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Influence of nutritional status on exertion-induced forearm amino acid metabolism in normal man
JOURNAL
OF SURGICAL
Influence
RESEARCH
36,438-445
(1984)
of Nutritional Status on Exertion-Induced Amino Acid Metabolism in Normal Man
STEPHEN F. LOWRY, M.D.,’
GLENN D. HOROWITZ, M.D., AND MURRAY F. BRENNAN, M.D.
DAVID
Forearm ROSE, M.D.,
Department of Surgery, The New York Hospital-Cornell Medical Center (NYH-CMC), and Department of Surgery, Memorial Sloan-Kettering Cancer Center, and Adult Clinical Research Center (NYH-CMC), New York, New York IO021
Presentedat the Annual Meeting of the Association for Academic Surgery, Syracuse, New York, November 2-5, 1983 Normal volunteers were evaluated in the postabsorptive state, following 10 days of protein-calorie starvation, and during intravenous feeding (ivf) to determine the impact of nutritional status upon exertion-induced muscle amino acid metabolism. An isolated forearm model allowed an evaluation of recovery of metabolism following 1 min of submaximal isotonic exercise. Forearm blood flow returned to near basal levels within 15 min after exertion during postabsorptive and ivf conditions, but remained 3 150%of basal at 1 hr after exercise during starvation. At 30 and 60 min after exercise, forearm plasma flux of total and essential amino acids were unchanged from basal in the postabsorptive state. However, the pattern of essential amino acid flux demonstrated a relative reduction in isoleucine and leucine efflux compared with basal, and this pattern persisted throughout 1 hr of recovery. During starvation, a significant (P i 0.05) increase in total and essential amino acid efflux was observed throughout the recovery period. Starvation was also associated with significant increases in alanine and lysine efflux during recovery. Intravenous feeding was associated with a significant (P < 0.05) uptake of essential amino acids with respect to basal levels at 30 min after exercise. At 60 min, there was a shift to total amino acid efflux but no change from basal flux for essential amino acids. During ivf, the pattern of essential amino acid uptake returned to basal within 1 hr after exertion. INTRODUCTION
The protein catabolic response to stressful stimuli may be modulated by antecedent nutritional status [S-l 1, 14, 261. The capacity of nutritional support to ameliorate the proteolysis of peripheral muscle tissue under clinical conditions of stress has been widely implied by improvements in nitrogen balance and 3-methylhistidine excretion [4,9, 14, 171. Recent studies of post-traumatic limb release of amino acids and 3-methylhistidine have not correlated with urinary 3-methylhistidine excretion [6]. As a consequence, the clinical utility of these indices of muscle protein catabolism remains controversial [4, 231. The present study was designed to examine a specific muscle catabolic stress under varying ’ Author to whom requests for reprints should be addressedat: New York Hospital-Cornell Medical Center, 525 East 68th Street, F-2016, New York, N. Y. 10021. 0022-4804/84 %1SO Copyright 8 1984 by Academic FTess, Inc. AU rights of reproduction in any form reserved.
nutritional conditions. The capacity of intravenous feeding to alter exertion-induced muscle amino acid metabolism was also examined in unstressed normal subjects during hospitalization. MATERIALS
AND
METHODS
Male volunteers, aged 20-29, were screened as outpatients by complete history, physical examination, and blood chemistries. Five subjects who were judged to be normal by the above criteria and were within 10% normal weight for sex, age, and height, were admitted to the Adult Clinical Research Center (CRC) of the New York Hospital-Cornell Medical Center (NYH-CMC) for 24 consecutive days. Informed, written consent under protocol approved by the Institutional Review Board of NYH-CMC was obtained. The activity of all subjects was strictly confined to the CRC and
438
LOWRY
ET AL.: MUSCLE
they were not permitted to leave the ward for work or recreational purposes. Upon admission to the CRC, each subject was placed on a defined formula diet (Sustacal, Mead-Johnson, Evansville, Ind.) which provided -0.35 g N/Kg-day and each subject consumed a300 g of carbohydrate per day. The diet was consumed at six intervals during the first 3 days, at which time subjects were restricted from all protein-calorie intake (starvation) for Days 4-14. During the starvation period, subjects were permitted ad Zibitum accessto no-calorie soda, as well as receiving daily oral supplements of sodium chloride. At the completion of the IO-day starvation period, all subjects were refed totally by vein using a percutaneous antecubital vein catheter (Intrafusor, Sorenson Research,Salt Lake City, Utah) threaded into the superior vena cava. The intravenous refeeding regimen included a commercially available amino acid source (Aminosyn10%)and a nonprotein caloric source of either 100% dextrose or of 50% dextrose and 50% of lipid emulsion (Liposyn-10%). The refeeding regimen achieved the targeted level of intake (0.29 g N/Kg-day, 35 + 2 nonprotein Kcal/kg-day) within 3 days after the start of intravenous feeding and was maintained at this rate for the duration of the lo-day refeeding phase. All subjectswere weighed daily after voiding at 0600, and had consecutive 24-hr urine collections analyzed for total nitrogen and creatinine (Technicon Auto Analyzer II; Technicon, Tarrytown, N. Y.). Blood was analyzed on Days 4, 9, 14, 19, and 24 for complete blood count, electrolytes, liver function tests, and proteins using standard automated techniques available in the NYH-CMC Clinical Biochemistry Laboratories. In the supine resting state at 0700 on Days 4, 14, and 24 each subject had percutaneous placement of a Teflon radial artery catheter and a retrograde, antecubital deep basilic vein catheter. Subjectsremained supine for the duration of the study. Simultaneous arterial and venous bloods were drawn according to previously described methods [2, 151.Venous oc-
AMINO
ACID
METABOLISM
439
elusion electrocapacitance plethysmography was performed immediately following blood withdrawal and forearm blood flow calculated by standard methods [2]. Following completion of the determinations in the basal state, each subject underwent a 60-set period of isotonic forearm exercise (1 cycle/3 set) using a hand-grip ergometry. Blood withdrawal and determination of forearm blood flow was performed at 1, 15, 30, and 60 min following completion of forearm exercise.Plasmaamino acids were determined using column chromatography [ 161and flux of amino acids within the plasma compartment were calculated as: (arterial concentration - deep vein concentration) X blood flow (1 - Hct). As the magnitude of change in flux of forearm amino acids may be influenced by the basal level [20] most data are presented below as the net change from basal (-: relative efflux, +: relative uptake). Results are expressed as mean f SEM and Student’s unpaired t test was used for statistical comparison [25]. RESULTS
