Journal of Surgical Oncology 9:493-501 (1977)
The Adverse Effects of Elemental Diets on Tolerance for 5-FU Toxicity in the Rat ....................................................................................... .......................................................................................
JOHN R. STANFORD, M.D.,DENNIS KING, M.D.,LARRY CAREY, M.D., and GREGG ANDERSON,M.D. The (effects of a group of elemental diets on the gastrointestinal toxicity of 5-FU in the SpragueDawley rat were evaluated. Diarrhea, stomatitis, hypoalbuminemia, and early deaths were more frequent in the animals on elemental diets than in those consuming standard rat chow. Sepsis and hypoalbuininemia were directly related t o the extent of protein hydrolysis of the particular elemental diet.
....................................................................................... ....................................................................................... Key words: 5-fluorouracil, elemental diet, protein hydrolysis, blood cultures, serum albumin
INTRODUCTION Five-Fluorouracil(5-FU) is a potent chemotherapeutic agent that has been used extensively in clinical practice for over 15 years. At present, the drug is recommended in th.e palliative management of a wide variety of malignancies, including those of the colon, rectum, breast, stomach, and pancreas. The effectiveness of 5-FU is t o a great extent limited by its toxicity. The rapidly dividing cells of the bone marrow and gastrointestinal mucosa are particularly vulnerable to 5-FU and, indeed, its main toxicity is found in these tissues. Leukopenia, stomatitis, and diarrhea are definite indications t o discontinue therapy. It has been suggested that elemental diets provide some protection against the gastrointestinal toxicity of 5-FU (6). T h s hypothesis was tested in the following study.
MATERIALS AND METHODS A total of 350 male Sprague-Dawley rats weighing 300-320 g were housed in pairs in wire-bottom cages. A 12 hour light-dark cycle was utilized in the room in which the From the Department of Surgery, Ohio State University College of Medicine, Columbus Address reprint requests to John R. Stanford, M.D., Ohio State University, College of Medicine, 410 'West Tenth Avenue, Columbus, OH 4321 0.
493
0 1977 Alan R. Liss, Inc., 150 Fifth Avenue, New York, NY 10011
494
Stanford e t at.
animals were housed. Five different diets were evaluated: Purina Rat Chow, Flexical, Precision L-R, Vivonex, and Warren-Teed Low Residue. Each diet was fed to a group of 70 animals. Six days after weight stabilization was achieved on the various diets 50 animals in each diet group were given 5-FU at 25 mg/kg subcutaneously for 6 consecutive days. The remaining 20 animals in each group served as controls. Daily weight was recorded and the presence or absence of diarrhea and stomatitis were noted both before and after 5-FU therapy. All animals were killed after 6 full days of 5-FU therapy. Blood was drawn aseptically for culture, hemoglobin, WBC, and platelet counts, and for serum protein electrophoresis. Histologic sections were prepared from colon specimens and stained with hematoxylin and eosin.
RESULTS Diarrhea and Stomatitis Prior t o 5-FU treatment, diarrhea was noted in 2 (4%) of the Chow rats, 11 (22%) of the Flexical rats, 2 (4%) of the Precision rats, 19 (38%) of the Vivonex rats, and 3 (6%) of the Warren-Teed rats. Following 5-FU treatment, these percentages increased t o 26% for Chow, 96% for Flexical, 91% for Precision, 100% for Vivonex, and 86% for WarrenTeed (Fig. 1). Stomatitis was not seen in any groups prior to 5-FU treatment; following treatment it was seen in 6 (1 2%) of the Chow rats, 47 (94%) of the Flexical rats, 1 5 (32%) of the Precision rats, 36 (72%) of the Vivonex rats, and 26 (52%) of the Warren-Teed rats (Fig. 2).
Chow
Prec
Flex
Viv
W-T
Fig. 1. Incidence of diarrhea in diet groups.
Weight Changes Weight data were analyzed with the aid of an IBM 370/165 computer. All the animals receiving elemental diets experienced an initial 2-3 day period of weight loss. After 6 days on the experimental diets, weight stability had been achieved. Accordingly, weight change was calculated from Day 7 to Day 13, at which time 5-FU treatment was begun. All groups gained weight in this pretreatment period. Net weight gain for the
Elemental Diets Enhance 5-FU Toxicity
Chow
Prec
Viv
Flex
495
W-T
Fig. 2. Incidence of stomatitis in diet groups.
