Influence of Diet on Growth in the Rat
ABSTRACT Twenty-eight-day old male Sprague Dawley rats were fed, either ad libitum or in restricted amounts, isoenergetic diets containing 2%, 5%, 10%, 15%, 25%, or 50% lactalbumin protein and 5%, 11.97o, or 21.1% fat for 8 weeks and were then killed. Weekly food consumption and body weight, terminal weight, body water and lipid, and liver weight, DNA, RNA, protein, and lipid were measured. The growth rate increased progressively with each increase in the level of dietary protein up to 25% protein and then declined. Growth was also accelerated by a high fat diet but was retarded by restriction of energy intake. Total body lipid correlated directly with the level of fat in the diet. Multiple regression analysis of the type: Y = ß0 + ßÄ+ 02X2 + ßsX3 + j34X4where Y - rate of weight gain, Xi = dietary protein level, X2 = protein efficiency ratio, X3 = appetite fac tor, and X4 = energy/protein ratio, showed that the maximum rate of weight gain of 58.8 g/week occurred when the diet contained 23% protein. Growth rate declined when the diet contained a higher protein level. J. Nutr. JOS: 282-290, 1978. INDEXING KEY WORDS protein •dietary fat •food restriction •maximum growth rate In the economically developed countries, there has been an increase in mean stature and weight of children and adults over suc ceeding generations (1, 2). At the same time, children from economically favored segments within these countries are taller and heavier than children from poorer eco nomic backgrounds (3-5). Infants and children in the developing countries are generally lighter and shorter for their age than their counterparts in the developed countries (6, 7). Furthermore, within the developing countries, economically priv ileged children grow at a faster rate than economically disadvantaged children (8, 9 ). A trend toward accelerated growth rate has also become evident in countries, such as Japan, that have experienced a rapid economic growth during the past 30 years (10, 11). It is generally believed that these differences in growth rate are related to environmental factors and to the protein intake in particular (11, 13). A number of 282
studies (9, 14, 15) have demonstrated that food supplements improve the growth of poor children. There is evidence that the trend toward accelerated growth of children which oc curred in the developed countries during the past 100 years has abated or even ceased (16, 17). The virtual cessation of this trend in growth suggests a limit to the stimulation of growth by increases in the intake of protein. Therefore, this study was undertaken to investigate the effects of in takes of protein, fat and energy on growth in the rat and in particular to determine whether a limit exists to the acceleration of growth associated with increases in pro tein intake. MATERIALS AND METHODS
Male Sprague-Dawley rats1 weighing 70 to 80 g were purchased and fed a laboRecelved for publication May 6, 1977. 1 Charles River Laboratories, Wilmington, chusetts.
Massa
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JOSEPH C. EDOZIEN ANDBOYD R. SWITZER Department of Nutrition, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514
PROTEIN
283
LEVEL FOR MAXIMUM RAT GROWTH
TABLE 1 Composition of 15% lactalbumin protein diets with 6%, 11.9% and 21.1% fat Percentage of fat in diet1'2 Food component Lactalbumin3 Dextrin, white technical4 Sucrose (cane sugar)4 Salt mix« Vitamin mixture5 Choline chloride (100%) Cottonseed oil Fiber«
5
11.9
21.1
g/kgdiet 187.50187.50187.50 477.83459.85282.34 238.92195.65141.17 50.00 50.00 50.00 5.00 5.00 5.00 2.00 2.00 2.00 38.75 100.00200.03 — — 131.97
1Diets were made up, as specified, by General Biochemicals, Laboratory Park, Chagrin Falls, Ohio 44022. a All diets provide about 4.0 kcals/g. The ME values used were in kcals/g: dextrin, 3.64; sucrose, 4.0; and cottonseed oil, 9.0. The percentages of total energy derived from fat in the 5%, 11.9%, and 21.1% fat diets were 11.4%, 22.0%, and 45.0% respectively. The diets containing 2%, 5%, 10%, 25%, and 50% protein and 5%, 11.9%, or 21.1% fat were made by varying the protein and carbohydrate contents of each of the above three diets. For example, the diet with 2% protein and 11.9% fat contained per kg: 25.0 g lactalbumin, 546.30 g dextrin, 271.70 g sucrose and 100 g cotton seed oil. 3Each 100 g of lactalbumin contains (in g) : protein, 80; ash, 4- moisture, 6; lactose, 3; either extractables, 6; other, 1. 'The dextrin: sucrose ratio was determined by the optimal amounts for pelletability whenever pellets were desired. Only powdered diets, however, were used in the experiments reported in this paper. 6Rogers and Harper (18). 6Non-nutritive fiber was added to the diets containing 21.1% fat to make up bulk. The fiber is a white, finely ground product purified from wood by Brown Company, New York, New York. It contains, in g/100 g, crude fiber, 64.85; pentosans, hexosans, and galactosans, 26.99; moisture, 7.7; ash, 0.3, and reducing substances, trace.
cient E\%m= 260 [32 /ig/ml = 1.0 A]. Liver protein concentrations were esti mated according to Lowry et al. (23) with bovine serum albumin as the reference. Total liver lipid was determined by ex tracting the lipids from a weighed portion of liver with a 2:1 solution or chloroformmethanol. The extract was washed with water by the method of Folch et al. (24) and an aliquot dried in a hot air oven. The "Purina Laboratory chow, Ralston Pnrlna Co., Checkerboard Square, St. Louis, Missouri. »Nembutal, Abbott Laboratories, North Chicago, Illinois.
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ratory stock diet2 until attaining a weight of 90 to 110 g. They were then fed isoenergetic diets containing 2%, 5%, 10%, 15%, 25%, or 50% lactalbumin protein given freely or in restricted amounts for 8 weeks. At each protein level, the diets of the ad libitum fed animals contained 5%, ll.Q^b, or 21.1% fat as cottonseed oil. Only the diets containing 11.9% fat were given in restricted amounts. The composition of the diets is shown in table 1. Rats on re stricted intake were fed either 50% or 75% of the amount of diet consumed ad libitum by rats of similar weight irrespec tive of their age; their diet was usually provided each day between 15:00 and 17:00 hours and all of it was generally consumed by the early evening. The rats were housed individually in wire-mesh cages located in a room with automatically controlled light and dark for 12 hours every day and were given water ad libitum. Four rats from each dietary group were transferred to a metabolic cage for 2 days of each week for the measure ment of food intakes. The food dishes were filled and weighed, and the amount con sumed was calculated from the difference in weight 24 hours later. All spilled food was collected and weighed, and was taken into account in calculating food intakes. The food consumption of each rat was taken as the average of the 2 days food intake study period. The food consumption data were used to adjust the amount of diet fed to the rats on restricted intakes. The rats were killed during the ninth week. Diet was removed from each rat 12 to 15 hours before it was anesthetized by intraperitoneal injection of phénobarbital,8 5 mg/100 g body weight. The abdominal wall was opened and the liver quickly ex cised and weighed. It was then placed in a Elastic bag and immediately frozen in a irge beaker containing alcohol and dry ice. It was kept frozen at —70° until the time of analysis. Liver DNA was measured by the diphenylamine method of Burton (19) against a standard of calf thymus DNA after sepa ration of RNA from DNA by the method of Munro and Fleck (20). RNA was esti mated by the technique of Schmidt and Thannhauser (21) as modified by Fleck and Munro (22) using extinction coeffi-
284
JOSEPH C. EDOZIEN AND BOYD R. SWITZER TABLE 2
% lactalbumin in diet1Week1234a/*4
hr25101525609.6±1.714.2 ±2.313.2 ±1.812.3 ±1.412.5 ±2.114.4 ±4.416.5 ±5.216.7 ±3.216.5±2.314.7 ±1.215.2 ±3.914.9 ±5.714.8±1.714.6 ±2.014.9 ±1.914.6±1.713.6 ±0.914.4±1.613.2±1.111.2 ±1.613.5±3.19.9 ±3.013.5±5.710.6 ±2.7 1Mean of four rats per group ±8E.All diets contained 11.9% fat.
