Nitrogen Excretion in Cancer Cachexia and Its …...[CANCER RESEARCH 49. 3800-3804. July 15. 1989] Nitrogen Excretion in Cancer Cachexia and Its Modification by a High Fat Diet in

[CANCER RESEARCH 49. 3800-3804. July 15. 1989]

Nitrogen Excretion in Cancer Cachexia and Its Modification by a HighFat Diet in Mice1

Susan A. Beck and Michael J. Tisdale2

CRC Experimental Chemotherapy Croup, Pharmaceutical Sciences Institute, Aston University, Birmingham B4 7ET, United Kingdom

ABSTRACT

Animals transplanted with the MAC16 colon adenocarcinoma showeda loss of body weight as the tumor weight increased, without a reductionin food intake. Both adipose tissue and muscle mass decreased in tumor-bearing animals, although loss of body fat exceeded that of muscle massfor given tumor weight. Urinary nitrogen excretion was significantlyelevated when the weight loss did not exceed 3 to 4 g, but above thisweight loss there was a conservation of nitrogen and the excretion levelfell to or below that found in non-tumor-bearing animals. The presenceof a tumor alone was not sufficient to account for the elevated nitrogenexcretion, since animals bearing a related colon adenocarcinoma(MAC13) that did not induce weight loss had a nitrogen excretion patternsimilar to that of non-tumor-bearing controls. Feeding an isocaloricisonitrogenous diet in which 80% of the calories were supplied as mediumchain triglycérides,which significantly elevated plasma levels of ketonebodies, reduced both tumor weight and host weight loss and restoredboth the nitrogen balance and urea excretion to that of non-tumor-bearinganimals. The plasma levels of amino acids, which were reduced in thecachectic state, were also restored to control values in animals fed themedium chain triglycéridediet. These results suggest that excessivenitrogen catabolism in the cachectic state can be prevented by suitabledietary modification.

INTRODUCTION

The principal endogenous energy and nitrogen sources duringevolution of weight loss in cancer are adipose tissue triglycéridesand skeletal muscle proteins (1). The total protein content ofthe muscles is significantly reduced, as is the synthetic ability(2, 3). Whole-body protein turnover has been shown to be aboutone-third higher in malnourished cancer patients that in non-cancer patients and fasted normal subjects (4). The rates ofwhole-body protein turnover have also been shown to be increased in children with newly diagnosed leukemia or lym-phoma (5). Increased muscle proteolysis in weight-losing cancerpatients is also suggested by an increased venous level of alaninefrom forearm muscles (6). A negative nitrogen balance has alsobeen demonstrated in both animals and humans with a varietyof tumors (7). Thus even when nitrogen intake was sufficientlyhigh to provide for both the host and the tumor, nitrogen waslost from the carcass to the tumor (8). Hypoalbuminemia isalso a feature of cancer cachexia and is the result of both adecreased rate of albumin synthesis and an increased fractionalcatabolic rate (9).

Although the biochemical mechanisms underlying these phenomena are still unclear, Lazo (10) has suggested that concentration gradients are established between the free amino acidpools, with a net flux of amino acids taking place towards thetumor cell. Using mice transplanted with Ehrlich ascites tumorcells, Carrascosa et al. (11) have shown a net nitrogen movement from the host to the tumor, which may account for the

Received 7/20/88; revised 3/1/89; accepted 4/11/89.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.

1This work has been supported by a grant from the Cancer Research Campaign. S. A. B. gratefully acknowledges the receipt of a Research Studentshipfrom the Cancer Research Campaign.

! To »hornrequests for reprints should be addressed.

negative nitrogen balance observed in cancer patients.As an experimental model of cachexia, we have used the

M AC 16 adenocarcinoma of the colon transplanted into NMRImice (12). This tumor produces extensive weight loss withouta reduction in food or water intake. This weight loss, which isdirectly proportional to the weight of the tumor, is associatedwith a decrease of both the fat and non-fat carcass mass. Recentresults suggest that weight loss may arise from the productionby the tumor of lipolytic and proteolytic factors (13). This studyinvestigates the effect of the M AC 16 tumor on nitrogen excretion and plasma amino acid levels and the effect of dietarymanipulation on these parameters.