various groups (as percentage of weight on Day 7) was: Chow +8.1%, Flexical +6.5%, Precision L-R +8.8%,Vivonex +6.2%, and Warren-Teed +7.5% (Fig. 3). The Chow and Precision L-R rats gained significantly more weight (P = 0.05) than did the Flexical or Vivoiiex rats. All the groups lost weight following 5-FU treatment. Net weight loss for the various groups (percentage of weight on Day 13) was Chow -23.3%, Flexical -24.2%, Precision L-R -22.3%, Vivonex -24.1%, and Warren-Teed -21.5%. The Vivonex and Flexical rats lost significantly more weight (p = 0.05) than did the Precision L-R or WarrenTeed rats. No other comparisons among the five groups attain statistical significance. Mortality Some rats died on the 4th or 5th day but most of the deaths occurred following 6 full days of 5-FU treatment. Total mortality for the various groups was: Chow 2 (4%),, Flexical 10 (20%), Precision 4 (9%), Vivonex 18 (36%), and Warren-Teed 5 (10%) (Fig. 4).
% Control
5 FU
2
-30'
-
Chow Prec Flex
Viv
Fig. 3. Weight changes in diet groups.
W-T
496
Stanford et al.
YO
'ool Chow
Prec
Flex
Viv
W-T
Fig. 4. Mortality prior to termination of study.
Blood Cultures Positive blood cultures were obtained in 12% of the controls and 70% of the 5-FU rats. The incidence of positive cultures in the 5-FU-treated rats was: Chow 50%, Flexical 7376, Precision L-R 4.576, Vivonex 94%, and Warren-Teed 97% (Fig. 5 ) . The extent of protein hydrolysis into individual amino acids of the various diets correlates extremely well with the incidence of positive blood cultures: Chow and Precision L-R (0%protein hydrolysis) had approximately 50% positive blood cultures, Flexical(66% protein hydrolysis) had 75% positive cultures, and Vivonex and Warren-Teed (100%protein hydrolysis) had almost 100%positive blood cultures. Hemogram
The average hemoglobin value in the control rats was 15.0 and in the 5-FU-treated rats was 17.8. This difference is significant (P = 0.01). There were no statistically significant differences between the groups. Platelet counts were also depressed in each diet group by 5-FU.
% Hydro. -
Chow Prec Flex
Viv
W-T
Fig. 5 . Incidence of positive blood cultures related to degree of protein hydrolysis of diet.
Elemental Diets Enhance 5-FU Toxicity
497
Serum Protein Values Control total serum protein levels were similar in all diet groups. All groups had lowered total protein values with 5-FU treatment. Serum albumin fell after administration of 5-FU and the fall was greatest (P = 0.05) in the animals receiving completely hydrolyzed protein (Fig. 6). Gamma globulin levels fell in all animals receiving 5-FU. The fall in gamma globulm was similar in all groups.
OControl -5
Chow
Prec
Flex
FU
Viv
W-T
Fig. 6 . Serum albumin levels in diet groups.
Colon Histology The control rat colon on histologic examination with B hematoxylin and eosin stain resembled normal human colon. Treatment with 5-FU resulted in glandular atrophy, subrnucosal edema, large nuclei showing maturation arrest, lack of nuclear polarity, and ulcers with areas of necrosis. Colonic goblet cell counts were significantly lower (P = 0.05) in the groups consuming elemental diets.
1)ISCUSSION The prudent physician of the 19th century advised his patients to eat a well-balanced diet, composed of protein, carbohydrate, and fat. He knew that adequate amounts of a diet so balanced would support optimal growth and maintenance, but he had no scientific evidence for this conclusion. Over the past 100 years, a vast number of nutritional investigations have been conducted. Early work led to the acceptance of 3 basic measurements of the nutritional quality of foodstuffs: biological value (BV), net protein utilization (NPU), and true digestibility (TD), described by the formulas: N intake -(fecal N-metabolic N) N-int ake N intake - (fecal N-metabolic N) - (urinary N-endogenous N) BV = N-intake - (fecal N-metabolic N)
TD =
NPU = (TD) (BV) 100
x
100
498
Stanford et al.
