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Quantitative nutritional studies with water-soluble, chemically defined diets. IX. Further studies on d -glucosamine-containing diets
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
89,
521-527 (1959)
Quantitative Nutritional Studies with Water- Soluble, Chemically Defined Diets. IX. Further Studies on D-Glucosamine-Containing Diets Takashi Sugimura, Sanford M. Birnbaum, Milton Wink Jesse P. Greenstein From the Laboratory of Health, Public
and
of Biochemistry, National Cancer Institute, National Institutes Health Service, United States Department of Health, Education, and Welfare, Bethesda, Maryland
Received January 19, 1959
An earlier report from this laboratory (1) was based on the effect on the food intake and growth of rats induced by incorporationg n-glucosamine at various levels into a water-soluble, chemically defined diet. It appeared from this study that the essential effect of the glucosamine was manifested solely in a diminished food intake yielding growth rates which varied inversely with the glucosamine concentration. This simple phenomenon then allowed a linear plot of weight change in grams gained per day against food intake in grams per day. Extrapolation of such a plot to the base line (zero weight gain) gave a figure which represented the average maintenance requirement for the animals over the particular period of investigation. Subtraction of this maintenance requirement (B) from the average daily total food intake (1) then gave a figure that was directly proportional to growth (G), and the G/(1 - B) values calculated over each successive growth period were quite constant for that period. The present report is concerned with the response of normal and tumorbearing rats to similar levels of glucosamine added to the more satisfactory diet No. 26 (2) which contains a wider variety of non-essential amino acids than the mixture earlier employed (diet No. 3) (2), and with the effect of such diets on the growth of a transplantable tumor. Inasmuch as the same phenomenon of depressed food intake was observed at the higher levels of glucosamine, the forced-feeding technique was employed in the latter studies to obviate the effects of decreased caloric intake on tumor growt,h and to observe any possible toxic effect of glucosamine on the organism. Incidental observations on the extreme hourly range of weight fluctuation exhibited by rats on various dietary regimens are included in 521
522
SUGIMURA,
BIRNBAUM,
WINITZ
AND
GREENSTEIN
order to emphasize the need for precise timing in the measurement of daily weight increments. EXPERIMENTAL Water-soluble, chemically defined diets prepared as transparent solutions containing 50% solids were employed exclusively as before (1, 2). The basal diet was that referred to previously (2) as diet No. 26 which contains 9.5 g. N/kg. solids derived from the essential amino acids and 15.7 g. N supplied by the non-essential amino Dacids in the same relative proportions in which they exist in casein. Crystalline glucosamine (1) replaced an equal weight of n-glucose in the experimental diets in the amounts of 25, 50, 80, 120, 160, and 200 g./kg. solids in diets No. 60-65, respectively. The free base was neutralized in each case with an equivalent amount of acetic acid which was likewise compensated by deletion of an equal weight of glucose. All mixtures were thus closely isocaloric and contained identical amino acid concentrations. Diets were supplied either ad Zibitum in inverted drinking tubes (2) or were forcefed in divided doses at 9 a.m., 1 p.m., 5 p.m., and 9 p.m. according to the procedure outlined in a previous report (3). Tap water was always supplied ad Zibitum. (N.I.H.) The animals employed were male albino rats of the Sprague-Dawley strain and were weaned from stock mothers which were reared on the Purina Laboratory Chow diet. Where larger animals were employed, they were transferred from the Chow diet and maintained ad libitum on diet No. 26 for a period of from 10 days to 2 weeks prior to t,he initiation of the experimental period. For the tumor growth studies, the Walker carcinosarcoma 256 was implanted subcutaneously by trochar into the right axillary region of animals so acclimated and allowed to grow for 6 days until the tumor was barely palpable. Following this induction period, the animals were divided into groups of equal average weight value and subjected to the experimental regimen. RESULTS
AND
DISCUSSION
Growth Studies In Fig. 1, the average daily growth response of weanling male rats over a 24-day period is plotted against the average daily food intake for a series of six diets ranging in glucosamine concentration from 0 % (diet No. 26) to 20 %. Table I illustrates that all of these dietary glucosamine concentrations are much less inhibitory at a given level in the present experiments than they were when the simpler mixture No. 3 was used as a base (l), and at the lower concentrations appear now even to exhibit a stimulatory effect. Again, however, the straight-line relationship between growth and food intake observed in Fig. 1 allows a simple extrapolation of the curve to the base line yielding a value for B of 2.6 g. food as the average daily requirement for the maintenance of body weight over the 24-day period involved. This value is compared to the figure of 2.2 g./day obtained over IL 25-day period using the otherwise less eficient dietary mixture No. 3 [cf. (1, 2)]. The apparent discrepancy arises, at least in part, from the
NUTRITIONAL
I
2
DIET
3
STUDIES.