total lipid was then determined gravimetrically. Total body water was determined by drying the rats to constant weight in a constant temperature oven at 100°.To ob tain total lipid, the lipids in the dried rats were blended six times in a Waring blender with four volumes ( w/v ) of a 2:1 solution of chloroform-methanol. The extracts were combined. After mixing thoroughly, a 50 ml aliquot was washed with water by the method of Folch et al. (24). An aliquot was dried in a hot air oven and the total lipid determined gravimetrically. The data were analyzed by multiple re gression analysis (25). The statistical sig nificance of differences between groups was indicated by a P value which is the probability associated with the F-statistic that means differ after adjusting for all
RESULTS
The average daily food consumption by week of rats fed the diets which contained 11.9% fat ad libitum is shown in table 2, while illustrative examples of weekly weight data are reported in tables 3-5. Table 3 presents the weights of rats fed unlimited amounts of diets containing 11.9% fat and 2%, 5%, 10%, 15%, 25%, or 507c protein. The weights of rats fed the diets which contained 15% protein and 5%, 11.9%, or 21.% fat ad libitum are reported in table 4 while table 5 presents the weights of rats fed diets which con tained 15% protein and 11.9% fat at 50%, 75%, or 100% of ad libitum intake. The results of multiple regression analy ses to show the effects of levels of dietary protein, fat and energy on body weight, are reported in table 6. Body weight in creased steeply with increases in the level of dietary protein up to 15% protein. Above this protein level, changes in body weight were relatively small. However, when the body weight of rats fed the 15% and 25% protein diets ad libitum were
TABLE 3 Mean weights of rats fed diets containing 11.9% fat and varying levels of protein ad libitum for 8 weeks
Protein content of diet g
15N Dâvs fßddiet0714212835424(1562N1 80110±15298±1197±1199±11100±12100 = 12110±10143±17174±22214±23243 = 60112±10119±10139±13151 = 60g112±10155±13210±23248±24300 = 12102± = 12100 = 10N
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other independent variables in the regres sion. The magnitude of the differences be tween groups was shown by calculating the mean values for each group after ad justments for all other factors (25). Differ ences between means were considered sig nificant at P < 0.05.
Weekly mean food intake of ad libitum-fed rats
7168±11218±13271±16314±23356±27386 6156±14203 ± ±20253 ±13168±14182±15201 ±28288 ±24276 ±30331 ±33315±36341 ±12102 ±36304 ±34366 ±12102±13105±125N ±17208±19221 ±42325 ±31400 ±45356±44421 ±38384±40424 ±43342 ±33437 ±21g/t( ±4390 ±4925N ±4850N ±49 1Number of rats.
2Results are means ±SD.
PROTEIN
285
LEVEL FOR MAXIMUM RAT GROWTH
TABLE 4 Mean weights of rats fed diets containing 15% protein and either 6% or 21.1% fat ad libitum
220
200
Level of fat in diet ISO
diet11.9N 100 g JayICQdiet07142128354249565N1 o
8105=
8110± = 609110±10155±13210±23248 =
82159±13204±19247 ± ±24285 ±29321 ±32345 ±40359 ±40400 ±40g/ 1Number of rats.
6174±12216±20268±28305
±24300 ±34341 ±30331 ±41367 ±34366 ±46392 ±38384±40424 ±50425 ±4921.1N ±45
2 Results are means ±SD.
compared by multiple regression analysis with days fed, level of fat and level of protein as the independent variables, it was found that the rats fed the 15% pro tein diets were significantly lighter. A sim ilar comparison of rats fed the 25% and 50% protein diets indicated that the rats fed the 50% protein diets were lighter. The conclusion was that the rats attained maximum growth when fed a 25% protein diet and that the growth rate declined when the percentage of protein in the diet was increased to 50%. There was a small progressive increase in body weight with increases in the level of fat in the diet.
20
40
LACTALBUMIN
60
80
100
120
140
PROTEIN EATEN U) g
Fig. 1 Utilization of lactalbumin protein at various dietary protein levels. The slope of the linear regressions is the PER. Protein utilization was reduced when the diet contained 25% or 50% protein.
Rats fed the diets ad libitum were heavier than those fed the diets at 75% of ad lib itum intake and the latter group, in turn, TABLE 5 were heavier than those fed the diets at Mean weights of rats fed diets containing 15% protein 50% of ad libitum intake. and 11.9% fat either ad libitum, 50% of ad It is evident from figure 1 that when the libitum intake or 76% of ad libitum intake diet contained 5%, 10%, or 15% lactal Level of energy consumption bumin protein and 11.9% fat, protein utilization was linearly related to the Ad 50% of ad 75% of ad libitum libitum libitum amount of protein consumed. Weight gain Da was also linearly related to the amount of N> = 10 N = 10 N = 60 iet die protein eaten when diets contained 25% or 50% protein but the slope of the line was different for each of these protein 071421283542495695±5"96 5121± levels; the efficiency with which protein ±4108±4113±6126 7143 7160±10179±13209 ± was utilized declined with each increase ±24300 in dietary protein level. ±6135±9139±8146 ±30331 Weight gain was also linearly related to ±16225 ±34366 ±18230 ±38384 energy consumption (fig. 2) but the ef ±8150±8995± ±21240±22112±10155±13210±23248 ±40424 ficiency of energy utilization depended on ±49 dietary protein level. Diets containing 25% or 50% protein were utilized most effi1Number of rats. 2 Results are means ±SD.
