56, -0.99 and -0.81, respectively. A balanced amino acid solution with an adequate energy supply has an optimal nitrogen-sparing efect. Branched chain amino acids or a-ketoisocaproate did not improve nitrogen balance or reduce protein degradation.
Nutritional regimens Surgical trauma is followed by a catabolic state with an increase in resting energy expenditure and a net loss of body protein'. The TPN regimen was designed to provide 146 kJ (35 kcal) kg-' 24 h - of non-protein calories as isocaloric parts of glucose and fat Most of the excreted nitrogen is derived from skeletal muscle (Intralipid 20%; Kabi Pharmacia, Stockholm, Sweden). The different protein by mobilization of amino acids for energy and visceral nutritional regimens were isonitrogenous and designed to provide 0.2 g synthesis of proteins'. A negative nitrogen balance occurs nitrogen kg- 24 h- I. The group receiving conventional TPN (group immediately after surgery, with losses of up to 2 0 g nitrogen 1, n = 8 ) was given Vamin 14 (Kabi Pharmacia) containing day- despite adequate nutritional s u p p o d . To reduce net 18.4 per cent BCAAs. Group 2 ( n = 8 ) was given 60 mmol (17 g ) protein degradation, branched chain amino acids (BCAAs ) a-ketoisocaproate in a balanced amino acid solution. Group 3 ( n = 8 ) have been given. These stimulate protein synthesis and reduce received an enriched solution containing 37 per cent BCAAs (7 g protein degradation in vitro in the liver and skeletal m ~ s c l e ~ . ~ . isoleucine, 14g leucine and 9 g valine lOOOml-') with a balanced Unlike leucine its a-keto analogue, a-ketoisocaproate, reduces amino acid solution. Electrolytes, trace elements and vitamins were added. No other nutrients were allowed. TPN was given for 16 h a the postoperative negative nitrogen balance when given in day up to 78 h after operation. addition to a hypocaloric nutritional regimen6. Clinical studies have shown favourable'** and unfavourable9 results of Protocol BCAA-enriched nutritional regimens in the control of protein Patients were asked not to eat meat for the 2 days before admission. metabolism after operation. Blood samples were taken on the morning of operation, 72 h after This randomized, controlled study evaluated the effects of operation in the morning (6 h after that day's infusion had stopped), infusion of a-ketoisocaproate and BCAAs on protein and 78 h after operation while undergoing TPN. Urine was collected metabolism in the first 3 days after major surgery. The results as 24-h specimens for 3 days beginning on the first day after operation. were compared with those from a control group who received conventional total parenteral nutrition (TPN).
',
Table 1 Patient details of the three groups
Patients and methods Twenty-four patients undergoing major abdominal surgery were studied (Table I ). Those with diabetes, cardiac failure or renal, hepatic or thyroid disease were excluded. The patients were randomized to one of three postoperative nutritional regimens. The study was approved by the local ethics committee.
0007-1323/92/030217-04
Q 1992 Butterworth-Heinemann Ltd
Mean(s.e.m. ) age (years) Sex ratio ( M: F ) Mean(s.e.m.) weight (kg) Disease Benign Malignant
Group 1 (n=8)
Group 2 (n=8)
Group 3 (n=8)
61.4(4.6) 6:2 72.3(3.9)
57.7(6.9) 7: 1 743(3.9)
55.4(8.3) 5:3 693(4.6)
4 4
3 5
3
5
217
Nutritional supplementation in surgical patients:
S.Sandstedt et al.
Biochemical variables
Serum concentrations of glucose, urea, creatinine, bilirubin and albumin were measured with an automatic flow analyser (SMAC; Technicon, Stockholm, Sweden). Prealbumin concentration was measured by electroimmunoassay" and fibronectin by immunochemical precipitation and laser nephelometry l . Samples of arterial blood for estimation of 3-hydroxybutyrate concentration were taken in duplicate and immediately precipitated using ice-cold perchloric acid. After centrifugation the protein-free extract was frozen at - 80°C. Analyses were performed by microfluorometry as previously described' '.