Weight decreasedfrom 73.3 f 2.0 to 67.5 f 1.7 kg during the 1O-day starvation period. By completion of the intravenous refeeding phase, weight had increased to 72.3 + 1.5 kg. Twenty-four-hour creatinine excretion decreased from 1.57 to 0.09 g/day to 1.49 to 0.06 g/day during starvation, and was 1.52 + 0.1 I at completion of intravenous refeeding. During the 24 hr immediately preceding the forearm studies, uncorrected 24-hr urinary nitrogen balances were +7.1 + 0.1 g/day on Day 3, -6.7 f 0.1 g/day on Day 13 (end starvation), and +7.5 + 0.8 g/day on Day 23 (intravenous feeding). Arterial plasma total alpha amino acid (TAA) was 2821 + 190 @mole/liter in the postabsorptive state of Day 4, 2686 f 222 pmole/liter at completion of starvation on Day 14, and 4483 + 239 pmole/liter during intravenous feeding on Day 24. Arterial plasma essential amino acid (EAA) levels were 833 t 68 pmole/liter on Day 4,907 f 111 pmole/
440
JOURNAL OF SURGICAL RESEARCH: VOL. 36, NO. 5, MAY 1984
liter on Day 14, and 1528 f 92 r.rmole/liter on Day 24. Basal forearm plasma flow decreasedfrom 3.8 f 0.5 ml/100 ml tissue-min to 2.2 +- 0.3 ml/ 100 ml tissue-min after 10 days of starvation, and was 3.6 + 0.8 ml/100 ml tissuemin during intravenous refeeding. The percentages of basal flow observed at specified time points following forearm exercise are shown in Fig. 1. Mean total forearm blood flow was consistently <2000/o of basal only during the 30- and 60-min postexercise periods. We have chosen to examine these time points as most representative of forearm muscle amino acid metabolism. The forearm plasma amino acid flux, expressed as net change from basal, during the 30- and 60-min postexercise recovery periods is shown in Table 1. While the postabsorptive forearm demonstrated no significant difference from basal flux for TAA within 30 min postexertion, the forearm in starved subjects demonstrated increased(P < 0.05) TAA efflux with no tendency to return toward preexerciseTAA flux within 1 hr. Subjectsreceiving total parenteral nutrition (TPN) demonstrated efflux of TAA by comparison to basal levels (P < 0.05 versus basal at 60 min). This level of efflux, however, rep500
resents near total nitrogen equilibrium across forearm tissues at 30 and 60 min after exertion [ 181. The forearm flux of EAA did not demonstrate a significant efflux change at 30 or 60 min following exercisein either postabsorptive or intravenously fed subjects. However, starved subjects continued to demonstrate an efflux of EAA with respect to basal levels throughout the recovery period (P < 0.05 versus basal at 60 min). The net change from basal of the predominant nitrogen transfer amino acids glutamine (Gln) and alanine (Ala) are shown in Table 2. As was evident for both TAA and EAA, starved subjects demonstrated significantly increased efflux over basal without a trend toward normalization within the 1 hr postexercise recovery period. Neither postabsorptive nor intravenously fed subjects demonstrated significant changes from basal flux of glutamine or alanine at 30 and 60 min after exercise. The net change from basal flux for lysine and tyrosine are also shown in Table 2. These are essential amino acids whose appearance from tissues is commonly used as an index of net protein catabolism [5, 191.Thirty and sixty minutes following cessation of exercise, the
T
q Starvation 4OC
&j
TPN
2 G z 0 ii =: 2
3oc
1 200
z s 100
l
I
153060
Minutes
I
153060
post
153060
exercise
FIG. I. Postexercisetotal foream blood flow as percentage of basal (preexercise) flow. Mean and standard error.
441
LOWRY ET AL.: MUSCLE AMINO ACID METABOLISM TABLE 1
Essential amino acids
Total amino acids
Postabsorptive Starve TPN
30 min
60 min
30 min
60 min
+35 k 483 -614 + 286 -80 + 247
+100 f 103 718 f 255* -360 f 103*
+59 + 104 -130 + 91 +271 + 99*
-49 f 46 -192 f 75* -41 f 83
iVotes.J?_’ SEM. Flux is expressedas nmole/lOO ml-tissue-min. + = uptake relative to basal. - = releaserelative to basal. 30 min = 30 min after exercise ceased.60 min = 60 min after exercise ceased. * P < 0.05 versus basal.
forearms of starved subjectshad a significantly increased efflux of lysine (P < 0.05). A significant uptake of lysine is observed acrossthe forearms of intravenously fed subjects 30 min postexercise(P -C0.05) and this coincides with uptake of all EAA observedat this time period. Sixty minutes following exercise, however, neither lysine nor tyrosine flux were altered from basal levels in this group. A further analysis of the forearm EAA fluxes is presented in Figs. 2 and 3. As there can be no net synthesis of essential amino acids by forearm tissues,the relationship of their efflux or uptake may provide insight into the disposition or source of these amino acids. We have previously documented the pattern of forearm essentialamino acid efflux and uptake to be anticipated during basal conditions in starved and intravenously fed subjects [ 181. During starvation, the basal pattern of EAA efflux is similar to the known concentrations
of EAA in actinomysin or mixed muscle protein. However, in response to intravenous feeding in such subjects, the branched chain amino acids are taken up in excess of their concentrations in muscle proteins during basal conditions, while the nonbranched chain amino acid uptake is relatively lessthan muscle protein content. The percentage efflux of individual EAA in relation to total EAA flux during postexercise recovery demonstrates a pattern of forearm EAA efflux similar to the known EAA concentration in mixed muscle proteins. This is particularly evident in the starved subjects at 60 min postexercise (Fig. 3). A reduced percentage uptake of isoleucine and leucine in relation to the known basal pattern was observed at 30 min in intravenously fed subjects (P < 0.05) while at 60 min postexercisea pattern of EAA uptake identical to that observed in the basal state was noted in this group. By 60 min after exercise, post-
TABLE 2 NET CHANGEFROMBASALINFOREARMGLUTAMINE, AND TYROSINE FLUX AFTER SUBMAXIMAL Gln
Postabsorptive StalVe TPN
ALANINE, EXERCISE
Ala
LYSINE,
LYS
TY~
30 min
60 min
30 min
60 min
30 min
60 min
+I k82 -100+52 fl2 + 91
-18 -t 82 -225 57 91’ -51 + 47
-125 f 102 -144 + 40; ?I03 f 76
-40 * 70 -123 f 50* -9 k 58
-4 * 22 -38 + 17’ t52 t 8*
-15 f 18 -38 + 18’ -7 f 26
Notes. J? + SEM. Flux expressed as nmole/lOO ml tissue-min. Gin = glutamine; Ala = &nine; *P < 0.05 vel~us basal.
