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versity of Florida College of Medicine, Gainesville, FL 32610. Accepted for publication June 4, 1991. From the Laboratories of Surgical Nutrition and Metabolism,.
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Effects of Glutamine-enriched Total Parenteral Nutrition on Tumor Growth and Host Tissues The

THOMAS R. AUSTGEN, M.D., PAUL S. DUDRICK, M.D., HARRY SITREN, PH.D., KIRBY 1. BLAND, M.D., EDWARD COPELAND, M.D., and WILEY W. SOUBA M.D., Sc.D.

The effects of glutamine-enriched total parenteral nutrition (TPN + GLN) were studied in tumor-bearing rats because glutamine can benefit host tissues but also may stimulate tumor growth. Rats were implanted with the methylcholanthrene-induced fibrosarcoma (MCA sarcoma) and were studied when the tumor constituted less than 5% of carcass weight (small tumor) and when the tumor constituted 10% of carcass weight (large tumor). Provision of 20% of TPN protein as glutamine produced a significant increase in the arterial glutamine level and maintained the skeletal muscle intracellular glutamine concentration (2.02 ± 0.1 versus 1.39 ± 0.07 ,umol/g, p < 0.01). Concurrently, hindquarter GLN fractional release increased nearly threefold (p < 0.05) in the TPN + GLN group. Glutamine-enriched total parenteral nutrition did not affect carcass weight, tumor weight, tumor DNA content, or tumor glutaminase activity. Furthermore, DNA flow cytometric analysis did not demonstrate any difference in percentage of aneuploid tumor cells within the GI, S, or G2M cell cycles. However, the ratio of aneuploid to diploid cells within the tumor mass increased by 20% in animals receiving glutamine. Glutamine-enriched total parenteral nutrition had no effect on tumor glutathione (GSH) levels. No increase in hepatic GSH levels was observed, but gut mucosal GSH levels were 20% greater in the TPN + GLN group (p < 0.05). The provision of glutamine-enriched TPN may be beneficial to the host by maintaining skeletal muscle glutamine stores and by supporting gut GSH biosynthesis. In this tumor model, TPN + GLN does not appear to increase tumor size, tumor DNA content, or tumor glutamine metabolism, but the ratio of tumor cells to host infiltrating cells within the tumor mass appears to be increased.

G_ LUTAMINE IS THE primary amino acid used by rapidly proliferating tissues, including tumors.1-3 Glutamine serves both as an important respiratory fuel2 and as a requisite substrate for de novo nucleotide biosynthesis.4 In fact, the rate of tumor glutamine Supported by NIH Grants T32 CA09605-01 (to T. R. Austgen and P. S. Dudrick), and CA45327 and an American Cancer Society Career Development Award (to W. W. Souba). Address reprint requests to Wiley W. Souba, M.D. Sc.D., Assistant Professor of Surgery, Biochemistry, and Physiology, Director, Surgical Metabolism Laboratories, Box J-286, J Hillis Miller Health Center, University of Florida College of Medicine, Gainesville, FL 32610. Accepted for publication June 4, 1991.

From the Laboratories of Surgical Nutrition and Metabolism, Department of Surgery, University of Florida, Gainesville, Florida

usage, as measured by the activity of the primary enzyme of glutamine hydrolysis, glutaminase, has been demonstrated to correlate with the rate of tumor growth and histologic differentiation.5 In animals with large tumors, the cancer can behave as a glutamine "sink" and result in profound changes in host glutamine metabolism. Previous studies have demonstrated that the tumor-bearing state causes changes in interorgan glutamine flow such that glutamine is redistributed from the visceral organs and skeletal muscle to the tumor.68 Presumably, this supports neoplastic metabolism and growth. One detrimental sequela of these changes is host glutamine depletion, which is especially pronounced in skeletal muscle, the principle site of glutamine synthesis and storage. This may be of central importance in understanding how the tumor causes cachexia in the host, because skeletal muscle glutamine concentration has been shown to directly correlate with skeletal muscle protein synthesis and indirectly with muscle protein degradation.9 The previous observations that exogenously delivered glutamine can have beneficial nutritional effects for both catabolic humans and animals are factors that prompted the current study. In postoperative patients, the provision of a glutamine-enriched total parenteral nutrition (TPN) diet maintained muscle glutamine concentrations, decreased muscle catabolism, and improved nitrogen balance when compared with postoperative patients fed standard glutamine-free TPN.'0 In another experiment employing the same tumor model used in the current study, rats fed a glutamine-enriched elemental oral diet had increased muscle glutamine concentration and maintained hindquarter glutamine efflux compared with rats fed a glutamine-free elemental diet." An additional benefit of glutamine-enriched TPN was the recent finding