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-p» fnrìL
286
JOSEPH
C. EDOZIEN
AND BOYD R. SWITZER
TABLE 6 Regression analysis to show the effects of levels of dietary protein, fat and energy intake on the biweekly weights of rats DietDF2468A.
fed
Independent variablesWeeks
on diet2 Level of protein Level of fat Level of energy1
Pl 5 2 20.0001
0.0001 0.0319 0.00010.0018
0.0001 0.0216 0.00010.0105
0.0001 0.0012 0.00010.9216
0.0001 0.0194 0.0001
B Adjusted mean weights3 (a) protein2%5%10%15%25%50%(b) Level of
(100)'106 (96)138 (48)170 (96)171 (48)163 (48)131
(99)113 (96)180 (48)228 (96)228 (48)214 (48)160
(96)135 (95)226 (48)278 (96)285 (48)258 (47)190
(96)130 (90)228 (43)280 (92)285 (48)260 (47)187
(40)139 (356)140 (40)110
(40)173 (355)177 (40)124
(40)197 (351)220 (39)137
(39)202 (321)215 (39)116
(60)134 (60)166 (316)58
(60)161 (60)225 (315)51
(56)199 (60)275 (314)51
(49)186 (49)301 (301)
fat5%11.9%21.1%(c) Level of
Level energy50% of libitum75% ad Ad libitumAd libitum74«
1P values after adjustments for all other factors in the regression. 2 All the rats were not weighed on exactly the same number of days from the time they were first fed the diets. This variable adjusts for the small differences in the time period the rats were fed the diet. Hence lack of statistical significance did not imply that body weight was independent of the duration of study. *Multiple regression analysis indicates significant differences among means for protein, fat and energy levels at each week tested (P < 0.05). 4Adjusted mean weight in g. *Number of rats.
ciently and the efficiency declined with each succeeding drop in protein level. The growth rate of rats fed diets containing 11.9% fat at 50% or 75% of ad libitum intake was accelerated by increasing the level of dietary protein up to 25% protein (figs. 3 and 4). The body composition data reported in table 7 show that total body lipid was di rectly related to the level of fat in the diet while total body water had a reciprocal relationship to the fat level. There was no difference in the percentage of water in lean tissue between rats fed 25% protein and those fed 50% protein. The influence of level of dietary protein on the weight and composition of the liver is shown in table 8. Maximum weight was attained when the diet contained 15% protein; an increase above this level had
no effect on liver weight. However, rats fed 50% protein had fewer cells (total liver DNA) and larger cells (liver weight/ mg DNA) than those fed 15% or 25% protein. DISCUSSION The results confirm other reports that the intakes of protein and energy have a profound effect on the growth of rats ( 26 ). The growth rate increased progressively with increases in the level of dietary pro tein up to 25% protein and then declined. Since the experimental diets provided 4.0 kcal/g, this level of dietary protein repre sented an intake of 25% of energy from protein. Body weight also correlated di rectly with the level of dietary fat. The increase in body weight with the level of dietary fat was associated with a relative
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valuesDuration
287
PROTEIN LEVEL FOR MAXIMUM RAT GROWTH
220 r
200
o>
Z
es
125
o E 100
S 75
10
AGE (WEEKS)
y=-5.0+
M8x
120 /
y=39+072x
100
LU
80
60
40
ISO
r =.995 r =.998 r =.999
140
O
% PROTEIN
50 •5% Protein o 10% Protein ••15% Protein o 25% Protein
180 160
175
y=-7.7+042x
20
500 1000 1500 2000 ENERGY CONSUMED (x)= k calories
Fig. 2 Utilization of energy for growth at various dietary protein levels. Efficiency of energy utilization was reduced when the diet contained 15% or less of lactalbumin protein.
Fig. 3 Body weight of rats fed iso-energetic diets containing 11.9% fat at 50% of ad libitum intakes for 8 weeks starting at 4 weeks of age. The differences between rats fed 2%, 5%, 10%, 15%, and 25% are statistically significant (P< 0.05). Rats fed 25% and 50% protein were sim ilar. Growth was accelerated by an increase in dietary protein level up to 25% protein.
due to methodological differences since they considered only one set of measure ments at three weeks whereas our rats were investigated for 8 weeks. The results presented in figure 1 which are based on food consumption during the first 4 weeks of feeding the experimental diets agree with the finding of Hegsted and Neff (29) that below a limiting value characteristic for each protein source, protein utilization is independent of the level in the diet. The limiting level for lactalbumin protein lies between 15% and 25%. The decrease in efficiency of utilization in diets containing 25% or 50% lactalbumin protein was not related to the cumulative protein consump tion, since the quantities of the 25% pro tein diet eaten in weeks 1 and 2 and of the 50% protein diet eaten in week 1 con tained less protein than was eaten in 4 weeks by rats fed the 15% protein diet. The composition of the liver was investi gated for possible clues to explain the re-
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increase in the fat content of the body. These findings suggest that the increase in the proportion of food energy derived from fat, which has also occurred in the de veloped countries during the last century (27, 28), may have contributed to the trend toward increased body weight. Hegsted and Neff (40) found no differ ence in body water and hence total body protein of rats fed 23% to 30% lactalbumin, casein or soy protein isolate and those fed diets containing 50% to 58.5% of the same protein source. However, their body weight and body water data showed a similar trend to our body weight data and the contradictory conclusions are probably
288
JOSEPH
C. EDOZEEN AND BOYD R. SWITZER % PROTEIN
250
200
50
6
8
K)
12
AGE (WEEKS)
Fig. 4 Body weight of rats fed iso-energetic diets containing 11.9% fat at 75% of ad libitum intakes for 8 weeks starting at 4 weeks of age. The differences between rats fed 2%, 5%, 10%, 15%, and 25% are statistically significant ( P < 0.0). Rats fed 25% and 50% protein were sim ilar. Growth was accelerated by an increase in dietary protein level up to 25% protein.
duced growth rate in rats fed a 50% pro tein diet. There was no decline in liver protein synthesis as measured by total liver RNA; on the contrary, the increase in protein: RNA ratio suggested an improved efficiency of protein synthesis. Rats fed
TABLE 7 Body composition of rats fed 25% or 60% protein and 5%, 11.9%, or 31.1% fat ad libitum Total Dietary protein level
Dietary fat level
N1
2550511.921.1511.921.155666664.5
Body water : body weight
Lipid : body weight
Lean mass : body weight
Body water : lean mass
±0.7262.9±2.059.9±4.463.3±1.761.1±1.461.2 ±3.184.6±3.484.4±3.083.2 ±0.273.8±1.573.4±1.4 ±2.883.4±3.673.4±1.074.0±0.670.8±4.875.1 ±2.212.1±1.214.9±3.115.4±3.415.6±3.016.8±2.816.6±3.687.9±1.285.1
1Number of rats. 2 Results are means±SD. Rats fed 5% differed significantly (25) at the 0.05 level from other rats in body water, total lipid and lean mass. Rats fed 11.9% and 21.1% fat were similar. Rats fed 50% protein had more total lipid and less lean tissue than rats fed 25% protein. Differences in water content of lean tissue were not statistically significant.
