(CANCER RESEARCH 48, 2698-2702, May 15, 1988)

Effects of Branched Chain Amino Acid-enriched Total Parenteral Nutrition onAmino Acid Utilization in Rats Bearing Yoshida Sarcoma1

Lisa E. Crosby, Bruce R. Bistrian,2 Pei-ra Ling, Nawfal W. Istfan, George L. Blackburn, and Stacy B. Hoffman

Laboratory of Nutrition/Infection, Cancer Research institute, New England Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02215

ABSTRACT

We have studied the ability of branched chain ammo-acid enriched

total parenteral nutrition solutions to improve nutritional status withoutstimulating tumor growth. Protein kinetics, nitrogen balance, tumorkinetics, fractional synthetic rates of individual tissues, and albuminsynthesis were compared in male Sprague-Dawley rats (125-145 g) thathad either s.c. Yoshida sarcoma (n = 15) or sham implantations (n =

18). Ten days postinjection, rats were randomly assigned to 2 diet groupsand given parenteral infusions of 4 days at 170 kcal/kg-body wi day asdextrose and 2 g N/kg-body Ht day as either 19 or 50% branched chainamino acid-enriched diet. During the last 4 h of feeding, protein kineticvalues were studied using a constant infusion of [l4C]tyrosine. Plasma

tyrosine appearance, synthesis, and breakdown were unchanged bybranched chain amino acid infusion. Percentage of tyrosine flux oxidizedand tyrosine oxidation decreased (/' < 0.05) and net tyrosine balanceimproved (/' < 0.05) in rats receiving the branched chain amino acid-

enriched diet. Greater nitrogen balance and lower tumor growth rateswere also found in branched chain amino acid-infused rats although not

statistically significant. Tumor ¡ntracellular specific activity was significantly higher in tumor animals receiving crystalline infusions, suggestinggreater tumor protein breakdown with branched chain amino acid-en

riched infusion. Fractional synthetic rates of liver, muscle, and tumorwere unchanged. Hence, branched chain amino acid-enriched total par

enteral nutrition increases amino acid utilization for net protein synthesisprincipally by reducing oxidation without stimulating tumor growth.

INTRODUCTION

Cancer cachexia is recognized as a primary cause of death inmany types of cancer (1). In fact, protein malnutrition is thesingle most common secondary diagnosis in cancer patients (2).Failure to prevent protein malnutrition increases the risk ofdeveloping complications, aggravates antineoplastic therapy,reduces the quality of life, and increases morbidity and mortality(2-5).

Although malnutrition may result from interference withgastrointestinal function, the usual cachexie syndrome developing in the course of malignant disease often cannot be explained by depression of food intake due to local factors aloneand behaves as if it were the result of a systemic alterationincluding anorexia induced by the tumor (6). Furthermore, theweight loss accompanying cancer in humans bears no consistentrelationship with the type, duration, site, size, or number ofmétastases(7).

Recent evidence indicates a similarity between the metabolicresponse to injury and active tumor growth (8). This similarityraises interest in a possible role for BCAA3 in modifying themetabolic response of the tumor-bearing host. When BCAAwere given as the sole source of amino nitrogen after injury it

Received 5/4/87; revised 10/13/87, 1/11/88; accepted 2/8/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

'Supported in part by Grants CA35352 and CA41535 from the National

Cancer Institute.3To whom requests for reprints should be addressed, at Laboratory of Nutri

tion/Infection, New England Deaconess Hospital, 194 Pilgrim Road, Boston,MA 02215.

3The abbreviations used are: BCAA, branched chain amino acids; TPN, total

parenteral nutrition.

reduced the loss of body protein by 15-80% in rats (9-12) andman (13, 14). In vitro, BCAA inhibit protein degradation andpromote protein synthesis (15) and have specific regulatoryfunctions in the turnover of skeletal muscle protein (16). Hence,the regulatory effect of BCAA on muscle protein turnover needsto be further characterized in relationship to host tissues aswell as the tumor itself.