'
Arieriovenous difference and net eflux of 3-meihylhisiidine
Arterial and venous blood samples were drawn simultaneously. The leg blood flow was measured by automatic mercury strain gauge plethysmography (Medimatic; Copenhagen, Denmark). Five measurements of leg blood flow were made at the maximal circumference of the calf immediately before and after blood sampling. The mean blood flow of the ten measurements was calculated and multiplied by the arteriovenous difference. The uptake and the release of 3-
methylhistidine was given in nmoles per 100 gram of tissue per minute, assuming the mean density of leg tissue to be 1.0 g m l - '. The method for analysing the concentration of 3-methylhistidine in plasma has previously been de~cribed'~. Urine The 24-h specimens of urine were analysed for creatinine, 3-methylhistidine and total nitrogen concentrations. The total nitrogen concentration was measured by the microKjeldahl methodI4; the non-urinary nitrogen losses were estimated to be 1.5 g day- l . The daily and cumulative whole body nitrogen balance was calculated and corrected for the change in blood urea. The concentration of 3-methylhistidine in urine was measured using an automated amino acid analyser (Kontron, Basel, Switzerland)15. Statistical analysis The significance of differences between the means of the groups was assessed by variance analyses and Student's unpaired t test, and within the groups by Student's paired t test. P < 0.05 was accepted as significant.
Table 2 Serum glucose, urea, creatinine, bilirubin and plasma protein concentrations before and 72 h afier major operaiion Group 2
Serum glucose (mmol I - ' ) Serum urea (mmol 1- ' ) Serum creatinine (pmol 1- ) Serum bilirubin (pmol I - ' ) Plasma albumin (g 1 ) Plasma prealbumin (g 1- ' ) Plasma fibronectin ( % )
Group 3
Before
After 72 h
Before
After 72 h
Before
After 72 h
6,5(0:4) 4.4(0.6 ) 87.9(6.5) 10.4(1.4) 35.0( 1.4) 0.22(0.02) 1l6(6.5)
8.8( 1.2) 4.9( 0.5 ) 75.0(6.3 ) 10.5(1.2) 27.0( I.5)t 0.13(0.02)* 96( 18)
6.6(0.6) 4.7(0.7) 90.0(4.2) 11.4( 1.4) 37.1 (0.7) 0.20(0.01) 124(9.3 )
9.5( 1.5) 8.9( 1.5)* 93.0( 8.9) 15.6(4.8) 27.7( l . 7 ) t 010(0~01)$ 65(4.4)f
5.7(05) 4.3 (0.2 ) 1W8(5-2) 10.6(0.9 ) 35.5( 1.6) 0.20(0.03) 92(6.1 )
9.8( 1.7) 5.4(0.7) 86-017.3) 1330.9) 29.3(2.0)* 0.11(0.01)*
75( 6.8 )
Values are mean(s.e.m.). * P < 0.05; t P < 0.01; $ P < 0401 (before surgery versus after 72 h, Student's paired t test)
Table 3 Arteriovenous diference and net eflux of bloodgow from ihe leg for 3-methylhisiidine and concentrations of 3-hydroxybuiyrate in arterial blood before and 72 and 78 h afier operalion in patienis receiving ioial parenteral nuiriiion Group 2
Group 1
Group 3 -
After 72 h
Before
2.39(029)
Blood flow (ml 100 g tissue-' min-') 3-Methylhistidine Arteriovenous difference (pmol I - ' ) Net efflux (nmol 1 0 0 g tissue-' m i n - l )
After 78 h
1.91(0.15)
Before
1.95(0.14)
After 72 h
1.99(0.22)
2,14(0.24)
After 78 h
2.72(0.25)
Before
3.07(0.69)
After 72h
2.59(0.37)
After 78 h
3.18(0.42)
-0.23(003)
-024(006)
-0.29(0.06)
-0.22(0.03)
-021(0.05)
-0.38(0.07)
-0.20(0Q4)
-0.23(0.05)
-023(0.05)
- 0.54(0.10)
-0.42(0.09)
-0.56(0,11)
- 0.42(0.07)
- 0.5 l(0.17)
- 0.99(0.19)*
-O.W(O.08)
-0.66(0.2I )
- 0 8 l(0.23)
0.05(0.01)
0,51(012)
011(0.06)
1.22(0.47)
0.04(0.0 I )
0,08(0.03)
3-H ydroxybutyrate (mmol I - ' )
0.55(012)
0-02(0QO)
0.20(0.09)
Values are mean(s.e.m.). * P < 0.05 (after 78 h uersus before and 72 h after operation, Student's paired r test)
Table 4 Daily and cumulaiive nitrogen balance and cumulaiive urinary 3-meihylhistidine excretion on days I , 2 and 3 afier major operaiion Group 1
Nitrogen balance (g day-') Cumulative nitrogen balance (g on days 1-3) Urinary 3-methylhistidine excretion (pmol mmol creatinine- day- ') Cumulative urinary 3methylhistidine excretion (pmol mmol creatinine- ')
Group 2
Group 3
Day 1
Day 2
Day 3
Day 1
Day 2
Day 3
Day I
Day 2
Day 3
-2.8(1.5)
-2.0(1.2)
-0.8(0.7)
- l.O(l.5)
-2.7(1.4)
-0.1(1.2)
-O.S(O.S)
-0.6(0.8)
-0.6(0.8)
-2.8(1.5)
-4.8(2.4)
-5.6(2.4)
- l.O(l.5)
-3.7(1.3)
-3.8(2.2)
-0.5(0.8)
-1'1(1'3)
- 1.7(1.8)
25.2(1.4)
25.1(1.7)
25.6(1.8)
27.8(2.3)
25.9(2.9)
34.3(11.1)
25.2(1.4)
503(3.8)
75.9(7.1)
27.8(2.3)
53.7(5.0)
88.0(14.8)
22.1(2.7)
22.1(2.7)
23q2.2)
21q1.3)
46.0(4.2)
67.9(7.2)
Values are mean(s.e.m.)
