NUTRITION RESEARCH, Vol. 3, pp. 195-202, 1983 0271-5317/83/020195-08503.00/0 Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.
PROTEIN QUALITY COMPARISON OF A NEW ISOLATED SOY PROTEIN AND MILK IN CHILEAN PRESCHOOL CHILDREN Juan I.
Egaga 1, ~.V.M., M. Sc~, Angel Fuentes 1, M.D., Fred H. Steinke 2, Ph.D.
and Ricardo Uauy-, M.D., Ph..
I
Institute of Nutrition and_Food Technology (INTA), Clinical Research Center University of Chile and ZCentral Research Ralston Purina Co. ABSTRACT The protein quality of a newly developed soybean protein isolate (SPF-200) and milk were compared using apparent N digestibility (AND), N balance (ANB) and serum biochemical response to graded levels of N intake (NI). Seven healthy well nourished children aged 35 to 62 months, were given for 8 day periods 1.5, 1.25, 1.0 and 0.75 g protein/kg/day of SPF and milk in decreasing order at a constant energy intake of 100 Kcal/kg/day and a vitamin mineral supplement. AND for SPF ranged from 86 to 70% and for milk 83 to 75%, corresponding to highest and lowest NI, ANB for SPF ranged from 63 to 0 mg/kg/day and for milk from 90 to 28 mg/kg/day. Mean individual regressions of daily ANB and NI were ANB = 0.49 NI- 54 for SPF and ANB = 0.51NI -34 milk. The slopes were not significantly different. Non significant decreases in serum albumin and urea were observed. A significant increase in transaminases was noted at the lowest NI for both proteins. Daily protein allowances to retain 39 mg N/kg/day were 1.27 and 0.91 g protein/kg/day for SPF a~d milk respectively. KEY WORDS: Soy, protein, requirements, children, milk.
INTRODUCTION Soy has been one of the most widely used of all vegetable protein sources due to its high biological value together with its technological versatility. Over the last ten years soybean technology has been able to create products with high protein content such as soybean concentrates and isolates. These are used as simulators of animal food and in the manufacturing of hypoallergenic i~ fant formula. Recent interest in isolated soybean protein has increased because its nutritive and technological attributes allow them to be used in wide ra~ ge of alternatives in today's food system. In the present study the protein quality of a newly developed filament li ke isolated soybean protein (SPF-200) Ralston Purina Co., St. Louis, Missouri was evaluated and compared to milk as a reference protein source. The study al so examined the protein and energy requirements of Chilean preschool children used as experimental subjects in the study.
PROCEDURES AND METHODS The physical, chemical and nutritional properties of SPF-200 are shown in Table 1.
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TABLE I Physical, Chemical and Nutritional Properties of Soybean Protein Isolated (SPF200) Elaborated by Ralston Purina Co. I,- Physical Characteristics Color Flavor pH
Light Buff Bland 7.1
2,- Chemical Composition: Dry Basis Protein (Nx6.25) Crude Fat Crude Fiber Ash Calcium Phosphorus
93.0 0.17 0.09 2.29 0.20 0.57
3.- Amino Acid Composition (g AA/I00 g protein) Cystine Isoleucine Leucine L~rsine Methionine Phenylalanine Threonine Tryptophan Tyrosine Valine
1.3 4.8 8.0 6.3 1.4 5.5 3.6 1.5
3.8 4.6
4.- Nutritional Evaluations PER in Rats:
Casein Soybean Isolate
Baby Pigs
Casein
Biological Value N.P.U.