True digestibility describes the fraction of nitrogen absorbed across the gastrointestinal mucosa from a particular diet, and biological value describes the fraction of nitrogen which is utilized by the body for growth and maintenance (19). These values have been determined for a wide variety of foodstuffs. Later work focused on the various amino acids that are not adequately synthesized by the human body. These were termed “essential” amino acids and a list of them and the minimum amounts necessary for optimal nutrition was published by Rose et al. in 1955 (21). The Food and Agriculture Organization of the United States subsequently used Rose’s initial formulation - called the “Rose pattern” - as the basis for its recommendations for protein nutrition for the underdeveloped countries (10). Since protein synthesis requires that all constituent amino acids be present in adequate amounts, Block and Mitchell (5) proposed that the nutritional value of a given protein was proportional t o the amount of its most limited amino acid. Miller and Payne (17) then used the percentage deficit of the limiting amino acid to calculate an “amino acid score.” Generally egg protein has been used as the reference standard and assigned a score of 100, with other proteins having somewhat lesser values (7). Basic problems arise: however, in attempting to calculate nutritional quality of proteins based on BV determinations for the amino acid score. The measured BV of a given protein depends on the type of carbohydrate in the meal (12). Eggum (7) showed that BV of skim milk powder was higher than casein in spite of the greater content of amino acids in casein. He proposed that this is due to the higher content of lactose in skim milk powder. Forbes et al. (8) reported that BV depends on the concentration of protein in the diet. The protein-to-calorie ratio of the diet (18) and the adequacy of caloric intake (9) also affect the BV. Problems also arise in attempting to use the amino acid score in assessing protein quality. Hegsted and co-workers questioned 2 basic assumptions of Miller and Payne: 1) that NPU is equivalent to amino acid score as calculated from the most limiting amino acid, and 2) that NPU falls in a linear and predictable fashion as dietary protein is increased. They showed that NPU for a given protein depends upon which amino acid is limiting (1 5). They also showed that NPU is constant over an appreciable range of intake of dietary protein (14). Barnes and Valle-Riestra speculated that the discrepancies between amino acid scores and actual nutritional value might be due to the fact that amino acids present in the protein were not available to the animal for protein synthesis. They showed this to be the case for heat-damaged egg albumin (4). Since Rose, other estimations of the human and rat requirements for essential amino acids have appeared. Comparison of published data from various investigations yields the data in Table I (derived from Refs. 16 and 22). It will be immediately noted that there is extremely wide variation. Indeed, for tryptophan (often taken as a reference point for amino acid scores) the requirement varies among various reports by a factor of 5 . Harper even questions the policy of classifying amino acids as “essential” or “nonessential” (13). He states that “evidence so far available indicates that no single nitrogenous compound is as effective as a mixture of all of the amino acids that can be synthesized by the body.” In their review article, Irwin and Hegsted state that the data on amino acid requirements, protein requirements, and the nutritional quality of protein d o not provide a consistent, understandable pattern: “Until such information is available, the data on amino acid requirements will remain of doubtful value in the development of practical nutrition standards” (1 6);
Elemental Diets Enhance 5-FU Toxicity
499
TABLE I. Variation in Estimated Amino Acid Requirements Among Various Reports Amino acid Arginine Histidine Isoleucine Leucine Lysine Methionine
Phenylalanine
Threonine Tryptophan Valine
Adult human “possibly essential” “possibly essential” 250-700 mg/day 170-1,100 mg/day 300-1,200 mg/day 75-350 mg/day (with 100 mg cystine) 80-1,100 mg/day (no cystine) 300-600 mg/day (with 400 nig tyrosine) 800-1,100 mg/day (no tyrosine) 100-1,500 mg/day 50-240 mg/day 230-800 mg/day
Adult rat 7 mg/day 100 mg/day 150 mg/day 500 mg/day 600 mg/day 100 mg/day
200 nig/day
150 mg/day 150 mg/day 220 mg/day
With the above observations in mind, an attempt was made to ascertain the optimum diet for protection of rats against the gastrointestinal toxicity of a standard chemotherapeutic agent, 5-FU. Although the vast majority of nutritional experiments have used the albino rat as the experimental animal, the exact correlation between rat and human nutrition remains unknown. All points of view are well represented in the literature. We were therefore gratified t o observe positive weight gain in all the groups of animals consuming an elemental diet designed for human nutrition. This has been observed by other investigators (20). The hematologic and serum protein values observed in control rats in the present experiment were identical to those of man. Indeed, the rats receiving Precision L-R gained inore weight than did the Rat Chow group (Fig. 3). In order t o adequately remove the fiber from the rats on the elemental diets, it was necessary to use wire-bottom cages t o prevent coprophagy, since rats may consume as much as one-half of their excreted feces (3). Although some reports indicate that this may be associated with Vitamin B12 (19), Biotin (l), and essential fatty acid deficiency ( 2 ) , Giovanetti (1 1) observed that the prevention of coprophagy had no significant effect on the availability of amino acids t o rats, and no effect on weight gain. ‘The extent of protein hydrolysis of the particular elemental diet used in this study was associated with increasing incidence of positive blood cultures and progressive decline in serum albumin values. Rat Chow and Precision L-R, which contain no hydrolyzed protein, had the least incidence of positive cultures and the least decline in serum albumin concentration. Vivonex and Warren-Teed Low Residue, which are 100%hydrolyzed, had the greatest incidence of positive blood cultures and decrease in albumin. If these results were due t o purely local phenomena (affecting dietary intake), e.g., the high osmolality of 100%hydrolyzed diets, then one would expect to see this reflected in the animals’ weight changes. However, the weight changes of the rats consuming Warren-Teed L-R were identical t o those of the animals on Rat Chow (Fig. 3).