21 8
Br. J. Surg., Vol. 79, No. 3, March 1992
Nutritional supplementation in surgical patients: S. Sandstedt et al.
Results Biochemical variables (Table 2 ) The serum glucose concentration did not increase significantly and there were no differences among the groups 72 h after operation. The serum urea concentration increased 72 h after surgery in group 2 by 89 per cent ( P < 0.05), but not in the other two groups. The plasma albumin concentration fell significantly in all groups, but the values did not differ significantly at 72 h. The plasma fibronectin concentration had fallen in all groups at 72 h, but significantly only in group 2 ( P < 0,001), the mean value being below the lower limit of the reference range for healthy subjects. Arteriovenous diferences and net efJlux of 3-methylhistidine (Table 3 ) All patients receiving T P N had a net efflux of 3-methylhistidine from the leg before and 7 2 and 78 h after operation. In group 2, the net efflux on the third day 78 h after operation was significantly increased compared with values obtained before and 72 h after operation. In groups 1 and 3 there were no significant changes in net efflux from the leg after operation and 78 h later. Nitrogen balance (Table 4 ) The daily and cumulative nitrogen balances were negative in all three groups, but only slightly so, and no differences were found between them. Urinary excretion of 3-methylhistidine (Table 4 ) To correct excretion of 3-methylhistidine for variations in muscle mass, it was related to urinary creatinine excretion. The mean daily and cumulative values for 3-methylhistidine were similar in all three groups and did not differ significantly.
Discussion The metabolic response to trauma entails a net protein loss of up to 20 g nitrogen (equivalent to 500-600 g wet skeletal m ~ s c l e ) ' , ~The . nitrogen losses are the result of increased protein degradation and, although protein synthesis is stimulated, the net effect is negative16. Nutritional support reduces negative nitrogen balance16, and adequate calories to keep the patient in energy balance will keep nitrogen loss to a minimum". Giving nitrogen in the form of a balanced amino acid solution reduces, but does not abolish, negative nitrogen balance. Nitrogen was retained ( u p to 0.2 g kg- 24 h - ) during the first week after trauma". Other nitrogen-saving techniques, such as using epidural analgesia, have been proposed but are difficult to apply in those with extensive trauma". BCAAs, particularly leucine, reduce protein breakdown and stimulate protein synthesis at physiological concentrations in vitro in skeletal muscle and liver20.21. Their effects in vivo are controversial. Decreased protein degradation is not an effect of leucine alone but requires its transamination to ketoleucine. Ketoleucine has been shown to spare nitrogen in obese fasting subjectsz2. In a study of surgical patients, ketoleucine caused significant ketosis, and ketosis may inhibit protein degradation6. In this study ketoleucine was given to assess its effect on protein degradation in muscle in three comparable groups of patients. Albumin and prealbumin concentrations decreased after operation and there was a decline in fibronectin in group 2. Leucine and ketoleucine may stimulate hepatic protein synthesis6.'' and hepatic synthesis of acute phase proteins in critically ill and septic rats was stimulated by an amino acid solution enriched with 45 per cent BCAAs2'. In this study the levels of plasma proteins with short half-lives, prealbumin (48 h ) and fibronectin (8 h ) both declined, more in group 2 than the other groups.
