FOLLOWING PHYSICAL TRRUMR(U) BRIGHAM AIND NOMEN'SHOSPITAL BOSTON MA DUNILMORE 96 DEC 97

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1A PROGRAM FOR THE STUDY OF SKELETAL MUSCLE CATABOLISMFOLLOWING PHYSICAL TRAUMA

.. ANNUAL REPORT

0 DTlCDouglas Wilmore, M.D. C ELECTEfl

December 6, 1987 SUA 98DSupported by

U.S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMANDFort Detrick, Frederick, Maryland 21701-5012

Contract No. DAMD17-81-C-1201

Harvard Medical School and the Brigham and Women's Hospital

75 Francis Street, Boston, Massachusetts 02115

Approved for public release; distribution unlimited

The findings of this report are not to be construed as an official Department

of the Army position unless so designated by other authorized documents.

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6c ADDRESS (Cy, State, and ZIP Code) 7b. ADDRESS (City, State, and ZIP Code)

75 Francis Street ., T IT: r

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8a. NAME OF FUNDING/SPONSORING 8b. OFFICE SYMBOL 9. PROCUREMENT INSTRUMENT IDENTIFICATION NUMBERORGANIZATION U.S. Army Medical (If applicable)Research & Development Comman. DAMD17-81-C-1201

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62772A 62772A874 AA 27811. TITLE (include Security Clanifation)A program for the Study of Sketetak Muscle Catabolism Following Physical Trauma...w.

12. PERSONAL AUTHOR(S)Douglas W. Wilmore, M.D.

13a. TYPE OF REPORT 13b. TIME COVERED 14. DATE OF REPORT (Yea, Month, Day) 15. PAGE COUNTAnnual FROM82/09 TO 849 . / ? " I 38

16. SUPPLEMENTARY NOTATION

17. COSATI CODES 18. SUBJECT TERMS (Contnue on reverse if necessary and identify by block number)FIELD GROUP SUB-GROUP Skeletal muscle proteolysis, glutamine, branched chain6- 11 amino acids, adrenergic regulation - -

IABSTRACT (Continue on reverse if necessary and identify by block number)The purpose of this work was to attenuate skeletal muscle proteolysis in the post-traumatic period. In the initial study, amino acid solutions were administered withor wit out lutamine supplimentation. Amino Acid administration at the dose of 0.624grams19kgour was associated with near nitrogen balance, maintenance of skeletal muscleintraclellular stores, and attenuation of hindquarter nitrogen loss

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Mrs. Vir~inia M. Millar ini 16AA-71?7 , 1 GRD-RMT-q

DO Form 1473. JUN 86 Previous etions are obsolete. SECURITY CLASSIFICATION OF THIS PAGE

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PAGE

I. Summary 1

-II. Foreword 2

III. The Effect of Supplemental Amino Acids in Altering 3Skeletal Muscle Proteolysis

A. Glutamine Administration 31. Materials and Methods 42. Results 93. Discussion 11

B. Branched Chain Amino Acid Supplementation 13

A. Materials and Methods 131. Results2. Discussion 14

IV. The Effect of Adrenergic Blockade on Skeletal Muscle 15Proteolysis

Introduction 15Epidermal Anesthesia 17Adrenergic Blockade 19Discussion 19

V. The Effect of Epinephrine Infusion on Muscle 20

Proteolysis and Intracellular Amino Acid Concentrations

VI. References 24 - 29

VII. Tables and Figures

1. Plasma Amino Acid Concentrations 302. Muscle Amino Acid Concentrations 313. Hindquarter Nitrogen Flux 324. Nitrogen Balance 335. Effects of Varying Concentrations of Branched Chain 34

Amino Acids on Nitrogen Metabolism6. Effects of Adrenergic Blockade on Nitrogen Metabolism 35

Following Operation7. The Effect of Hormonal Infusion on Plasma and Muscle 36 I]

Intracellular Glutamine Concentrations in the Dog8. The Effects of Infusion of Saline or Counterregulatory 37

Hormones Over 6 Hours into "Normal" Dogs

Figure 1: The Relationship Between Nitrogen Intake 38and Nitrogen Balance

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1.

I. SUMMARY

The purpose of this study was to attenuate skeletal muscle

proteolysis in the postraumatic period. In the initial study, amino

acid solutions were administered with or without glutamine supple-

mentation. Amino acid administration at the dose of 0.624 grams N/

kg . hour was associated with near nitrogen balance, maintenance

of skeletal muscle intracellular stores, and attenuation of hind-

quarter nitrogen loss. Glutamine enriched solutions were as

effective as standard balanced formulas in sparing body protein.

In a second study, amino acid formulas were constructed to

provide a range of concentrations of branched chain amino acids

(from 12%-44%). Isonitrogenous infusions were administered to animals

following operations and the effects of the various formulas

determined. No benefit was derived from branched chain enriched

solutions over a standard formula.

In additional studies, adrenergic blockade was achieved by

administering phentolamine and propranolol or utilizing high

epidural anesthesia. While blockade did not reduce nitrogen excre-

tion in the posttraumatic period, nitrogen efflux from the hind-

quarter was markedly attenuated. This is the first demonstration

of a relationship between the adrenergic nervous system and accel-

erated proteolysis. The significance of these findings is discussed.

IB I

2i

FOREWORD

In conducting the research described in this report. the investigator adheredthe "Guide for the Care and Use of Laboratory Animals", prepared by theCommittee on Care and Use of Laboratory Animals of the Institute of laboratoryAnimal Resources, National Research Council (DHEW Publication No. (NIH) 78-23,Revised 1978).

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I. V

3.

III. The Effect of Supplemental Amino Acids inAltering Skeletal Muscle Proteolysis

A. Glutamine Administration

Accelerated nitrogen excretion following moderate or severeinjury was first described by Cuthbertson over 50 years ago (1)and has since been recognized as a characteristic feature of themetabolic response to trauma. The site of the protein loss wasthought to be skeletal muscle, an hypothesis that was initiallyconfirmed by Levinson, who demonstrated the translocation ofskeletal muscle nitrogen to visceral protein in experiments using15N-labeled amino acids in injured rats (2). Subsequent investi-gations in humans documented an increased skeletal muscle releaseof amino acids from the uninjured extremities of thermally injuredpatients (3), a finding that corresponded with a marked uptake ofamino acids across the splanchnic bed (4).