500
Stanford et al.
A significant number of animals died prior t o completion of the 6 day course of 5-FU administration (Fig. 4). The 2 diet groups with the greatest number of early deaths were also the groups with the greatest incidence of stomatitis (Fig. 2). This finding is confirmed clinically, stomatitis being a definite indication t o discontinue 5-FU therapy. A toxic dose of 5-FU was given t o these animals so that possible differences in the various diets would be optimally demonstrated. Also a generally accepted principle of cancer chemotherapy is that the greatest amount of chemotherapeutic agent which the patient can tolerate should be given. We conclude that protein hydrolysis of the diet given t o animals receiving toxic doses of 5-FU is associated with increased morbidity and mortality.
ACKNOWLEDGMENTS The authors would like t o express their appreciation to the Mead-Johnson, Doyle Pharmaceutical, Morton-Norwich (Eat on Laboratories), and Warren-Teed Pharmaceutical companies for their generous support of this project.
REFERENCES 1. Barnes, R. H., Kwong, J., and Fiala, GI: Effects of prevention of coprophagy in the rat. Biotin. J. Nutr. 61:599, 1999. 2. Barnes, R. H., Tuthill, T,, Kwong, J., aad Fiqla, G.: &€fpc$s of prevention of coprophagy in the rat: Essential fatty acid deficiency. J. Netr. 68rl21, $959: 3. Barnes, R. H. and Fialp, G.: Effects of prevention of coprophagy in the rat. J. Nutr. 65: 103, 1958. 4. Barnes, R. H. and Valle-Riestra, J.: Digestion of heat-damaged egg albumin by the rat. J. Nutr. 100:873, 1970. 5. Block, R. J. and Mitchell, H. H.: The correlation of the amino acid compositions of proteins with their nutritive value. Nutr. Abst. Rev. 16:249, 1946-47. 6. Bounous, G., Hugson, J., and Gentile, U.: Elemental diet in the management of the intestinal lesion produced by 5-fluorouracil in the rat. Can. J. Surg. 14:298, 1971. 7. Eggum, B.O.: “A Study of Certain Factors Influencing Protein Utilization in Rats and Pigs.” Copenhagen, 1973. 8. Forbes, R. M., et al.: Dependence of biological value on protein concentration in the diet of the growingrat. J. Nutr. 64:291, 1958. 9. Forbes, R. M. and Yoke, M.: Effect of energy intake on the biological values of protein fed to rats. J. Nutr. 55:499, 1954. 10. Food and Agriculture Organization: “Protein Requirements.” F A 0 Nutritional Studies #16, Report of the F A 0 Committee, Rome, 1957. 11. Giovanetti, P. M., et al.: Coprophagy prevention and availability of amino acids in wheat for the growing rat. Canad. J. Animal Sci. 50:269. 12. Guggenheim, K., et al.: Levels of lysine and methionine in portal blood of rats following protein feeding. Arch. Biochem. Biophys. 91:6, 1960. 13. Harper, A. E.: Nonessential amino acids. J. Nutr. 104:965, 1974. 14. Hegsted, D. M. and Neff, R.: Efficiency of protein utilization in young rats and various levels of intake. J. Nutr. 100:1173, 1970. 15. Hegsted, D. M. and Saik, A. K.: Response of adult rats t o low dietary levels of essential amino acids, J. Nutr. 100:1363, 1970. 16. Irwin, M. I. and Hegsted, D. M.: A conspectus of research in amino acid requirements of man. J. Nutr. 101:539, 1971,
Elemental Diets Enhance 5-FUToxicity
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17. Miller,.D. S. and Payne. P. R.: Problems in the prediction of protein concentration. Brit. J. Nutr. 15:1b, 1961. 18. Miller. D S. and Payne, P. R.: Problems in the prediction of protein values of diets: The use of food composition tables. J. Nutr. 74:413, 1961. 19. Mitchell, H. H.: A method of determination of the biological value of protein. J. Biol. Chem. 58:873, 1924. 20. Nakogawa, I. and Masana, Y . : Effects of protein nutrition on growth and lifespan in the rat. J. Nutr. 101:613, 1971. 21. Rose, W. C.,et al.: Amino acid requirement of man. J. Biol. Chem. 217:997, 1954. 22, ?YiiIeke, H. L., et al.: Evaluation of linear programming techniques in formulating human diets with rat feeding tests. J. Nutr. 103:179, 1973.