'
Br. J. Surg.. Vol. 79, No. 3. March 1992
'
The whole body nitrogen balance was not affected by either BCAAs or ketoleucine. In a study of patients after operation, Sapir et showed that ketoleucine plus glucose improved nitrogen balance and suppressed proteolysis, which was indicated by reduced urinary 3-methylhistidine excretion. Leucine did not seem to have the same effect. The increase in urea concentrations in the keto group at 7 2 h in the present study probably represented impaired breakdown of the ketoleucine solution. 3-Methylhistidine is found exclusively in actin and myosin and is released during proteolysis, after which it is neither reused for protein synthesis nor catabolized further. In the present study, excretion of 3-methylhistidine in urine was increased in all three groups", which indicates that myofibrillar proteolysis is not influenced by either BCAAs or balanced amino acids enriched with ketoleucine. It has been shown that 3methylhistidine excretion is not influenced by the supply of balanced amino acids16, but is related to the degree of injury and is mostly of skeletal muscle origin23. The significantly improved nitrogen balance in patients after operation that was reported by Sapir et aL6 was attributed to a reduced excretion of 3-methylhistidine. The arteriovenous difference in 3methylhistidine excretion and the net release from one leg were increased during T P N in group 2, indicating that ketoleucine had, on the contrary, a proteolytic effect. In the group receiving only a balanced amino acid solution (group 1 ) there was no such effect. Provided that adequate balanced nutritional support is given, supplementary ketoleucine and BCAAs d o not seem to have any beneficial effects on protein metabolism.
Acknowledgements This study was supported by grants from the Swedish Medical Research Council (no. 04139), Henning and Jone Throne-Holsts Foundation and Kabi Pharmacia. We gratefully acknowledge the technical assistance of Mrs Ninna Elfstrom.
References 1.
2.
3.
4. 5. 6. 7.
8. 9. 10. 11.
12. 13.
Kinney JM. The metabolic response to injury. In: Richards JR, Kinney JM, eds. Nutritional Aspecrs of Care in the Critirallj~Ill. Edinburgh: Churchill Livingstone, 1977: 95- 133. Cuthbertson DP. The distribution of nitrogen and sulphur in the urine during conditions of increased catabolism. Biorhrrn J 1931; 25: 236-44. Vinnars E, Holmstrom B, Schildt B, Odebdck A-C, Fiirst P. Metabolic effects of four intravenous regimens undergoing elective surgery. 11. Muscle amino acids and energy-rich phosphates. Clin Nutr 1983; 2: 3-11. Buse MG, Reid SS. Leucine: a possible regulator of protein turnover in muscle. J Clin Inuesi 1975; 56: 1250-61. Fulks RM, Li JB, Goldberg AL. Effects of insulin, glucose and amino acids on protein turnover in rat diaphragm. J Biol Cheni 1975; 250: 290-8. Sapir DG, Walser M, Moyer ED et al. Effects of alphaketoisocdproate and of leucine on nitrogen metabolism in postoperative patients. Lancet 1983; 7: 1010-14. Kern KA, Bower RH, Atamian S, Matarese LE, Shory M J , Fisher JE. The effect of a new branched chain enriched amino acid solution on postoperative catabolism. Surgery 1982; 92: 780-5. Cerra FB, Upson D, Angelic0 R ei ul. Branched chains support postoperative protein synthesis. Surgery 1982; 92: 192-9. Freund H, Yoshimura N, Lunetta L, Fischer JE. The role of the branched chain amino acids in decreasing muscle catabolism in uiuo. Surgery 1978; 83: 611-18. Laurel1 C-B. Electroimmunoassay.Scand J Clin Lab Intresi 1972; 29(Suppl 124): 21-37. Brodin B, Cederblad G , Larsson J et a/. Rapid determination of cold insoluble globulin by laser nephelometry. Application in patients receiving preoperative total parenteral nutrition. JPEN J Parenrer Enieral Nutr 1982; 6 : 214-17. Jorfeldt L, Juhlin-Dannfeldt A. The influence of ethanol on human splanchnic and skeletal muscle metabolism in man. Metabolism 1978; 27: 97-106. Andersson E, Hbkansson E, Larsson J , Mbrtensson J. A simple,
219
Nutritional supplementation in surgical patients: S.Sandstedt at al.
14. 15. 16. 17. 18. 19.
220
rapid and sensitive method for determination of arterio-venous differences and leg efflux of 3-methylhistidine using ion-pair HPLC and postcolumn derivatization. J Cbromatogr ,1987; 414: 174-9. Archibald RM. In: Seligson D, ed. Standard Methods of Clinicul Chemistry. New York: Academic Press, 1958: 2-3. Neuhauser M, Furst P. An automatic method for determination of urinary 3-methylhistidine. Normal values. Ann Biochem 1979; 92: 294. Shenkin A, Neuhauser M, Bergstrom J et al. Biochemical changes associated with severe trauma. Am JClin Nutr 1980;33: 2219-27. McDougal WS, Wilmore DW, Pruitt BA. Effects of intravenous near isosmotic nutrient infusions on nitrogen balance in critically ill injured patients. Surg Gynecol Obstet 1977; 145: 408. Larsson J, Lennmarken C, Mirtensson J, Sandstedt S, Vinnars E. Nitroeen requirements in severely injured patients. Br J Surg 1990; 17: 413-16. Kehlet H. The stress response to anaesthesia and surgery: release
20. 21.