With the development of microanalytic techniques and method-ology for sampling human skeletal muscle and other tissues, ithas been possible to define the alterations in plasma and freeamino acid pools in various tissues following injury (5). Wholeblood and skeletal muscle intracellular glutamine concentrationshave been found to decline markedly in response to a variety ofstresses (6), including sepsis (7) and elective operations (8).These alterations occur even when solutions containing glucose andamino acids are infused (9). The restoration of muscle intracellularglutamine levels is slow, with a return to normal concentrations,possibly signaling an end to post-injury convalescence. The declinein muscle glutamine is especially significant, since 80% of thefree amino acids of the body reside within the intracellularcompartment of skeletal muscle, and glutamine alone accounts forapproximately two-thirds of this free amino acid pool (excludingtaurine) (10). Whole blood glutamine concentrations also arehigher than levels of any other amino acid. Although a storageform of body nitrogen is not generally recognized, muscle andplasma glutamine represents a large free amino acid pool that appearsto be an important available form of amino acid nitrogen (11).

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Intracellular glutamine concentrations in skeletal muscledecrease rapidly in the dog following a standard surgical pro-cedure, similar to the response observed in humans (12). Thereis a 40% fall in intracellular glutamine concentrations within24 hours of standard operation; this depression is maintainedfor 72 hours and returns gradually to normal by the eighth post-operative day. Studies in pair-fed animals have demonstratedthat these postoperative alterations are not the result of shortterm nutritional depletion. The present study was designed: (a)to determine if the postoperative decline in muscle glutamineconcentrations in dogs could be prevented by the infusion of glutamineand/or standard intravenous amino acid solutions, and (b) to charac-terize the influence of amino acid infusion on nitrogen balance andon the efflux of amino acids from the hindquarter skeletal muscle.

1.Materials and Methods

Animal Care and Operative Procedures

Twenty-two mongrel dogs, weighing between 20 and 40 kg, wereobtained from a farm where they had been regularly exercised andscreened for parasites. All female animals were nonpregnant.While in our kennel, they were maintained in accordance with theguidelines of the Committee on Animals at Harvard Medical School andthe Committee on Care and Use of Laboratory Animals of the Institutefor Laboratory Animal Resources, the National Research Council (DHEWPublication #NIH 78-23, reviewed 1978). The animals were kept inindividual kennels at a constant temperature of 20'C, with 24-hourlight exposure. They were exercised for two hours every morning,provided with water ad lib, and given a single daily feeding between1:00 and 3:00 p.m. of Agway Respond 2000 Dry Dog Chow® (containsat least 25% protein, 10% fat, and the remaining calories as carbo-hydrate). Five to seven days were allowed for the dogs to acclimateto the kennel conditions, during which time they were trained torest quietly in a Pavlov stand. On the day before obtaining basalsamples, all food was removed from the kennel at 5:00 p.m. Afteran overnight fast, the dog was walked for at least 20 minutes, placedin a Pavlov stand, and a foreleg vein was cannulated. After the doghad rested in the stand for at least 20 minutes, a venous bloodsample was obtained for amino acid determination. Following rapidinduction of anesthesia with intravenous sodium thiopental (AbbottLaboratories, 5 mg/kg body weight), a biopsy of the vastus lateralis

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5.

muscle was obtained by the method of Bergstrom (5). The animalwas then taken out of the stand and a 5-ml sample of arterialblood was obtained from the femoral artery via percutaneouspuncture.

The animal was allowed to recover from the biopsy for aminimum of two days prior to the standard operative procedure.On the day prior to surgery all food was again removed from thekennel at 5:00 p.m. At 7:00 a.m. the dog was walked for 20minutes and then taken to the operating suite where it wasanesthetized with intravenous sodium pentobarbital (AbbottLaboratories, 30 mg/kg body weight). An andotracheal tube wasplaced, and the animal was allowed to spontaneously breathe amixture of oxygen and room air. The dog was placed on an operatingtable in a supine position, and a cannula placed by percutaneouspuncture into the external jugular vein and directed into thesuperior vena Cava. After noting the starting time, the infusionsolution was administered via this cannula by constant infusion(IMED pump t San Diego, CA) at 4 ml/hour . kg. Penicillin G(E.R. Squibb, Princeton, NJ; 600 mg) and Keflin ® (Eli Lilly,Indianapolis, IN; 1 gram) were given intravenously. The urinarybladder was catheterized, the initial urine sample discarded, andthe catheter was connected to a closed urine bag for 24-hourcollection. The abdomen and flanks of the dog were shaved, andthe skin washed with soap and water, and prepared with a povidoneiodine p.rep solution (Clinipad Corporation, Guilford, CT). Thedog was draped with sterile sheets and the abdominal cavity enteredvia a vertical infra-umbilical incision in females and a rightparamedial incision in males. The bowel was retracted into theupper abdomen, and the exposed retroperitoneum incised. The rightdeep circumflex iliac artery and vein and the medial sacral arterywere isolated by sharp and blunt dissection. A specially preparedcatheter consisting of a 6-cm segment of polyethylene tubing (2.08

mm OD) coated with silastic and linked to a 2.8 mm OD polyethylenecatheter was inserted 6 cm cranially into the aorta via the right

deep circumflex iliac artery. A similar catheter was inserted into

the middle sacral artery, its tip being positioned approximately 1

cm proximal to the bifurcation of the aorta, but distal to theinferior mesenteric artery. A third catheter was inserted into theinferior vena Cava via the right deep circumflex iliac vein and

positioned distal to the renal vein. All catheters were secured

and exteriorized through stab wounds in the flank. The abdomen

was then closed, and the animal was turned onto its left side. The

exterior catheters were cut to appropriate lengths, plugged with

blunt needles connected to intermittent injection ports (Jelco® ,

I

6.

Critikon, Inc., Tampa, FL), flushed with saline, filled withheparin (1,000 units/ml), and buried subcutaneously. Theinjection ports were positioned high on the flank of the animalunder the skin and in the approximate vicinity of the vertebralcolumn. This allowed access to the aorta and vena cava by per-cutaneous puncture of the injection ports of the catheters. Twofurther doses of Keflin ® (1 gram) were given 8 and 24 hours post-operatively via the venous catheter.