Studies by Schaur and his associates demonstrated that continuous p.o. administration of BCAA to rats bearing Yoshidasarcoma significantly increased survival time by 32% and reduced tumor size by 33% after 3 wk of growth (17). Their workalso demonstrated that a shift in nitrogen balances was delayed,but not prevented, by BCAA administration (17). In our laboratory, Kawamura et al. found that BCAA-enriched solutionsinfused into animals bearing RNC-fibrosarcoma limited thereduction in skeletal protein synthesis seen in tumor-bearinganimals receiving standard amino acid formulas (18). Also,Tayek and his coworkers gave parenteral infusions containing50% BCAA-enriched solutions to malnourished patients withintraabdominal metastatic adenocarcinoma and improvedwhole body leucine flux, oxidation, leucine balance, and fractional synthetic rates of albumin (19). However, there wasconcern in the latter study that the choice of leucine as thetracer when leucine-enriched solutions are studied might leadto underestimates of leucine oxidation and therefore overestimates of leucine balance. Thus, we sought to confirm thebenefits of parenterally administered BCAA for kinetic estimates of protein metabolism using tyrosine as the amino acidtracer. Since the tyrosine content of both formulas is very lowand lower in the BCAA-enriched solution, a finding of improvement in parameters of protein metabolism with the BCAA-enriched solution using tyrosine as the tracer amino acid in asecond animal tumor model would lend further support to theirpotential value for nutritional repletion in cancer cachexia.

MATERIALS AND METHODS

Animal Preparation and Nutrient Infusion. Thirty-three specific pathogen-free male Sprague-Dawley rats (35-40 g) were obtained fromTaconic Farms (Germantown, NY). The study was conducted underthe approval of the Animal Care Committee at the New EnglandDeaconess Hospital and in compliance with their established rules andguidelines. Prior to the experiment, rats were housed, for a minimumof 5 days, 2 to a cage and maintained on a 12/12 h light/dark photo-period at an ambient temperature of 27 ±1"C. Tap water and rodent

chow (Charles River D-3000; Agway Agricultural Products, Minneapolis, MN) were provided ad libitum.

The Yoshida sarcoma tumor cell line was used to induce cancercachexia. The study was conducted from day 10 to 14 which is on thelinear portion of the tumor growth curve and was determined to be acachectic period (Fig. 1). On day 0 of the study, 15 animals wereinoculated with IO7 cells of viable Yoshida sarcoma into the s.c. areaof the right flank. The non-tumor-bearing rats were given an identicalsham injection with sterile saline. The animals were weighed, returnedto their cages, and allowed to consume standard laboratory chow andtap water ad libitum. The animals were not pair fed and the rats bearingrapidly growing tumors had diminished food intake, reflecting thepresence of tumor. On day 10, the tumor-bearing animals weighed less

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BRANCHED CHAIN AMINO ACIDS AND TUMOR-BEARING RATS

DflYS POST

12 14

NOCULATION

Fig. 1. Weight gain of tumor-bearing and control rats. The Yoshida sarcomatumor cell line was used. Tumors were palpable by day 5 in all animals. Tumorgrowth followed first order kinetics and the study was conducted from day 10 to14 which was previously determined to be the cachetic period. •,control; +,Yoshida sarcoma.

Table 1 Dietary intake

Group BCAA(kcal/kg/body

weight)Control

groupTest group 51.6Crystalline

Glucoseamino acids (kcal/kg/body

(kcal/kg/body weight)weight)45.6

174.4168.4Total

calories(kcal/kg/body

weight)220

220Diets with one half dextrose calories

Regular dietAmino acid = 3.7%

Branched chain enriched dietAmino acid = 4.2%

Diets with full dextrose caloriesRegular diet

Amino acid = 3.7%Branched chain enriched diet

Amino acid = 4.2%

Dextrose = 10.7%

Dextrose = 10.4%

Dextrose = 21.4%

Dextrose = 20.9%

Additive per 1500 mlNaCINaAcKC1-KAcKPhosCaGluMgSO4Trace minerals

45 mEq45mEq45 mEq37 mEq23 mEq12.5 mEq12.0 mEq15.2 mEq

Plus 1.0 ml of MVC 9 + 3 vitamins per 100 ml of hyper»!imental ion solution.