The Adverse Effects of Elemental Diets on Tolerance for 5-FU Toxicity in the Rat ....................................................................................... .......................................................................................
JOHN R. STANFORD, M.D.,DENNIS KING, M.D.,LARRY CAREY, M.D., and GREGG ANDERSON,M.D. The (effects of a group of elemental diets on the gastrointestinal toxicity of 5-FU in the SpragueDawley rat were evaluated. Diarrhea, stomatitis, hypoalbuminemia, and early deaths were more frequent in the animals on elemental diets than in those consuming standard rat chow. Sepsis and hypoalbuininemia were directly related t o the extent of protein hydrolysis of the particular elemental diet.
....................................................................................... ....................................................................................... Key words: 5-fluorouracil, elemental diet, protein hydrolysis, blood cultures, serum albumin
INTRODUCTION Five-Fluorouracil(5-FU) is a potent chemotherapeutic agent that has been used extensively in clinical practice for over 15 years. At present, the drug is recommended in th.e palliative management of a wide variety of malignancies, including those of the colon, rectum, breast, stomach, and pancreas. The effectiveness of 5-FU is t o a great extent limited by its toxicity. The rapidly dividing cells of the bone marrow and gastrointestinal mucosa are particularly vulnerable to 5-FU and, indeed, its main toxicity is found in these tissues. Leukopenia, stomatitis, and diarrhea are definite indications t o discontinue therapy. It has been suggested that elemental diets provide some protection against the gastrointestinal toxicity of 5-FU (6). T h s hypothesis was tested in the following study.
MATERIALS AND METHODS A total of 350 male Sprague-Dawley rats weighing 300-320 g were housed in pairs in wire-bottom cages. A 12 hour light-dark cycle was utilized in the room in which the From the Department of Surgery, Ohio State University College of Medicine, Columbus Address reprint requests to John R. Stanford, M.D., Ohio State University, College of Medicine, 410 'West Tenth Avenue, Columbus, OH 4321 0.
493
0 1977 Alan R. Liss, Inc., 150 Fifth Avenue, New York, NY 10011
494
Stanford e t at.
animals were housed. Five different diets were evaluated: Purina Rat Chow, Flexical, Precision L-R, Vivonex, and Warren-Teed Low Residue. Each diet was fed to a group of 70 animals. Six days after weight stabilization was achieved on the various diets 50 animals in each diet group were given 5-FU at 25 mg/kg subcutaneously for 6 consecutive days. The remaining 20 animals in each group served as controls. Daily weight was recorded and the presence or absence of diarrhea and stomatitis were noted both before and after 5-FU therapy. All animals were killed after 6 full days of 5-FU therapy. Blood was drawn aseptically for culture, hemoglobin, WBC, and platelet counts, and for serum protein electrophoresis. Histologic sections were prepared from colon specimens and stained with hematoxylin and eosin.
RESULTS Diarrhea and Stomatitis Prior t o 5-FU treatment, diarrhea was noted in 2 (4%) of the Chow rats, 11 (22%) of the Flexical rats, 2 (4%) of the Precision rats, 19 (38%) of the Vivonex rats, and 3 (6%) of the Warren-Teed rats. Following 5-FU treatment, these percentages increased t o 26% for Chow, 96% for Flexical, 91% for Precision, 100% for Vivonex, and 86% for WarrenTeed (Fig. 1). Stomatitis was not seen in any groups prior to 5-FU treatment; following treatment it was seen in 6 (1 2%) of the Chow rats, 47 (94%) of the Flexical rats, 1 5 (32%) of the Precision rats, 36 (72%) of the Vivonex rats, and 26 (52%) of the Warren-Teed rats (Fig. 2).
Chow
Prec
Flex
Viv
W-T
Fig. 1. Incidence of diarrhea in diet groups.
Weight Changes Weight data were analyzed with the aid of an IBM 370/165 computer. All the animals receiving elemental diets experienced an initial 2-3 day period of weight loss. After 6 days on the experimental diets, weight stability had been achieved. Accordingly, weight change was calculated from Day 7 to Day 13, at which time 5-FU treatment was begun. All groups gained weight in this pretreatment period. Net weight gain for the
Elemental Diets Enhance 5-FU Toxicity
Chow
Prec
Viv
Flex
495
W-T
Fig. 2. Incidence of stomatitis in diet groups.