MATERIALS AND METHODS

Pure strain NMRI mice were purchased from Banting and Kingman(Hull, United Kingdom) and were fed a rat and mouse breeding diet(Pilsbury, Birmingham, United Kingdom) and water ad libitum. Fragments of the MAC 16 tumor (1 x 2 mm) were implanted into the flanksof male NMRI mice by means of a trocar, as described (12). Animalsbearing the MAC 16 tumor develop their tumors at different times aftertransplantation and, therefore, the results have been expressed as afunction of tumor weight or weight loss rather than days after transplantation. Blood was removed from animals, using a heparanizedsyringe, by cardiac puncture, under anesthesia with a mixture of halo-thane, oxygen, and nitrous oxide. Plasma was prepared by centrifugingwhole blood in a Beckman microfuge for 30 s and amino acid profileswere obtained by the Macromolecular Analysis Service at BirminghamUniversity, using a Locarte Automatic Amino Acid Analyser.

Dietary Studies. All animals were given free access to rat and mousebreeding diet for 14 days after transplantation, at which time the tumorswere palpable but weight loss had not occurred. They were then randomly divided into three groups and weighed. The standard diet contained 50% carbohydrate and supplied 11.5% of the energy as fat. Anisonitrogenous isocaloric diet supplying 80% of the calories as MCT'

was formulated as a paste to minimize food scatter, as previouslydescribed (14). One group was given sodium D-(—)-3-hydroxybutyrate

in the drinking water, at a concentration of 30 ¿imol/ml.The averagedaily water consumption for all groups was 4.1 ml/mouse. Body weightsand food and water intake were measured daily during the course ofthe study and food scatter was subtracted. Body weights were measuredat the same time of day. After 8 days the mice were put into Metabowlsand urine was collected for 24 h.

Urine Analysis. Urine was collected from the Metabowls and analyzed quantitatively for ammonia and urea using a Sigma diagnostic kit(Sigma Chemical Co., Dorset, U.K.) which depends on the colorimetriedetermination of ammonia at 570 nm (15). Creatinine nitrogen excretion was determined colorimetrically at 500 nm using a Sigma diagnostic kit (16).

Metabolite Assays. Whole blood (0.2 ml) was used and glucose wasdetermined using the o-toluidine reagent kit (Sigma). Acetoacetate and3-hydroxybutyrate levels were measured by the method of Mellanbyand Williamson (17) and Williamson and Mellanby (18), respectively.

Body Composition Analysis. Each carcass was placed in an oven at80°Cuntil constant weight was reached. Carcasses were then reweighed

and the total fat content was determined by the method of Lundholmet al. (19). The residue was the non-fat mass. The water content wascalculated from the wet and dry weights.

1The abbreviation used is: MCT, medium chain triglycérides.

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Statistical Analysis. The results were analyzed statistically using theanalysis of variance or F ratio.

RESULTS

The effect of the M AC 16 tumor on the total body weight andthe body composition of male NMRI mice is shown in Fig. 1.Although food intake remained the same (12), total body weightdecreased as the tumor weight increased and there was a corresponding reduction in the size of the individual body compartments. The carcass weight loss has been previously shownto be directly proportional to tumor weight (r = —¿�0.91)(13).

The rate of loss of body fat exceeded that of muscle, particularlyat the lower tumor weights. The effect of tumor weight on thethigh plus gastrocnemius muscle dry weights is shown in Table1. Both muscle weights decreased as the tumor weight increased, suggesting extensive protein mobilization.

Concomitant with the increase in size of the tumor, there wasa decrease in the plasma concentration of all amino acids exceptfor taurine, although the percentage decrease varied for theindividual amino acids (Table 2). The maximum decrease of54% was observed for valine and isoleucine. Most of the otheramino acids were present in tumor-bearing mice at only 60%of the levels found in non-tumor-bearing animals. One of thehallmarks of chronic malnutrition is an elevation of plasmaglycine. However, in the M AC 16 tumor-bearing animals glycinelevels were reduced to 60% of that of non-tumor-bearing animals, again confirming that the animals were not malnourished.