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that hepatic levels of the antioxidant glutathione could be maintained and that survival could be improved in rats treated with a median lethal dose of the chemotherapeutic drug 5-fluorouracil (5-FU).'2 Furthermore, a glutamine-enriched enteral diet has been demonstrated to increase gut mucosal DNA content in tumor-bearing animals.' 3 Although beneficial effects of glutamine-enriched diets have been previously reported, the effect of glutamineenriched TPN solutions on both host and tumor growth and metabolism is unclear. This is an important clinical question because alterations in tumor growth can be caused by other methods of nutritional manipulation.'1'6 The purpose of this study was to examine the effects of glutamine-enriched parenteral nutrition on tumor growth, glutamine metabolism, and antioxidant production, and on host growth, glutamine stores, and antioxidant production.

the central line insertion, day 7 after tumor implantation. At this timepoint of tumor growth, the tumor masses were minimally palpable, and planimetric determination of tumor size was not possible. Therefore, animals were excluded from the study if palpable differences of tumor size were noted. On day 7, the animals were anesthetized with an intraperitoneal injection of ketamine (0.75 mg/ 100 g body weight) and acepromazine (0.075 mg/ 100 g). Using sterile technique and sterilized equipment, a central venous line (Silastic, 0.020 in. inner diameter X 0.037 in. outer diameter, Dow Coming Corp., Midland, MI) was inserted under direct vision into the right external jugular vein and advanced 1.5 cm into the chest. The tubing was tunneled subcutaneously to an exit site between the scapulae. A spring and swivel apparatus, which was sutured to the animal's back, was used to protect the lines. From day 7 to day 11, the lines were kept patent with 0.45% normal saline infused at a rate of 0.5 to 1.0 mL/hr, using cassette pump (Manostat Cassette Pump, New York, NY). During this period, the animals were allowed free access to chow and water. Food intake was monitored during this period, and no differences between groups were noted. On day 11, the animals had all chow removed and were started on TPN. Over a 6-hour period, the delivery rate of TPN was increased to its final flow rate of 2.5 mL/ hr. During the period of TPN, the animals were allowed free access to water. On day 15, the animals were studied. On day 15, in the morning, the rats were anesthetized with an intraperitoneal injection of ketamine (0.75 mg/ 100 g) and acepromazine (0.075 mg/100 g), weighed, and secured to a heated animal board. A midline abdominal incision was made. The abdominal aorta was identified and 1 mL aortic blood was obtained and processed for determination of glutamine concentration. Next, the ligament of Treitz was identified, and a 20-cm portion of proximal jejunum was removed. The mucosa was obtained by gently scraping the opened bowel with a glass slide and processed for determination of glutathione content. A portion of the anterior quadriceps of the right hindlimb was removed for determination of intracellular glutamine concentration. The tumor masses were dissected free from the flanks and weighed. Separate portions were removed for determination of glutathione content, DNA content, glutaminase activity, and DNA flow cytometric analysis. In the second experiment (large tumor study), 24 rats were randomized to a TPN + GLN group and a TPN - GLN group. Randomization ofanimals was done such that animal weight and tumor size were equivalent between groups at the time of the central line insertion. In this experiment, tumor size at the time of central line insertion was determined planimetrically.1' This experiment was done twice; 12 rats were studied each time. On day 14 after tumor implantation, central venous lines were a

Materials and Methods Model

Male Fischer 344 rats (250 g), obtained from the National Cancer Institute (NCI), were used for this study. The rats were housed in the Animal Care facility at the University of Florida for at least 5 days before the study. The rats were exposed to alternate 12-hour light and dark cycles and fed standard rat chow. On day 0, all rats had bilateral flank implantations of a 2-mm cube of viable methylcholanthrene-induced fibrosarcoma (MCA sarcoma). Description of Tumor