various groups (as percentage of weight on Day 7) was: Chow +8.1%, Flexical +6.5%, Precision L-R +8.8%,Vivonex +6.2%, and Warren-Teed +7.5% (Fig. 3). The Chow and Precision L-R rats gained significantly more weight (P = 0.05) than did the Flexical or Vivoiiex rats. All the groups lost weight following 5-FU treatment. Net weight loss for the various groups (percentage of weight on Day 13) was Chow -23.3%, Flexical -24.2%, Precision L-R -22.3%, Vivonex -24.1%, and Warren-Teed -21.5%. The Vivonex and Flexical rats lost significantly more weight (p = 0.05) than did the Precision L-R or WarrenTeed rats. No other comparisons among the five groups attain statistical significance. Mortality Some rats died on the 4th or 5th day but most of the deaths occurred following 6 full days of 5-FU treatment. Total mortality for the various groups was: Chow 2 (4%),, Flexical 10 (20%), Precision 4 (9%), Vivonex 18 (36%), and Warren-Teed 5 (10%) (Fig. 4).
% Control
5 FU
2
-30'
-
Chow Prec Flex
Viv
Fig. 3. Weight changes in diet groups.
W-T
496
Stanford et al.
YO
'ool Chow
Prec
Flex
Viv
W-T
Fig. 4. Mortality prior to termination of study.
Blood Cultures Positive blood cultures were obtained in 12% of the controls and 70% of the 5-FU rats. The incidence of positive cultures in the 5-FU-treated rats was: Chow 50%, Flexical 7376, Precision L-R 4.576, Vivonex 94%, and Warren-Teed 97% (Fig. 5 ) . The extent of protein hydrolysis into individual amino acids of the various diets correlates extremely well with the incidence of positive blood cultures: Chow and Precision L-R (0%protein hydrolysis) had approximately 50% positive blood cultures, Flexical(66% protein hydrolysis) had 75% positive cultures, and Vivonex and Warren-Teed (100%protein hydrolysis) had almost 100%positive blood cultures. Hemogram
The average hemoglobin value in the control rats was 15.0 and in the 5-FU-treated rats was 17.8. This difference is significant (P = 0.01). There were no statistically significant differences between the groups. Platelet counts were also depressed in each diet group by 5-FU.
% Hydro. -
Chow Prec Flex
Viv
W-T
Fig. 5 . Incidence of positive blood cultures related to degree of protein hydrolysis of diet.
Elemental Diets Enhance 5-FU Toxicity
497
Serum Protein Values Control total serum protein levels were similar in all diet groups. All groups had lowered total protein values with 5-FU treatment. Serum albumin fell after administration of 5-FU and the fall was greatest (P = 0.05) in the animals receiving completely hydrolyzed protein (Fig. 6). Gamma globulin levels fell in all animals receiving 5-FU. The fall in gamma globulm was similar in all groups.
OControl -5
Chow
Prec
Flex
FU
Viv
W-T
Fig. 6 . Serum albumin levels in diet groups.
Colon Histology The control rat colon on histologic examination with B hematoxylin and eosin stain resembled normal human colon. Treatment with 5-FU resulted in glandular atrophy, subrnucosal edema, large nuclei showing maturation arrest, lack of nuclear polarity, and ulcers with areas of necrosis. Colonic goblet cell counts were significantly lower (P = 0.05) in the groups consuming elemental diets.
1)ISCUSSION The prudent physician of the 19th century advised his patients to eat a well-balanced diet, composed of protein, carbohydrate, and fat. He knew that adequate amounts of a diet so balanced would support optimal growth and maintenance, but he had no scientific evidence for this conclusion. Over the past 100 years, a vast number of nutritional investigations have been conducted. Early work led to the acceptance of 3 basic measurements of the nutritional quality of foodstuffs: biological value (BV), net protein utilization (NPU), and true digestibility (TD), described by the formulas: N intake -(fecal N-metabolic N) N-int ake N intake - (fecal N-metabolic N) - (urinary N-endogenous N) BV = N-intake - (fecal N-metabolic N)
TD =
NPU = (TD) (BV) 100
x
100
498
Stanford et al.