The total urinary nitrogen excretion in tumor-bearing animals varied according to the extent of weight loss and wassignificantly elevated at small tumor masses, when the weightloss did not exceed 3 to 4 g (Fig. 2). However, above this weight

S 20.0.

10.0-

ooJ

—¿�

0.0 0.02 0.18 0.20 0.35 0.36 0.51 0.51 0.60 0.61

Tumor weight (g)

Fig. 1. Effect of the MAC 16 tumor on the body composition of male NMRImice. Animals weighing 30 g were transplanted with fragments of the MAC 16tumor and the body composition was determined at intervals as the tumor grew.®,H¡O(g); •¿�fat (g); D, non-fat (g).

Table 1 Effect of the MACI6 tumor on the thigh plus gastrocnemius muscleweights of male NMRImiceResults

are fromindividualTumor

wt(g)0.1790.3300.3460.3500.3600.4000.5090.5400.5400.604animals.Wtloss(g)0.03.23.53.63.67.16.07.08.89.5Musclewt(g)0.1110.0800.0670.0810.0920.0640.0670.0770.0550.028

Table 2 Plasma concentrations of amino acids (mean ±SE) in control (C) andtumor-bearing (TB) NMRI mice fed either normal laboratory pellets, 80% MCT,

or 80% MCT plus sodium 3-hydroxybutyrate (3HB)

Values have been averaged over the period when weight loss is 2 to 6 g andare the pooled values of 5 animals/group.

Plasma amino acid concentration inumimi)

Normal 80% MCT 80% MCT + 3HB

AminoacidTaurineThrSerGlyAlaGinValIleLeuLysMetTyrPheHis32519514530744523537615519326374899992C±45±

5±5±

12±40±5±9±

12±20±9±4±

1±7±iTB310

±109±92±185±285±185±165±75±120±180±44±54±72±77

±5011815355259101029115C430

±220±150±280±500±240±360±170±240±440±64±100±120±120

±45195184551915351857615TB580

±210±150

±290±430±190±390±180±250±380±55±71±110±100±402010222551517254348714C420

±200±140±300±445±390±370±170±235

±255±61±88±105±115±101010104510105551155TB530

±16210±12150±53IO±10480

±35420±45480±15200±10310±15220±1859

±2100±6120±790

±3

0.00

-0.04

0246

weight loss (g)Fig. 2. Change in total nitrogen excretion in male NMRI mice bearing the

MAC16 tumor. Urine was collected for a 24-h period at times after transplantation corresponding to the stated weight loss. The individual lines represent pointsfrom a single animal. Values have been expressed relative to the nitrogen excretionwhen no weight loss was observed.

loss there was a conservation of nitrogen and the excretion levelfell to or below that found in non-tumor-bearing animals. Thetotal nitrogen output in non-tumor-bearing animals or in ani

mals bearing a related colon adenocarcinoma (MAC 13) thatdid not induce weight loss did not change with time during thecourse of the study (Table 3) and is not included in Figs. 2 to6. There was no difference in the nitrogen excretion in the fecesbetween control animals and those bearing the M AC 16 adenocarcinoma. Although animals bearing the M AC 16 adenocarcinoma remained in positive nitrogen balance throughout thestudy, there was an initial sharp drop in nitrogen balance whenweight loss started to occur, followed by a shift towards a morepositive nitrogen balance as weight loss increased (Fig. 3), againsuggesting some mechanism for conservation of body nitrogen.The contributors to the total urinary excretion, urea, ammonia,and creatinine, were all elevated in tumor-bearing animals and

followed a pattern with weight loss similar to that of the totalnitrogen excretion (Figs. 4, 5, and 6).