The MCA sarcoma was obtained initially from the NCI. It is passed by sequential implantation in male Fischer 344 rats. This tumor displays aggressive local behavior, rarely metastasizes, and never regresses spontaneously. Previous studies have demonstrated this tumor to be an avid glutamine consumer3"6 and that it induces a cachectic state in the host. In the rats, food intake uniformly falls by 14 to 16 days after tumor implantation, and death occurs between 35 and 40 days after implantation. Study Procedure Two separate experiments were conducted. In the first experiment (small tumor study), 36 rats were randomized to receive one of two intravenous diets, glutamine-enriched total parenteral nutrition (TPN + GLN) or standard glutamine-free total parenteral nutrition (TPN - GLN). This experiment was done three separate times, with 12 rats involved in each separate study. Randomization of animals was done such that animal weight and tumor size were equivalent between groups at the time of

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placed as described above. From day 14 to day 18, the central venous lines were kept patent with 0.45% saline at an infusion rate of 0.5 to 1 mL/hr. During this period, the animals were allowed free access to both chow and water. Food intake was monitored, and no differences between groups was noted. On day 18, all chow was removed and the animals were begun on TPN. Again the TPN delivery rate was increased over 6 hours to its final flow rate of 2.5 mL/hr. The TPN was continued for 5 days from days 18 through 23. On day 23, the animals were studied. On day 23, in the morning, the rats were anesthetized, weighed, and secured to a heated animal board. A midline abdominal incision was done and the aorta and distal inferior vena cava were identified. A l-mL sample was obtained from both sites and was processed for determination of glutamine concentration. A portion of the right lobe of the liver then was removed for determination of glutathione content. A portion of the right anterior quadriceps was removed for determination of intracellular glutamine content. The tumors were dissected free of the flanks and weighed. Separate portions were removed and processed for determination of glutathione content, glutaminase activity, DNA content, and DNA flow cytometric analysis. Diet

The TPN diets were isonitrogenous, isocaloric, and isovolumic. Each rat received 60 cc/day and 60 nonnitrogen kcal/day. Eighty per cent of calories was provided as dextrose, and 20% was provided as lipid (McGaw/Nutrilipid, Kendall McGaw Lab., Irvine, CA). The rats received 2 g protein per day. In the both groups, 80% of the nitrogen load was provided with a commercially available crystalline amino acid solution (10% Aminosyn, Abbott Laboratories, Chicago, IL). In the rats maintained on TPN + GLN, the other 20% of nitrogen was provided as glutamine. In the TPN - GLN groups, the other 20% of nitrogen was provided as an equimolar mixture of three other nonessential amino acids already found in Aminosyn and not considered precursors to glutamine: serine, proline, and glycine. The animals also received standard electrolytes, trace minerals, and vitamins every day (Table 1). Processing of Blood and Tissues Whole blood was processed as described previously." Samples were stored at -20 C until glutamine concentration was determined fluorometrically.'8 Muscle samples were processed immediately after biopsies were done, and intracellular glutamine concentration was determined as previously described.' 1"18 Liver, gut, and tumor glutathione content were measured using a spectrophotometric

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TABLE 1. Composition ofParenteral Diets TPN + GLN (per liter)

TPN - GLN (per liter)

Protein (2 g/day)

267 mL of 10% aminosyn 222 mL of 3% glutamine

Carbohydrate (48 Kcal/day) Fat (12 Kcal/day) Vitamins* Electrolytes and trace mineralst

342 mL of 70% dextrose

267 mL of 10% aminosyn 74 mL of 3.2% glycine 74 mL of 4.7% proline 74 mL of 4.2% serine 342 mL of 70% dextrose

100 mL of 20% lipid 5 mL 50 mL

100 mL of 20% lipid 5 mL 50 mL

Component

* MVI 12 (Rorer Pharmaceuticals, Ft. Washington, PA). t Concentration of electrolytes/L: Na+ 40 mEq, K+ 80 mEq, Cl- 40 mEq, Ca+2 18 mEq, P04-3 16 mEq, Mg+2 4 mEq, Zn+2 2 mg, Cu12 0.8 mg, Mn+2 0.2 mg, Cr+ 8 lAg. TPN, total parenteral nutrition; GLN, glutamine.