Following the operative procedure the animal was placed onits side, and body temperature was maintained with heat lampsand blankets during recovery from anesthesia. Approximately fivehours after the start of the infusion the animal was placed in aPavlov stand, and a solution of para-aminohippuric acid (PAH,0.5% w/v in saline) was infused at a rate of 0.76 ml/minute with aHarvard pump into the distal aorta through the medial sacral arterycatheter. After 40 minutes of dye infusion, simultaneous arterialand venous samples were obtained for measurement of amino acid andPAR concentrations. Three sample sets were drawn at 10-minuteintervals over a period of 20 minutes. The catheters were thenflushed, filled with heparin, and the animal was kept in a Pavlovsling. Twenty-three hours following the initiation of theexperiment, the hindquarter flux studies were repeated. After 24hours, the urine collection was terminated. The animal receivedintravenous sodium thiopental, as previously described, and biopsyof the vastus lateralis muscle in the leg not previously biopsiedwas performed. The intravenous infusion was terminated, and theanimal placed in a metabolic cage for the ensuing 24 hours whereit was offered water ad lib and no food.

Infusion Solutions

All animals received an infusion at a rate of 4 ml/hour • kg.Five control animals received 0.9% saline. Other animals were givencommercially available amino acid solution (FreAmine III ® , AmericanMcGaw) at two different concentrations designed to deliver approxi-mately 0.312 (N-2) or 0.624 (N-6) grams of nitrogen/24 hr • kg bodyweight. The higher dose was designed to provide the equivalent of4 grams of protein/24 hr • kg body weight. Three animals receiveda solution containing glutamine at 0.312 grams nitrogen/24 hr • kg.A final group (N-6) received an equal mixture of glutamine andFreAmine , providing nitrogen at 0.624 grams/24 hr • kg. The glutaminesolutions were made by dissolving L-glutamine (Sigma, St. Louis, MO)in distilled water to form a 0.157 M solution which was then adjustedto pH 6.8 with sodium hydroxide. This solution was sterilized byfiltration through a 0.22 PM membrane and stored at 40C for less

7.

than 24 hours. On the morning of utilization the solutions wereformulated at required concentrations in 2-liter bags (AmericanMcGaw) and maintained at 4*C until use. A 10-ml sample was takenfrom each bag at the end of the infusion and stored at -20°C foranalysis of nitrogen content. An additional 10-ml sample wasadjusted to pH 4.75 as described below and stored frozen foranalysis of glutamine content.

Preparation and Analysis of Bloodand Tissue Samples

Whole blood and plasma samples were deproteinized by com-bining with equal volume of ice cold 10% (w/v) perchloric acidand then centrifuging at 3000 rpm at 4*C-for 20 minutes. A 2-ml aliquot of the supernatant was buffered with 0.3 ml of 2Nsodium acetate (pH 4.75), adjusted to pH 4.75-4.90 with 5N potas-sium hydroxide, and brought to a final volume of 4 ml with distilledwater. The samples were stored at -20*C for later batch analysis ofglutamine and glutamate concentrations, with an enzymatic microfluoro-metric assay modified from the method of Lund (13).

During the muscle biopsy procedure, a stop watch was startedimmediately when the tissue was removed. The muscle was dissectedfree of fat and connective tissue and divided into two unequalportions. Both samples were weighed at least four times over theensuing two minutes, and the weight and time following biopsy wererecorded. Actual muscle wet weight at time zero was calculated fromthe best fit linear regression of weight plotted against time. Thesmaller sample (approximately 15 mg) was dried to a constant weight ina 900 oven, and the weight of dry, fat-free solids was obtained afterextraction in petroleum ether. This sample was then digested in250 Ul of IN nitric acid, and the chloride content was measured bytitration with silver nitrate using a semi-automated titrator(Radiometer, Copenhagen). Plasma chloride was also determined andintra- and extracellular water calculated by the method of Bergstrom(5). The second muscle sample (approximately 100 mg) was homogenizedin 0.5 ml of ice cold perchloric acid (10% w/v) with a PolytronHomogenizer (Brinkman, Westbury, NY). The homogenate was centrifugedand the supernatant prepared for enzymatic glutamine and glutamateanalysis.

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At the start of this study plasma and intracellular glutamineand glutamate concentrations were determined by an enzymatic methodpreviously described (11). Concentrations of other amino acids weredetermined by automated high performance liquid chromatography(HPLC) after pre-column derivatization with o-phethaldehyde. Allamino acids commonly found in proteins were quantitated exceptglutamine, glutamate, proline, cystine, and lysine. As the studyprogressed, techniques were developed for glutamine-glutaminatemeasurement using HPLC. Samples measured by the two techniques(enzymatic and HPLC) yielded comparable glutamine-glutamate concen-trations; hence, only HPLC analysis was utilized in the latter portionof the study. The concentration of PAH in the arterial and venoussamples was determined spectrophotometrically following deprotein-ization with 5% trichloroacetic acid (11-.

Nitrogen Determinations

Urine excreted during the 24 hours of infusion was collectedin the closed urinary collecting system and stored in acidified,refrigerated containers. Aliquots were stored frozen at -20*C forbatch analysis. The nitrogen content of the infusion solution andurine was determined in the same batch by the macro-Kjeldahl method(14).

Statistical Analysis and Calculations

Statistical calculations were performed on an IBM 4341 Computerutilizing a standard statistical package (Minitab, The PennsylvaniaState University, State College, PA, 1983). The results are expressedas mean±SEM. Paired and unpaired Student t-tests were used asappropriate. Analysis of variance was used for multiple group com-parisons. Regression analysis was performed using methods of leastsquares. Because of the small sample size in the groups receiving0.312 grams of nitrogen/24 hr • kg, most statistical comparisonswere only performed between the other groups.

Hindquarter bloodflow was calculated as previously described(11), and the rate was expressed per kg body weight to account forvariation in size of the animals. Amino acid flux rates werecalculated as the product of bloodflow and arterial-venous concen-tration differences. Three sets of samples were drawn, flux wascalculated for each set, and the mean of the three values determined(11). Total amino acid nitrogen in whole blood, plasma, and intra-cellular water was calculated by taking into account the nitrogencontent of each amino acid and summing the individual differences.

9.