4
5
INTAKE
6
523
IX
7
6
9
IO
IN GRAMS PER DAY
FIG. 1. Plot of average daily weight gain against diet intake of male weanling rats on diet No. 26 in which n-glucosamine at various levels had replaced an equal weight of n-glucose. Symbols represent average values of six animals per group at glucosamine levels in terms of per cent of dry diet as follows: 0, 0%; 0, 2.5%; A,
5%; A, 8%; q ,12%; n , 16%; and @, 20%. greater size attained by the animals in a similar time period on the more efficient mixture No. 26 leading to increased average basal weight maintenance requirements. The calculated values of G/(I - B) [cf. (l)] for the seven mixtures studied exhibit the remarkable constancy of 0.56, 0.55, 0.54, 0.55, 0.54, 0.57, and 0.53 g. gained/g. of food intake above that required for weight TABLE I Growth Per cent glucosamine in diet 0 2.5 5.0 8.0 12.0 16.0 20.0
Response”
of Weanling Basal
Diet
No. 3 17 18 19 20 21 16
mixture
Rats to Glucosamine-Containing No. 3b Relative growth rate 100 93 81 60 33 10 -d
Basal Diet
No.
Diets
mixture
No. 26c Relative growth rate
26 60 61 62 63 64 65
a Expressed as per cent of growth rate on unsubstituted diet. b Data from Ref. (l), average of five rats per group for 25-day period. c Average of six rats per group for 24-day period. d Diet completely rejected.
100 102 114 95 80 56 33
524
SUGIMURA,
BIRNBAUM,
WINITZ
AND
GREENSTEIN
maintenance, for the diets containing 0, 2.5, 5.0, 8.0, 12.0, 16.0, and 20.0% glucosamine, respectively. The average value obtained with the basal diet No. 3 substituted with varying amounts of glucosamine was 0.58 g. (1). That the depressed growth rate of animals ingesting these glucosaminecontaining diets is easily reversible was shown by the attainment of growth rates of 3.85 and 4.05 g./day by animals switched after 28 days on diets 64 and 65 to the basal diet No. 26. These figures represent the averages of six rats in each group over a period of 10 days following the changeover. Tumor Studies
The depressive effect of higher concentrations of glucosamine on food intake invalidates the ad libitum technique as a means of investigating the effects of dietary glucosamine on the growth rate of a transplantable tumor. By means of the force-feeding procedures, total caloric intake and total amino acid nitrogen intake were maintained constant, and the only variable between the test groups and their controls was the actual glucosamine concentration in the diet. Table II shows the response of normal and tumor-bearing rats to 20 ml. (10 g.) per day of the control diet No. 26 and diets 62 and 63 which contain 8 and 12%, respectively, of glucosamine. Although the daily glucosamine intake for animals on these diets was thus 800 and 1200 mg./rat, the effect of these amounts on both tumor growth and upon carcass growth was essentially inappreciable. The original findings of Quastel and Canter0 (4) on the inhibition by TABLE Efects
of Forced-Fed
Glucosamine and Tumor-Bearing
COUcentration of glucosamine
II Diet” on Growth of Normal Animals
Weight change for 12 day9 Animals”
--
,”
--
control
Over-all
Tumor
N
ChKWS
--
No. No. No. No. No. No.
26 26 62 62 63 63
% 0 0 8 8 12 12
g.
Tumor-bearing Normal Tumor-bearing Normal Tumor-bearing Normal
53.2 33.1 54.0 29.5 56.2 31.2
a Twenty milliliters of diet was at 9:06 a.m., 5 ml. at 1:09 p.m., 5 b The initial body weight was animals. c Data for 12 days between 6th
f f f f f zk
g.
1.425.8 1.2 2.729.7 2.0 3.424.2 0.6
f k f -
g.