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150 X O
50% protein had a higher protein :lipid ratio than rats fed 25% protein but the normal lipid:RNA ratio as well as the higher percentage of body fat indicated that there was no impairment of fat syn thesis. Factors other than metabolic impairment must have been responsible for the reduced efficiency of protein utilization in rats fed the 25% and 50% protein diets. A possible explanation is that the daily rate of pro tein ingestion might have exceeded the maximum capacity of the body to synthe size new protein. Another more likely ex planation is that the energy :protein ratio was not optimal for maximal retention of the ingested nitrogen. The decrease in ef ficiency of energy utilization in rats fed low-protein diets (fig. 2) was probably related to the relatively long time it took to gain weight when such diets were fed with consequent expenditure of a high percentage of ingested energy for daily maintenance. In addition to dietary protein level, pro tein efficiency ratio (PER) and energy: grotein ratio, another factor which inuenced the rate of growth was "appetite"; table 2 shows, for example, that ad libitumfed rats reduced their food intake when the diet contained more than about 15% protein. In order to avoid problems arising out of differences in body size, the amount of food eaten during the first week may be taken as a measure of appetite since the
PROTEIN LEVEL FOR MAXIMUM RAT GROWTH
289
TABLE 8 Effects of dietary protein level on size and composition of rat liver1*1 levelVariableNWeight,
Dietary protein
iance of d588.53.40.32»3.2»26.2»7.159.42.2226»1,93471.7 ¡fferenceVVVVVVVVVVVVVVVVV Downloaded from https://academic.oup.com/jn/article-abstract/108/2/282/4770424 by Tulane University user on 02 December 2018
gWeight, weightWeight, % body DNADNA, g/mg liverDNA, mg/g liverRNA, mg per liverRNA, mg/g liverRNA, mg per DNAProtein, mg/mg liverProtein, mg/g liverProtein, mg per DNAProtein, mg/mg RNALipid, mg/mg liverLipid, mg/g DNALipid, mg/mg RNAProtein/Lipid, mg/mg ratio2891.53.70.195.410.67.810.71.517422233.522.676.913.89.93.05973.63.00.244.315.78.229.92.020070847.724.554.713. 1Results are mean values adjusted for levels of dietary fat and energy. 2Multiple regression analysis indicates significant differences among group means for each variable determined. 3Significantly different (0.05 level) from rats fed 25% protein ad libitum.
respectively, and that X4 = 400/Xj kcal/g protein, it was found that the maximum rate of weight gain of 58.8 g/week oc curred when the diet contained 23% pro tein. The present study does not, of course, give any indication as to whether or not the differences in growth rate of rats fed Y = ßo + j8i X! + X2 + ß3 X3 + 04 X4 diets containing 15% to 50% protein will be reflected in their ultimate body size. where An increase in the efficiency of energy utilization can account for the increase in Xi = dietary protein level ( % ) the rate of weight gain when human sub X2 = PER ( weight gain in g/g protein con jects are fed protein supplements but do sumed ) Xs = amount of food in grams consumed in not consume more energy. For instance, marginally protein deficient children gained the first week more weight when they were fed supple X4 = energy/protein ratio in kcals/g pro ments of skimmed milk which provided an tein additional 1 g/kg/day protein but negli Y = weight gained in grams/week. gible amounts of energy (19). Similarly, The results of the analysis showed that pregnant women and children participating Y = -54.1 - 0.53 Xj - 17.4 X2 + 1.66 X3 in the Special Supplemental Food Pro —0.51 X4. The regression correlation co gram for Women, Infants and Children efficient of 0.9988 suggests that the four (WIC) gained more weight when their factors mentioned above can explain most supplemented diets provided more protein of the variation in the rate of weight gain. When values of Y were calculated for without change in energy content (15). different values of Xi, assuming that X2 Conversely, the level of energy consump and X3 were linearly related to dietary tion profoundly affects protein require ments (30). These results reemphasize the protein level between 15% and 25% pro tein and between 25% and 50% protein, importance of energy :protein balance in
average weight of rats in each group was about 110 g at the beginning of the ex periment. The rate of weight gain can be deter mined by multiple linear regression analy sis of the type:
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JOSEPH C. EDOZIEN AND BOYD R. SWITZER
studies of protein requirements and protein quality. ACKNOWLEDGMENTS
LITERATURE CITED 1. Tanner, J. M. (1962) Growth at Adoles cence, 2nd edition, Oxford, Blackwell Sci. Pubi. 2. Meredith, H. V. (1963) Changes in the Stature and Body Weight of North American Boys During the Last 80 years. The Maple Press, York, Pa. 3. Hundley, J. M., Mickelsen, O., Mantel, N., Weaver, R. N. & Taber, R. C. (1955) Height and weight of first-grade children as a potential index of nutritional status. Am. J. Pub. Health 45, 1454-1461. 4. National Center for Health Statistics (1973) Height and Weight of Children, Socioeconomic Status, United States. Vital and Health Statistics, Series 11, No. 119. DHEW Pub. No. (HSM) 73-1606. U.S. Government Print ing Office. 5. Scott, R. B., Cardoza, W. W., Smith, A. DeG. & Dehitty, M. R. (1950) Growth and development of negro infants. III. Growth during the first year of life as observed in private pediatrie practice. J. Pediat. 37, 885893. 6. Jelliffe, D. B. ( 1968 ) Infant nutrition in the subtropics and the tropics. WHO monogr. Ser. 29, 2nd edition. 7. National Center for Health Statistics (HEW). (1971) Height and Weight of Children in the United States, India, and the United Arab Republic. PHS Publication No. 1000 Series 3, No. 14. U.S. Government Printing Office, Washington, D. C. 8. Kahn, E. & Freedman, M. L. (1959) The physical development of a privileged group of African children. S. Afr. Med. J. 33, 934936. 9. Edozien, J. C., Khan, M. A. R. & Waslien, C. J. (1976) Human protein deficiency: Results of a Nigerian village study. J. Nutr. 106, 312-328. 10. Mitchel, H. S. (1962) Nutrition in relation to stature. J. Am. Diet. Assoc. 40, 521-524. 11. Meredith, H. V. (1976) Findings from Asia, Australia, Europe, and North America on secular change in mean height of children, youths and young adults. Am. J. Phys. Anthropol. 44, 315-325. 12. Greulich, W. W. (1957) A comparison of the physical growth and development of American-born and native Japanese children. Am. J. Phys. Anthropol. 15, 489-515. 13. Greulich, W. W. (1958) Growth of chil dren of the same race under different environ mental conditions. Science 127, 515-516.
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We are grateful for the expert technical assistance of Nelsen Niehaus and MeiHeng Mar. The statistical analyses were performed by Henry Lee. The investigation was supported by a grant from General Research Support.