2.Results

Plasma and Intracellular AminoAcid Concentrations

Plasma amino acid concentrations were measured preoperativelyand 24 hours following the standard operation. In the saline-treated animals, the total nitrogen content of the plasma wasunchanged by the operative procedure (Table I). The glutamineconcentration remained constant, but the branched chain aminoacids rose, the sum of their concentrations increasing from 326±21to 501t9 umol/l (p <0.01). In the animals receiving 0.624 gramsN/24 hr • kg, there was an upward trend in the plasma nitrogenconcentration that was statistically significant only in the groupreceiving the mixture of amino acids plus glutamine. The plasmaglutamine concentration also rose in this group. Branched chainamino acids were elevated in all animals receiving amino acidinfusions.

Skeletal muscle nitrogen concentrations declined during salineinfusion (Table II). This decrease in total amino acid nitrogenwas reflected primarily by a fall in glutamine from 21.48±3.21pmol/l intracellular water to 15.86±3.80 (p <.05). Although the sumof-the concentrations of non-essential amino acids diminished, thesum of total essential amino acids in the intracellular poolremained unchanged. No change in intracellular nitrogen or glutamineoccurred in animals receiving the larger dose of amino acid nitrogen(Table II). There was an upward trend in the intracellular concen-tration of branched chain amino acids with infusion of the higheramino acid loads, although statistical significance was achievedonly in the animals receiving the mixture of amino acids and gluta-mine. There was not a significant change in the total concentra-tions of essential and non-essential amino acids in these twogroups fullowing operation. In contrast to the animals receivingthe higher dose of nitrogen, the five animals infused with 0.312gm N/24 hr - kg did not consistently maintain the skeletal muscleintracellular nitrogen pool, regardless of the solution infused.Intracellular glutamine fell in three of the animals, remainedunchanged in one, and increased in one (data not shown).

Thus, providing amino acids at 0.624 gm N/24 hr • kg as anamino acid mixture with or without glutamine, maintained the skeletalmuscle intracellular amino acid pool. A decrease in the intra-cellular pool, which was characterized by a fall in intracellularglutamine, occurred consistently in the animals receiving salineand was variable in the animals receiving the lower dose of aminoacids.

10.

Hindquarter Nitrogen Flux

Net hindquarter amino acid flux, calculated as the sum ofthe nitrogen flux of the individual amino acids, averaged-19.05±4.06 .mol N/minute kg when measured at 6 hours post-

operation in the animals receiving saline. This was significantlygreater than the efflux rates of -7.70±5.90 and -6.50±1.81 pmol NIminute - kg observed in the two groups of animals receiving thehigher doses of amino acids (Table III). However, glutamine effluxfrom the hindquarter was unchanged among these three groups. Incontrast, branched chain amino acids were released in the salinedogs, but taken up in both groups of animals receiving the higherdoses of amino acids. Hindquarter exchange of branched chainamino acids appeared to be related to the rate of branched chainamino acid administration; the hindquarter demonstrated branchedchain amino acid release in the saline-treated group, balance withthe solution containing amino acids plus glutamine, and greateruptake in the group receiving the highest branched chain amino aciddose. In the five animals receiving 0.312 gm N/24 hr • kg, there wasnot a significant alteration in hindquarter nitrogen efflux comparedto the saline-treated dogs. However, there was considerable varia-tion in these flux data, and the number of animals studied was small.Hindquarter amino acid flux studies 24 hours following operationdemonstrated no differences between groups (Table III).

Nitrogen Excretion

Nitrogen excretion in the five animals infused with saline was0.492±0.022 gm N/24 hr kg. In the 6 animals receiving the highestdose of the commercial amino acid mixture, measured nitrogen intakewas 0.632±0.001 gm N/24 hr • kg, and nitrogen excretion averaged0.684±0.031 (Table IV). In the 6 animals receiving the solutionmade up of one-half commercial amino acid solution and one-halfglutamine, nitrogen intake was comparable but excretion was greater,averaging 0.775±0.019 gm N/24 hr • kg (p <0.05). Nitrogen balancein these two groups was significantly less negative than in theanimals receiving saline, averaging--0.052±0.031 and -0.140±0.022 gmN/24 hr • kg, respectively. In the five animals that receivedapproximately 0.312 gm N/24 hr • kg, the average nitrogen excretionwas intermediate between that observed in the saline controls and inthe animals receiving the larger quantity of infused nitrogen. Takentogether, these studies demonstrated that nitrogen balance approachedequilibrium as the quantity of administered nitrogen increased(Figure ). When glutamine was combined with a commercial glutamine-free amino acid solution, the effects on nitrogen balance were addi-tive. When summed together, the nitrogen retained in response tothe infusion of commercial amino acids or glutamine alone accountedfor the nitrogen retained when the solutions were combined.

11.

3. Discussion

Operative stress indogs stimulates net skeletal muscleprotein breakdown, as evidenced by negative nitrogen balanceand increased amino acid efflux from the hindquarter in asso-ciation with a fall in the intracellular skeletal muscle free

amino acid pool. Previous studies have demonstrated thatprotein wasting is not related to fasting or anesthesia, but isclearly a response to the operative stress (12). The releaseof amino acids from the hindquarter 6 hours postoperation inthe saline-treated group was approximately 6 to 8 times thatobserved in chronically-catheterized, postabsorptive dogs studiedunder basal conditions (11). Moreover, the rate of hindquarternitrogen release cannot be accounted for by depletion of theintracellular free amino acid pool and therefore must reflectnet skeletal muscle proteolysis.

Provision of amino acids in the perioperative period offsetthe nitrogen loss, maintained or increased plasma amino acidconcentrations, and diminished the fall in the skeletal muscleintracellular free amino acid pool. These effects appear to berelated to the quantity of amino acid nitrogen infused. Wholebody and hindquarter nitrogen losses were greatly decreased atthe highest amino acid doses, which also maintained intracellularpools of glutamine and other amino acids. These results differfrom the findings reported by Askanazi and associates (9), whodescribed a decline in the intracellular concentrations of glutamineand other amino acids in patients after hip replacement that couldnot be reversed by the infusion of dextrose and amino acids. Ourstudy would suggest that this may in part be related to the quantityof amino acids infused and/or the lack of glutamine in the infusate.Infusion of lower concentrations of amino acids (0.312 gm N/24 hrkg), either as glutamine alone or as FreAmine ® , failed to maintainthe intracellular amino acid pool in three of the five animalsstudied. In contrast, the higher rate of amino acid infusionstabilized or increased the intracellular pool. Thus, it appearsthat an adequate quantity of administered nitrogen can maintainthe skeletal muscle intracellular amino acid pool postoperatively.