Induction of ketosis in tumor-bearing animals by feeding a

diet in which 80% of the calories were supplied as MCT, eitherwith or without 3-hydroxybutyrate supplementation, signifi

cantly reduced the weight loss produced by the M AC 16 tumor,despite the fact that the total calorie and nitrogen input did notchange (Table 3). Concomitant with the reduction in weightloss, there was also a significant reduction in tumor weight in

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Table 3 Effect of tumor type and dietary modification on weight loss and nitrogen balanceResults are given as mean ±SE for 7 to 9 animals/group. The total nitrogen input was 0.048 ±0.007 g/24 h and did not vary between the individual dietary

groups.

TumortypeNone

MAC13MAC16MACI6MAC16DietNormal

NormalNormal80% MCT80% MCT + 3HBInitial

wt(g)25.38

±0.2325.89 ±0.2525.92 ±0.1925. 14 ±0.3524.78 ±0.38Final

wt(g)26.1

3 ±0.226.08 ±0.1619.68 ±0.9721.69 ±0.5922.08 ±0.66Wt

loss (-)or gain(+)+0.75

±0.23-HO.19 ±0.25-5.68 ±0.54-3.46 ±0.59r-2.45 ±0.36''Tumor

wt(g)0.70

±0.060.66 ±0.100.23 ±0.03"0.19 ±0.04''Total

nitrogenoutput

(g/24h)0.032±0.003°

0.023 ±0.004*

0.044 ±0.0030.028 ±0.004*0.034 ±0.004°Nitrogen

balance(g/24h)0.016 ±0.007*

0.025 ±0.007*0.004 ±0.007*0.020 ±0.004*0.014 ±0.007"

" F < 0.05 when compared with MAC 16 tumor-bearing animals fed the normal diet.* P < 0.003 when compared with MAC 16 tumor-bearing animals fed the normal diet.' P< 0.01 when compared with MAC16 tumor-bearing animals fed the normal diet.' /' - 0.0005 when compared with MACI6 tumor-bearing animals fed the normal diet.

0246

weight loss (g)

Fig. 3. Effect of weight loss on nitrogen balance in male NMRI mice bearingthe MAC 16 tumor. The individual lines represent points from a single animal,and the symbols represent the same animals as depicted in Fig. 2.

8 10 12

weight loss (g)

Fig. 4. Change in urea nitrogen excretion in male MNRI mice bearing theMACI6 tumor. Urine was collected for a 24-h period at times after transplantation corresponding to the stated weight loss. The individual lines represent pointsfrom a single animal, and the symbols represent the same animals as depicted inFig. 2. Values have been expressed relative to the urea excretion when no weightloss was observed.

weight loss (g)

Fig. 5. Change in ammonia nitrogen excretion in male NMRI mice bearingthe MAC16 tumor. The individual liin-\ represent points from a single animal,and the symbols represent the same animals as depicted in Fig. 2. Values havebeen expressed relative to the ammonia excretion when no weight loss wasobserved.

io ,_

s sO) Cd

.E O)

.C —¿�

weight loss (g)

Fig. 6. Change in creatinine nitrogen excretion in male NMRI mice bearingthe M AC 16 tumor. The individual linc\ represent points from a single animal,and the symbols represent the same animals as depicted in Fig. 2. Values havebeen expressed relative to the creatinine excretion when no weight loss wasobserved.

animals fed the MCT diets. The effect of the MCT diet was toreduce the total nitrogen output, which did not differ significantly from non-tumor-bearing controls. Also the nitrogenbalance was restored to that of non-tumor-bearing controls(Table 3).

The contributions to the total urinary nitrogen output aregiven in Table 4. Urea is the main end product of nitrogenmetabolism and was the main contributor to the total nitrogenoutput in all groups of animals. Mice bearing the MAC16tumor have a significantly elevated urea excretion when compared with either non-tumor-bearing controls or animals bearing the M AC 13 tumor, but this was reduced to control values

when the normal diet was substituted with 80% MCT with orwithout inclusion of sodium 3-hydroxybutyrate. Ammonia excretion was not significantly elevated in animals bearing theMAC 16 tumor but was increased in the urine of animals consuming 80% MCT plus 3-hydroxybutyrate. This coincided withthe appearance of both 3-hydroxybutyrate and acetoacetate inthe urine and increased levels of these ketone bodies in theplasma (Table 5). Creatinine excretion was significantly elevated in MAC16 tumor-bearing animals fed the MCT diets.