14. Mora, J. O., Amezquita, A., Castro, L., Chris tiansen, N., Clement-Murphy, J., Cobos, L. R., Crémier,H. D., Dragistin, S., Elias, M. F., Franklin, D., Herrera, M. G., Ortiz, N., Pardo, F., deParedes, B., Ramos, C., Riley, R., Rodriguez, H., Vuori-Christiansen, L., Wag ner, M. & Stare, F. J. (1974) Nutrition, Health and social factors related to intellectual performance. World Review of Nutrition and Dietetics, Vol. 19, p. 205. 15. Select Committee on Nutrition and Human Needs, U.S. Senate. (1976) Medical Evalu ation of the Special Supplemental Food Pro gram for Women, Infants and Children. U.S. Printing Office, Washington, D.C. 16. Barwin, H. & McLaughlin, S. D. (1964) Secular increase in height. Is the end in sight? Lancet 2, 1195-1196. 17. Damon, A. (1974) Larger body size and earlier menarche: The end may be in sight. Soc. Biol. 21, 8-12. 18. Rogers, Q. R. & Harper, A. E. (1965) Amino acid diets and maximal growth in the rat. J. Nutr. 87, 267-273. 19. Burton, K. (1956) A study of the condi tions and mechanisms of the diphenylamine reaction for the colorimetrie estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323. 20. Munro, H. N. & Fleck, A. (1966) Recent developments in the measurement of nucleic acids in biological materials. Analyst 91, 78-88. 21. Schmidt, G. & Thannhauser, S. J. (1945) Method for the determination of desoxyribonucleic acid and phosphoproteins in animal tissues. J. Biol. Chem. 161, 83-89. 22. Fleck, A. & Munro, H. N. (1962) The pre cision of ultraviolet absorption measurements in the Schmidt-Thannhauser procedure for nucleic acid estimation. Biochem. Biophys. Acta 55, 571-583. 23. Lowry, O. H., Rosebrough, N. J., Fair, A. L. & Randall, R. J. (1951) Protein measure ment with the Folin phend reagent. J. Biol. Chem. 193, 265-275. 24. Folch, J., Lees, M. & Stanley, G. H. S. ( 1957 ) A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497-509. 25. SAS. (1972) A User's Guide to the Statisti cal Analysis System. SAS Institute Inc. Post Office Box 10522, Raleigh, North Carolina. 26. National Academy of Sciences (1972) Nu trient Requirements of Laboratory Animals. National Academy of Sciences, Publication No. 10, Washington, D.C. pp. 56-93. 27. Friend, B. (1967) Nutrients in United States Food Supply. A review of trends 19091913 to 1965. Am. J. Clin. Nutr. 20, 907-914. 28. FAO/WHO. (1973) Energy and Protein Requirements. FAO Rome. 29. Hegsted, D. M. & Neff, R. (1970) Effi ciency of protein utilization in young rats at various levels of intake. J. Nutr. 100, 11731179. 30. Scrimshaw, N. S. (1976) An analysis of past and present recommended dietary al lowances for protein in health and disease. New Engl. J. Med. 294, 136-142 and 198203.
ABSTRACT Twenty-eight-day old male Sprague Dawley rats were fed, either ad libitum or in restricted amounts, isoenergetic diets containing 2%, 5%, 10%, 15%, 25%, or 50% lactalbumin protein and 5%, 11.97o, or 21.1% fat for 8 weeks and were then killed. Weekly food consumption and body weight, terminal weight, body water and lipid, and liver weight, DNA, RNA, protein, and lipid were measured. The growth rate increased progressively with each increase in the level of dietary protein up to 25% protein and then declined. Growth was also accelerated by a high fat diet but was retarded by restriction of energy intake. Total body lipid correlated directly with the level of fat in the diet. Multiple regression analysis of the type: Y = ß0 + ßÄ+ 02X2 + ßsX3 + j34X4where Y - rate of weight gain, Xi = dietary protein level, X2 = protein efficiency ratio, X3 = appetite fac tor, and X4 = energy/protein ratio, showed that the maximum rate of weight gain of 58.8 g/week occurred when the diet contained 23% protein. Growth rate declined when the diet contained a higher protein level. J. Nutr. JOS: 282-290, 1978. INDEXING KEY WORDS protein •dietary fat •food restriction •maximum growth rate In the economically developed countries, there has been an increase in mean stature and weight of children and adults over suc ceeding generations (1, 2). At the same time, children from economically favored segments within these countries are taller and heavier than children from poorer eco nomic backgrounds (3-5). Infants and children in the developing countries are generally lighter and shorter for their age than their counterparts in the developed countries (6, 7). Furthermore, within the developing countries, economically priv ileged children grow at a faster rate than economically disadvantaged children (8, 9 ). A trend toward accelerated growth rate has also become evident in countries, such as Japan, that have experienced a rapid economic growth during the past 30 years (10, 11). It is generally believed that these differences in growth rate are related to environmental factors and to the protein intake in particular (11, 13). A number of 282
studies (9, 14, 15) have demonstrated that food supplements improve the growth of poor children. There is evidence that the trend toward accelerated growth of children which oc curred in the developed countries during the past 100 years has abated or even ceased (16, 17). The virtual cessation of this trend in growth suggests a limit to the stimulation of growth by increases in the intake of protein. Therefore, this study was undertaken to investigate the effects of in takes of protein, fat and energy on growth in the rat and in particular to determine whether a limit exists to the acceleration of growth associated with increases in pro tein intake. MATERIALS AND METHODS
Male Sprague-Dawley rats1 weighing 70 to 80 g were purchased and fed a laboRecelved for publication May 6, 1977. 1 Charles River Laboratories, Wilmington, chusetts.
Massa
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JOSEPH C. EDOZIEN ANDBOYD R. SWITZER Department of Nutrition, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514
PROTEIN
283
LEVEL FOR MAXIMUM RAT GROWTH
TABLE 1 Composition of 15% lactalbumin protein diets with 6%, 11.9% and 21.1% fat Percentage of fat in diet1'2 Food component Lactalbumin3 Dextrin, white technical4 Sucrose (cane sugar)4 Salt mix« Vitamin mixture5 Choline chloride (100%) Cottonseed oil Fiber«
5
11.9
21.1
g/kgdiet 187.50187.50187.50 477.83459.85282.34 238.92195.65141.17 50.00 50.00 50.00 5.00 5.00 5.00 2.00 2.00 2.00 38.75 100.00200.03 — — 131.97
1Diets were made up, as specified, by General Biochemicals, Laboratory Park, Chagrin Falls, Ohio 44022. a All diets provide about 4.0 kcals/g. The ME values used were in kcals/g: dextrin, 3.64; sucrose, 4.0; and cottonseed oil, 9.0. The percentages of total energy derived from fat in the 5%, 11.9%, and 21.1% fat diets were 11.4%, 22.0%, and 45.0% respectively. The diets containing 2%, 5%, 10%, 25%, and 50% protein and 5%, 11.9%, or 21.1% fat were made by varying the protein and carbohydrate contents of each of the above three diets. For example, the diet with 2% protein and 11.9% fat contained per kg: 25.0 g lactalbumin, 546.30 g dextrin, 271.70 g sucrose and 100 g cotton seed oil. 3Each 100 g of lactalbumin contains (in g) : protein, 80; ash, 4- moisture, 6; lactose, 3; either extractables, 6; other, 1. 'The dextrin: sucrose ratio was determined by the optimal amounts for pelletability whenever pellets were desired. Only powdered diets, however, were used in the experiments reported in this paper. 6Rogers and Harper (18). 6Non-nutritive fiber was added to the diets containing 21.1% fat to make up bulk. The fiber is a white, finely ground product purified from wood by Brown Company, New York, New York. It contains, in g/100 g, crude fiber, 64.85; pentosans, hexosans, and galactosans, 26.99; moisture, 7.7; ash, 0.3, and reducing substances, trace.