The change in the intracellular free amino acid pool in saline-infused animals, largely attributable to a rapid fall in glutamine,was prevented when adequate nitrogen was provided. This occurredeven when glutamine was not present in the commercially availablesolution. The mechanism by which intracellular glutamine was maintained

12.

under these circumstances is unclear, although it seems probablethat glutamate substrate for glutamine synthesis was derived fromthe branched chain amino acids via transamination. For unexplainedreasons, net glutamine efflux was similar in all groups. Hind-quarter release was not accelerated by branched chain amino acidsor attenuated by the provision of glutamine in the amino acidsolution. The results in this postoperative model differ fromreported effects of branched chain amino acid infusion in normalhumans, in whom branched chain amino acid forearm uptake wasassociated with accelerated glutamine release (15).

Although there were marked differences in composition of thetwo amino acid solutions administered at the rate of 0.624 gm NI24 hr • kg, hindquarter nitrogen efflux-Vas comparable in bothgroups of animals. This occurred even though the quantity ofessential amino acids and branched chain amino acids in thebalanced solution was twice that in the glutamine-containingsolution. Thus, in this experimental model of operative stress,glutamine supplementation of a balanced amino acid formula was atleast as effective as standard balanced formula in diminishinghindquarter nitrogen loss.

In the dogs that received saline, branched chain amino acidswere released from skeletal muscle. Quantitative transfer ratescalculated from these data suggest that a marked uptake of branchedchain amino acids must have occurred in visceral organs, mostprobably the liver, during the early postoperative period. Theprovision of branched chain amino acids appeared to offset thistranslocation by perhaps both meeting visceral requirements andreversing skeletal muscle efflux. A quantitative relationship alsoexisted between hindquarter nitrogen balance and preservation ofthe intracellular nitrogen pool. When intracellular pools weremaintained, the hindquarter was near nitrogen equilibrium; when salinewas administered, amino acid concentrations in the intracellularpool were markedly depleted and there was a marked loss of hind-quarter nitrogen. Although the relationship between skeletalmuscle proteolysis and nitrogen concentration in the free aminoacid pool is unknown, these data suggest that skeletal musclenitrogen balance is related to the intracellular amino acidconcentration. Further studies are necessary to determine ifthis relationship is causal or circumstantial.

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13.

B. Branched Chain Amino Acid Supplementation

As previously noted, accidental injury, major operativeprocedures, and sepsis are characterized by negative nitrogenbalance, skeletal muscle amino acid efflux, and a fall inintracellular free amino acid concentrations. Branched chainamino acids (BCAA - leucine, isoleucine, and valine) are theonly essential amino acids that are primarily oxidized in skeletalmuscle (16). The amino group is transaminated with pyruvateor a-ketoglutarate to synthesize alanine or glutamate (17). Thisreaction yields branched chain ketoacids, compounds which exertspecial regulatory effects on skeletal muscle protein catabolismand synthesis, and thus minimize net protein breakdown (17).While it is clear that BCAA (primarily leucine) can reduce netprotein degradation in vitro, the effect of amino acid formulassupplemented with additional BCAA on skeletal muscle breakdownin catabolic patients remains controversial. For example, Freundand Cerra have administered solutions containing up to 45% BCAAand reported beneficial effects on plasma amino acid concentrationsand nitrogen balance (19,20). In contrast, Daly and associatescompared the effects of a standard amino acid solution containing25% BCAA and a 45% BCAA enriched solution in a randomized, blindedstudy in postoperative patients. Nitrogen balance was comparablein the two groups of patients regardless of the composition of theamino acids infused (21).

The hypothesis that administration of BCAA diminishes theskeletal muscle catabolic response was tested by infusing varyingconcentrations of BCAA and studying skeletal muscle amino acidexchange and nitrogen metabolism following a standard catabolicstress.

I.Materials and Methods

Mongrel dogs were utilized in this experiment as previouslydescribed. The dogs weighed between 18 and 35 kg and underwenta preoperative muscle biopsy of the vastus lateralis to quantitateintracellular free amino acid levels. Five days later a standardlaparotomy and retroperitoneal dissection were performed with theimplantation of polyethylene catheters into the aorta and venacava. During the 24-hour perioperative period, the dogs wereinfused via an external jugular catheter with either saline ordifferent isonitrogenous amino acid solutions, containing increasingquantities of essential and decreasing quantities of non-essentialamino acids. The BCAA varied in concentration between 12 and 42.4%

of the total amino acids infused. Hindquarter amino acid flux was

14.

determined at 6 and 24 hours postoperation, as previouslydescribed. A second muscle biopsy was obtained 24 hourspostoperation. Urine was collected via an indwelling bladdercatheter for the 24-hour perioperative period and was analyzedfor total nitrogen content.

2. Results

Nitrogen balance was improved with amino acid infusionbut only minimal differences were noted between the varioussolutions utilized (Table V). At 6 hours postop, nitrogenefflux (calculated as the sum of individual amino acid fluxesmultiplied by their respective number of nitrogens) was reducedin all treatment groups compared to saline controls. BCAA werereleased from skeletal muscle during saline infusion, but hind-quarter BCAA uptake occurred at 6 hours in all treatment groupsand was related to BCAA concentration. At 6 hours hindquarterBCAA uptake correlated with skeletal muscle nitrogen retention.This effect was small and unrelated to nitrogen balance. At 24hours hindquarter flux was similar in all groups. Skeletal muscleintracellular free amino acids were maintained at preoperativelevels in all animals infused with amino acids but fell in the salinetreated group.

3. Discussion

This standard operative procedure increases skeletal musclenitrogen efflux six to eight times more than that which occurs inthe basal, postabsorptive animal. In this catabolic model wholebody nitrogen loss was minimized, skeletal muscle amino acidefflux diminished, and intracellular free amino acid concentrationspreserved with amino acid infusions. Despite alterations in concen-trations of BCAA from 12 to 42%, no major effects on total bodynitrogen economy were observed. These results are similar to thosefindings of Daly et al. in catabolic surgical patients (21). Presentavailable balanced intravenous formulas appear to maximize theanti-catabolic effects of amino acid administration.

15.