Coincident with the increased plasma concentrations of acetoacetate and 3-hydroxybutyrate, there was an increase in theplasma concentrations of all amino acids, towards control val-

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Table 4 Effect of tumor type and dietary modification on the excretion of urea,ammonia, and creatinine

Results are given as mean ±SE for 7 to 9 animals/group.

Urea Ammonia CreatinineTumor type Diet (g/24 h) (10-g/24h) (10^g/24h)

NoneMAC13M AC 16MAC 16M AC 16Normal

NormalNormal80% MCT80% MCT +0.028

0.0150.0410.023

3HB 0.028£++-t-0.003°

0.001*

0.0030.004'0.002'3.91

13.84.156.14

14.48-t-

±

+±-+-0.50

5.10.47

1.654.21°6.44

3.878.66

14.6018.20-t-

+

-t-±0.86

0.09°

1.152.52"3.34°

°P < 0.02 when compared with MAC 16 tumor-bearing animals fed the normal

diet.b P < 0.001 when compared with M AC 16 tumor-bearing animals fed the

normal diet.c P < 0.003 when compared with MAC16 tumor-bearing animals fed the

normal diet.d P< 0.05 when compared with MACI 6 tumor-bearing animals fed the normal

diet.' P < 0.006 when compared with M AC 16 tumor-bearing animals fed the

normal diet.

ues (Table 2). Thus, in animals fed a diet in which 80% of thecalories were supplied as MCT the plasma concentration ofamino acids did not differ significantly from non-tumor-bearingcontrols fed the same diet. These results suggest a normalization of nitrogen metabolism in cachectic animals fed high fatdiets.

DISCUSSION

The elevated nitrogen excretion observed during the progressof cancer cachexia is indicative of an increased mobilization ofbody proteins for the production of glucose for use by thetumor. Alterations in host carbohydrate metabolism include anincreased glucose turnover (20), gluconeogenesis (21), and increased glucose recycling (22). Increased rates of láclate production have also been observed in cancer patients and contribute to the elevated Cori cycle activity (23). Stein (24) hassuggested that the greater avidity of the tumor for certain aminoacids could lead to an amino acid imbalance and an increasedgluconeogenesis to dispose of the remaining amino acids. Theresults obtained from animals bearing the M AC 16 tumor wouldtend to detract from this argument, since the serum level ofmost amino acids is reduced and none are increased. Theelevated urea excretion in animals bearing the M AC 16 tumorprobably arises from an increased gluconeogenesis from aminoacids to provide glucose for the tumor, which is known to havean elevated glucose requirement (25). However, in the cachecticanimals there appears to be some compensatory mechanism,since the total nitrogen excretion is only elevated at weightlosses less than 3 to 4 g in male mice. Greater weight loss isaccompanied by nitrogen retention and movement towards amore positive nitrogen balance, suggesting a compensatorynitrogen retention.

Depletion of muscle protein in animals bearing the M AC 16tumor has been attributed to the production by the tumor of acirculatory proteolytic factor, the activity of which is suppressedby both insulin and 3-hydroxybutyrate (13). In addition toincreased protein catabolism, there must be increased aminoacid utilization since the plasma concentration of most aminoacids decreases at a time when weight loss occurs. However, itis unlikely that the presence of a tumor alone is sufficient toaccount for the increased nitrogen excretion, since in animalsbearing the MAC 13 adenocarcinoma, which do not developweight loss, the tumor mass is not significantly different fromthat of the MAC 16 tumor, although the total nitrogen excretionis similar to that of non-tumor-bearing animals. An increasedgluconeogenesis is thought to counteract the tendency towardshypoglycemia in tumor-bearing animals (26), although animalsbearing the M AC 16 tumor do develop hypoglycemia within 16days after tumor transplantation (12). The absence of ketosisin cancer patients (27) and in animals bearing the M AC 16tumor (12) would allow continuation of gluconeogenesis, sinceketone bodies are thought to protect muscle protein duringstarvation, possibly by inhibiting the rate of oxidation ofbranched-chain amino acids in muscle (28).