cient E\%m= 260 [32 /ig/ml = 1.0 A]. Liver protein concentrations were esti mated according to Lowry et al. (23) with bovine serum albumin as the reference. Total liver lipid was determined by ex tracting the lipids from a weighed portion of liver with a 2:1 solution or chloroformmethanol. The extract was washed with water by the method of Folch et al. (24) and an aliquot dried in a hot air oven. The "Purina Laboratory chow, Ralston Pnrlna Co., Checkerboard Square, St. Louis, Missouri. »Nembutal, Abbott Laboratories, North Chicago, Illinois.
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ratory stock diet2 until attaining a weight of 90 to 110 g. They were then fed isoenergetic diets containing 2%, 5%, 10%, 15%, 25%, or 50% lactalbumin protein given freely or in restricted amounts for 8 weeks. At each protein level, the diets of the ad libitum fed animals contained 5%, ll.Q^b, or 21.1% fat as cottonseed oil. Only the diets containing 11.9% fat were given in restricted amounts. The composition of the diets is shown in table 1. Rats on re stricted intake were fed either 50% or 75% of the amount of diet consumed ad libitum by rats of similar weight irrespec tive of their age; their diet was usually provided each day between 15:00 and 17:00 hours and all of it was generally consumed by the early evening. The rats were housed individually in wire-mesh cages located in a room with automatically controlled light and dark for 12 hours every day and were given water ad libitum. Four rats from each dietary group were transferred to a metabolic cage for 2 days of each week for the measure ment of food intakes. The food dishes were filled and weighed, and the amount con sumed was calculated from the difference in weight 24 hours later. All spilled food was collected and weighed, and was taken into account in calculating food intakes. The food consumption of each rat was taken as the average of the 2 days food intake study period. The food consumption data were used to adjust the amount of diet fed to the rats on restricted intakes. The rats were killed during the ninth week. Diet was removed from each rat 12 to 15 hours before it was anesthetized by intraperitoneal injection of phénobarbital,8 5 mg/100 g body weight. The abdominal wall was opened and the liver quickly ex cised and weighed. It was then placed in a Elastic bag and immediately frozen in a irge beaker containing alcohol and dry ice. It was kept frozen at —70° until the time of analysis. Liver DNA was measured by the diphenylamine method of Burton (19) against a standard of calf thymus DNA after sepa ration of RNA from DNA by the method of Munro and Fleck (20). RNA was esti mated by the technique of Schmidt and Thannhauser (21) as modified by Fleck and Munro (22) using extinction coeffi-
284
JOSEPH C. EDOZIEN AND BOYD R. SWITZER TABLE 2
% lactalbumin in diet1Week1234a/*4
hr25101525609.6±1.714.2 ±2.313.2 ±1.812.3 ±1.412.5 ±2.114.4 ±4.416.5 ±5.216.7 ±3.216.5±2.314.7 ±1.215.2 ±3.914.9 ±5.714.8±1.714.6 ±2.014.9 ±1.914.6±1.713.6 ±0.914.4±1.613.2±1.111.2 ±1.613.5±3.19.9 ±3.013.5±5.710.6 ±2.7 1Mean of four rats per group ±8E.All diets contained 11.9% fat.
total lipid was then determined gravimetrically. Total body water was determined by drying the rats to constant weight in a constant temperature oven at 100°.To ob tain total lipid, the lipids in the dried rats were blended six times in a Waring blender with four volumes ( w/v ) of a 2:1 solution of chloroform-methanol. The extracts were combined. After mixing thoroughly, a 50 ml aliquot was washed with water by the method of Folch et al. (24). An aliquot was dried in a hot air oven and the total lipid determined gravimetrically. The data were analyzed by multiple re gression analysis (25). The statistical sig nificance of differences between groups was indicated by a P value which is the probability associated with the F-statistic that means differ after adjusting for all
RESULTS
The average daily food consumption by week of rats fed the diets which contained 11.9% fat ad libitum is shown in table 2, while illustrative examples of weekly weight data are reported in tables 3-5. Table 3 presents the weights of rats fed unlimited amounts of diets containing 11.9% fat and 2%, 5%, 10%, 15%, 25%, or 507c protein. The weights of rats fed the diets which contained 15% protein and 5%, 11.9%, or 21.% fat ad libitum are reported in table 4 while table 5 presents the weights of rats fed diets which con tained 15% protein and 11.9% fat at 50%, 75%, or 100% of ad libitum intake. The results of multiple regression analy ses to show the effects of levels of dietary protein, fat and energy on body weight, are reported in table 6. Body weight in creased steeply with increases in the level of dietary protein up to 15% protein. Above this protein level, changes in body weight were relatively small. However, when the body weight of rats fed the 15% and 25% protein diets ad libitum were
TABLE 3 Mean weights of rats fed diets containing 11.9% fat and varying levels of protein ad libitum for 8 weeks
Protein content of diet g
15N Dâvs fßddiet0714212835424(1562N1 80110±15298±1197±1199±11100±12100 = 12110±10143±17174±22214±23243 = 60112±10119±10139±13151 = 60g112±10155±13210±23248±24300 = 12102± = 12100 = 10N
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other independent variables in the regres sion. The magnitude of the differences be tween groups was shown by calculating the mean values for each group after ad justments for all other factors (25). Differ ences between means were considered sig nificant at P < 0.05.
Weekly mean food intake of ad libitum-fed rats
7168±11218±13271±16314±23356±27386 6156±14203 ± ±20253 ±13168±14182±15201 ±28288 ±24276 ±30331 ±33315±36341 ±12102 ±36304 ±34366 ±12102±13105±125N ±17208±19221 ±42325 ±31400 ±45356±44421 ±38384±40424 ±43342 ±33437 ±21g/t( ±4390 ±4925N ±4850N ±49 1Number of rats.
2Results are means ±SD.
PROTEIN
285
LEVEL FOR MAXIMUM RAT GROWTH
TABLE 4 Mean weights of rats fed diets containing 15% protein and either 6% or 21.1% fat ad libitum
220
200
Level of fat in diet ISO
diet11.9N 100 g JayICQdiet07142128354249565N1 o
8105=
8110± = 609110±10155±13210±23248 =
82159±13204±19247 ± ±24285 ±29321 ±32345 ±40359 ±40400 ±40g/ 1Number of rats.