IV. The Effect of Adrenergic Blockadeon Skeletal Muscle Proteolysis

A. Introduction

With the stress of injury, or injury complicated byinfection, a variety of neurohormonal responses occur. Thesechanges characterized by a rise in circulating levels ofglucagon, glucocorticoids, and catecholamines, are associatedin time by a series of well recognized responses affectingthe metabolism of carbohydrate, protein, and fat. Skeletalmuscle proteolysis and negative nitrogen balance reflect loss ofan important component of body protein that limits optimalfunction and may impair recovery. One method of minimizingskeletal muscle breakdown following injury is to provide adequatenutrients which will optimize net skeletal muscle synthesisduring stress, and thus minimize skeletal muscle breakdown.This approach has been tested by evaluating the effects ofadministered nutrients on protein catabolism. The nutrientsstudied to date include "ketone" bodies (22) (see Annual Report#2)A and specialized amino acid formulations (see SectionI.aofthis report). Although effects of nutrient administration couldbe observed, such approaches were not without their own limita-tions (these include the energy costs of nutrients, transport,and metabolism, and the potential side effects of the therapy).Moreover, the particular advantages gained by administeringbalanced amino acid formulas occurred in large part becauseof the effects of the "mass balance" of nitrogen. Thebenefit(s) of amino acid administration on skeletal musclemetabolism were achieved by infusing relatively large quantitiesof nitrogen (4 grams protein equivalent/kg . day), which wasaccompanied by the increased urinary excretion of nitrogen. Evenwith amino acid administration, glutamine efflux from skeletalmuscle remained accelerated. These data support nitrogen turnoverstudies performed in traumatized and septic patients which demon-strated that protein degradation was accelerated; feeding increasedsynthesis rates to approach or match accelerated rates of proteincatabolic rates (23). The catabolic process continued unabatedfollowing feeding, but net body protein loss was minimized by food(nitrogen) intake.

* Annual Report ,August 1981- September 1982

16.

Because of the limitations associated with the food intakeand the lack of specific regulatory effects on protein metabolismin the catabolic period using nutrients such as ketone bodies orspecialized amino acid formulas, we have attempted to define therole of specific mediators and modulators on the protein catabolicresponse to injury. Once these mediators can be identified andtheir role defined, it should be possible to modulate the proteincatabolic response and evaluate the benefits of such treatment onprotein catabolism, skeletal muscle function, and the eventual

outcome of patients receiving such therapy.

Hormones exert major regulatory effects on protein metabolism.Of the counter-regulatory hormones, glucagon exerts its effectsprimarily on the liver and is not thought to mediate skeletal muscleproteolysis (24). Cortisone is known to increase following stressand is associated with accelerated gluconeogenesis and proteolysis(25). However, the administration of glucocorticoids alone does notaccount for the alterations in protein metabolism which occurfollowing injury; synergistic effects of all three counter-regulatoryhormones appear necessary to produce most of the posttraumaticresponses commonly observed (26).

Catecholamines are elevated following injury and have beenassociated with posttraumatic hypermetabolism (27). Unlike mosttissues of the body, however, skeletal muscle does not possessdetectable sympathetic innervation. Yet, it contains adrenergicreceptors and is responsive to adrenergic agents (28). It is possiblethat circulating catecholamines (primarily epinephrine) secretedby the adrenal medulla are major effectors. Also, norepinephrinereleased from sympathetic nerve endings may "spill over" into thebloodstream and mediate skeletal muscle adrenergic responses.Additional effects could result from neurotransmitters releasedfrom the innervation of the nearby smooth muscle cells of theskeletal muscle vasculature which diffuse through the local inter-stitial fluid compartment to affect skeletal muscle. Yet, anotherexplanation of possible action is that catecholamines may activateprostaglandins in regional skeletal muscle beds (29). Thesecompounds then serve as a (the) signal which initiates proteolysis(30).

Regardless of the source of the catecholamines and mechanismof action, adrenergic stimulation appears to be primarily respon-sible for some of the major alterations observed in skeletal musclefollowing injury. For example, dogs were infused with glucose tomaintain fixed hyperglycemia (using the glucose clamp technique)

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17.

and then received isoproterenol, a 5-agonist. Although insulinconcentrations increased two-fold, total body glucose disposalwas reduced to one-half mean control value. Skeletal muscle glucosedisposal determined by simultaneous hindquarter flux was reduced60% (31). In seriously injured patients the maximal glucosedisposal rate during euglycemia and hyperinsulinemia was 9.17±0.87 mg/kg • minute, significantly less than the 14.3±0.78 observedin the age-matched normal controls (32). Using forearm fluxtechniques a major portion of this tissue insulin resistance waslocalized to skeletal muscle (33). These total body and forearmchanges have been simulated in normals with epinephrine infusion (34).

B. Epidural Anesthesia

While a number of techniques are available to block sympatheticnervous system outflow, one particularly -applicable approach ishigh epidural anesthesia. This technique requires placement of asmall cati&ater in the epidural space which is used to administerregional anesthesia. Utilizing this technique in patients under-going abdominal hysterectomy, the elaboration of cortisol and cate-cholamines was significantly attenuated and many of the commonlyobserved features of the posttraumatic response blocked (35). Inthis study we administered high epidural anesthesia to our standardposttraumatic model to evaluate the effects of sympathetic blockadeon posttraumatic responses.

Materials and Methods

Eleven mongrel dogs were studied. Approximately two weeksprior to study, six of the animals underwent anesthesia and opera-tive placement of an epidural catheter. Using short actinggeneral anesthetic (pentothal 0.3 mg/kg) and strict aseptic tech-nique, a 2-cm vertical incision was made over the L7-Sl vertebraeand deepened until the ligamentum flavum was reached. A 17-gaugeTuohy needle was inserted into the forearm between L7-Sl, and thebevel at the end of the needle positioned to allow the epiduralcatheter to be directed to the desired position. A 20-gauge teflonepidural catheter (Deseret, Utah) was passed through the needle anddirected cranially until approximately 5 cm lay within the epiduralspace. This position was chosen because our early experiencesshowed it to be the most reliable for achieving anesthesia to theheight desired while still maintaining the adequate caudal block.The catheter was tethered to the ligament by a silk suture toprevent displacement, and the suture was secured to the catheterwith a drop of epoxy glue. A piece of sterile teflon tubing wasused as an outer sleeve around the catheter to prevent kindking.The catheter was trimmed to the appropriate length and attached to

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18.

a blunt needle which, in turn, was connected to an intermittentinjection port (Jelco). This was placed subcutaneously alongthe lateral aspect of the spinal column to allow repeated injectionof anesthesia via percutaneous puncture. The skin wound was closedand the animal allowed to recover for at least 10 days. Three tofive days prior to operation, biopsy of the vastus lateralis musclewas performed in all animals. On the day of study, all animalsunderwent general anesthesia, a standard abdominal exploration,retroperitoneal dissection, and implantation of aortic and cavalcatheters as previously described. The animals with the epiduralcatheters received epidural anesthesia before the induction of thegeneral anesthesia.