30
min
+1 f 9 -10 * 5 f7 f 5
60
min
+5 f 6 -11 k5 -5 * 5
Lys = lysine; Tyr = tyrosine.
442
JOURNAL
OF SURGICAL
RESEARCH:
VOL. 36, NO. 5, MAY
1984
H
Muscle
0
Post-absarptwe
proteln content
/-J Storvoilon
THR
MET
PHE
VAL
LYS
efflux
eff Iux
LEU
ILE
FIG. 2. Forearm plasma flux of essential amino acids as percentage of total essential amino acid flux at
30 min after exercise. THR = threonine, MET = Methionine, = valine, ILE = isoleucine, LEU = leucine.
absorptive subjects demonstrated continued reduction of isoleucine and leucine efflux with respect to basal levels [3, 241. The importance of the branched chain amino acids to forearm metabolism in starved subjects is demonstrated by the fact that these amino acids represent 36 f 4% of the total EAA efflux at 30 min and 55 f 5% of EAA efflux at 60 min following exercise. The variation in branched chain amino acid flux is in contrast to the relatively constant relationship of lysine flux to total EAA flux. In the postabsorptive state, lysine accounted for 37-40% of total EAA flux at 30 and 60 min postexercise, while at these time points during star-
PHE = phenylalanine,
LYS = lysine, VAL
vation lysine efflux was 25-33% of net EAA loss by the forearm. During intravenous refeeding at a time when net EAA uptake was occurring, lysine uptake was 15 + 3% of total EAA flux at 30 min, and 8 + 3% of EAA uptake at 60 min following exercise. DISCUSSION
Skeletal muscle representsthe predominant source of labile protein for mobilization during injury or diseasestates. The balance between muscle protein synthesis and catabolism may be modulated by many factors, including nutritional status. Recent measurements of in
H
Muscle
2
tf
Post-absorptlve,efflux
= z
rJ
Starvation
40
pmteln
content
efflux
0 30 0 .5 5 3 20 ; z 5 =5
IO
% z THR
MET
PHE
LYS
VAL
ILE
LEU
FIG. 3. Forearm plasma flux of essential amino acids as percentage of total essential amino acid flux at 60 min after exercise.
LOWRY
ET AL.: MUSCLE
AMINO
viva muscle protein synthesis have demonstrated a decline in mixed muscle protein synthetic rates during the postabsorptive phase [22] and in response to prolonged periods of submaximal exercise [21]. The regulation of muscle protein balance is seldom examined under conditions which are independent of altered hormonal or substrate levels. This study was designed to examine the acute effects of submaximal exertion in an isolated human forearm under conditions in which the intrinsic regulation of forearm metabolism resulting from nutritional status could be evaluated. Previous efforts to determine tissue-specific responses to catabolic stimuli have not differentiated substrate availability or hormonally mediated factors. Acute exercise has been utilized as a catabolic model where in viva tissue kinetic studies have suggested that prolonged submaximal exertion causes an inhibition of mixed muscle protein synthesis [ 12,2 11.The systemic effect of acute exercise causes a negative muscle nitrogen balance to mobilize gluconeogenic precursors [l]. This effect is evidenced by the discrepancy between forearm glutamine and alanine output during isolated [3] or whole body exertion [ 131.In contrast to net extremity efflux of gluconeogenic precursors during exercise, prolonged (40-240 min) exercise may result in uptake of many amino acids [ 131. We have utilized acute exercise as a catabolic stimulus at a submaximal, abbreviated level to minimize systemic responses. It was demonstrated by Cuthbertson that the extent of body nitrogen excretion in response to exercise was larger when exercise is performed after a meal than when a similar amount of exercise is performed prior to eating [ 111. Aoki has shown that muscle nitrogen efflux from exercised arm as glutamine is increased over efflux from a nonexercised area in postprandial subjects [3]. Our observations suggest that both the postabsorptive and intravenously fed conditions favorably affect an exercise-induced imbalance of protein catabolism over synthesis such that recovery is observed within 60 min. This is not the case in
ACID
METABOLISM
443
starvation where continued efflux of total and essential amino acids over basal levels were observed at 1 hr. This study also examines possible regulatory mechanisms of muscle protein metabolism. Despite equivalent arterial levels of total and essential amino acids, the discrepancy between the patterns of efflux observed during the postabsorptive and starvation states suggests that extracellular amino acid availability is not a prominent feature in the recovery from exertional stress. Our results also suggest that insulin does not exert a major role on muscle recovery from submaximal exertion. For both the postabsorptive and fasted conditions, where plasma insulin levels were =G15 ctU/ml, there was no correlation of either basal or postexercise forearm amino acid flux to arterial insulin concentration. The branched chain amino acids are presumed to play an important role in muscle protein metabolism during exercise [ 131. Leutine oxidation is known to increase in absolute terms as a primary energy source [2 1] during exercise. This study, although not directly measuring rates of branched chain amino acid transamination or oxidation, supports the central role of leucine as an important muscle metabolite during stress. At 30 min following recovery from exercise, during both postabsorptive and starvation conditions, the efflux of leucine is below the level seen in basal conditions [ 181.Within 1 hr of recovery, the efflux of leucine continues to be depressed, especially in the postabsorptive state, suggesting that increased leucine transamination and/or oxidation is occurring. Valine flux appears from our data to be little affected by exercise, while that of isoleucine is intermediate to that of valine and leucine. These alterations in branched chain amino acid uptake occurred under conditions which are independent of known hormonal effects on their metabolism [7]. The favorable impact of concurrent intravenous nutritional support upon muscle amino acid metabolism following submaximal exercise is demonstrated by the recovery
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JOURNAL OF SURGICAL RESEARCH: VOL. 36, NO. 5, MAY 1984
within 1 hr to basal flux patterns for total and essential amino acids. While it is not possible to differentiate whether substrate availability alone may have influenced the continued forearm uptake of amino acids at 1 hr, the study design did allow an assessmentof insulinization upon forearm amino acid metabolism. A differential insulin response (2040 pU/ml) was obtained with the 100% dextrose nonprotein calories versus the 50% dextrose/50% lipid calorie source. Although the number of observations obtained with these regimens was limited (n = 2, glucose; II = 3, glucose/lipid), we were unable to observe any differences in either the basal or postexercise recovery patterns of forearm amino acid flux during intravenous feeding. The present observations are of clinical interest in that an abbreviated period of submaximal exertion resulted in major deviations of forearm nitrogen balance in nonnutritionally supported subjects. While postabsorptive subjects demonstrated a return to basal flux patterns within 1 hr after exertion, this still represents a loss of approximately 1 pmole of protein/l00 ml of tissue per minute. Starved subjects, despite a reduction in basal output to approximately 0.2 pmole protein per minute [ 181,responded to submaxial exercisewith a 2300% increase in forearm amino acid loss that had not decreased within 1 hr after exercise.,Only the intravenously fed subjects attained forearm nitrogen balance during the postexercise recovery period and only at 60 min after recovery did the pattern of essential amino acid flux resemble that in the basal state [ 181.These observations imply that nutritional status affectsboth the magnitude and rate of recovery in exertion-induced muscle amino acid metabolism. Intravenous nutritional support appearsto impact favorably on this process although complete recovery may require a longer period than is generally appreciated. REFERENCES
1. Ahlborg, G., Felig, P., Hagenfeldt, L., Hendler, R., and Wahren, J. Substrate turnover during prolonged exercise in man. J. Clin. Invest. 53: 1080, 1974.