method.'9 Tumor for DNA content was processed by the method of Labarca and Paigen.20 Tumor glutaminase activity was determined using a modification ofthe method of Pinkus and Windmueller.2' Protein content was determined using the Bio-Rad protein assay (Bio-Rad Lab., Richmond, CA). Tumor Flow Cytometric Analysis DNA flow cytometric analysis was performed using a Becton Dickinson Facstar Plus flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) on approximately 30,000 cells per tumor. Tissue preparation was performed using a low-salt, detergent-trypsin method.22 Reagents were obtained from a commercial kit, Cycletest (Becton Dickinson, San Jose, CA). DNA flow cytometric data analysis was performed using Multicycle software (Phoenix Flow System, San Diego, CA).

Statistics and Calculations Carcass weight change was determined by subtracting the carcass weight at the time of central line insertion from the carcass weight at the end of TPN administration. To determine carcass weight, tumor weight was subtracted from total body weight. Tumor weight change was calculated by subtracting the tumor weight at the time of central line insertion from the tumor weight at the end of the study. Hindquarter arteriovenous glutamine difference was determined using the formula: arterial (Art) glutamine inferior vena cava (IVC) glutamine. The hindquarter fractional release rate was determined using the formula: (IVC glutamine - Art glutamine) / (Art glutamine). All concentration differences were analyzed for statistical significance with 0 to determine uptake or release of a substance. Data analysis between groups was done with Stu-

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AUSTGEN AND OTHERS

110 TABLE 2. Arterial Glutamine Levels (MM)

Small Tumor TPN-GLN TPN + GLN

TABLE 3. Tumor and Carcass Weights

(n= 15)

Large Tumor (n= 10)

609 27 728 ±28*

579 24 675 ±24t

Mean ± SEM. * p < 0.01 vs. TPN - GLN. t p < 0.05. TPN, total parenteral nutrition; GLN, glutamine.

dent's t test, using a MacIntosh Plus Computer and Statview 512 software (Apple Computers Inc., Cupertino, CA). A p value less than 0.05 was considered significant. Results

The provision of glutamine-enriched TPN produced a significant elevation in the arterial glutamine concentration in both studies (Table 2). Glutamine-enriched total parenteral nutrition produced a 20% greater arterial glutamine concentration in the small tumor study (p < 0.0 1) and a 17% greater arterial glutamine concentration in the large tumor study (p < 0.05). In the small tumor study, the muscle intracellular glutamine concentration was not different between groups (2.28 ± 0.3 umol/g in TPN + GLN versus 2.18 ± 0.13 ,mol/g in the TPN - GLN group). In the large tumor study, the intracellular concentration of glutamine was maintained in those rats receiving TPN + GLN (2.02 ± 0.1 ,umol/g), but fell by 31% in those animals receiving TPN - GLN (1.39 ± 0.07 ,umol/g, p < 0.001, Fig. 1). Concurrently in these rats, the hindquarter arteriovenous glutamine concentration difference (-87 ± 24 ,mol/L in TPN + GLN versus -24 + 13 ,mol/l in TPN - GLN) and the hindquarter glutamine fractional release rate (14.2 ± 4% in TPN + GLN versus 4.8 ± 2.7% in TPN - GLN, p < 0.05, Fig. 1) were greater in the GLN + TPN animals. In addition, the ratio of the intracellular glutamine concentration to arterial glutamine concentration ([,umol/g wet muscle] + [,umol/ mL whole blood]) was 30% greater in those rats maintained on TPN + GLN in the large tumor study (3.22

Tumor

Small tumor TPN - GLN (n = 15) TPN + GLN (n = 15) Large tumor TPN - GLN (n = 10) TPN + GLN (n = 10)

Tumor Weight*

Carcass Weight

Weight Changet

(g)

Change (g)

(g)

6.9 ± 0.6

2.0 ± 3.5

6.9 ± 0.6

7.1 ± 0.8

3.0 ± 3.1

7.1 ± 0.8

25.2 ± 2.5

-5.7 ± 2.1

18.2 ± 2.5

23.7 ± 2.9

-7.1 ± 3.4

16.7 ± 2.9

Mean ± SEM. * Tumor weight determined at the end of the study. t Tumor weight change during TPN. TPN, total parenteral nutrition; GLN, glutamine.