3.627.4 33.1 1.424.3 29.5 2.832.0 31.2
f f f f f f
4.6 1.2 4.3 2.0 4.6 0.6
%
%
100 114 94 -
100 88 95
given by four times forced-feeding per day (4 ml. ml. at 5:00 p.m., and 6 ml. at 9:00 p.m.). 149 g. on average. Each group consisted of six and 18th day after
tumor
transplantation.
NUTRITIONAL
STUDIES.
IX
525
glucosamine of tumor growth in mice appear to have been substantiated in studies employing human (5) and mouse (6) tumor cells cultured in vitro. Studies of the influence of this amino sugar on tumor growth in viva have, however, yielded generally negative findings [cf. (7)]. Ball, Wick, and Sanders (8) reopened the question of the chemotherapeutic efficacy of glucosamine by reporting some 27 % depression of the growth rate of the Walker carcinosarcoma 256 implanted in 120-160-g. Wistar rats. Their procedure involved intraperitoneal injection of 400-600 mg. glucosamine/ rat/day in 4-6 divided doses, the treatment being initiated on the day following tumor implantation and continued daily for 18 days. They attributed the failure of the majority of investigators [cf. (7)] to obtain positive tumor growth inhibition by glucosamine to the fact that almost invariably single daily injections were employed. The findings obtained in the present work, even though divided dosages were employed and the total intake was the highest yet reported, indicate that the effect of dietary glucosamine on tumor growth is of minor importance, if any, from a chemotherapeutic point of view and that any observable effects however slight are highly dependent upon conditions of administration, animal strain, stage of tumor development, etc. However, the appreciable breakdown of glucosamine by bacteria [cf. (4)] in the gastrointestinal tract cannot, at present, be entirely precluded. Comparison of the growth response of normal animals force-fed with equal amounts of diets containing 0, 8, and 12 % glucosamine reveals insignificant differences in rate of gain among the three groups (Table II). No toxic symptoms were noted among the normal rats receiving the glucosamine diets, and all remained clean and active. These findings confirm the hypothesis (1) that the dietary glucosamine exerts solely a self-limiting effect on food intake in ad libitum experiments without complicating toxic side reactions. Attempts to force-feed diets containing more than 12% glucosamine in the same volumes did, however, lead to a quickly appearing but transient diarrhea thus invalidating any results obtained. Hourly Weight Fluctuations Although animal weights hitherto reported from this laboratory have invariably been those recorded at 9 a.m., it became of interest in connection with the present experiments to observe the cyclic daily weight fluctuations of rats following a period of acclimatization to the various dietary regimens. Figure 24 illustrates the typical hourly weight response of such animals eating ad libitum of the solid Purina Chow stock diet and of the soluble diet No. 26. Although the two curves are not strictly comparable owing to the initial weight differences between the two groups of animals, similar responses to the two widely variant diets are evident. The average maximal
526
SUGIMURA,
BIRNBAUM,
WINITZ
a
AND
0. force
9A.M.
I PM
5 PM.
9RM.
GREENSTEIN
feeding
9 A.M.
FIG. 2. (A) Refers to average 24-hr. weight fluctuations under ad libitum feeding schedule. Six rats per group were observed over a S-day period. 0, Chow; 0, diet
No. 26. (B) Refers to average daily weight fluctuations of rats force-fed diets No. 26 and 64. Feeding schedule as in Table II, with weights taken just before each feeding. Six rats per group observed over a 3-day period. 0 diet No. 64; l diet No. 26. fluctuation of 12 g. for the animals on diet No. 26 compares with the 9 g. observed for the group on the Chow diet, the difference in range being at least partially attributable to the differences in initial body weight. On the forced-feeding routines (Fig. 2B) the schedule is of course reversed, nutriment being received for the most part during the daylight hours contrary to the normal nocturnal feeding habits of the rat. Consequently, the 9 a.m. reading is minimal rather than maximal. The response of the partially starved rats to the first 4-ml. dose is most striking, resulting in an apparent increment of up to 18 g. over the ensuing 4-hr. period when only water is available. This increase is much more pronounced in the group receiving the diet No. 64, which contains 16% glucosamine, than in the group receiving an equal amount of diet No. 26. The difference is a reflection of the voluntary increased water intake of animals on the glucosamine diet, where an average daily intake of 28 ml. water was recorded, as opposed to an intake of only 15.5 ml. by the animals fed diet No. 26.