Bupivacaine (Marcain, Sterling Drugs) 0.5% was the anestheticagent used for epidural anesthesia because it possessed a relativelylong duration of action. The dose required to produce anesthesiato the level of T4 was 0.3 ml/kg administered over 2-3 minutes;repeated doses of 0.15 ml/kg at 2-1/2 hour intervals were necessaryfor maintenance. Adequacy of anesthesia was judged by neurologicalassessment which confirmed loss of reflexes and sensation to theappropriate dermotomes. Barbiturate anesthesia was then administeredand the standard operative procedure and catheter implantation per-formed in the manner previously described. In the postoperativeperiod, assessment of the level of the epidural anesthesia was madeat hourly intervals and additional anesthesia administered tomaintain high epidural anesthesia, including the T4 through SI segments,for 24 hours. Blood levels of cortisol and glucose were monitored toconfirm the adequacy of the epidural blockade.

At 6 and 24 hours postoperation, all animals underwent hindquarterflux studies as previously described. At 24 hours a muscle biopsywas taken for amino acid analysis, as previously described, and theresults compared with preoperative values. Urine was collected via abladder catheter and urinary nitrogen quantitated.

Results

All animals receiving the epidural anesthesia were maintainedwithout difficulty and tolerated the operative procedure andanesthesia well. In addition to the neurological signs of highepidural anesthesia (bilateral Homer's sign was frequently observed),blood glucose failed to rise following operation (6-hour glucose was120 mg% in the control animals versus 90 mg% in the epidural group)and cortisol at 4 and 6 hours postoperative remained near normal

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limits (4-6 ug/dL versus 16-25 ug/dL in controls). That both markersremained normal indicates An adequate postoperative sympathetic blockade(35).

At 6 hours postoperation, the blood amino acid nitrogen levelswere similar in the epidural and saline control groups (Table VI).However, hindquarter nitrogen efflux was markedly reduced in the

animals receiving epidural anesthesia, averaging less than 50% ofthe amino acid nitrogen released in controls. This was primarily

due to the decrease in alanine flux; release of glutamine and theBCAA was similar between groups. Skeletal muscle intracellularconcentrations of glutamine fell in both groups. In spite of themarked effect of epidural anesthesia on hindquarter flux, urinarynitrogen excretion was similar in both groups.

C. a- and $-Adrenergic Blockade

Combined adrenergic blockade was carried out in five animals._The blockade was initiated 30 minutes before operation and maintainedthroughout the 24-hour study period. Phentolamine was administeredas the c-blocker (given as a 0.6 mg/kg loading dose followed by0.45 mg/hr • kg constant infusion), and propranolol was used as theB-blocker (2.8 g/kg loading dose, 2.1 mg/hr • kg constant infusion).These doses have been shown to adequately block a- and B-responses inthe dog (36).

Results

The blockade was tolerated well by the animals. At 6 hourswhole blood amino acid nitrogen content was markedly reduced (TableVI). Hindquarter nitrogen efflux was also significantly less thanthe controls, and this was primarily reflected in a decrease inalanine release. Intracellular glutamine concentration diminishedin three of the five animals studied and increased in the other twoanimals. In spite of these changes, however, urinary nitrogen losswas not significantly altered by the a- and B-blockade.

D. Discussion

This is the first report that net skeletal muscle proteolysiscan be decreased following a major catabolic insult by altering communi-cation between the sympathetic nervous system and skeletal muscle.Not all aspects of skeletal muscle catabolism were affected; glutamineefflux continued and intracellular glutamine concentration consistentlyfell. Simultaneously, the demand for alanine (presumably as a gluco-enogenic precursor) was markedly reduced. That diminished net skeletalmuscle breakdown occurred following adrenergic blockade is confirmedby reduced excretion of creatinine in the experimental animals.Creatinine is produced by muscle and reflects, in part, skeletal musclebreakdown rate (37). Creatinine excretion averaged 0.039±0.002grams/day • kg in the control animals and fell to 0.030±0.002 and0.031±0.001 in the two groups receiving adrenergic blockade (p <0.01).

20.

This occurred at a time when blood creatinine concentrations remainedconstant or fell during the perioperative period.

In spite of the ability to affect skeletal muscle proteolysis,urinary excretion of nitrogen remained elevated. This may be dueto multiple mechanisms which affect hepatic gluconeogenesis andureagenesis which were not affected by skeletal muscle adrenergicblockade. One such mediator is glucagon which has potent gluco-neogenic effects and may be the mediator of the accelerated ureaproduction. The nitrogen precursors for the urinary nitrogen excretedmust have originated from other sources (visceral organs) or werederived from plasma amino acid (and possibly plasma proteins). Thismay have been the reason for the marked fall in the plasma aminoacids in the animals undergoing a- and B-tlockade. While furtherstudies are necessary to determine the source of the excreted nitrogenand mediators of the visceral changes observed, the importance ofthese findings should be emphasized: skeletal muscle proteolysis canbe diminished in the posttraumatic state by physiologic manipulationof the adrenergic nervous system.