than the non-tumor-bearing animals, indicating their cachectic state.The rats were anesthetized with ether and a Silastic catheter (0.025inside diameter x 0.047 in outside diameter; Dow-Corning Laboratories, Corning, NY) was inserted through the internal jugular vein aspreviously described (20). The animals were placed in metabolic unitsand i.v. infused with saline (approximately 20 ml of sodium chloride(154 HIM)overnight from a Holler pump ((rit ikon Inc., Tampa, FL)).On day 11, the animals were randomly assigned to 2 groups and i.v.administered diets containing amino acids and one half the dextrosecalories (Table 1) to allow adaptation to the glucose. There was nosignificant difference in the size of the tumors, as indicated by measurements of tumor volume, between the two diet groups. The animalswere given the full calorie diet on day 12 and feeding continued for anadditional 2 days. At the full calorie level both diets contained 220calories/kg •body wt-day, of which 75% was hydrous dextrose and 25%was amino acids (2 g amino N/kg-body wt day as either 19% BCAAor 50% BCAA (Table 1). The diets were isonitrogenous, isovolemic,and isocaloric and were formulated in the hospital pharmacy underaseptic conditions. An infusion of 40 ml per day was sufficient to deliverthe total amount of calories. The animals were infused for 3.5 days

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which has been demonstrated to be a sufficient time to reveal differencesif they indeed exist. Hyperalimentation in a small tumor-bearing ratfor a longer period of time (i.e., 10-14 days) is difficult to accomplishdue to technical failures and an increase in mortality. Twenty four-hurine was collected for nitrogen balance determination.

Isotopie Turnover Design. On day 14, ("Cjtyrosine (350 mCi/mmol;ICN, Irvine, CA) was added to the diets and a 4-h constant infusionwas conducted to investigate protein kinetics. Each animal received 1.7/tCi/h of [14C]tyrosine at a volume rate of 1.67 ml/h through a syringe

pump (±1%;Harvard Apparatus Co., S. Natick, MA).Total carbon dioxide production, O2 consumption, respiratory quo

tient, and resting energy expenditure were determined during the infusion as previously described (20). Vo2 and VCo2 were measured bymethods previously published by our laboratory (20).

At the end of the infusion, the animals were sacrificed by decapitationand the blood was collected in heparinized tubes. Plasma was separatedby centrifugation and stored at -25°C. Immediately following decapi

tation, the body was quickly dissected, and the liver, tumor, and portionsof the abdominus rectus muscle were removed. The liver was weighedand 1-g pieces were placed in 5 ml of 10% sulfosalicyclic acid and 5 mlof saline and then completely frozen in liquid nitrogen (-180°C) to

halt all metabolic processes. Two 1-g pieces of muscle were similarlyfrozen. The tumor was weighed and also frozen intact in liquid nitrogen.All samples were stored at —25°Cuntil analysis. The total time between

decapitation and sample freezing in liquid nitrogen was 2-3 min.Analytical Methods. Vo2, VCo2, respiratory quotient, and resting

energy expenditure were derived from the equations of Weir (21).Total urinary nitrogen was determined following a micro-Kjeldahl

digestion.In preparation for the tyrosine and albumin specific activity assays,

the plasma was thawed and deproteinized with 10% trichloroacetic acid(2:1 v/v; plasma: trich loroacetic acid). Following centrifugation, thesupernatant was analyzed for free tyrosine specific activity as previouslydescribed (22). Tyramine concentration was determined by the methodof Waalkes and Undenfriend (23).

The protein pellet was analyzed for albumin specific activity by meansof an ethanol-water extraction. One ml of the extracted supernatantwas added to 10 ml of commercial scintillant (Monofluor: NationalDiagnostics, Manville, NJ) with 100 »\of glacial acetic acid to supresschemiluminescence and analyzed, in duplicate, for 14C radioactivity(LS-8000 spectrometer; Beckman, Fullerton, CA). To determine albumin concentration, 25 »\of the ethanol supernatant were transferred,in duplicate, into test tubes containing 6 ml of a bromocresol greensolution (Albustrate; General Diagnostics, Morris Plains, NJ) anddistilled water (1:5 v/v solution) and vortexed for 10 sec. The sampleswere analyzed spectrophotometrically at a wavelength of 630 nm.Albumin concentrations were determined by linear regression of knownstandards, and unknowns were determined from the regression line.