NUTRITIONAL
STUDIES.
IX
527
SUMMARY
The effect of incorporating n-glucosamine at various levels into the water-soluble, chemically defined diet No. 26 fed ad Mn’tum to rats has been compared with that observed earlier under the same conditions when the inferior diet No. 3 was employed. As before, a linear graphical relationship between growth and food intake was observed which, by extrapolation, permitted a calculation of the average daily requirement of food for the maint,enance of body weight over the entire course of the experiment. On the superior diet, No. 26, there was (a) a slightly higher maintenance requirement, and (b) appreciably less depression in the growth rate at equal levels of glucosamine, than on diet No. 3. No toxic symptoms were evident, and it is apparent that the dietary glucosamine simply exerts a self-limiting effect on food intake. Force-feeding of diet No. 26 containing 8 and 12 % of glucosamine to rats bearing the transplanted Walker tumor resulted in no symptoms of toxicity nor any effect upon tumor or carcass growth. Observations on the cyclic daily weight fluctuations of animals ingesting soluble diets as compared with Purina Chow are described. REFERENCES
1. WINITZ, M., BIRNBAUM, S. M., AND GREENSTEIN, J. P., Arch. Biochem. 2. 3. 4. 5.
6. 7.
8.
Biophys. 72, 437 (1957). GREENSTEIN, J. P., BIRNBAUM, S. M., WINITZ, M., AND OTEY, M. C., Arch. Biothem. Biophys. 73, 396 (1957). SUOIMURA, T., BIRNBAUM,~. M., WINITZ, M., AND GREENSTEIN,J. P., Arch.Biothem. Biophys. 81, 439 (1959). QUASTEL, J. H., AND CANTERO, A., Nature 171, 252 (1953); FAULKNER, P., AND QUASTEL, J. H., Nature 177, 1216 (1956). FJELDE, A., SORKIN, E., AND RHODES, J. M., Ezptl. Cell Research 10, 88 (1956). RUBIN, A., SPRINGER, G. F., AND HOGUE, M. J., Cancer Research 14, 456 (1954). Cancer Research, Suppl. No. 3 (1955). BALL, H. A., WICK, A. N., AND SANDERS,C., Cancer Research 17, 235 (1957).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
89,
521-527 (1959)
Quantitative Nutritional Studies with Water- Soluble, Chemically Defined Diets. IX. Further Studies on D-Glucosamine-Containing Diets Takashi Sugimura, Sanford M. Birnbaum, Milton Wink Jesse P. Greenstein From the Laboratory of Health, Public
and
of Biochemistry, National Cancer Institute, National Institutes Health Service, United States Department of Health, Education, and Welfare, Bethesda, Maryland
Received January 19, 1959
An earlier report from this laboratory (1) was based on the effect on the food intake and growth of rats induced by incorporationg n-glucosamine at various levels into a water-soluble, chemically defined diet. It appeared from this study that the essential effect of the glucosamine was manifested solely in a diminished food intake yielding growth rates which varied inversely with the glucosamine concentration. This simple phenomenon then allowed a linear plot of weight change in grams gained per day against food intake in grams per day. Extrapolation of such a plot to the base line (zero weight gain) gave a figure which represented the average maintenance requirement for the animals over the particular period of investigation. Subtraction of this maintenance requirement (B) from the average daily total food intake (1) then gave a figure that was directly proportional to growth (G), and the G/(1 - B) values calculated over each successive growth period were quite constant for that period. The present report is concerned with the response of normal and tumorbearing rats to similar levels of glucosamine added to the more satisfactory diet No. 26 (2) which contains a wider variety of non-essential amino acids than the mixture earlier employed (diet No. 3) (2), and with the effect of such diets on the growth of a transplantable tumor. Inasmuch as the same phenomenon of depressed food intake was observed at the higher levels of glucosamine, the forced-feeding technique was employed in the latter studies to obviate the effects of decreased caloric intake on tumor growt,h and to observe any possible toxic effect of glucosamine on the organism. Incidental observations on the extreme hourly range of weight fluctuation exhibited by rats on various dietary regimens are included in 521
522
SUGIMURA,
BIRNBAUM,
WINITZ
AND
GREENSTEIN