V. The Effect of Epinephrine Infusion on MuscleProteolysis and Intracellular Amino AcidConcentrations

Several studies indicate that the counter-regulatory hormones,cortisol, glucagon, and the catecholamines, affect the body in amanner which is consistent with what is found in injured patients.It has been hypothesized that the alteration in the hormonal milieuis the cause of the metabolic response to injury. It is believedthat elevated levels of these hormones have a synergic effect on thebody which causes accelerated muscle protein degradation, an effluxof amino acids from the periphery to the viscera, and increasedhepatic uptake of amino acids and gluconeogenesis. This conceptis gaining more support from in vivo studies in which investigatorshave infused these hormones into healthy humans and animals. It istheorized that such infusion will create a pseudo-injury state inhealthy individuals if these hormones are responsible, at leastin part, for the metabolic alterations that occur following injury.Sacca and his associates (38), from studies in which glucagon,epinephrine, and cortisol were infused into normal dogs, concludedthat"changes in glucose metabolism in circumstances in which severalcounter-regulatory hormones are elevated (e.g. 'stress hyperglycemia')are a consequence of synergic interactions among these hormones."Bessey et al (26) found that infusion of these three hormones intohealthy volunteers resulted in negative nitrogen balance. Compari-son of these results with control studies in which normal saline wasinfused into subjects receiving the same nitrogen intake as thevolunteers above received, indicates that the results were not due to

I

21.

bedrest alone or the experimental conditions. Bessey andassociates also found that whole-body protein catabolism waselevated relative to synthesis in volunteers receiving thehormonal infusion. Hence, present evidence supports thehypothesis that the synergism of the counter-regulatoryhormones is a major mediator of the metabolic response to injury.This concept is supported by the findings of studies utilizingadrenergic blockade (Section IV) which demonstrate that skeletalmuscle breakdown is diminished following interruption ofcommunication between the sympathetic nervous system and skeletalmuscle. This experiment evaluates the effect of infusion ofepinephrine with or without other counter-regulatory hormones(glucagon and cortisol) on skeletal muscle amino acid metabolism.

Materials and Methods

Experimental Protocol

Male and non-gravid female dogs weighing between 20 and 35kilograms were used. Each dog had been operated upon aspreviously described (Section II?)for chronic implantation ofaortic and vena caval catheters capped with subcutaneous injectionports. The dogs were fed Agway Respond Dry Dog Chow andreceived water ad libitum. All dogs used in the experimentshad completely recovered from their operations, were healthy,and had been trained to rest quietly in the Pavlov stand.

Each dog was studied on two days separated by three to fourdays. On the night before each study day, food was withdrawnfrom the dogs at 5:00 p.m. so that they could be studied in thepostabsorptive state. On the "Basal Study" day the dog was walkedand then placed in a Pavlov dog stand, and temperature taken.Following this, the injection ports of the catheters were enteredby subcutaneous puncture with a 21-gauge needle. The catheterswere cleared with saline, and a venous blood sample was taken formeasurement of plasma glutamine concentration and hematocritdetermination. Finally, the dogs were anesthetized with sodiumpentobarbital, and a needle biopsy of the vastus lateralis wastaken for measurement of basal muscle intracellular glutamine con-centration.

On the "Infusion Day" the dogs were treated as they were onthe Basal Study day, up to and including the point where a venous

%blood sample was taken. After this, one of five infusions was6begun and continued for 6 hours (see below). Venous blood samples

were taken at two, four, and six hours from the start of theinfusion for measurement of amino acids and glucose concentrations.

!r

22.

At six hours after commencement of the infusion, the dogs wereanesthetized, and a needle biopsy of the vastus lateralis ofthe leg which had not been biopsied on the Basal Study day wastaken. This was done for measurement of post-infusion muscleintracellular glutamine concentration.

Infusions

1. Isotonic saline (4 ml/kg dog weight . hour).

2. Isotonic saline with epinephrine (50 ng/kg dogweight • minute).

3. Isotonic saline with glucagon (3 ng/kg dog weightminute).

4. Isotonic saline with epinephrine + glucagon (samedoses as above).

5. Isotonic saline with epinephrine + glucagon (samedoses as above) + cortisol (200 Ug/kg dog weighthour).

Results

With infusion, plasma glutamine concentration did not changewith saline, epinephrine or glucagon alone (Table VII). Plasmaglutamine concentration fell significantly with epinephrine plusglucagon or "triple hormones" (epinephrine + glucagon + cortisol).Muscle intracellular glutamine concentration tended to fall duringthe 6-hour study but did not change significantly with any treatment.

Because the major alterations appeared to be associated withthe triple hormonal infusion, the alterations which occurred wereevaluated in more detail (Table VIII). Hormonal infusion wasassociated with a fall in concentration of whole blood and intra-cellular amino acid nitrogen. During this same time period, plasmaglucose rose from 111±5 mg/dl to 180±12 (p <0.001). However, noalteration in efflux of hindquarter amino acids was noted withhormonal infusion; all flux values were indistinguishable fromzero, suggesting that the hindquarter remained in amino acid balanceduring these experiments.

Discussion

These results demonstrate that some but not all of the metaboliceffects observed in the postoperative model can be mimicked by theinfusion of epinephrine in combination with other counter-regulatoryhormones. The effects of hypoaminoacidemia must be due to accelerated

•S

23.

amino acid clearance by visceral tissues mediated via thesehormones. Glucagon has been described as mediating aminotransport into the liver; this effect appears augmented byepinephrine and cortisol.

In spite of these hormonal effects, posttraumatic hind-quarter nitrogen efflux observed at 6 hours could not reproducethese changes in hormonal environment. While some skeletalmuscle effects may have occurred (note decrease in skeletal muscleintracellular nitrogen), these changes could be explained byaugmented skeletal muscle bloodflow which could lower intracellularconcentrations via changes in concentration gradients and "washouteffects."

This study does not support the hypothesis that epinephrineserves as the mediator of skeletal muscle- proteolysis (in vitrostudies suggest B-stimulation of skeletal muscle augments synthesis,not breakdown, of protein) (39), and coupled with the findingspresented in Section 2, points to norepinephrine mediation as thestimulus for skeletal muscle catabolism.

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TABLE VII

The Effect of Hormonal Infusion on Plasma andMuscle Intracellular Glutamine Concentrations in the Dog

(Mean±SEM)

- PLASMA (Vjmol/L) MUSCLE (mmol/L)

-N Before After Before After

Saline 7 879t98 893±54 18.07±1.71 15.33±1.4

Epinephrine 2 727±211 691±111 24.28±7.16 20.91±4.56

Glucagon 4 843±113 753±121 23.62±3.45 25.40±1.38

Epi + Glucagon 6 673±55 514±57* 20.56±3.10 18.65±1.85

Epi + Glucagon 7 771±57 467±49**- 18.81±1.75 14.05±2.90*wx

+ Cortisol

-*p<O.OS, different from before by paired t-test.

**p<O.O0l, different from before by paired t-test.

***p<O.O 54 , different from before by paired t-test.

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DISTRIBUTION LIST

4 copies CommanderLetterman \rmy Institute of

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