The tissue samples, liver, abdominus rectus muscle, and tumor werethawed and homogenized (Polytron; Brinkmann Instruments, West-bury, NY). Nitrogen content of tissue samples stored in saline wasdetermined by micro-Kjeldahl digestion. Homogenized tissue samplesstored in sulfosalicylic acid were centrifuged at 4500 rpm to separatethe acid-soluble amino acids (Si) from protein-bound precipitate (Sb).The acid-soluble amino acid supernatant for each sample was analyzedfor tyrosine specific activity as stated previously. The protein-boundprecipitate was washed with sulfosalicylic acid and recentrifuged. Theprotein pellet was then dried at 100*C.

To determine the amount of tyrosine in the protein-bound precipitate, 200-mg samples of wet tissue (liver, muscle, and tumor) wereobtained from noninfused animals. These samples were hydrolyzed in10 ml of 6 N HC1 at 115'C for 24 h. Tyrosine concentration (^mol

tyrosine/mg protein) of the hydrolysate was determined on a reverse-phase column (CIS ¿iBondapack;Waters Associates, Milford, MA) byhigh performance liquid chromatography using gradient elution withphosphate buffer and 65% met hunt>1and precolumn deriviti/ut ion witho-phthaldialdehyde and fluorescence detection. Nitrogen content (N/g)of 40 mg of dried protein-bound precipitate was determined by micro-Kjeldahl digestion. Two ml of Tissue Solubilizer-450 (Beckman Instruments, Inc., Fullerton, CA) were added to another 40-mg aliquot of

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BRANCHED CHAIN AMINO ACIDS AND TUMOR-BEARING RATS

dried protein-bound precipitate and the samples were incubated overnight at 50'C. Ten ml of commercial scintillant (Beckman NA) were

added to the vials with 200 n\ of glacial acetic acid to suppresschemiluminescence and analyzed for I4Cradioactivity (dpm/mg) (Beckman LS-8000 spectrometer).

Rate of whole-body i>rosine appearance, oxidation, percentage oftyrosine oxidized, synthesis, breakdown, and tyrosine balance wereestimated from the equations of Waterlow (24). It was assumed that aplateau labeling (steady state) of the plasma compartment was achievedwhen the Sa,„„was reached in the expired breath (between 2 to 3 h ofcontinuous [l4C]tyrosine infusion) (25). Plasma tyrosine appearance

can be attributed to the degradation of body protein, dietary intake, orhydroxylation of phenylalanine (Fig. 1). It was previously shown in ourlaboratory that approximately 22% of tyrosine flux is derived fromphenylalanine (26). Studies done by other investigators (27) have shownsimilar proportions of phenylalanine-derived plasma tyrosine, suggesting that this fraction is relatively stable in animals with normal liverfunction. In this experiment, most of the plasma phenylalanine flux isderived from protein breakdown since dietary intake was negligible(Table 1). Under these conditions, it can be shown that a 50% increasein the rate of conversion to tyrosine will result in only a 10% increasein tyrosine appearance in excess of what is derived from breakdown.Therefore, it is appropriate to consider that changes in plasma tyrosineappearance in this study are closely related to changes in whole-bodyprotein breakdown. The protein fractional synthetic rates in the liver,abdominus rectus muscle, and tumor were derived from the equationsof Garlickrt al. (28).

Estimates of fractional tumor growth, A,, were derived from tumorvolume measurements on days 10 and 14. Tumor volumes were estimated from measurements of tumor length, width, and depth in millimeters as previously described (29). These measurements bear a closerelationship to tumor weight. Tumor protein breakdown rates wereestimated as the difference between tumor protein synthesis, measuredisotopically, and tumor growth.

Statistical Analysis. Data are presented as mean ±SEM. The datafrom groups one through four were compared for statistical differencesusing two-way and one-way analysis of variance using statistical software (BMI)I' Statistical Software, Los Angeles, CA).