2. Andres, R. K., Zierler, K. L., and Anderson, H. N. Measurement of blood flow with volume in the forearm of man; with notes on the theory of indicator dilution and on production of turbulence, haemolysis and vasodilation by introvascular injection. J. Clin. Invest. 33: 482, 1954. 3. Aoki, T. T., Brennan, M. F., Fitzpatrick, G. F., and Knight, D. C. Leucine meal increasesglutamine and total nitrogen release from forearm muscle. J. Clin. Invest. 68: 1522, 1981. 4. Ballard, F. J., and Tomas, F. M. 3-Methylhistidine as a measure of skeletal muscle protein breakdown in human subjects: The case for its continued use. Clin. Sci. 65: 209, 1983. 5. Bennegard, K., Eden, E., Ekman, L., Schersten, T., and Lundholm, K. Metabolic balance acrossthe leg in weight-losing cancer patients compared to depleted patients without cancer. Cancer Rex 42: 4293, 1982. 6. Benneard, K., Rennie, M. J., Eden, E., Emery, P. W., and Lundholm, K. Influence of major surgical trauma on urinary excretion and efflux from the leg of 3methylhistidine. Proc. 5th Eur. Sot. Parent Ent. Nutr. A-103, 1983. 7. Buse, M. G., Biggers, J. F., Drier, C., and Buse, J. F. The effect of epinephrine, glucagon, and the nutritional state on the oxidation ofbranched chain amino acids and pyruvate by isolated hearts and diaphragms of the rat. J. Biol. Chem. 248: 697, 1973. 8. Calloway, D. H., Grossman, M. I., Bowman, J., and Calhoun, W. K. The effect of previous level of protein feeding on wound healing and the metabolic response to injury. Surgery 37: 935, 1955. 9. Clague, M. B., Keir, M. J., Wright, P. D., and Johnston, I. D. A. The effectsof nutrition and trauma on whole-body protein metabolism in man. Clin. Sci. 65: 165, 1983. 10. Cuthbertson, D. P. Observations on the disturbance of metabolism produced by injury to the limbs. Quart. J. Med. 1: 223, 1932. 11. Cuthbertson, D. P., McGin, J. L., and Munro, H. N. A study of the effect of overfeeding on the protein metabolism of man. IV. The effect of muscular work at different levels of energy intake, with particular reference to the timing of the work in relation to the taking of food. Biochem. J. 31: 2293, 1937. 12. Dohm, G. L., Kasparek, G. J., Tapscott, E. B., and Beecher, G. R. Effect of exercise on synthesis and degradation of muscle protein. Biochem. J. 188: 255, 1980. 13. Felig, P., and Wahren, J. Amino acid metabolism in exercising man. J. Clin. Invest. 50: 2703, 1971. 14. Holbrook, I. B., Gross, E., Milewski, P. J., Shipley, K., and Irving, M. H. N-methylhistidine excretion and myofibrillar protein breakdown in patients receiving intravenous or enteral nutrition. C/in. Sci. 59: 211, 1980. 15. Kontos, H. A., Richardson, D. W., and Patterson, J. L. Blood flow and metabolism of forearm muscle
LOWRY ET AL.: MUSCLE AMINO ACID METABOLISM in man at restand during sustainedcontraction. Amer. J. Physiol. 211: 869, 1966. 16. Lee, P. L. Y. Single-column system for accelerated amino acid analysis of physiologic fluids using five lithium buffers. Biochem. Med. 10: 107, 1974. 17. Long, C. L., Birklohn, R. H., Geiger, J. W., Betts, J. E., Schiller, W. R., and Blakemore, W. S. Urinary excretion of 3-methylhistidine: An assessment of musclecatabolism in adult normal subjectsand during malnutrition, sepsis,and skeletal trauma. Metabolism 30: 765, 1981. 18. Lowry, S. F., Horowitz, G. D., Rose,D., and Brennan, M. F. Forearm amino acid metabolism following starvation and intravenous refeeding in normal man. Surg. Forum 34: 93, 1983. 19. Lundholm, K., Bylund, A. C., Holm, G., and Schersten, T. Skeletal muscle metabolism in patients with malignant tumor. Eur. J. Cancer 12: 465, 1976. 20. Pozefsky, T., Felig, P., Tobin, J. D., Soeldner, J. S., and Cahill, G. F. Amino acid balance acrosstissues of the forearm in postabsorptiveman. Effectsof insulin at two dose levels. J. Clin. Invest. 48: 2273, 1969.
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21. Rennie, M. J., Edwards, R. H. T., Krywauryck, S., Davies, C. T. M., Halliday, D., Waterlow, J. C., and Millward, D. J. Effect of exercise on protein turnover in man. Clin. Sci. 61: 627, 1981. 22. Rennie, M. J., Edwards, R. H. T., HaIliday, D., Matthews, D. E., Wolman, S. L., and Millward, D. J. Muscle protein synthesis measured by stable isotope techniques in man: The effectsof feeding and fasting. Clin. Sci. 63: 519, 1982. 23 Rennie, M. J., and Millward, D. J. 3-Methylhistidine excretion and the urinary 3-metbylhistidine/creatinine ratio are poor indicators of skeletal muscle protein breakdown. Clin. Sci. 65: 217, 1983. 24. Ruderman, N. B. Muscle amino acid metabolism and gluconeogeneis. Ann. Rev. Med. 26: 245, 1975. 25. Snedecor, G. W., and Cochran, W. G. Statistical Methods, 6th ed. Ames, Iowa: Iowa Univ. Press,1967. 26. Tomkins, A. M., Garlick, I’. J., Schofield, W. N., and Waterlow, J. C. The combined effects of infection and malnutrition on protein metabolism in children. Clin. Sci. 65: 313, 1983.