± 0.27 in TPN + GLN versus 2.48 ± 0.21 in TPN -GLN, p < 0.05). There was no difference in carcass weight change or tumor weight change during TPN in either study (Table 3). Likewise, there was no difference in tumor size, tumor DNA content, or tumor glutaminase activity between groups in either the small tumor study or the large tumor study (Tables 3 and 4). Representative DNA flow cytometric histograms of the MCA sarcoma, Fischer 344 rat peripheral blood leukocytes (predominantly lymphocytes obtained by Phycoll extraction of rat blood), and an internal standard (mixture of lymphocytes and tumor cells) are demonstrated in Figure 2. The early DNA peak on the histogram of the tumor samples correspond to the peak generated by DNA cytometric analysis of the peripheral blood lymphocytes. This is further corroborated by the fact that, in the internal standard histogram (equal mixture of lymphocytes and tumor cells), an augmentation of the early GI peak is produced. Therefore, these early peaks are thought to represent host cells (lymphocytes, stromal cells, inflammatory cells) that are commonly found to infiltrate tumor masses. The late peaks on the DNA histogram are thought to rep20

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FIG. 1. Muscle glutamine metabolism in tumor-bearing rats (large tumor study). (Left) Effects of glutamine-enriched TPN on muscle glutamine concentration. (Center) Effects of GLN + TPN on intracellular/arterial glutamine gradient [ 1 unit = (Mmol GLN/g wet muscle) + (,umol GLN/mL whole blood)]. (Right) Effects of GLN + TPN on hindquarter fractional glutamine release. *p < 0.05, **p < 0.001 vs. TPN - GLN.

VOL. 215 * NO. 2

III

GLUTAMINE-ENRICHED TPN IN THE TUMOR-BEARING RAT TABLE 5. Tumor DNA Flow Cytometrics

TABLE 4. Tumor DNA Content, Glutaminase Activity, and Glutathione Content Tumor Glutaminase Activity

Tumor DNA Content mg/g

Small tumor TPN - GLN (n = 15) TPN + GLN (n= 15) Large tumor TPN - GLN (n= 10) TPN + GLN (n = 10)

Aneuploid/

Tumor Glutathione Content gsmol/g

4mol/mg prot/hr

6.7 ± 0.4

1.37 ± 0.16

2.1 ± 0.2

6.7 ± 0.3

1.35 ± 0.17

2.1 ± 0.2

S (%)

Diploid

54 ± 3

29 ± 3

0.94 ± 0.14

51±2

30±2

1.1 ±0.12

62 ±2

22 ±2

1.55 ±0.043

64 ± 2

24 ± 2

1.86 ± 0.088*

Mean ± SEM. * p < 0.05 vs. TPN - GLN. TPN, total parenteral

2.4±0.1

1.99 ±0.15

4.4±0.2

Small tumor TPN - GLN (n = 6) TPN + GLN (n=6) Large tumor TPN - GLN (n = 6) TPN + GLN (n = 6)

GI (%)

nutrition; GLN, glutamine. 4.1 ± 0.2

2.1 ± 0.2

2.16 ± 0.15

Mean ± SEM. TPN, total parenteral nutrition; GLN, glutamine.

hepatic glutathione content was not different between groups.

resent aneuploid tumor cells. The DNA flow cytometric analysis results are depicted in Table 4. The percentage of aneuploid cells in the G1, S, or G2M cell cycles were not different between groups in either the small tumor study or the large tumor study. Although not shown, the percentage of diploid cells within the specific cell cycles was not different between groups. Of note, the ratio of aneuploid cells to diploid cells within the tumor mass is greater in those animals receiving TPN + GLN, statistically significantly so in the large tumor study. The glutathione data are displayed on Table 5 and Figure 3. The tumor glutathione content is not different between groups in either study. In the small tumor study, however, gut mucosal glutathione content was 20% greater in the TPN + GLN group than in those animals maintained on TPN - GLN (2.19 ± 0.14 ,umol/g versus 1.82 ± 0.09 ,mol/g, p < 0.05). In the large tumor study, the