True digestibility describes the fraction of nitrogen absorbed across the gastrointestinal mucosa from a particular diet, and biological value describes the fraction of nitrogen which is utilized by the body for growth and maintenance (19). These values have been determined for a wide variety of foodstuffs. Later work focused on the various amino acids that are not adequately synthesized by the human body. These were termed “essential” amino acids and a list of them and the minimum amounts necessary for optimal nutrition was published by Rose et al. in 1955 (21). The Food and Agriculture Organization of the United States subsequently used Rose’s initial formulation - called the “Rose pattern” - as the basis for its recommendations for protein nutrition for the underdeveloped countries (10). Since protein synthesis requires that all constituent amino acids be present in adequate amounts, Block and Mitchell (5) proposed that the nutritional value of a given protein was proportional t o the amount of its most limited amino acid. Miller and Payne (17) then used the percentage deficit of the limiting amino acid to calculate an “amino acid score.” Generally egg protein has been used as the reference standard and assigned a score of 100, with other proteins having somewhat lesser values (7). Basic problems arise: however, in attempting to calculate nutritional quality of proteins based on BV determinations for the amino acid score. The measured BV of a given protein depends on the type of carbohydrate in the meal (12). Eggum (7) showed that BV of skim milk powder was higher than casein in spite of the greater content of amino acids in casein. He proposed that this is due to the higher content of lactose in skim milk powder. Forbes et al. (8) reported that BV depends on the concentration of protein in the diet. The protein-to-calorie ratio of the diet (18) and the adequacy of caloric intake (9) also affect the BV. Problems also arise in attempting to use the amino acid score in assessing protein quality. Hegsted and co-workers questioned 2 basic assumptions of Miller and Payne: 1) that NPU is equivalent to amino acid score as calculated from the most limiting amino acid, and 2) that NPU falls in a linear and predictable fashion as dietary protein is increased. They showed that NPU for a given protein depends upon which amino acid is limiting (1 5). They also showed that NPU is constant over an appreciable range of intake of dietary protein (14). Barnes and Valle-Riestra speculated that the discrepancies between amino acid scores and actual nutritional value might be due to the fact that amino acids present in the protein were not available to the animal for protein synthesis. They showed this to be the case for heat-damaged egg albumin (4). Since Rose, other estimations of the human and rat requirements for essential amino acids have appeared. Comparison of published data from various investigations yields the data in Table I (derived from Refs. 16 and 22). It will be immediately noted that there is extremely wide variation. Indeed, for tryptophan (often taken as a reference point for amino acid scores) the requirement varies among various reports by a factor of 5 . Harper even questions the policy of classifying amino acids as “essential” or “nonessential” (13). He states that “evidence so far available indicates that no single nitrogenous compound is as effective as a mixture of all of the amino acids that can be synthesized by the body.” In their review article, Irwin and Hegsted state that the data on amino acid requirements, protein requirements, and the nutritional quality of protein d o not provide a consistent, understandable pattern: “Until such information is available, the data on amino acid requirements will remain of doubtful value in the development of practical nutrition standards” (1 6);
Elemental Diets Enhance 5-FU Toxicity
499
TABLE I. Variation in Estimated Amino Acid Requirements Among Various Reports Amino acid Arginine Histidine Isoleucine Leucine Lysine Methionine
Phenylalanine
Threonine Tryptophan Valine
Adult human “possibly essential” “possibly essential” 250-700 mg/day 170-1,100 mg/day 300-1,200 mg/day 75-350 mg/day (with 100 mg cystine) 80-1,100 mg/day (no cystine) 300-600 mg/day (with 400 nig tyrosine) 800-1,100 mg/day (no tyrosine) 100-1,500 mg/day 50-240 mg/day 230-800 mg/day
Adult rat 7 mg/day 100 mg/day 150 mg/day 500 mg/day 600 mg/day 100 mg/day
200 nig/day
150 mg/day 150 mg/day 220 mg/day
With the above observations in mind, an attempt was made to ascertain the optimum diet for protection of rats against the gastrointestinal toxicity of a standard chemotherapeutic agent, 5-FU. Although the vast majority of nutritional experiments have used the albino rat as the experimental animal, the exact correlation between rat and human nutrition remains unknown. All points of view are well represented in the literature. We were therefore gratified t o observe positive weight gain in all the groups of animals consuming an elemental diet designed for human nutrition. This has been observed by other investigators (20). The hematologic and serum protein values observed in control rats in the present experiment were identical to those of man. Indeed, the rats receiving Precision L-R gained inore weight than did the Rat Chow group (Fig. 3). In order t o adequately remove the fiber from the rats on the elemental diets, it was necessary to use wire-bottom cages t o prevent coprophagy, since rats may consume as much as one-half of their excreted feces (3). Although some reports indicate that this may be associated with Vitamin B12 (19), Biotin (l), and essential fatty acid deficiency ( 2 ) , Giovanetti (1 1) observed that the prevention of coprophagy had no significant effect on the availability of amino acids t o rats, and no effect on weight gain. ‘The extent of protein hydrolysis of the particular elemental diet used in this study was associated with increasing incidence of positive blood cultures and progressive decline in serum albumin values. Rat Chow and Precision L-R, which contain no hydrolyzed protein, had the least incidence of positive cultures and the least decline in serum albumin concentration. Vivonex and Warren-Teed Low Residue, which are 100%hydrolyzed, had the greatest incidence of positive blood cultures and decrease in albumin. If these results were due t o purely local phenomena (affecting dietary intake), e.g., the high osmolality of 100%hydrolyzed diets, then one would expect to see this reflected in the animals’ weight changes. However, the weight changes of the rats consuming Warren-Teed L-R were identical t o those of the animals on Rat Chow (Fig. 3).