OF SURGICAL
Influence
RESEARCH
36,438-445
(1984)
of Nutritional Status on Exertion-Induced Amino Acid Metabolism in Normal Man
STEPHEN F. LOWRY, M.D.,’
GLENN D. HOROWITZ, M.D., AND MURRAY F. BRENNAN, M.D.
DAVID
Forearm ROSE, M.D.,
Department of Surgery, The New York Hospital-Cornell Medical Center (NYH-CMC), and Department of Surgery, Memorial Sloan-Kettering Cancer Center, and Adult Clinical Research Center (NYH-CMC), New York, New York IO021
Presentedat the Annual Meeting of the Association for Academic Surgery, Syracuse, New York, November 2-5, 1983 Normal volunteers were evaluated in the postabsorptive state, following 10 days of protein-calorie starvation, and during intravenous feeding (ivf) to determine the impact of nutritional status upon exertion-induced muscle amino acid metabolism. An isolated forearm model allowed an evaluation of recovery of metabolism following 1 min of submaximal isotonic exercise. Forearm blood flow returned to near basal levels within 15 min after exertion during postabsorptive and ivf conditions, but remained 3 150%of basal at 1 hr after exercise during starvation. At 30 and 60 min after exercise, forearm plasma flux of total and essential amino acids were unchanged from basal in the postabsorptive state. However, the pattern of essential amino acid flux demonstrated a relative reduction in isoleucine and leucine efflux compared with basal, and this pattern persisted throughout 1 hr of recovery. During starvation, a significant (P i 0.05) increase in total and essential amino acid efflux was observed throughout the recovery period. Starvation was also associated with significant increases in alanine and lysine efflux during recovery. Intravenous feeding was associated with a significant (P < 0.05) uptake of essential amino acids with respect to basal levels at 30 min after exercise. At 60 min, there was a shift to total amino acid efflux but no change from basal flux for essential amino acids. During ivf, the pattern of essential amino acid uptake returned to basal within 1 hr after exertion. INTRODUCTION
The protein catabolic response to stressful stimuli may be modulated by antecedent nutritional status [S-l 1, 14, 261. The capacity of nutritional support to ameliorate the proteolysis of peripheral muscle tissue under clinical conditions of stress has been widely implied by improvements in nitrogen balance and 3-methylhistidine excretion [4,9, 14, 171. Recent studies of post-traumatic limb release of amino acids and 3-methylhistidine have not correlated with urinary 3-methylhistidine excretion [6]. As a consequence, the clinical utility of these indices of muscle protein catabolism remains controversial [4, 231. The present study was designed to examine a specific muscle catabolic stress under varying ’ Author to whom requests for reprints should be addressedat: New York Hospital-Cornell Medical Center, 525 East 68th Street, F-2016, New York, N. Y. 10021. 0022-4804/84 %1SO Copyright 8 1984 by Academic FTess, Inc. AU rights of reproduction in any form reserved.
nutritional conditions. The capacity of intravenous feeding to alter exertion-induced muscle amino acid metabolism was also examined in unstressed normal subjects during hospitalization. MATERIALS
AND
METHODS
Male volunteers, aged 20-29, were screened as outpatients by complete history, physical examination, and blood chemistries. Five subjects who were judged to be normal by the above criteria and were within 10% normal weight for sex, age, and height, were admitted to the Adult Clinical Research Center (CRC) of the New York Hospital-Cornell Medical Center (NYH-CMC) for 24 consecutive days. Informed, written consent under protocol approved by the Institutional Review Board of NYH-CMC was obtained. The activity of all subjects was strictly confined to the CRC and
438
LOWRY
ET AL.: MUSCLE
they were not permitted to leave the ward for work or recreational purposes. Upon admission to the CRC, each subject was placed on a defined formula diet (Sustacal, Mead-Johnson, Evansville, Ind.) which provided -0.35 g N/Kg-day and each subject consumed a300 g of carbohydrate per day. The diet was consumed at six intervals during the first 3 days, at which time subjects were restricted from all protein-calorie intake (starvation) for Days 4-14. During the starvation period, subjects were permitted ad Zibitum accessto no-calorie soda, as well as receiving daily oral supplements of sodium chloride. At the completion of the IO-day starvation period, all subjects were refed totally by vein using a percutaneous antecubital vein catheter (Intrafusor, Sorenson Research,Salt Lake City, Utah) threaded into the superior vena cava. The intravenous refeeding regimen included a commercially available amino acid source (Aminosyn10%)and a nonprotein caloric source of either 100% dextrose or of 50% dextrose and 50% of lipid emulsion (Liposyn-10%). The refeeding regimen achieved the targeted level of intake (0.29 g N/Kg-day, 35 + 2 nonprotein Kcal/kg-day) within 3 days after the start of intravenous feeding and was maintained at this rate for the duration of the lo-day refeeding phase. All subjectswere weighed daily after voiding at 0600, and had consecutive 24-hr urine collections analyzed for total nitrogen and creatinine (Technicon Auto Analyzer II; Technicon, Tarrytown, N. Y.). Blood was analyzed on Days 4, 9, 14, 19, and 24 for complete blood count, electrolytes, liver function tests, and proteins using standard automated techniques available in the NYH-CMC Clinical Biochemistry Laboratories. In the supine resting state at 0700 on Days 4, 14, and 24 each subject had percutaneous placement of a Teflon radial artery catheter and a retrograde, antecubital deep basilic vein catheter. Subjectsremained supine for the duration of the study. Simultaneous arterial and venous bloods were drawn according to previously described methods [2, 151.Venous oc-
AMINO
ACID
METABOLISM
439
elusion electrocapacitance plethysmography was performed immediately following blood withdrawal and forearm blood flow calculated by standard methods [2]. Following completion of the determinations in the basal state, each subject underwent a 60-set period of isotonic forearm exercise (1 cycle/3 set) using a hand-grip ergometry. Blood withdrawal and determination of forearm blood flow was performed at 1, 15, 30, and 60 min following completion of forearm exercise.Plasmaamino acids were determined using column chromatography [ 161and flux of amino acids within the plasma compartment were calculated as: (arterial concentration - deep vein concentration) X blood flow (1 - Hct). As the magnitude of change in flux of forearm amino acids may be influenced by the basal level [20] most data are presented below as the net change from basal (-: relative efflux, +: relative uptake). Results are expressed as mean f SEM and Student’s unpaired t test was used for statistical comparison [25]. RESULTS