22.
23.
mechanisms and modifying factors. Clin Anesthesiol 1984; 2: 315-39. Buse MG, Reid SS. A possible regulator of protein turnover in muscle. J Clin Invest 1975; 56: 1250-61. Mori E, Hasebe M, Kobayashi K, Suzuki H. Immediate stimulation of protein metabolism in burned rats by total parenteral nutrition enriched in branched-chain amino acids. JPEN J Parenter Enteral Nutr 1989; 13: 484-9. Mitch WE, Walser M, Sapir DG. Nitrogen sparing induced by leucine compared with that induced by its keto analogue, alpha-ketoisocaproate, in fasting obese man. J Clin Invest 1981; 67: 553-62. Sjolin J, Stjernstrom H, Henneberg S, Anderson E, Mirtensson J, Larsson J. Splanchnic and peripheral release of 3methylhistidine in relation to the urinary excretion in human infection. Metabolism 1988; 38: 23-29.
Paper accepted 11 November 1991
Br. J. Surg.. Vol. 79, No. 3, March 1992
S. Sandstedt, L. Jorfeldt* and J. Larsson-f Departments of Anaesthesiology, *Clinical Physiology and ?Surgery, University Hospital, S-581 85 Linkoping, Sweden Correspondence to: Associate Professor J. Larsson
Randomized, controlled study evaluating effects of branched chain amino acids and alpha-ketoisocaproate on protein metabolism after surgery The efSect of nutritional supplementation with branched chain amino acids or the ketoacid a-ketoisocaproate on protein metabolism after surgery was studied in 24 patients with gastrointestinal cancer. They were randomized to receive one of three nutritional regimens. Allpatients received a balanced amino acid solution and group 1 ( n = 8 ) received no further supplements, group 2 ( n = 8 ) received supplementation with a-ketoisocaproate (17 g day-') andgroup 3 (n = 8 ) receiveda branched chain solution including leucine, isoleucine and valine, corresponding to 3.3 g nitrogen day- I . Plasma albumin, prealbumin, fibronectin and serum urea concentrations, nitrogen balance and 3-methylhistidine release from the leg and its excretion in the urine were measured. Albumin and prealbumin concentrations fell after surgery in all groups, andJibronectin levels fell in group 2 ( P < 0.001). In group 2 there was also a signiJicant increase in serum urea concentration after operation ( P < 0.05). Cumulative nitrogen balance after 3 days was -5.6 g (group I ) , -3.8 g .(group 2 ) and - 1.7 g (group 3). The release of 3-methylhistidine (nmol100 g - min- ') from the leg after operation, following an overnight fast, was -0.42 (group I ) , -0-51 (group 2 ) and -0.66 (group 3). During infusion the release was -0.56, -0.99 and -0.81, respectively. A balanced amino acid solution with an adequate energy supply has an optimal nitrogen-sparing efect. Branched chain amino acids or a-ketoisocaproate did not improve nitrogen balance or reduce protein degradation.
Nutritional regimens Surgical trauma is followed by a catabolic state with an increase in resting energy expenditure and a net loss of body protein'. The TPN regimen was designed to provide 146 kJ (35 kcal) kg-' 24 h - of non-protein calories as isocaloric parts of glucose and fat Most of the excreted nitrogen is derived from skeletal muscle (Intralipid 20%; Kabi Pharmacia, Stockholm, Sweden). The different protein by mobilization of amino acids for energy and visceral nutritional regimens were isonitrogenous and designed to provide 0.2 g synthesis of proteins'. A negative nitrogen balance occurs nitrogen kg- 24 h- I. The group receiving conventional TPN (group immediately after surgery, with losses of up to 2 0 g nitrogen 1, n = 8 ) was given Vamin 14 (Kabi Pharmacia) containing day- despite adequate nutritional s u p p o d . To reduce net 18.4 per cent BCAAs. Group 2 ( n = 8 ) was given 60 mmol (17 g ) protein degradation, branched chain amino acids (BCAAs ) a-ketoisocaproate in a balanced amino acid solution. Group 3 ( n = 8 ) have been given. These stimulate protein synthesis and reduce received an enriched solution containing 37 per cent BCAAs (7 g protein degradation in vitro in the liver and skeletal m ~ s c l e ~ . ~ . isoleucine, 14g leucine and 9 g valine lOOOml-') with a balanced Unlike leucine its a-keto analogue, a-ketoisocaproate, reduces amino acid solution. Electrolytes, trace elements and vitamins were added. No other nutrients were allowed. TPN was given for 16 h a the postoperative negative nitrogen balance when given in day up to 78 h after operation. addition to a hypocaloric nutritional regimen6. Clinical studies have shown favourable'** and unfavourable9 results of Protocol BCAA-enriched nutritional regimens in the control of protein Patients were asked not to eat meat for the 2 days before admission. metabolism after operation. Blood samples were taken on the morning of operation, 72 h after This randomized, controlled study evaluated the effects of operation in the morning (6 h after that day's infusion had stopped), infusion of a-ketoisocaproate and BCAAs on protein and 78 h after operation while undergoing TPN. Urine was collected metabolism in the first 3 days after major surgery. The results as 24-h specimens for 3 days beginning on the first day after operation. were compared with those from a control group who received conventional total parenteral nutrition (TPN).
