Responses of hepatoma-bearing rats to total parenteral hyperalimentation and to ad libitum feeding

JOURNAL

OF SURGICAL

RESEARCH

23, 189-195 (1977)

Responses of Hepatoma-Bearing Rats to Total Parenteral Hyperalimentation and to ad Libitum Feeding1 IVAN L. CAMERON, PH.D., WILLIAM J. ACKLEY, AND WAID ROGERS, M.D.,PH.D. Departments of Anatomy and Surgery, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284 Submitted for publication July 8, 1976

Total parenteral feeding has been reported to be of value to cancer patients who are undergoing therapy, be it irradiation, chemotherapy, or surgery [5, 6, 8, 9, 15, 17, 191. It is understandably difficult to do controlled clinical studies on cancer patients undergoing total parenteral feeding because of numerous variables. Therefore, we have turned our attention to the study of total parenteral hyperalimentation on unrestrained BUF strain rats bearing a transplantable nonmetastasizing Morris hepatoma (No. 7777) to learn more about this increasingly popular clinical procedure. Parenteral feeding of rats allows precise control of nutritional intake and therefore allows a more accurate assessment of the role of nutrition on the tumor-host interaction. For example, recent reports indicate that the rate of tumor growth can be influenced and even stimulated depending on the level and type of parenteral diet given [3, 5, 191. The present report evaluates differences between hepatoma-bearing rats which are fed (i) solid food (lab blocks) ad libitum, (ii) complete liquid diet ad libitum, and (iii) a total parenteral diet of the composition of the liquid diet. The parameters followed include body weight changes, tumor growth rate, calorie and fluid intake, urine output, and necropsy observations. The findings answer a number of questions about the role of nutrition in tumor-host interactions and help form a basis for understand’ Supported by PHS Research Grant CA 16831 from the National Cancer Institute.

ing parenteral feeding as an adjunct to cancer therapy. METHODS AND MATERIALS The equipment, techniques, and solutions for total parenteral feeding of rats have been described previously [3, 4, 14, 18, 201. Seven-week-old female BUF rats were randomized into four experimental groups: intravenous feeding with tumor (IVT), liquid diet with tumor (LT), intravenous feeding without tumor (IV), and solid food with tumor (ST). Immediately thereafter, the three groups (IVT, LT, and ST) of rats were inoculated with the Morris No 7777 hepatoma. The various experimental feeding regimens were started 5 weeks after tumor inoculation. Briefly, parenterally fed rats had a Silastic catheter tube inserted into the superior vena cava or the right atrium via the external jugular vein. The other end of the catheter exited from the body between the scapulae. The rat was placed in a padded harness and attached to the fluid infusion swivel apparatus (Instec Labs, Philadelphia, Pennsylvania) via a tightly wound steel shielding cable housing the plastic infusion tubing. These unrestrained rats were housed individually in metabolic cages during the study and were infused by a Model 903 Holter pump. A 27% solution of glucose and 3.9% solution of Freamine (McGaw Laboratories) with electrolytes and multivitamins were infused. These rats were fed 13 kcal/ 100 g body weight the first day of infusion, 20 kcaV100 g on the second day, and 26 kcaU

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JOURNALOFSURGICALRESEARCH:VOL.23,NO.3,SE~EMBER1977

100 g from Day 3 until the end of the experiment. The Morris hepatoma No 7777 enlarges subcutaneously and is palpable by 4 weeks after tumor inoculation. Normally, at 5 to 6 weeks after the inoculation of this tumor dose these animals on oral feedings begin to lose weight and at about 8 weeks they die. Three and one-half weeks following tumor cell inoculation, daily measurements were made of body weight and water and food consumption (solid food consisted of Wayne Lab Blox for rats, Allied Mills, Chicago, Illinois, which has a caloric value of 3.53 k&g weight). Liquid diet was given orally ad libitum and was of the same composition as the parenterally delivered diet mentioned above. Daily caliper measurements of the length and width of each tumor were tabulated and a skin thickness correction (a subtraction of 2 mm from the length and the width measurements) applied prior to calculation of the cross-sectional area of the tumor using the formula for an ellipse