6174±12216±20268±28305
±24300 ±34341 ±30331 ±41367 ±34366 ±46392 ±38384±40424 ±50425 ±4921.1N ±45
2 Results are means ±SD.
compared by multiple regression analysis with days fed, level of fat and level of protein as the independent variables, it was found that the rats fed the 15% pro tein diets were significantly lighter. A sim ilar comparison of rats fed the 25% and 50% protein diets indicated that the rats fed the 50% protein diets were lighter. The conclusion was that the rats attained maximum growth when fed a 25% protein diet and that the growth rate declined when the percentage of protein in the diet was increased to 50%. There was a small progressive increase in body weight with increases in the level of fat in the diet.
20
40
LACTALBUMIN
60
80
100
120
140
PROTEIN EATEN U) g
Fig. 1 Utilization of lactalbumin protein at various dietary protein levels. The slope of the linear regressions is the PER. Protein utilization was reduced when the diet contained 25% or 50% protein.
Rats fed the diets ad libitum were heavier than those fed the diets at 75% of ad lib itum intake and the latter group, in turn, TABLE 5 were heavier than those fed the diets at Mean weights of rats fed diets containing 15% protein 50% of ad libitum intake. and 11.9% fat either ad libitum, 50% of ad It is evident from figure 1 that when the libitum intake or 76% of ad libitum intake diet contained 5%, 10%, or 15% lactal Level of energy consumption bumin protein and 11.9% fat, protein utilization was linearly related to the Ad 50% of ad 75% of ad libitum libitum libitum amount of protein consumed. Weight gain Da was also linearly related to the amount of N> = 10 N = 10 N = 60 iet die protein eaten when diets contained 25% or 50% protein but the slope of the line was different for each of these protein 071421283542495695±5"96 5121± levels; the efficiency with which protein ±4108±4113±6126 7143 7160±10179±13209 ± was utilized declined with each increase ±24300 in dietary protein level. ±6135±9139±8146 ±30331 Weight gain was also linearly related to ±16225 ±34366 ±18230 ±38384 energy consumption (fig. 2) but the ef ±8150±8995± ±21240±22112±10155±13210±23248 ±40424 ficiency of energy utilization depended on ±49 dietary protein level. Diets containing 25% or 50% protein were utilized most effi1Number of rats. 2 Results are means ±SD.
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-p» fnrìL
286
JOSEPH
C. EDOZIEN
AND BOYD R. SWITZER
TABLE 6 Regression analysis to show the effects of levels of dietary protein, fat and energy intake on the biweekly weights of rats DietDF2468A.
fed
Independent variablesWeeks
on diet2 Level of protein Level of fat Level of energy1
Pl 5 2 20.0001
0.0001 0.0319 0.00010.0018
0.0001 0.0216 0.00010.0105
0.0001 0.0012 0.00010.9216
0.0001 0.0194 0.0001
B Adjusted mean weights3 (a) protein2%5%10%15%25%50%(b) Level of
(100)'106 (96)138 (48)170 (96)171 (48)163 (48)131
(99)113 (96)180 (48)228 (96)228 (48)214 (48)160
(96)135 (95)226 (48)278 (96)285 (48)258 (47)190
(96)130 (90)228 (43)280 (92)285 (48)260 (47)187
(40)139 (356)140 (40)110
(40)173 (355)177 (40)124
(40)197 (351)220 (39)137
(39)202 (321)215 (39)116
(60)134 (60)166 (316)58
(60)161 (60)225 (315)51
(56)199 (60)275 (314)51
(49)186 (49)301 (301)
fat5%11.9%21.1%(c) Level of
Level energy50% of libitum75% ad Ad libitumAd libitum74«
1P values after adjustments for all other factors in the regression. 2 All the rats were not weighed on exactly the same number of days from the time they were first fed the diets. This variable adjusts for the small differences in the time period the rats were fed the diet. Hence lack of statistical significance did not imply that body weight was independent of the duration of study. *Multiple regression analysis indicates significant differences among means for protein, fat and energy levels at each week tested (P < 0.05). 4Adjusted mean weight in g. *Number of rats.
ciently and the efficiency declined with each succeeding drop in protein level. The growth rate of rats fed diets containing 11.9% fat at 50% or 75% of ad libitum intake was accelerated by increasing the level of dietary protein up to 25% protein (figs. 3 and 4). The body composition data reported in table 7 show that total body lipid was di rectly related to the level of fat in the diet while total body water had a reciprocal relationship to the fat level. There was no difference in the percentage of water in lean tissue between rats fed 25% protein and those fed 50% protein. The influence of level of dietary protein on the weight and composition of the liver is shown in table 8. Maximum weight was attained when the diet contained 15% protein; an increase above this level had
no effect on liver weight. However, rats fed 50% protein had fewer cells (total liver DNA) and larger cells (liver weight/ mg DNA) than those fed 15% or 25% protein. DISCUSSION The results confirm other reports that the intakes of protein and energy have a profound effect on the growth of rats ( 26 ). The growth rate increased progressively with increases in the level of dietary pro tein up to 25% protein and then declined. Since the experimental diets provided 4.0 kcal/g, this level of dietary protein repre sented an intake of 25% of energy from protein. Body weight also correlated di rectly with the level of dietary fat. The increase in body weight with the level of dietary fat was associated with a relative
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valuesDuration
287
PROTEIN LEVEL FOR MAXIMUM RAT GROWTH
220 r
200
o>
Z
es
125
o E 100
S 75
10
AGE (WEEKS)
y=-5.0+
M8x
120 /
y=39+072x
100
LU
80
60
40
ISO
r =.995 r =.998 r =.999
140
O
% PROTEIN
50 •5% Protein o 10% Protein ••15% Protein o 25% Protein
180 160
175
y=-7.7+042x
20
500 1000 1500 2000 ENERGY CONSUMED (x)= k calories
Fig. 2 Utilization of energy for growth at various dietary protein levels. Efficiency of energy utilization was reduced when the diet contained 15% or less of lactalbumin protein.