order to emphasize the need for precise timing in the measurement of daily weight increments. EXPERIMENTAL Water-soluble, chemically defined diets prepared as transparent solutions containing 50% solids were employed exclusively as before (1, 2). The basal diet was that referred to previously (2) as diet No. 26 which contains 9.5 g. N/kg. solids derived from the essential amino acids and 15.7 g. N supplied by the non-essential amino Dacids in the same relative proportions in which they exist in casein. Crystalline glucosamine (1) replaced an equal weight of n-glucose in the experimental diets in the amounts of 25, 50, 80, 120, 160, and 200 g./kg. solids in diets No. 60-65, respectively. The free base was neutralized in each case with an equivalent amount of acetic acid which was likewise compensated by deletion of an equal weight of glucose. All mixtures were thus closely isocaloric and contained identical amino acid concentrations. Diets were supplied either ad Zibitum in inverted drinking tubes (2) or were forcefed in divided doses at 9 a.m., 1 p.m., 5 p.m., and 9 p.m. according to the procedure outlined in a previous report (3). Tap water was always supplied ad Zibitum. (N.I.H.) The animals employed were male albino rats of the Sprague-Dawley strain and were weaned from stock mothers which were reared on the Purina Laboratory Chow diet. Where larger animals were employed, they were transferred from the Chow diet and maintained ad libitum on diet No. 26 for a period of from 10 days to 2 weeks prior to t,he initiation of the experimental period. For the tumor growth studies, the Walker carcinosarcoma 256 was implanted subcutaneously by trochar into the right axillary region of animals so acclimated and allowed to grow for 6 days until the tumor was barely palpable. Following this induction period, the animals were divided into groups of equal average weight value and subjected to the experimental regimen. RESULTS
AND
DISCUSSION
Growth Studies In Fig. 1, the average daily growth response of weanling male rats over a 24-day period is plotted against the average daily food intake for a series of six diets ranging in glucosamine concentration from 0 % (diet No. 26) to 20 %. Table I illustrates that all of these dietary glucosamine concentrations are much less inhibitory at a given level in the present experiments than they were when the simpler mixture No. 3 was used as a base (l), and at the lower concentrations appear now even to exhibit a stimulatory effect. Again, however, the straight-line relationship between growth and food intake observed in Fig. 1 allows a simple extrapolation of the curve to the base line yielding a value for B of 2.6 g. food as the average daily requirement for the maintenance of body weight over the 24-day period involved. This value is compared to the figure of 2.2 g./day obtained over IL 25-day period using the otherwise less eficient dietary mixture No. 3 [cf. (1, 2)]. The apparent discrepancy arises, at least in part, from the
NUTRITIONAL
I
2
DIET
3
STUDIES.
4
5
INTAKE
6
523
IX
7
6
9
IO
IN GRAMS PER DAY
FIG. 1. Plot of average daily weight gain against diet intake of male weanling rats on diet No. 26 in which n-glucosamine at various levels had replaced an equal weight of n-glucose. Symbols represent average values of six animals per group at glucosamine levels in terms of per cent of dry diet as follows: 0, 0%; 0, 2.5%; A,
5%; A, 8%; q ,12%; n , 16%; and @, 20%. greater size attained by the animals in a similar time period on the more efficient mixture No. 26 leading to increased average basal weight maintenance requirements. The calculated values of G/(I - B) [cf. (l)] for the seven mixtures studied exhibit the remarkable constancy of 0.56, 0.55, 0.54, 0.55, 0.54, 0.57, and 0.53 g. gained/g. of food intake above that required for weight TABLE I Growth Per cent glucosamine in diet 0 2.5 5.0 8.0 12.0 16.0 20.0
Response”
of Weanling Basal
Diet
No. 3 17 18 19 20 21 16
mixture
Rats to Glucosamine-Containing No. 3b Relative growth rate 100 93 81 60 33 10 -d
Basal Diet
No.
Diets
mixture
No. 26c Relative growth rate
26 60 61 62 63 64 65
a Expressed as per cent of growth rate on unsubstituted diet. b Data from Ref. (l), average of five rats per group for 25-day period. c Average of six rats per group for 24-day period. d Diet completely rejected.