Weight decreasedfrom 73.3 f 2.0 to 67.5 f 1.7 kg during the 1O-day starvation period. By completion of the intravenous refeeding phase, weight had increased to 72.3 + 1.5 kg. Twenty-four-hour creatinine excretion decreased from 1.57 to 0.09 g/day to 1.49 to 0.06 g/day during starvation, and was 1.52 + 0.1 I at completion of intravenous refeeding. During the 24 hr immediately preceding the forearm studies, uncorrected 24-hr urinary nitrogen balances were +7.1 + 0.1 g/day on Day 3, -6.7 f 0.1 g/day on Day 13 (end starvation), and +7.5 + 0.8 g/day on Day 23 (intravenous feeding). Arterial plasma total alpha amino acid (TAA) was 2821 + 190 @mole/liter in the postabsorptive state of Day 4, 2686 f 222 pmole/liter at completion of starvation on Day 14, and 4483 + 239 pmole/liter during intravenous feeding on Day 24. Arterial plasma essential amino acid (EAA) levels were 833 t 68 pmole/liter on Day 4,907 f 111 pmole/
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liter on Day 14, and 1528 f 92 r.rmole/liter on Day 24. Basal forearm plasma flow decreasedfrom 3.8 f 0.5 ml/100 ml tissue-min to 2.2 +- 0.3 ml/ 100 ml tissue-min after 10 days of starvation, and was 3.6 + 0.8 ml/100 ml tissuemin during intravenous refeeding. The percentages of basal flow observed at specified time points following forearm exercise are shown in Fig. 1. Mean total forearm blood flow was consistently <2000/o of basal only during the 30- and 60-min postexercise periods. We have chosen to examine these time points as most representative of forearm muscle amino acid metabolism. The forearm plasma amino acid flux, expressed as net change from basal, during the 30- and 60-min postexercise recovery periods is shown in Table 1. While the postabsorptive forearm demonstrated no significant difference from basal flux for TAA within 30 min postexertion, the forearm in starved subjects demonstrated increased(P < 0.05) TAA efflux with no tendency to return toward preexerciseTAA flux within 1 hr. Subjectsreceiving total parenteral nutrition (TPN) demonstrated efflux of TAA by comparison to basal levels (P < 0.05 versus basal at 60 min). This level of efflux, however, rep500
resents near total nitrogen equilibrium across forearm tissues at 30 and 60 min after exertion [ 181. The forearm flux of EAA did not demonstrate a significant efflux change at 30 or 60 min following exercisein either postabsorptive or intravenously fed subjects. However, starved subjects continued to demonstrate an efflux of EAA with respect to basal levels throughout the recovery period (P < 0.05 versus basal at 60 min). The net change from basal of the predominant nitrogen transfer amino acids glutamine (Gln) and alanine (Ala) are shown in Table 2. As was evident for both TAA and EAA, starved subjects demonstrated significantly increased efflux over basal without a trend toward normalization within the 1 hr postexercise recovery period. Neither postabsorptive nor intravenously fed subjects demonstrated significant changes from basal flux of glutamine or alanine at 30 and 60 min after exercise. The net change from basal flux for lysine and tyrosine are also shown in Table 2. These are essential amino acids whose appearance from tissues is commonly used as an index of net protein catabolism [5, 191.Thirty and sixty minutes following cessation of exercise, the
T
q Starvation 4OC
&j
TPN
2 G z 0 ii =: 2
3oc
1 200
z s 100
l
I
153060
Minutes
I
153060
post
153060
exercise
FIG. I. Postexercisetotal foream blood flow as percentage of basal (preexercise) flow. Mean and standard error.
441
LOWRY ET AL.: MUSCLE AMINO ACID METABOLISM TABLE 1
Essential amino acids
Total amino acids
Postabsorptive Starve TPN
30 min
60 min
30 min
60 min
+35 k 483 -614 + 286 -80 + 247
+100 f 103 718 f 255* -360 f 103*
+59 + 104 -130 + 91 +271 + 99*
-49 f 46 -192 f 75* -41 f 83
iVotes.J?_’ SEM. Flux is expressedas nmole/lOO ml-tissue-min. + = uptake relative to basal. - = releaserelative to basal. 30 min = 30 min after exercise ceased.60 min = 60 min after exercise ceased. * P < 0.05 versus basal.
forearms of starved subjectshad a significantly increased efflux of lysine (P < 0.05). A significant uptake of lysine is observed acrossthe forearms of intravenously fed subjects 30 min postexercise(P -C0.05) and this coincides with uptake of all EAA observedat this time period. Sixty minutes following exercise, however, neither lysine nor tyrosine flux were altered from basal levels in this group. A further analysis of the forearm EAA fluxes is presented in Figs. 2 and 3. As there can be no net synthesis of essential amino acids by forearm tissues,the relationship of their efflux or uptake may provide insight into the disposition or source of these amino acids. We have previously documented the pattern of forearm essentialamino acid efflux and uptake to be anticipated during basal conditions in starved and intravenously fed subjects [ 181. During starvation, the basal pattern of EAA efflux is similar to the known concentrations
of EAA in actinomysin or mixed muscle protein. However, in response to intravenous feeding in such subjects, the branched chain amino acids are taken up in excess of their concentrations in muscle proteins during basal conditions, while the nonbranched chain amino acid uptake is relatively lessthan muscle protein content. The percentage efflux of individual EAA in relation to total EAA flux during postexercise recovery demonstrates a pattern of forearm EAA efflux similar to the known EAA concentration in mixed muscle proteins. This is particularly evident in the starved subjects at 60 min postexercise (Fig. 3). A reduced percentage uptake of isoleucine and leucine in relation to the known basal pattern was observed at 30 min in intravenously fed subjects (P < 0.05) while at 60 min postexercisea pattern of EAA uptake identical to that observed in the basal state was noted in this group. By 60 min after exercise, post-
TABLE 2 NET CHANGEFROMBASALINFOREARMGLUTAMINE, AND TYROSINE FLUX AFTER SUBMAXIMAL Gln
Postabsorptive StalVe TPN
ALANINE, EXERCISE
Ala
LYSINE,
LYS
TY~
30 min
60 min
30 min
60 min
30 min
60 min
+I k82 -100+52 fl2 + 91
-18 -t 82 -225 57 91’ -51 + 47
-125 f 102 -144 + 40; ?I03 f 76
-40 * 70 -123 f 50* -9 k 58
-4 * 22 -38 + 17’ t52 t 8*
-15 f 18 -38 + 18’ -7 f 26
Notes. J? + SEM. Flux expressed as nmole/lOO ml tissue-min. Gin = glutamine; Ala = &nine; *P < 0.05 vel~us basal.