Fig. 3 Body weight of rats fed iso-energetic diets containing 11.9% fat at 50% of ad libitum intakes for 8 weeks starting at 4 weeks of age. The differences between rats fed 2%, 5%, 10%, 15%, and 25% are statistically significant (P< 0.05). Rats fed 25% and 50% protein were sim ilar. Growth was accelerated by an increase in dietary protein level up to 25% protein.
due to methodological differences since they considered only one set of measure ments at three weeks whereas our rats were investigated for 8 weeks. The results presented in figure 1 which are based on food consumption during the first 4 weeks of feeding the experimental diets agree with the finding of Hegsted and Neff (29) that below a limiting value characteristic for each protein source, protein utilization is independent of the level in the diet. The limiting level for lactalbumin protein lies between 15% and 25%. The decrease in efficiency of utilization in diets containing 25% or 50% lactalbumin protein was not related to the cumulative protein consump tion, since the quantities of the 25% pro tein diet eaten in weeks 1 and 2 and of the 50% protein diet eaten in week 1 con tained less protein than was eaten in 4 weeks by rats fed the 15% protein diet. The composition of the liver was investi gated for possible clues to explain the re-
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increase in the fat content of the body. These findings suggest that the increase in the proportion of food energy derived from fat, which has also occurred in the de veloped countries during the last century (27, 28), may have contributed to the trend toward increased body weight. Hegsted and Neff (40) found no differ ence in body water and hence total body protein of rats fed 23% to 30% lactalbumin, casein or soy protein isolate and those fed diets containing 50% to 58.5% of the same protein source. However, their body weight and body water data showed a similar trend to our body weight data and the contradictory conclusions are probably
288
JOSEPH
C. EDOZEEN AND BOYD R. SWITZER % PROTEIN
250
200
50
6
8
K)
12
AGE (WEEKS)
Fig. 4 Body weight of rats fed iso-energetic diets containing 11.9% fat at 75% of ad libitum intakes for 8 weeks starting at 4 weeks of age. The differences between rats fed 2%, 5%, 10%, 15%, and 25% are statistically significant ( P < 0.0). Rats fed 25% and 50% protein were sim ilar. Growth was accelerated by an increase in dietary protein level up to 25% protein.
duced growth rate in rats fed a 50% pro tein diet. There was no decline in liver protein synthesis as measured by total liver RNA; on the contrary, the increase in protein: RNA ratio suggested an improved efficiency of protein synthesis. Rats fed
TABLE 7 Body composition of rats fed 25% or 60% protein and 5%, 11.9%, or 31.1% fat ad libitum Total Dietary protein level
Dietary fat level
N1
2550511.921.1511.921.155666664.5
Body water : body weight
Lipid : body weight
Lean mass : body weight
Body water : lean mass
±0.7262.9±2.059.9±4.463.3±1.761.1±1.461.2 ±3.184.6±3.484.4±3.083.2 ±0.273.8±1.573.4±1.4 ±2.883.4±3.673.4±1.074.0±0.670.8±4.875.1 ±2.212.1±1.214.9±3.115.4±3.415.6±3.016.8±2.816.6±3.687.9±1.285.1
1Number of rats. 2 Results are means±SD. Rats fed 5% differed significantly (25) at the 0.05 level from other rats in body water, total lipid and lean mass. Rats fed 11.9% and 21.1% fat were similar. Rats fed 50% protein had more total lipid and less lean tissue than rats fed 25% protein. Differences in water content of lean tissue were not statistically significant.
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150 X O
50% protein had a higher protein :lipid ratio than rats fed 25% protein but the normal lipid:RNA ratio as well as the higher percentage of body fat indicated that there was no impairment of fat syn thesis. Factors other than metabolic impairment must have been responsible for the reduced efficiency of protein utilization in rats fed the 25% and 50% protein diets. A possible explanation is that the daily rate of pro tein ingestion might have exceeded the maximum capacity of the body to synthe size new protein. Another more likely ex planation is that the energy :protein ratio was not optimal for maximal retention of the ingested nitrogen. The decrease in ef ficiency of energy utilization in rats fed low-protein diets (fig. 2) was probably related to the relatively long time it took to gain weight when such diets were fed with consequent expenditure of a high percentage of ingested energy for daily maintenance. In addition to dietary protein level, pro tein efficiency ratio (PER) and energy: grotein ratio, another factor which inuenced the rate of growth was "appetite"; table 2 shows, for example, that ad libitumfed rats reduced their food intake when the diet contained more than about 15% protein. In order to avoid problems arising out of differences in body size, the amount of food eaten during the first week may be taken as a measure of appetite since the
PROTEIN LEVEL FOR MAXIMUM RAT GROWTH
289
TABLE 8 Effects of dietary protein level on size and composition of rat liver1*1 levelVariableNWeight,
Dietary protein
iance of d588.53.40.32»3.2»26.2»7.159.42.2226»1,93471.7 ¡fferenceVVVVVVVVVVVVVVVVV Downloaded from https://academic.oup.com/jn/article-abstract/108/2/282/4770424 by Tulane University user on 02 December 2018
gWeight, weightWeight, % body DNADNA, g/mg liverDNA, mg/g liverRNA, mg per liverRNA, mg/g liverRNA, mg per DNAProtein, mg/mg liverProtein, mg/g liverProtein, mg per DNAProtein, mg/mg RNALipid, mg/mg liverLipid, mg/g DNALipid, mg/mg RNAProtein/Lipid, mg/mg ratio2891.53.70.195.410.67.810.71.517422233.522.676.913.89.93.05973.63.00.244.315.78.229.92.020070847.724.554.713. 1Results are mean values adjusted for levels of dietary fat and energy. 2Multiple regression analysis indicates significant differences among group means for each variable determined. 3Significantly different (0.05 level) from rats fed 25% protein ad libitum.
respectively, and that X4 = 400/Xj kcal/g protein, it was found that the maximum rate of weight gain of 58.8 g/week oc curred when the diet contained 23% pro tein. The present study does not, of course, give any indication as to whether or not the differences in growth rate of rats fed Y = ßo + j8i X! + X2 + ß3 X3 + 04 X4 diets containing 15% to 50% protein will be reflected in their ultimate body size. where An increase in the efficiency of energy utilization can account for the increase in Xi = dietary protein level ( % ) the rate of weight gain when human sub X2 = PER ( weight gain in g/g protein con jects are fed protein supplements but do sumed ) Xs = amount of food in grams consumed in not consume more energy. For instance, marginally protein deficient children gained the first week more weight when they were fed supple X4 = energy/protein ratio in kcals/g pro ments of skimmed milk which provided an tein additional 1 g/kg/day protein but negli Y = weight gained in grams/week. gible amounts of energy (19). Similarly, The results of the analysis showed that pregnant women and children participating Y = -54.1 - 0.53 Xj - 17.4 X2 + 1.66 X3 in the Special Supplemental Food Pro —0.51 X4. The regression correlation co gram for Women, Infants and Children efficient of 0.9988 suggests that the four (WIC) gained more weight when their factors mentioned above can explain most supplemented diets provided more protein of the variation in the rate of weight gain. When values of Y were calculated for without change in energy content (15). different values of Xi, assuming that X2 Conversely, the level of energy consump and X3 were linearly related to dietary tion profoundly affects protein require ments (30). These results reemphasize the protein level between 15% and 25% pro tein and between 25% and 50% protein, importance of energy :protein balance in
average weight of rats in each group was about 110 g at the beginning of the ex periment. The rate of weight gain can be deter mined by multiple linear regression analy sis of the type:
290
JOSEPH C. EDOZIEN AND BOYD R. SWITZER
studies of protein requirements and protein quality. ACKNOWLEDGMENTS
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We are grateful for the expert technical assistance of Nelsen Niehaus and MeiHeng Mar. The statistical analyses were performed by Henry Lee. The investigation was supported by a grant from General Research Support.
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