',
Table 1 Patient details of the three groups
Patients and methods Twenty-four patients undergoing major abdominal surgery were studied (Table I ). Those with diabetes, cardiac failure or renal, hepatic or thyroid disease were excluded. The patients were randomized to one of three postoperative nutritional regimens. The study was approved by the local ethics committee.
0007-1323/92/030217-04
Q 1992 Butterworth-Heinemann Ltd
Mean(s.e.m. ) age (years) Sex ratio ( M: F ) Mean(s.e.m.) weight (kg) Disease Benign Malignant
Group 1 (n=8)
Group 2 (n=8)
Group 3 (n=8)
61.4(4.6) 6:2 72.3(3.9)
57.7(6.9) 7: 1 743(3.9)
55.4(8.3) 5:3 693(4.6)
4 4
3 5
3
5
217
Nutritional supplementation in surgical patients:
S.Sandstedt et al.
Biochemical variables
Serum concentrations of glucose, urea, creatinine, bilirubin and albumin were measured with an automatic flow analyser (SMAC; Technicon, Stockholm, Sweden). Prealbumin concentration was measured by electroimmunoassay" and fibronectin by immunochemical precipitation and laser nephelometry l . Samples of arterial blood for estimation of 3-hydroxybutyrate concentration were taken in duplicate and immediately precipitated using ice-cold perchloric acid. After centrifugation the protein-free extract was frozen at - 80°C. Analyses were performed by microfluorometry as previously described' '.
'
Arieriovenous difference and net eflux of 3-meihylhisiidine
Arterial and venous blood samples were drawn simultaneously. The leg blood flow was measured by automatic mercury strain gauge plethysmography (Medimatic; Copenhagen, Denmark). Five measurements of leg blood flow were made at the maximal circumference of the calf immediately before and after blood sampling. The mean blood flow of the ten measurements was calculated and multiplied by the arteriovenous difference. The uptake and the release of 3-
methylhistidine was given in nmoles per 100 gram of tissue per minute, assuming the mean density of leg tissue to be 1.0 g m l - '. The method for analysing the concentration of 3-methylhistidine in plasma has previously been de~cribed'~. Urine The 24-h specimens of urine were analysed for creatinine, 3-methylhistidine and total nitrogen concentrations. The total nitrogen concentration was measured by the microKjeldahl methodI4; the non-urinary nitrogen losses were estimated to be 1.5 g day- l . The daily and cumulative whole body nitrogen balance was calculated and corrected for the change in blood urea. The concentration of 3-methylhistidine in urine was measured using an automated amino acid analyser (Kontron, Basel, Switzerland)15. Statistical analysis The significance of differences between the means of the groups was assessed by variance analyses and Student's unpaired t test, and within the groups by Student's paired t test. P < 0.05 was accepted as significant.