30
min
+1 f 9 -10 * 5 f7 f 5
60
min
+5 f 6 -11 k5 -5 * 5
Lys = lysine; Tyr = tyrosine.
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OF SURGICAL
RESEARCH:
VOL. 36, NO. 5, MAY
1984
H
Muscle
0
Post-absarptwe
proteln content
/-J Storvoilon
THR
MET
PHE
VAL
LYS
efflux
eff Iux
LEU
ILE
FIG. 2. Forearm plasma flux of essential amino acids as percentage of total essential amino acid flux at
30 min after exercise. THR = threonine, MET = Methionine, = valine, ILE = isoleucine, LEU = leucine.
absorptive subjects demonstrated continued reduction of isoleucine and leucine efflux with respect to basal levels [3, 241. The importance of the branched chain amino acids to forearm metabolism in starved subjects is demonstrated by the fact that these amino acids represent 36 f 4% of the total EAA efflux at 30 min and 55 f 5% of EAA efflux at 60 min following exercise. The variation in branched chain amino acid flux is in contrast to the relatively constant relationship of lysine flux to total EAA flux. In the postabsorptive state, lysine accounted for 37-40% of total EAA flux at 30 and 60 min postexercise, while at these time points during star-
PHE = phenylalanine,
LYS = lysine, VAL
vation lysine efflux was 25-33% of net EAA loss by the forearm. During intravenous refeeding at a time when net EAA uptake was occurring, lysine uptake was 15 + 3% of total EAA flux at 30 min, and 8 + 3% of EAA uptake at 60 min following exercise. DISCUSSION
Skeletal muscle representsthe predominant source of labile protein for mobilization during injury or diseasestates. The balance between muscle protein synthesis and catabolism may be modulated by many factors, including nutritional status. Recent measurements of in
H
Muscle
2
tf
Post-absorptlve,efflux
= z
rJ
Starvation
40
pmteln
content
efflux
0 30 0 .5 5 3 20 ; z 5 =5
IO
% z THR
MET
PHE
LYS
VAL
ILE
LEU
FIG. 3. Forearm plasma flux of essential amino acids as percentage of total essential amino acid flux at 60 min after exercise.
LOWRY
ET AL.: MUSCLE
AMINO
viva muscle protein synthesis have demonstrated a decline in mixed muscle protein synthetic rates during the postabsorptive phase [22] and in response to prolonged periods of submaximal exercise [21]. The regulation of muscle protein balance is seldom examined under conditions which are independent of altered hormonal or substrate levels. This study was designed to examine the acute effects of submaximal exertion in an isolated human forearm under conditions in which the intrinsic regulation of forearm metabolism resulting from nutritional status could be evaluated. Previous efforts to determine tissue-specific responses to catabolic stimuli have not differentiated substrate availability or hormonally mediated factors. Acute exercise has been utilized as a catabolic model where in viva tissue kinetic studies have suggested that prolonged submaximal exertion causes an inhibition of mixed muscle protein synthesis [ 12,2 11.The systemic effect of acute exercise causes a negative muscle nitrogen balance to mobilize gluconeogenic precursors [l]. This effect is evidenced by the discrepancy between forearm glutamine and alanine output during isolated [3] or whole body exertion [ 131.In contrast to net extremity efflux of gluconeogenic precursors during exercise, prolonged (40-240 min) exercise may result in uptake of many amino acids [ 131. We have utilized acute exercise as a catabolic stimulus at a submaximal, abbreviated level to minimize systemic responses. It was demonstrated by Cuthbertson that the extent of body nitrogen excretion in response to exercise was larger when exercise is performed after a meal than when a similar amount of exercise is performed prior to eating [ 111. Aoki has shown that muscle nitrogen efflux from exercised arm as glutamine is increased over efflux from a nonexercised area in postprandial subjects [3]. Our observations suggest that both the postabsorptive and intravenously fed conditions favorably affect an exercise-induced imbalance of protein catabolism over synthesis such that recovery is observed within 60 min. This is not the case in
ACID
METABOLISM
443
starvation where continued efflux of total and essential amino acids over basal levels were observed at 1 hr. This study also examines possible regulatory mechanisms of muscle protein metabolism. Despite equivalent arterial levels of total and essential amino acids, the discrepancy between the patterns of efflux observed during the postabsorptive and starvation states suggests that extracellular amino acid availability is not a prominent feature in the recovery from exertional stress. Our results also suggest that insulin does not exert a major role on muscle recovery from submaximal exertion. For both the postabsorptive and fasted conditions, where plasma insulin levels were =G15 ctU/ml, there was no correlation of either basal or postexercise forearm amino acid flux to arterial insulin concentration. The branched chain amino acids are presumed to play an important role in muscle protein metabolism during exercise [ 131. Leutine oxidation is known to increase in absolute terms as a primary energy source [2 1] during exercise. This study, although not directly measuring rates of branched chain amino acid transamination or oxidation, supports the central role of leucine as an important muscle metabolite during stress. At 30 min following recovery from exercise, during both postabsorptive and starvation conditions, the efflux of leucine is below the level seen in basal conditions [ 181.Within 1 hr of recovery, the efflux of leucine continues to be depressed, especially in the postabsorptive state, suggesting that increased leucine transamination and/or oxidation is occurring. Valine flux appears from our data to be little affected by exercise, while that of isoleucine is intermediate to that of valine and leucine. These alterations in branched chain amino acid uptake occurred under conditions which are independent of known hormonal effects on their metabolism [7]. The favorable impact of concurrent intravenous nutritional support upon muscle amino acid metabolism following submaximal exercise is demonstrated by the recovery
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JOURNAL OF SURGICAL RESEARCH: VOL. 36, NO. 5, MAY 1984
within 1 hr to basal flux patterns for total and essential amino acids. While it is not possible to differentiate whether substrate availability alone may have influenced the continued forearm uptake of amino acids at 1 hr, the study design did allow an assessmentof insulinization upon forearm amino acid metabolism. A differential insulin response (2040 pU/ml) was obtained with the 100% dextrose nonprotein calories versus the 50% dextrose/50% lipid calorie source. Although the number of observations obtained with these regimens was limited (n = 2, glucose; II = 3, glucose/lipid), we were unable to observe any differences in either the basal or postexercise recovery patterns of forearm amino acid flux during intravenous feeding. The present observations are of clinical interest in that an abbreviated period of submaximal exertion resulted in major deviations of forearm nitrogen balance in nonnutritionally supported subjects. While postabsorptive subjects demonstrated a return to basal flux patterns within 1 hr after exertion, this still represents a loss of approximately 1 pmole of protein/l00 ml of tissue per minute. Starved subjects, despite a reduction in basal output to approximately 0.2 pmole protein per minute [ 181,responded to submaxial exercisewith a 2300% increase in forearm amino acid loss that had not decreased within 1 hr after exercise.,Only the intravenously fed subjects attained forearm nitrogen balance during the postexercise recovery period and only at 60 min after recovery did the pattern of essential amino acid flux resemble that in the basal state [ 181.These observations imply that nutritional status affectsboth the magnitude and rate of recovery in exertion-induced muscle amino acid metabolism. Intravenous nutritional support appearsto impact favorably on this process although complete recovery may require a longer period than is generally appreciated. REFERENCES
1. Ahlborg, G., Felig, P., Hagenfeldt, L., Hendler, R., and Wahren, J. Substrate turnover during prolonged exercise in man. J. Clin. Invest. 53: 1080, 1974.