Discussion Maintenance of host nutritional status in the patient with advanced malignant disease can be a challenging clinical problem. The malnourished, cachectic patient oftentimes cannot withstand optimal rigorous anticancer regimens. Several studies demonstrate that tumor growth can be influenced by nutrient infusion and metabolic manipulation, however. 116,23,24 In the tumor model used in the present study, Popp et al.'4" 5 demonstrated that the rate of neoplastic growth varies directly with the caloric and protein infusion rate. In addition, the same investigators observed that specific amino acid manipulation could influence tumor growth.'5 Deletion of the two amino acids found to be required for MCA tumor cell growth in vitro,3 glutamine and asparagine, or the direct precursors of these amino acids (glutamate or aspartate) slowed tumor growth as compared with a TPN diet con-

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FIG. 2. DNA flow cytometric histograms from (left) a suspension of cells derived from the MCA sarcoma, (center) a suspension of lymphocytes derived from phycol extraction of rat blood, and (right) a suspension of cells consisting of equal parts MCA sarcoma and rat lymphocytes (internal standard). The early DNA peak seen on the histogram of the MCA sarcoma corresponds to the DNA peak generated by host lymphocytes. An augmentation of this peak is observed in the internal standard suspension, consisting of host lymphocytes and MCA cells.

AUSTGEN AND OTHERS

112

LIVER GSH CONTENT

TPN TPN +GLN -GLN

TPN TPN +GLN -GLN

FIG. 3. Effects of TPN + GLN on gut mucosal GSH levels (small tumor study) and hepatic glutathione concentration (large tumor study). *p < 0.05 vs. TPN - GLN.

taining these amino acids. Currently none of these amino acids are found in commercially available TPN. Similar studies have demonstrated also that preventing tumor glutamine metabolism with administration ofthe systemically toxic glutamine antimetabolite acivicin also slows or arrests MCA tumor growth.'6'25 Conversely, two enteral feeding studies, one using the MCA sarcoma" and one in a rat mammary carcinoma model,13 indicated that the provision of glutamine failed to stimulate tumor growth. In the present study, the provision of glutamine-enriched TPN (compared with a control TPN solution without glutamine) did not affect total tumor size or DNA content. There is, however, some evidence in the DNA flow cytometric analysis that may suggest that an alteration in tumor growth occurred in rats receiving TPN + GLN. Although the provision of glutamine-enriched total parenteral nutrition did not affect the percentage of aneuploid (tumor) cells within individual cell cycle fractions, the ratio of aneuploid cells to diploid cells (presumably host cells infiltrating the tumor) was slightly greater in the small tumor study, and 20% greater (p < 0.05) in those rats with a larger tumor burden. Thus the provision of glutamine-enriched TPN may not yield a measurable increase in tumor size, DNA content, or aneuploid cell cycle kinetics, but the actual amount of tumor cells within a specified amount of tumor mass may be increased. A similar finding was noted with patients with head and neck cancer who were treated with TPN versus an enteral diet.23

An alternative explanation exists. Although the early peaks on the DNA flow cytometric histograms are thought to represent host cell infiltration, (because they correspond to the peaks generated by DNA cytometric analysis of the host lymphocytes and the augmented early peak on the internal standard histogram), the possibility exists that these peaks actually represent an additional tumor cell population with a similar amount of DNA per cell as that