RESULTS

During a constant infusion of L-[t/-14C]tyrosine, radioisotope

excretion in expired breath generally reached a steady statewithin 2-4 h. Whole-body tyrosine kinetic values are shown inTable 2. There was no significant difference in mean whole-body protein flux due to BCAA infusion. The use of BCAA-enriched diets significantly reduced whole-body tyrosine oxidation and percentage of flux oxidized compared to animals

Table 2 Whole-body tyrosine kineticsThere was no significant difference in mean whole-body flux due to BCAA

infusion. Regardless of the presence or absence of tumor, there was a significantreduction in tyrosine oxidation and percentage of tyrosine oxidized in animalsgiven I«AA enriched infusions. Similar rates of synthesis and breakdown werefound. Net tyrosine balance was significantly greater in animals infused withBCAA-enriched solutions.

TumorFlux

% of fluxoxidized

OxidationSynthesisBreakdownTyrosine

balanceBCAA(7)42.1

±4.914.4±2.55.7

±0.8'

36.4 ±4.840.0 ±4.8-3.6 ±0.8*Crystalline

aminoacids(8)40.3

±5.816.1 ±1.36.6

±1.233.7 ±4.738.1 ±5.8-4.4 ±1.2NontumorBCAA(10)42.8

±7.611.4±3.44.6

±0.7a

38.2 ±7.140.6 ±7.6-2.4 ±0.7*CAA(8)41.8

±4.718.5 ±1.57.6

±1.134.1 ±4.039.5 ±4.7-5.4 ±1.1

given a standard formula containing 19% BCAA in both tumorand non-tumor-bearing animals. Although tyrosine incorporation into protein and release by tissue breakdown were notstatistically different between the two groups, tyrosine balancewas improved (P < 0.05) by BCAA administration in bothtumor and non-tumor-bearing animals. The greater sensitivityof tyrosine balance to detect differences over the individualcomponents incorporation and breakdown has been previouslynoted (30). The effectiveness of BCAA in non-tumor-bearinganimals may be attributable to the presence of a stress responserelated to the catheterization.

Nitrogen analysis revealed significantly higher nitrogen balance in tumor-bearing rats versus the nontumor controls (Fig.2). The greater nitrogen retention in the tumor-bearing animalsmay be due to the tumor functioning as a "nitrogen trap" (29)

particularly as the animals were studied during a rapid tumorgrowth phase. Also, the nitrogen balance of both the tumor andnon-tumor-bearing animals infused with BCAA-enriched solutions was numerically higher than those infused with crystallinesolutions.

All of the animals in the study lost weight, but there were nosignificant differences in final body weight or weight change.The weight loss may be explained by the small size of theanimals, the effects of surgical stress, and the administration ofonly half of the dextrose calories the first night of feeding toallow for adaptation to parenteral glucose.

Tumor measurements and kinetics are displayed in Table 3.There were no significant differences in tumor volume, weight,percentage of body weight, or fractional synthetic rates due todiet manipulation. The growth rate of the Yoshida sarcoma, asindicated by measurements of tumor volume, was decreased toan insignificant degree by BCAA infusion. Tumor breakdownrates, determined by the difference between tumor synthesisand tumor growth, were higher but also not significant in theanimals receiving BCAA-enriched infusions. Of note, however,tumor intracellular spedile activity was significantly higher (/'

< O.OS) in animals infused with crystalline amino acids whichis consistent with greater tumor breakdown with BCAA infusion.

The rates of fractional protein synthesis were calculated usingthe specific radioactivities of the plasma and the protein boundprecipitate. No change in the synthetic rates of liver, rectus

f

110

13O -

120 -

no

100

90

•o

30 -

20 -

II

" Mean ±SEM (jimol/h/g body weight). P < 0.05 two-way analysis of variance

(BCAA versusCAA).* P < 0.05 one-way analysis of variance (BCAA versus CAA).

TUMOR TUMOR NONTUMORNONTUMORBCAA CAA BCAA CAA

Fig. 2. Nitrogen balance. Nitrogen balance was determined to be significantlyhigher in tumor-bearing animals as compared to non-tumor-bearing animals. Theanimals infused with BCAA-enriched solutions were in a more positive state thanthose infused with crystalline solutions, but this did not reach a level of statisticalsignificance. Mean ±SEM, P < 0.05 tumor versus nontumor.