2. Andres, R. K., Zierler, K. L., and Anderson, H. N. Measurement of blood flow with volume in the forearm of man; with notes on the theory of indicator dilution and on production of turbulence, haemolysis and vasodilation by introvascular injection. J. Clin. Invest. 33: 482, 1954. 3. Aoki, T. T., Brennan, M. F., Fitzpatrick, G. F., and Knight, D. C. Leucine meal increasesglutamine and total nitrogen release from forearm muscle. J. Clin. Invest. 68: 1522, 1981. 4. Ballard, F. J., and Tomas, F. M. 3-Methylhistidine as a measure of skeletal muscle protein breakdown in human subjects: The case for its continued use. Clin. Sci. 65: 209, 1983. 5. Bennegard, K., Eden, E., Ekman, L., Schersten, T., and Lundholm, K. Metabolic balance acrossthe leg in weight-losing cancer patients compared to depleted patients without cancer. Cancer Rex 42: 4293, 1982. 6. Benneard, K., Rennie, M. J., Eden, E., Emery, P. W., and Lundholm, K. Influence of major surgical trauma on urinary excretion and efflux from the leg of 3methylhistidine. Proc. 5th Eur. Sot. Parent Ent. Nutr. A-103, 1983. 7. Buse, M. G., Biggers, J. F., Drier, C., and Buse, J. F. The effect of epinephrine, glucagon, and the nutritional state on the oxidation ofbranched chain amino acids and pyruvate by isolated hearts and diaphragms of the rat. J. Biol. Chem. 248: 697, 1973. 8. Calloway, D. H., Grossman, M. I., Bowman, J., and Calhoun, W. K. The effect of previous level of protein feeding on wound healing and the metabolic response to injury. Surgery 37: 935, 1955. 9. Clague, M. B., Keir, M. J., Wright, P. D., and Johnston, I. D. A. The effectsof nutrition and trauma on whole-body protein metabolism in man. Clin. Sci. 65: 165, 1983. 10. Cuthbertson, D. P. Observations on the disturbance of metabolism produced by injury to the limbs. Quart. J. Med. 1: 223, 1932. 11. Cuthbertson, D. P., McGin, J. L., and Munro, H. N. A study of the effect of overfeeding on the protein metabolism of man. IV. The effect of muscular work at different levels of energy intake, with particular reference to the timing of the work in relation to the taking of food. Biochem. J. 31: 2293, 1937. 12. Dohm, G. L., Kasparek, G. J., Tapscott, E. B., and Beecher, G. R. Effect of exercise on synthesis and degradation of muscle protein. Biochem. J. 188: 255, 1980. 13. Felig, P., and Wahren, J. Amino acid metabolism in exercising man. J. Clin. Invest. 50: 2703, 1971. 14. Holbrook, I. B., Gross, E., Milewski, P. J., Shipley, K., and Irving, M. H. N-methylhistidine excretion and myofibrillar protein breakdown in patients receiving intravenous or enteral nutrition. C/in. Sci. 59: 211, 1980. 15. Kontos, H. A., Richardson, D. W., and Patterson, J. L. Blood flow and metabolism of forearm muscle
LOWRY ET AL.: MUSCLE AMINO ACID METABOLISM in man at restand during sustainedcontraction. Amer. J. Physiol. 211: 869, 1966. 16. Lee, P. L. Y. Single-column system for accelerated amino acid analysis of physiologic fluids using five lithium buffers. Biochem. Med. 10: 107, 1974. 17. Long, C. L., Birklohn, R. H., Geiger, J. W., Betts, J. E., Schiller, W. R., and Blakemore, W. S. Urinary excretion of 3-methylhistidine: An assessment of musclecatabolism in adult normal subjectsand during malnutrition, sepsis,and skeletal trauma. Metabolism 30: 765, 1981. 18. Lowry, S. F., Horowitz, G. D., Rose,D., and Brennan, M. F. Forearm amino acid metabolism following starvation and intravenous refeeding in normal man. Surg. Forum 34: 93, 1983. 19. Lundholm, K., Bylund, A. C., Holm, G., and Schersten, T. Skeletal muscle metabolism in patients with malignant tumor. Eur. J. Cancer 12: 465, 1976. 20. Pozefsky, T., Felig, P., Tobin, J. D., Soeldner, J. S., and Cahill, G. F. Amino acid balance acrosstissues of the forearm in postabsorptiveman. Effectsof insulin at two dose levels. J. Clin. Invest. 48: 2273, 1969.
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21. Rennie, M. J., Edwards, R. H. T., Krywauryck, S., Davies, C. T. M., Halliday, D., Waterlow, J. C., and Millward, D. J. Effect of exercise on protein turnover in man. Clin. Sci. 61: 627, 1981. 22. Rennie, M. J., Edwards, R. H. T., HaIliday, D., Matthews, D. E., Wolman, S. L., and Millward, D. J. Muscle protein synthesis measured by stable isotope techniques in man: The effectsof feeding and fasting. Clin. Sci. 63: 519, 1982. 23 Rennie, M. J., and Millward, D. J. 3-Methylhistidine excretion and the urinary 3-metbylhistidine/creatinine ratio are poor indicators of skeletal muscle protein breakdown. Clin. Sci. 65: 217, 1983. 24. Ruderman, N. B. Muscle amino acid metabolism and gluconeogeneis. Ann. Rev. Med. 26: 245, 1975. 25. Snedecor, G. W., and Cochran, W. G. Statistical Methods, 6th ed. Ames, Iowa: Iowa Univ. Press,1967. 26. Tomkins, A. M., Garlick, I’. J., Schofield, W. N., and Waterlow, J. C. The combined effects of infection and malnutrition on protein metabolism in children. Clin. Sci. 65: 313, 1983.