[3]. Body and tumor growth rate data were subjected to computer analysis to obtain the best exponential fit for the data before and after the beginning of each feeding experiment. Data on differences in body weight and tumor growth slopes, before and after feeding treatments, were analyzed by a one-way analysis of variance. The standard error of differences between means was calculated and differences between means were tested using Student’s t values. Binomial expectancy statistics of paired data were also made. At necropsy, the abdomen and thorax were opened and visual observations made on various organs. RESULTS Analysis of Body Weight Changes before and after Different Feeding Regimens As summarized in Table 1 and illustrated in Fig. 1, there were no significant differences in the rate of body weight gain

TABLE1 ANALYSIS OF THE EXPONENTIAL SLOPES OF BODY WEIGHT CHANGES PER DAY (NATURAL LOG OF GRAMSBEFORE AND AFTER DIFFERENT FEEDING TRRATMENTS

Intravenous and tumor

Abbreviation

Liquid food (ad li6itum) and tumor

Intravenous

Solid food (ad libitum) and tumor

Before

At&

Before

After

Before

After

Before

After

BIVT

AIVT

BLT

ALT

BIV

AIV

BST

AST

Number of rats Mean of slope + SEM

10 10 0.00325 0.03047 -c o.ooo97 2 0.00505

16 16 0.00308 0.01245 f o.ooo83 f 0.00373

7 7 0.00388 0.02267 f O.ooo83 f 0.00123

5 5 0.00386 0.02158 t- O.lW46 2 0.00329

Mean of correlation coefficient -t SEM

0.42 f 0.25

0.79 2 0.26

0.57 f 0.27

0.53 f 0.24

0.44 + 0.31

0.26 2 0.52

0.68 * 0.41

Analysis of variance0

BIVT

AIVT

BLT

ALT

BIV

BST

AST




NS CO.01
AST BST AIV BIV ALT BLT AIVT BIVT


0.89 kO.08 AIV co.lw1
<0.001

a To use this table follow the desired comparison columns from the left and the top to the intercept to read the probability value. b Robability level. NS, not sign&ant.

CAMERON, ACKLEY,

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191

WEEKS AFTER TUMOR INOCULATION

FIG. 1. Plot of the average exponential slopes of body weight changes per day (natural log of grams) before and after different feeding treatments. Abscissa is the number of weeks after tumor inoculation. The abbreviations indicate the feeding regime, i.e., IVT = intravenous and tumor, average of 10 rats; LT = liquid food (ad libitum) and tumor, average of 16 rats; IV = intravenous, average of 7 rats; and ST = solid food and tumor, average of 5 rats. The data for this plot are summarized in Table 1.

among groups prior to applying the different regimens. As seen in Fig. 1 and Table 1, there were significant differences in growth rate after initiating the feeding regimen and among the different feeding groups. As anticipated, those animals given liquid or solid food ad libitum lost weight as their tumors enlarged. In contrast, rats receiving total parenteral nutrition showed a steady increase in body weight gain. The body weight change data were analyzed using binomial expectancy analysis to determine the effect of the feeding regimen on body growth. The growth rate of each rat was compared before and after treatment in a paired manner. The numbers of times the growth was higher or was lower after the feeding regimen was started were compared using the formula: J.!- (0.5)~(0.5)9, p!q!

where n = number of pairs, p = pairs with an increase after treatment, and q = pairs with a decrease following feeding treatment. This analysis shows statistically significant trends: Total parenteral feeding of tumorous rats resulted in a weight gain (P = O.OOlO), liquid diet ad libitum resulted in a weight loss (P = 0.0018), and solid food ad iibitum also resulted in a weight loss (P = 0.0313). The group of rats without tumors that was given total parenteral hyperalimentation increased in weight to a significant extent (P = 0.0078). Analysis of Tumor Growth Changes before and after Different Feeding Regimens Table 2 and Fig. 2 summarize tumor growth data. Before instituting different feeding regimens, the tumors of rats in the solid food group had a somewhat slower growth rate than those in the other groups and the rate was significantly lower than the

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JOURNAL OF SURGICAL RESEARCH: VOL. 23, NO. 3, SEPTEMBER 1977 TABLE 2 ANALYSISOFTHEEXPONENTIAL SLOPFSOFTUMORGROWTH PERDAY(NATURAL LOG OF AREA, SQUARECENTIMETERS)BEFOREANDAFTERDIFFERENTFEEDINGTREATMENTS Intravenous