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Table 3 Tumor measurements and kineticsThere were no significant differences in tumor volume, weight, percentage as

body weight, or fractional synthetic rate due to diet manipulation. Growth rateas indicated by measurements of tumor volume was decreased, but not at asignificant level. Tumor protein breakdown rates (fractional synthetic rate —growth rate) increased in rats infused with BCAA-enriched diets, but not significantly. Intracellular specific activity (normalized for total infúsate volume) wassignificantly lower in rats receiving BCAA infusions suggesting greater tumorprotein breakdown with BCAA-enriched infusions.

Tumor volume, day 10(mm3)Tumorvolume, day 14(mm3)Tumor

weight, day 14(g)Tumorweight/body weight,day14

(%)Tumorfractional syntheticrate(%/day)Tumor

growth rate(%/day)Tumorprotein breakdownrate(%/day)Intracellular

specificactivity(dpm/^mol/mlinfúsate)Tumor

BCAA(7)5336

±39907202±38746.0±4.94.3±3.321

±65.1

±5.416±71204±

151Tumor

crystalline amino acids

(8)4779

±22298167±53165.6

±5.14.3±3.920

±810.5

±3.39±211967

±268°'

P < 0.05.

Table 4 Fractional synthetic ratesThe rates of synthesis were calculated using the specific radioactivities of the

plasma and protein-bound precipitate. There were no significant changes in thefractional synthetic rates of the liver, muscle, or tumor due to diet manipulation.

Tumor BCAATumor crystalline amino acidsNontumor BCAANontumor crystalline amino

acidsLiver65

+ 7"

59 + 569 + 1363 + 6Muscle8+

18+ 19 + 28 + 2Tumor21+6

20 + 3

' Mean ±SEM (%/day).

Table 5 Albumin synthesisThere were no changes in albumin specific activity or plasma albumin levels,

suggesting no significant alterations in albumin synthetic rates

Tumor BCAATumor crystalline amino acidsNontumor BCAANontumor crystalline amino acidsSpecific

activity(dpm/umol)19,541

+ 4,497°

16,244 + 2,34117,058 + 81816,386 + 721Plasma

levels(g/100ml)3.1

+ 0.33.0 ±0.32.9 ±0.22.7

+ 0.2°Mean ±SEM.

abdominus muscle, and tumor was found after BCAA administration (Table 4).

Plasma albumin levels and specific radioactivities as reportedin Table 5 were unchanged by BCAA infusion.

DISCUSSION

Cancer-associated cachexia is the primary cause or an associated phenomenon in many tumor-related deaths (1). Earlyexperience suggested that poor nutrition in cancer patientsinterfered with therapy similar to the situation found in acuteor chronic nonmalignant illness, and that malnutrition couldinterfere with the delivery of the optimal oncological therapy(31). Moreover, other evidence had been gathered that nutritional repletion of the malnourished cancer patient with standard regimens was difficult, if not impossible (32). In consideringthe consequences of protein malnutrition in the tumor-bearinghost, we sought to develop a more effective nutritional supporttherapy through the use of BCAA.

In patients with actively growing neoplasms, plasma aminoacid profiles are characterized by reduced BCAA levels andarterial-venous differences across skeletal muscle reflect anincreased proportion of BCAA being oxidized (33). BCAA

administered p.o. were shown to increase survival time andnitrogen balance while reducing tumor growth (17). There isalso evidence for a beneficial effect of BCAA-enriched infusionon plasma leucine and albumin kinetics in humans (19) andmuscle protein turnover in animals with cancer (18).

This study examined the effects of BCAA on host proteinsynthesis, breakdown, and growth. Although tyrosine is not anessential amino acid and rates of tyrosine release from proteinbreakdown cannot be derived from its appearance withoutadditional assumptions, the rate of tyrosine oxidation andincorporation into protein can be evaluated (34). There was asignificant reduction in whole-body tyrosine oxidation and percentage of tyrosine flux oxidized in the animals given BCAAinfusions. Also, with the reasonable assumption that tyrosineappearance by de novo synthesis from phenylalanine does notdiffer between the two diet groups, net tyrosine balance wassignificantly greater in animals infused with BCAA-enrichedsolutions. Even without this assumption, the de novo synthesisof tyrosine is very small in relation to tyrosine appearance frombreakdown and is unlikely to seriously affect the tyrosine balance calculation.