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A significant number of animals died prior t o completion of the 6 day course of 5-FU administration (Fig. 4). The 2 diet groups with the greatest number of early deaths were also the groups with the greatest incidence of stomatitis (Fig. 2). This finding is confirmed clinically, stomatitis being a definite indication t o discontinue 5-FU therapy. A toxic dose of 5-FU was given t o these animals so that possible differences in the various diets would be optimally demonstrated. Also a generally accepted principle of cancer chemotherapy is that the greatest amount of chemotherapeutic agent which the patient can tolerate should be given. We conclude that protein hydrolysis of the diet given t o animals receiving toxic doses of 5-FU is associated with increased morbidity and mortality.
ACKNOWLEDGMENTS The authors would like t o express their appreciation to the Mead-Johnson, Doyle Pharmaceutical, Morton-Norwich (Eat on Laboratories), and Warren-Teed Pharmaceutical companies for their generous support of this project.
REFERENCES 1. Barnes, R. H., Kwong, J., and Fiala, GI: Effects of prevention of coprophagy in the rat. Biotin. J. Nutr. 61:599, 1999. 2. Barnes, R. H., Tuthill, T,, Kwong, J., aad Fiqla, G.: &€fpc$s of prevention of coprophagy in the rat: Essential fatty acid deficiency. J. Netr. 68rl21, $959: 3. Barnes, R. H. and Fialp, G.: Effects of prevention of coprophagy in the rat. J. Nutr. 65: 103, 1958. 4. Barnes, R. H. and Valle-Riestra, J.: Digestion of heat-damaged egg albumin by the rat. J. Nutr. 100:873, 1970. 5. Block, R. J. and Mitchell, H. H.: The correlation of the amino acid compositions of proteins with their nutritive value. Nutr. Abst. Rev. 16:249, 1946-47. 6. Bounous, G., Hugson, J., and Gentile, U.: Elemental diet in the management of the intestinal lesion produced by 5-fluorouracil in the rat. Can. J. Surg. 14:298, 1971. 7. Eggum, B.O.: “A Study of Certain Factors Influencing Protein Utilization in Rats and Pigs.” Copenhagen, 1973. 8. Forbes, R. M., et al.: Dependence of biological value on protein concentration in the diet of the growingrat. J. Nutr. 64:291, 1958. 9. Forbes, R. M. and Yoke, M.: Effect of energy intake on the biological values of protein fed to rats. J. Nutr. 55:499, 1954. 10. Food and Agriculture Organization: “Protein Requirements.” F A 0 Nutritional Studies #16, Report of the F A 0 Committee, Rome, 1957. 11. Giovanetti, P. M., et al.: Coprophagy prevention and availability of amino acids in wheat for the growing rat. Canad. J. Animal Sci. 50:269. 12. Guggenheim, K., et al.: Levels of lysine and methionine in portal blood of rats following protein feeding. Arch. Biochem. Biophys. 91:6, 1960. 13. Harper, A. E.: Nonessential amino acids. J. Nutr. 104:965, 1974. 14. Hegsted, D. M. and Neff, R.: Efficiency of protein utilization in young rats and various levels of intake. J. Nutr. 100:1173, 1970. 15. Hegsted, D. M. and Saik, A. K.: Response of adult rats t o low dietary levels of essential amino acids, J. Nutr. 100:1363, 1970. 16. Irwin, M. I. and Hegsted, D. M.: A conspectus of research in amino acid requirements of man. J. Nutr. 101:539, 1971,
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17. Miller,.D. S. and Payne. P. R.: Problems in the prediction of protein concentration. Brit. J. Nutr. 15:1b, 1961. 18. Miller. D S. and Payne, P. R.: Problems in the prediction of protein values of diets: The use of food composition tables. J. Nutr. 74:413, 1961. 19. Mitchell, H. H.: A method of determination of the biological value of protein. J. Biol. Chem. 58:873, 1924. 20. Nakogawa, I. and Masana, Y . : Effects of protein nutrition on growth and lifespan in the rat. J. Nutr. 101:613, 1971. 21. Rose, W. C.,et al.: Amino acid requirement of man. J. Biol. Chem. 217:997, 1954. 22, ?YiiIeke, H. L., et al.: Evaluation of linear programming techniques in formulating human diets with rat feeding tests. J. Nutr. 103:179, 1973.