Liquid food ad libitum

Solid food ad libitum

Before

After

Before

After

Before

After

BIVT

AIVT

BLT

ALT

BST

AST

Number of rats

10

10

16

16

5

5

Mean of slopes 2 SEM

0.0852 f 0.0124

0.0%9 * 0.0057

0.1062 + 0.0062

0.0831 f 0.0035

0.0759 + 0.0123

0.0327 + 0.0026

Mean of correlation coefficient 2 SEM

0.84 f 0.09

0.94 2 0.04

0.86 2 0.07

0.91 f 0.07

0.88 + 0.15

0.63 f 0.4%

Analysis of variancea

BIVT

AIVT

BLT

ALT

BST

<0.001* NS NS NS NS




co.01

Abbreviation

AST BST ALT BLT AIVT

AST

DTo use this table follow the desired comparison columns from the left and the top to the intercept to read the probability value. b Probability level. NS, not sign&ant.

I6

WEEKS AFTER TUMOR INCCULATION

FIG. 2. Blot of the average exponential slopes of tumor growth per day (natural log of tumor area, square centimeters) before and after different feeding treatments. Abscissa is the number of weeks after tumor inoculation. The abbreviations are the same as in Fig. 1 and the complete analysis of data is given in Table 2.

CAMERON, ACKLEY,

AND ROGERS: HYPERALIMENTATION

group selected for liquid diet (P < 0.05). Since animals in each group were selected at random and no other variables could be found to explain the differences between these two groups, it is proposed that chance and small numbers may be the explanation. At 5 weeks after tumor inoculation, those rats allowed solid or liquid food ad libitum as their feeding regimen showed decreasing rates of tumor growth as the animals simultaneously lost total body weight. This decrease in tumor growth rate was especially evident in those rats continuing on solid food. Rats on total intravenous hyperalimentation demonstrated an increase in tumor growth rate which was significantly higher than in the group continued on solid food ad libitum. Binomial expectancy analysis of the tumor growth rate on the paired data from

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each rat before and after the feeding regimen was started confirmed that parenteral hyperalimentation significantly stimulated tumor growth (P = 0.0439) and that on liquid or solid food diet (ad libitum ) tumor growth significantly decreased (P = 0.0018 and P = 0.0313, respectively). Water Intake, Urine output, and Caloric Intake during the last 7 Days of Feeding Table 3 summarizes the intake and output data on each feeding group during the last 7 days. As might be expected, the animals on solid food had a higher oral water intake than the other treatment groups. Rats receiving total parenteral feeding showed suppressed water drinking compared to the groups on solid or liquid food ad libitum. This is due to the high fluid intake in the iv feeding solution.

TABLE 3 WATER INTAKE, URINE OUTPUT, AND CALORIC INTAKE DURING LAST 7 DAYS OF TREATMENT Feeding treatment and tumor condition (N = number of rats used)

Water drunk (ml)

Total fluid intakeb (ml)

Total urine output (ml)

Solid food ad libitum , with hepatoma (n = 5) 92.4 k 16.7a 97.2 zt 18.3 29.6 i 6.1 Intravenous, without hepatoma (n = 7) 21.7 r 4.8 435.7 2 11.9 192.2 + 13.3 Intravenous, with 9.8 + 2.1 hepatoma (n = IO) 404.2 2 11.9 132.1 2 19.5 Liquid food ad libittm , with hepatoma (n = 16) 72.3 + 6.3 172.5 + 8.2 71.5 2 5.2 Statistical analysis of variance F value P value

26.4
P value of differences between means co.05 co.02 co.01
22.5c 27.0 30.1 39.7

Ratio, intake/ output

Difference, intake minus output

Caloric intake W-4

3.41 r .38

67.6

16.9 2 5.8

2.32 t .15

243.4

488.5 f 14.0

3.06 -c .28

272.1

465.3 t 23.3

2.51 + .I3

101.0

118.1 + 7.3

116.2
24.6
4.37 co.02

223.6
44.7 53.7 59.9 78.9

38.7 46.4 51.9 68.3

0.65 0.78 0.87 1.15

46.5 55.9 62.4 81.1

a Mean 2 SEM. ’ Including water drunk and in food. c Means which differ by these values are significantly different at this level of probability.