Table 2 Serum glucose, urea, creatinine, bilirubin and plasma protein concentrations before and 72 h afier major operaiion Group 2
Serum glucose (mmol I - ' ) Serum urea (mmol 1- ' ) Serum creatinine (pmol 1- ) Serum bilirubin (pmol I - ' ) Plasma albumin (g 1 ) Plasma prealbumin (g 1- ' ) Plasma fibronectin ( % )
Group 3
Before
After 72 h
Before
After 72 h
Before
After 72 h
6,5(0:4) 4.4(0.6 ) 87.9(6.5) 10.4(1.4) 35.0( 1.4) 0.22(0.02) 1l6(6.5)
8.8( 1.2) 4.9( 0.5 ) 75.0(6.3 ) 10.5(1.2) 27.0( I.5)t 0.13(0.02)* 96( 18)
6.6(0.6) 4.7(0.7) 90.0(4.2) 11.4( 1.4) 37.1 (0.7) 0.20(0.01) 124(9.3 )
9.5( 1.5) 8.9( 1.5)* 93.0( 8.9) 15.6(4.8) 27.7( l . 7 ) t 010(0~01)$ 65(4.4)f
5.7(05) 4.3 (0.2 ) 1W8(5-2) 10.6(0.9 ) 35.5( 1.6) 0.20(0.03) 92(6.1 )
9.8( 1.7) 5.4(0.7) 86-017.3) 1330.9) 29.3(2.0)* 0.11(0.01)*
75( 6.8 )
Values are mean(s.e.m.). * P < 0.05; t P < 0.01; $ P < 0401 (before surgery versus after 72 h, Student's paired t test)
Table 3 Arteriovenous diference and net eflux of bloodgow from ihe leg for 3-methylhisiidine and concentrations of 3-hydroxybuiyrate in arterial blood before and 72 and 78 h afier operalion in patienis receiving ioial parenteral nuiriiion Group 2
Group 1
Group 3 -
After 72 h
Before
2.39(029)
Blood flow (ml 100 g tissue-' min-') 3-Methylhistidine Arteriovenous difference (pmol I - ' ) Net efflux (nmol 1 0 0 g tissue-' m i n - l )
After 78 h
1.91(0.15)
Before
1.95(0.14)
After 72 h
1.99(0.22)
2,14(0.24)
After 78 h
2.72(0.25)
Before
3.07(0.69)
After 72h
2.59(0.37)
After 78 h
3.18(0.42)
-0.23(003)
-024(006)
-0.29(0.06)
-0.22(0.03)
-021(0.05)
-0.38(0.07)
-0.20(0Q4)
-0.23(0.05)
-023(0.05)
- 0.54(0.10)
-0.42(0.09)
-0.56(0,11)
- 0.42(0.07)
- 0.5 l(0.17)
- 0.99(0.19)*
-O.W(O.08)
-0.66(0.2I )
- 0 8 l(0.23)
0.05(0.01)
0,51(012)
011(0.06)
1.22(0.47)
0.04(0.0 I )
0,08(0.03)
3-H ydroxybutyrate (mmol I - ' )
0.55(012)
0-02(0QO)
0.20(0.09)
Values are mean(s.e.m.). * P < 0.05 (after 78 h uersus before and 72 h after operation, Student's paired r test)
Table 4 Daily and cumulaiive nitrogen balance and cumulaiive urinary 3-meihylhistidine excretion on days I , 2 and 3 afier major operaiion Group 1
Nitrogen balance (g day-') Cumulative nitrogen balance (g on days 1-3) Urinary 3-methylhistidine excretion (pmol mmol creatinine- day- ') Cumulative urinary 3methylhistidine excretion (pmol mmol creatinine- ')
Group 2
Group 3
Day 1
Day 2
Day 3
Day 1
Day 2
Day 3
Day I
Day 2
Day 3
-2.8(1.5)
-2.0(1.2)
-0.8(0.7)
- l.O(l.5)
-2.7(1.4)
-0.1(1.2)
-O.S(O.S)
-0.6(0.8)
-0.6(0.8)
-2.8(1.5)
-4.8(2.4)
-5.6(2.4)
- l.O(l.5)
-3.7(1.3)
-3.8(2.2)
-0.5(0.8)
-1'1(1'3)
- 1.7(1.8)
25.2(1.4)
25.1(1.7)
25.6(1.8)
27.8(2.3)
25.9(2.9)
34.3(11.1)
25.2(1.4)
503(3.8)
75.9(7.1)
27.8(2.3)
53.7(5.0)
88.0(14.8)
22.1(2.7)
22.1(2.7)
23q2.2)
21q1.3)
46.0(4.2)
67.9(7.2)
Values are mean(s.e.m.)
21 8
Br. J. Surg., Vol. 79, No. 3, March 1992
Nutritional supplementation in surgical patients: S. Sandstedt et al.