100 102 114 95 80 56 33
524
SUGIMURA,
BIRNBAUM,
WINITZ
AND
GREENSTEIN
maintenance, for the diets containing 0, 2.5, 5.0, 8.0, 12.0, 16.0, and 20.0% glucosamine, respectively. The average value obtained with the basal diet No. 3 substituted with varying amounts of glucosamine was 0.58 g. (1). That the depressed growth rate of animals ingesting these glucosaminecontaining diets is easily reversible was shown by the attainment of growth rates of 3.85 and 4.05 g./day by animals switched after 28 days on diets 64 and 65 to the basal diet No. 26. These figures represent the averages of six rats in each group over a period of 10 days following the changeover. Tumor Studies
The depressive effect of higher concentrations of glucosamine on food intake invalidates the ad libitum technique as a means of investigating the effects of dietary glucosamine on the growth rate of a transplantable tumor. By means of the force-feeding procedures, total caloric intake and total amino acid nitrogen intake were maintained constant, and the only variable between the test groups and their controls was the actual glucosamine concentration in the diet. Table II shows the response of normal and tumor-bearing rats to 20 ml. (10 g.) per day of the control diet No. 26 and diets 62 and 63 which contain 8 and 12%, respectively, of glucosamine. Although the daily glucosamine intake for animals on these diets was thus 800 and 1200 mg./rat, the effect of these amounts on both tumor growth and upon carcass growth was essentially inappreciable. The original findings of Quastel and Canter0 (4) on the inhibition by TABLE Efects
of Forced-Fed
Glucosamine and Tumor-Bearing
COUcentration of glucosamine
II Diet” on Growth of Normal Animals
Weight change for 12 day9 Animals”
--
,”
--
control
Over-all
Tumor
N
ChKWS
--
No. No. No. No. No. No.
26 26 62 62 63 63
% 0 0 8 8 12 12
g.
Tumor-bearing Normal Tumor-bearing Normal Tumor-bearing Normal
53.2 33.1 54.0 29.5 56.2 31.2
a Twenty milliliters of diet was at 9:06 a.m., 5 ml. at 1:09 p.m., 5 b The initial body weight was animals. c Data for 12 days between 6th
f f f f f zk
g.
1.425.8 1.2 2.729.7 2.0 3.424.2 0.6
f k f -
g.
3.627.4 33.1 1.424.3 29.5 2.832.0 31.2
f f f f f f
4.6 1.2 4.3 2.0 4.6 0.6
%
%
100 114 94 -
100 88 95
given by four times forced-feeding per day (4 ml. ml. at 5:00 p.m., and 6 ml. at 9:00 p.m.). 149 g. on average. Each group consisted of six and 18th day after
tumor
transplantation.
NUTRITIONAL
STUDIES.
IX
525
glucosamine of tumor growth in mice appear to have been substantiated in studies employing human (5) and mouse (6) tumor cells cultured in vitro. Studies of the influence of this amino sugar on tumor growth in viva have, however, yielded generally negative findings [cf. (7)]. Ball, Wick, and Sanders (8) reopened the question of the chemotherapeutic efficacy of glucosamine by reporting some 27 % depression of the growth rate of the Walker carcinosarcoma 256 implanted in 120-160-g. Wistar rats. Their procedure involved intraperitoneal injection of 400-600 mg. glucosamine/ rat/day in 4-6 divided doses, the treatment being initiated on the day following tumor implantation and continued daily for 18 days. They attributed the failure of the majority of investigators [cf. (7)] to obtain positive tumor growth inhibition by glucosamine to the fact that almost invariably single daily injections were employed. The findings obtained in the present work, even though divided dosages were employed and the total intake was the highest yet reported, indicate that the effect of dietary glucosamine on tumor growth is of minor importance, if any, from a chemotherapeutic point of view and that any observable effects however slight are highly dependent upon conditions of administration, animal strain, stage of tumor development, etc. However, the appreciable breakdown of glucosamine by bacteria [cf. (4)] in the gastrointestinal tract cannot, at present, be entirely precluded. Comparison of the growth response of normal animals force-fed with equal amounts of diets containing 0, 8, and 12 % glucosamine reveals insignificant differences in rate of gain among the three groups (Table II). No toxic symptoms were noted among the normal rats receiving the glucosamine diets, and all remained clean and active. These findings confirm the hypothesis (1) that the dietary glucosamine exerts solely a self-limiting effect on food intake in ad libitum experiments without complicating toxic side reactions. Attempts to force-feed diets containing more than 12% glucosamine in the same volumes did, however, lead to a quickly appearing but transient diarrhea thus invalidating any results obtained. Hourly Weight Fluctuations Although animal weights hitherto reported from this laboratory have invariably been those recorded at 9 a.m., it became of interest in connection with the present experiments to observe the cyclic daily weight fluctuations of rats following a period of acclimatization to the various dietary regimens. Figure 24 illustrates the typical hourly weight response of such animals eating ad libitum of the solid Purina Chow stock diet and of the soluble diet No. 26. Although the two curves are not strictly comparable owing to the initial weight differences between the two groups of animals, similar responses to the two widely variant diets are evident. The average maximal
526
SUGIMURA,
BIRNBAUM,
WINITZ
a
AND
0. force
9A.M.