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Total fluid intake is listed in Table 3 (e.g., oral and intravenous water as well as the water content of the food). In the case of solid food this was determined to be 7% of the weight of the Lab Blox. Fluid intake as well as urine output was much greater in the iv fed rats than in the rats allowed food and water ad libitum. Comparison of urine output in the two groups of intravenously fed rats shows that those with the hepatoma produced significantly less urine (P < 0.01). Analysis of the fluid intake to output ratios in the iv fed animals revealed that those with a growing hepatoma had a significantly decreased urine output in relation to fluid intake. It appears that the presence of the tumor influenced fluid balance, reflected as a greater fluid retention. The caloric intake data (Table 3) illustrate the strikingly decreased solid food consumption which is attributed to the presence of the tumor. Of interest, tumorbearing rats given liquid food ad libitum actually took in significantly more calories (>sevenfold) than those on solid food. The reason why the liquid diet was accepted better than the solid diet in tumor-bearing animals is not clear. Possibly, the thirst for fluid differed, or the greater energy expenditure required to chew the solid food vs drinking the liquid diet, or the difference in palatability of these two diets might be factors. Nevertheless, animals fed intravenously received a greater caloric load than either of the ad libitum groups. The control over dietary intake that can be obtained by total parenteral feeding in tumor-bearing animals is obvious.

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differ significantly between any of tumor-bearing groups (i.e., 13.56 f days for the solid food group, 12.60 + days for the iv fed rats, and 13.60 + days for the rats on liquid food).

the 1.16 1.48 1.75

DISCUSSION

Tumorous rats which are fed solid or liquid diets ad libitum decrease food intake and lose weight until death (present study and [l, 3, 7, 10-13, 16, 21, 221). Poor food intake with cancer cachexia or wasting of body weight associated with tumor growth can be avoided by use of total parenteral hyperalimentation. However, the present study as well as another recent report [3] indicate that parenteral nutrition results in a concomitant greater tumor growth rate when compared to the starving animal. In the present experiment, there were no significant differences in survival times among the feeding regimens in hepatomabearing rats. However, our observations indicate that death was associated with severe inanition or starvation in the rats given solid or liquid food ad libitum, but appeared to be associated with fluid overload in the parenterally fed rats. Examination of fluid intake and urine output data indicates an interesting fluid retention problem as an associated complication in the tumorous iv fed rats. The reason for this is not clear. If congestive heart failure and fluid retention problems indeed contribute to the death in tumor-bearing rats which are parenterally fed, it can be predicted that decreasing the fluid volume might significantly increase the survival time in this group. Such manipulations of Observations at Necropsy volume of water and nutrients are presently The different feeding regimens resulted in consistent differences in the tumor- under investigation. bearing rats. Those on solid and liquid SUMMARY ad libitum diets had little fat remaining in BUF rats with a transplantable Morris the body, whereas animals fed intravenously had large amounts of fat present, hepatoma No. 7777 were given three feeding regimens, (i) solid food ad libitum, (ii) but tissues were edematous. Survival time, expressed in days after the total parenteral hyperalimentation, and (iii) start of the feeding treatments, did not liquid diet ad libitum. Those rats on solid

CAMERON, ACKLEY,

AND ROGERS: HYPERALIMENTATION

AND CANCER

195

Oncology, Vol. III, pp. 154- 158. Year Book or liquid food ad libitum undergo body Medical, Chicago, 1970. weight loss or cancer cachexia with dell. Morrison, S. D. Feeding response to change in creased food intake. Total parenteral hyperabsorbable food fraction during growth of Walker alimentation prevented the loss in body 256 carcinosarcoma. Cancer Res. 32: %8, 1972. weight but stimulated tumor growth when 12. Morrison, S. D. Control of food intake during growth of a Walker 256 carcinosarcoma. Cancer compared to those fed ad libitum. Although there was no significant differ- 13,Res. 33: 526, 1973. Nichol, C. A. The manipulation of metabolism ence in survival time in the tumorous rats by drugs and nutrients. Cancer Res. 29: 2422, in the different feeding groups, the par1%9. enterally fed rats appeared to die with fluid 14. Pavlat, W. A., Rogers, W., and Cameron, 1. L. Morphometric analysis of pancreatic acinar cells overload while the ad libitum fed rats died from orally fed and intravenously fed rats. J. Surg. of inanition or starvation.

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