Results Biochemical variables (Table 2 ) The serum glucose concentration did not increase significantly and there were no differences among the groups 72 h after operation. The serum urea concentration increased 72 h after surgery in group 2 by 89 per cent ( P < 0.05), but not in the other two groups. The plasma albumin concentration fell significantly in all groups, but the values did not differ significantly at 72 h. The plasma fibronectin concentration had fallen in all groups at 72 h, but significantly only in group 2 ( P < 0,001), the mean value being below the lower limit of the reference range for healthy subjects. Arteriovenous diferences and net efJlux of 3-methylhistidine (Table 3 ) All patients receiving T P N had a net efflux of 3-methylhistidine from the leg before and 7 2 and 78 h after operation. In group 2, the net efflux on the third day 78 h after operation was significantly increased compared with values obtained before and 72 h after operation. In groups 1 and 3 there were no significant changes in net efflux from the leg after operation and 78 h later. Nitrogen balance (Table 4 ) The daily and cumulative nitrogen balances were negative in all three groups, but only slightly so, and no differences were found between them. Urinary excretion of 3-methylhistidine (Table 4 ) To correct excretion of 3-methylhistidine for variations in muscle mass, it was related to urinary creatinine excretion. The mean daily and cumulative values for 3-methylhistidine were similar in all three groups and did not differ significantly.
Discussion The metabolic response to trauma entails a net protein loss of up to 20 g nitrogen (equivalent to 500-600 g wet skeletal m ~ s c l e ) ' , ~The . nitrogen losses are the result of increased protein degradation and, although protein synthesis is stimulated, the net effect is negative16. Nutritional support reduces negative nitrogen balance16, and adequate calories to keep the patient in energy balance will keep nitrogen loss to a minimum". Giving nitrogen in the form of a balanced amino acid solution reduces, but does not abolish, negative nitrogen balance. Nitrogen was retained ( u p to 0.2 g kg- 24 h - ) during the first week after trauma". Other nitrogen-saving techniques, such as using epidural analgesia, have been proposed but are difficult to apply in those with extensive trauma". BCAAs, particularly leucine, reduce protein breakdown and stimulate protein synthesis at physiological concentrations in vitro in skeletal muscle and liver20.21. Their effects in vivo are controversial. Decreased protein degradation is not an effect of leucine alone but requires its transamination to ketoleucine. Ketoleucine has been shown to spare nitrogen in obese fasting subjectsz2. In a study of surgical patients, ketoleucine caused significant ketosis, and ketosis may inhibit protein degradation6. In this study ketoleucine was given to assess its effect on protein degradation in muscle in three comparable groups of patients. Albumin and prealbumin concentrations decreased after operation and there was a decline in fibronectin in group 2. Leucine and ketoleucine may stimulate hepatic protein synthesis6.'' and hepatic synthesis of acute phase proteins in critically ill and septic rats was stimulated by an amino acid solution enriched with 45 per cent BCAAs2'. In this study the levels of plasma proteins with short half-lives, prealbumin (48 h ) and fibronectin (8 h ) both declined, more in group 2 than the other groups.
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Br. J. Surg.. Vol. 79, No. 3. March 1992
'
The whole body nitrogen balance was not affected by either BCAAs or ketoleucine. In a study of patients after operation, Sapir et showed that ketoleucine plus glucose improved nitrogen balance and suppressed proteolysis, which was indicated by reduced urinary 3-methylhistidine excretion. Leucine did not seem to have the same effect. The increase in urea concentrations in the keto group at 7 2 h in the present study probably represented impaired breakdown of the ketoleucine solution. 3-Methylhistidine is found exclusively in actin and myosin and is released during proteolysis, after which it is neither reused for protein synthesis nor catabolized further. In the present study, excretion of 3-methylhistidine in urine was increased in all three groups", which indicates that myofibrillar proteolysis is not influenced by either BCAAs or balanced amino acids enriched with ketoleucine. It has been shown that 3methylhistidine excretion is not influenced by the supply of balanced amino acids16, but is related to the degree of injury and is mostly of skeletal muscle origin23. The significantly improved nitrogen balance in patients after operation that was reported by Sapir et aL6 was attributed to a reduced excretion of 3-methylhistidine. The arteriovenous difference in 3methylhistidine excretion and the net release from one leg were increased during T P N in group 2, indicating that ketoleucine had, on the contrary, a proteolytic effect. In the group receiving only a balanced amino acid solution (group 1 ) there was no such effect. Provided that adequate balanced nutritional support is given, supplementary ketoleucine and BCAAs d o not seem to have any beneficial effects on protein metabolism.
Acknowledgements This study was supported by grants from the Swedish Medical Research Council (no. 04139), Henning and Jone Throne-Holsts Foundation and Kabi Pharmacia. We gratefully acknowledge the technical assistance of Mrs Ninna Elfstrom.
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Paper accepted 11 November 1991
Br. J. Surg.. Vol. 79, No. 3, March 1992