Ann. Surg . February 1992

found in host lymphocytes. In this case, it would appear that glutamine-enriched TPN selectively stimulated one tumor cell population without stimulating another tumor cell population. The growing MCA tumor has a high glutamine requirement and consequently alters the normal balance of interorgan glutamine flow. Teleologically it appears that glutamine flow becomes redirected to supply glutamine for the growing tumor. The liver switches from an organ of net glutamine balance to one of glutamine release,6 and the normally observed acidosis-induced increase in kidney glutamine uptake fails to occur in the acidotic, tumor-bearing rat.7 In addition, the ability of the gut to use circulating glutamine is impaired.6 Furthermore, skeletal muscle glutamine release is augmented and intracellular glutamine stores become severely depleted.8 Although net uptake of glutamine by the tumor was not measured in this study, the elevation of arterial glutamine did not increase glutaminase activity in the growing sarcoma, as has been observed in other tissues.26 This suggests that the tumor may be less sensitive to substrateinduced changes in metabolism compared with depleted host tissues. In the skeletal muscle, the provision of TPN + GLN increased the muscle cytoplasmic glutamine concentration to a greater degree than the arterial glutamine concentration. The resultant increase in the muscle intracellular to blood glutamine gradient may account for the ability of the hindquarter in glutamine-fed tumorbearing animals to release glutamine at a faster rate than tumor-bearing animals fed glutamine-free diets. Such a relationship has been previously noted." Our data are consistent with a model whereby a significant fall in the intracellular glutamine concentration may become the rate-limiting step in the ability of muscle to release glutamine into the bloodstream. This theory was previously posited by Newsholme and Parry-Billings27 to explain the changes in muscle glutamine metabolism in states of significant intracellular depletion. Another recently recognized benefit of the use of glutamine-enriched TPN is maintenance of hepatic glutathione levels and increased survival in rats exposed to 5FU.12 Glutathione (GSH) is a tripeptide consisting ofglycine, glutamate, and cysteine. One function of GSH is as a oxygen free radical scavenger. Normally GSH biosynthesis is limited by cysteine availability.28 As proposed by Hong et al,'2 however, standard TPN solutions contain sufficient amounts of cysteine in the form ofits precursor methionine. Conversely, glutamate is not found in commercial TPN solutions, but may be available from glutamine hydrolysis in animals receiving glutamine-enriched TPN. In the present study, no change in hepatic glutathione levels was noted, but a significant increase in gut mucosal glutathione levels was observed in the animals receiving the glutamine-enriched solutions. Clinically this is significant, because it has been demonstrated that glu-

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tathione is required for intestinal function29and provides a protective effect against oxidant stresses.30 However, TPN + GLN does not stimulate tumor glutathione content. Tumor glutathione levels correlate with resistance to adjuvant therapy, both chemotherapy and radiotherapy, in a number of tumor types.3'32 In the present study there are a number of indices suggesting that glutamine-enriched TPN may be beneficial to the tumor-bearing host even though the duration of TPN was relatively short. The tumor displayed a rapid rate of growth during the periods of TPN, producing systemic effects on glutamine metabolism. Although the animals studied were not cachectic before initiating TPN, nor were they protein depleted before initiating TPN, distinct differences between groups were observed. Previous studies have demonstrated that the MCA sarcoma induces muscle glutamine depletion when tumor size approaches 8% to 10% of carcass weight.8 In the large tumor study, the rats that received standard glutamine-free TPN displayed glutamine depletion. These rats had a decrease in both the blood glutamine concentration as well as the muscle glutamine concentration. If tumor growth is allowed to progress, central necrosis within the tumor mass occurs, confounding the relationship of measured tumor size and viable, metabolically active tumor mass. Thus, the duration of TPN in this study was long enough to unmask differences produced by glutamine supplementation while maintaining the accuracy of physical tumor measurements. Furthermore, the provision of glutamine (in the amounts given) produced measurable changes in other, less metabolically active, slower growing tissues than the MCA tumor. Thus it stands to reason that if the provision of glutamine produces changes in tumor growth or tumor antioxidant production, it would be measurable by the means used in the present study. In summary, in the present study there are a number of indices suggesting that glutamine-enriched TPN does not stimulate tumor growth or tumor glutamine metabolism, but may affect intratumor cell population dynamics. Glutamine-enriched TPN may be beneficial to host tissues by repleting muscle intracellular glutamine concentration, by maintaining muscle glutamine efflux, and by supporting gut glutathione levels. References 1. Sauer LA, Stayman JW, Dauchy RT. Amino acid, glucose, and lactic acid utilization in vivo by rat tumors. Cancer Res 1982; 42:4090-4097. 2. Kovacevic Z, Morris HP. The role of glutamine in the oxidative metabolism of malignant cells. Cancer Res 1972; 32:326-333. 3. Miller TJ, Franco RS, Chance WT, et al. Amino acid requirements of a rat sarcoma as determined by a stem cell assay. JPEN 1987; 1 1(3):223-228. 4. Frisell WR. Synthesis and catabolism of nucleotides. In Frisell WR, ed. Human Biochemistry. New York: MacMillan, 1982, pp 292304. 5. Knox WE, Horowitz ML, Friedell GH. The proportionality of glutaminase content to growth rate and morphology of rat neoplasms. Cancer Res 1969; 29:669-680.

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