It has recently been shown (14) that animals bearing theMAC 16 adenocarcinoma that were fed a ketogenic diet had areduction in both weight loss and tumor size when comparedwith those fed a normal laboratory diet, which is low in fat andhigh in carbohydrate; this has been continued by the presentexperiments and also by clinical studies on a small group ofpatients (29), although the latter study did not examine theeffect on tumor growth. Such a diet has been shown to increasethe plasma concentration of all amino acids towards that foundin non-tumor-bearing animals. Induction of ketosis might be

expected to inhibit catabolism of muscle proteins and also toinhibit directly the tumor proteolytic activity (13). Thus, inanimals fed a diet isonitrogenous with normal laboratory foodbut in which 80% of the calories are supplied as MCT with orwithout 3-hydroxybutyrate supplementation, the nitrogen balance and the urea excretion are restored to that of non-tumor-bearing controls, suggesting a decrease in amino acid-cat abolizing activity. Ammonium ions are excreted as part of themechanism of pH regulation and the increased urinary excretion observed in animals fed 80% MCT plus 3-hydroxybutyratecoincides with the excretion of the weak acids acetoacetate and3-hydroxybutyrate. Creatinine excretion bears a direct relationship to the muscle mass and the increased urinary levels foundin animals fed the 80% MCT diets are probably related to theincreased muscle mass (14). These results contrast with theeffect of diet-induced systemic ketosis in a small group ofcachectic cancer patients, where no evidence was obtained foran alteration in whole-body nitrogen kinetics or the ability of

Table 5 Effect of dietary modification on the plasma and urinary concentrations of ketone bodies in animals bearing the MACI 6 adenocarcinomaResults are means ±SE of seven to nine animals/group.

PlasmaconcentrationsTumorNone

MAC16M AC 16MACI6DietNormal

Normal80% MCT80% MCT + 3HBGlucose

(mg/100ml)121.8

±4.4106.4 ±9.5100.8 ±6.5°91.3 ±3.2'Acetoacetate

(IHM)0.040

±0.0020.039 ±0.0020.074 ±0.007*0.060 ±0.004''3-Hydroxybulyrate

(HIM)0.078

±0.0070.094 ±0.0150.241 ±0.022'0.1 70 ±0.012''Urine

concentrationsAcetoacetate

(mg/24h)0.007

±0.0020.01 2 ±0.0020.01 1 ±0.0030.070 ±0.004'3-Hydroxybutyrate

(mg/24h)0.00

0.000.012 ±0.04"

1.09 ±0.19'

°P < 0.025 from non-tumor-bearing animals.* P < 0.002 from MAC 16 tumor-bearing animals fed the normal diet.c P < 0.0005 from MAC16 tumor-bearing animals fed the normal diet.d P< 0.01 from MAC16 tumor-bearing animals fed the normal diet.' P < 0.002 from non-tumor-bearing animals.f P < 0.00001 from MAC16 tumor-bearing animals fed the normal diet.

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NITROGEN EXCRETION IN CANCER CACHEX1A

the body to retain nitrogen, although all of the patients gainedweight (29).

These results suggest that it is possible to reverse the protein-mobilizing effect of a cachexia-inducing tumor by a ketogenicdiet. Such a diet should be tolerable clinically and has the addedadvantage that it causes an increase in host weight while simultaneously causing a reduction in tumor weight. It thus overcomes the potential problem of dietary supplementation causing an increase in tumor growth rate.

ACKNOWLEDGMENTS

The authors would like to thank M. Wynter for the tumor transplantation.

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