In injury and sepsis, the metabolic response is characterizedby the net degradation of muscle protein and the mobilizationof body fat for energy substrates and gluconeogenesis (35).BCAA are preferentially utilized in skeletal muscle and it hasbeen hypothesized that BCAA supplementation may provide aprotein-sparing metabolic fuel when energy demands increasedue to the metabolic response to stress or disease. Freund et al.hypothesized that BCAA spared nitrogen by providing substrates for muscle fuel catabolism, by providing amino groupsto produce alanine for hepatic gluconeogenesis, and by blockingamino acid efflux from muscle (36). Although the metabolicresponse in a tumor model is somewhat different from an injuryor trauma model, there are numerous similarities (8) and theseprinciples of BCAA utilization may apply in the tumor-bearinghost. Administration of TPN solutions enriched with BCAAreduces whole-body tyrosine oxidation and percentage of tyrosine flux oxidized and spares body protein by improving aminoacid reutilization through nonoxidative pathways. Thus, such adietary manipulation may favorably influence net protein metabolism in the carcass without enhancing tumor growth.

The tumor-bearing animals retained more nitrogen than thenon-tumor-bearing animals (/' < 0.05). This significant differ

ence may be due to the tumor functioning as a nitrogen trapwhere tumor breakdown is minimal, limiting systemic reentryof amino acids.

Tumor volume, weight, and percentage of body weight indicate a rapidly growing sarcoma. In the clinical setting, tumorsgrow at slower rates and rarely comprise more than 1% of totalbody weight when patients die from metastatic disease. Thework of Tayek et al. with the Walker 256 carcinosarcomademonstrated variable tumor growth rates of the same tumortype in different animals and postulated that accelerated tumorgrowth was related more to reduction of tumor protein breakdown rates than to stimulation of tumor protein synthesis (29).Tumor protein breakdown rates were numerically higher inanimals receiving BCAA-enriched infusions which is consistentwith the significant reduction in tumor intracellular specificactivity in animals receiving branched chain amino acid-enriched diets. The slightly lower tyrosine content in the BCAA-enriched diet would increase the specific activity of the infúsate,rather than lowering tumor intracellular specific activities, asseen in our study, thus lending support to our hypothesis thatBCAA-enriched infusions increase tumor protein breakdown.

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BRANCHED CHAIN AMINO ACIDS AND TUMOR-BEARING RATS

Thus, the data suggest that administration of TPN solutionsenriched with BCAA does not stimulate tumor growth but doessupport host protein metabolism and may play a role in retarding tumor growth by increasing tumor protein breakdown rates.

In conclusion, our results suggest that BCAA-enriched TPNsolutions increase amino acid utili/ation for net protein synthesis by the host principally by reducing oxidation without stimulating tumor growth. The site of the improved protein balancewas not specifically identified but presumably was in skeletalmuscle due to the large size of this component as well as beingthe usual major site of net nitrogen balance during feeding.Such a location would also be consistent with the mode ofaction of BCAA to reduce net skeletal protein catabolism. Theseobservations may be important for the development of optimalnutritional support therapies for cancer patients in the treatment of cancer cachexia. Improved support regimens mightafford a heretofore undetected benefit of nutritional support inrandomized trials of cancer patients receiving chemotherapy(37) and might also be an effective means to slow the progression of the cachexia syndrome and prolong life where effectivechemotherapy does not exist. Finally, the suggestive effect ofBCAA-enriched total parenteral nutrition to increase tumorbreakdown rates deserves further investigation over a longerstudy period.

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1988;48:2698-2702. Cancer Res   Lisa E. Crosby, Bruce R. Bistrian, Pei-ra Ling, et al.   Yoshida SarcomaParenteral Nutrition on Amino Acid Utilization in Rats Bearing Effects of Branched Chain Amino Acid-enriched Total

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