I PM
5 PM.
9RM.
GREENSTEIN
feeding
9 A.M.
FIG. 2. (A) Refers to average 24-hr. weight fluctuations under ad libitum feeding schedule. Six rats per group were observed over a S-day period. 0, Chow; 0, diet
No. 26. (B) Refers to average daily weight fluctuations of rats force-fed diets No. 26 and 64. Feeding schedule as in Table II, with weights taken just before each feeding. Six rats per group observed over a 3-day period. 0 diet No. 64; l diet No. 26. fluctuation of 12 g. for the animals on diet No. 26 compares with the 9 g. observed for the group on the Chow diet, the difference in range being at least partially attributable to the differences in initial body weight. On the forced-feeding routines (Fig. 2B) the schedule is of course reversed, nutriment being received for the most part during the daylight hours contrary to the normal nocturnal feeding habits of the rat. Consequently, the 9 a.m. reading is minimal rather than maximal. The response of the partially starved rats to the first 4-ml. dose is most striking, resulting in an apparent increment of up to 18 g. over the ensuing 4-hr. period when only water is available. This increase is much more pronounced in the group receiving the diet No. 64, which contains 16% glucosamine, than in the group receiving an equal amount of diet No. 26. The difference is a reflection of the voluntary increased water intake of animals on the glucosamine diet, where an average daily intake of 28 ml. water was recorded, as opposed to an intake of only 15.5 ml. by the animals fed diet No. 26.
NUTRITIONAL
STUDIES.
IX
527
SUMMARY
The effect of incorporating n-glucosamine at various levels into the water-soluble, chemically defined diet No. 26 fed ad Mn’tum to rats has been compared with that observed earlier under the same conditions when the inferior diet No. 3 was employed. As before, a linear graphical relationship between growth and food intake was observed which, by extrapolation, permitted a calculation of the average daily requirement of food for the maint,enance of body weight over the entire course of the experiment. On the superior diet, No. 26, there was (a) a slightly higher maintenance requirement, and (b) appreciably less depression in the growth rate at equal levels of glucosamine, than on diet No. 3. No toxic symptoms were evident, and it is apparent that the dietary glucosamine simply exerts a self-limiting effect on food intake. Force-feeding of diet No. 26 containing 8 and 12 % of glucosamine to rats bearing the transplanted Walker tumor resulted in no symptoms of toxicity nor any effect upon tumor or carcass growth. Observations on the cyclic daily weight fluctuations of animals ingesting soluble diets as compared with Purina Chow are described. REFERENCES
1. WINITZ, M., BIRNBAUM, S. M., AND GREENSTEIN, J. P., Arch. Biochem. 2. 3. 4. 5.
6. 7.
8.
Biophys. 72, 437 (1957). GREENSTEIN, J. P., BIRNBAUM, S. M., WINITZ, M., AND OTEY, M. C., Arch. Biothem. Biophys. 73, 396 (1957). SUOIMURA, T., BIRNBAUM,~. M., WINITZ, M., AND GREENSTEIN,J. P., Arch.Biothem. Biophys. 81, 439 (1959). QUASTEL, J. H., AND CANTERO, A., Nature 171, 252 (1953); FAULKNER, P., AND QUASTEL, J. H., Nature 177, 1216 (1956). FJELDE, A., SORKIN, E., AND RHODES, J. M., Ezptl. Cell Research 10, 88 (1956). RUBIN, A., SPRINGER, G. F., AND HOGUE, M. J., Cancer Research 14, 456 (1954). Cancer Research, Suppl. No. 3 (1955). BALL, H. A., WICK, A. N., AND SANDERS,C., Cancer Research 17, 235 (1957).