2.5 1.9 Soybean Isolate
90,4 88.8
79.3 76.3
Subjects: The study was carried out on seven "healthy" preschool children aged between 32 and 62 months (Table 2) who were admitted to INTA's Clinical Center and were kept under medical and nursing supervision during the whole period. A play therapist provided an educational and recreational program for the children throughout the study. TABLE 2 Individual Characteristics of Pre-School Participating in the Study Initials
Age (Months) JL 38 MA 36 MC 43 RD 56 JC 46 ER 34 AB** 62 Mean 45 S,D, ]0,5 * ~ of NCHS Standard (I)
Weight (k~) 13.80 12.90 14. O0
Height Weight/ Height* (cm) ~; 95.0 98 90.0 98 92.0 97 18,25 103.0 109 13.20 92.0 97 12.60 83.0 108 18.90 108.0 104 ]4, 8 ! 96.4 101.6 2,63 8.9 5.3 ** participated only in SPF-200 evaluation
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197
The study group was selected from children living in "Casa Nacional del Niffo" an institution that keeps temporary custody of children up for placement or adoption. All children were in good health, medical history did not reveal evidence of diseases such as tuberculosis, bacterial infections, renal, hepati~ digestive, metabolic or neoplastic conditions. Children did not have a history of acute illness during two weeks prior to the start of the study nor were drugs taken. Physical and anthropometric examination were whithin normal limits of the National Center for Health Statistics standard (I). The following laboratory screening tests were performed: total plasma protein and albumin, blood count, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), sedimentation rate, serum creatinine, fasting blood sugar, serum lipids, cholesterol and blood urea nitrogen (BUN), urinalysis including microscopic examination of sediment and search for ova and parasites in stool. Written consent for the 7 selected children were obtained from the legal guardians of the children, and the project had the approval of the INTA's Ethics Committee which regulates the use of humans as experimental subjects. Diets: During the initial week children were fed a free choice creatinine free diet which provided 100 Kcal/kg/day. This time was used to test the capacity of the diet to meet the caloric needs of all individuals. SPF-200 and full fat powdered spray dry milk (Purita R, Ministry of Healt~ Chile) were evaluated in decreasing levels of N intake aimed at 240, 200, 160 and 120 mg N/kg/day, which represent protein intake of 1.5, 1.25, 1.0 and 0.75 g/kg/day (Nx6.25) respectively. Energy intake was kept constant at 100 Kcal/ kg/day. Both proteins were provided in the diets as liquid formula and solid preparations. The rest of the ingredients used to adjust energy intake were: dextrin maltose, potato starch, sugar, corn oil, margarine, honey and sweeteners. Dai ly minerals and!vitamins supplementation were given to meet the NAS-NRC recommendations (2). Table 2 shows a typical isolated soy protein diet fed at 1.5 g/kg to a 15 kg child. TABLE 3 Composition if a Typical Soybean Protein Isolate Diet Fed at 1.5 g/kg to a 15 kg Child
Ingredient
g
Energy
Protein
CliO
Fat
SPF-200 Potato Starch
24.75 20.70
(Kcal) 92.24 68.72
(9) 21.56 0.12
(9) 17.09
(g) 0.02
Sugar Maltose-Dextrin Corn oil Margarine Corn starch St rawbe r ry f Iavored Maltose-Dextrin Honey Orange flavored Beverage Flavorings Water
71.40 49.50 40.50 3.0 55.50
274.89 189.09 358.02 21.60 194.81
0.44 -
71.04 47.03 -
0.21
48.11
40.50 2.43 0.11
30 27.6 22,5 2.07 1444.50
118.80 61.41 116.19 5,25
0.03 0.04 0.02 0.23
29.64 ]5.83 28.80 0.30
O.34
22.65 1.50
257.84 17.2
43.4 2.89
Total per day Total per kg per day
1501 1OO
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Egana et al.
Each protein level was Red for 8 days, the initial 4 days, were used as a stabilization period and the last 4 days for the balance period. Autoclaved carmine red and brillant blue were given orally to mark the beginning and end of each 4 day stool collection. After completion of the first study period with SPF-200 the children for 7 days ate ad lib creatine and creatinine free diet to allow for protein repletion. After this, subjects were switched to the milk protein diet and the second study period began. A daily record of clinical indices body weight and weekly arm circumferen ce, triceps fat fold and scapular fat for each subject was taken. Arm fat mus cle area (AMA) and fat a r e a (AFA) were calculated as suggested by Jellife (3, --
4). A l l c h i l d r e n mantained t h e i r usual a c t i v i t y p a t t e r n during the metabolic phase of the study. Urine and stool were c o l l e c t e d at 24 hour i n t e r v a l s during the 4 day balance p e r i o d . A homogeneized sample f o r the 4 day pooled fecal col lection was analyzed by the ~eldahl method for total nitrogen (5). A urine alliquot was obtained and creatinine, total N and urea N were measured. Total albumin, BUN, creatlnine, transaminase, cholesterol and serumlipids were measured in plasma after completion of the two dietary protein study periods at the lowest N intake. Nitrogen balance techniques were used to evaluate the quality of both pr~ tei ns. Regression equations between N intake or N absorbed to N retained were calculated (6,7). Apparent digestibility of both proteins were also determined. Individual regression equations were obtained using daily N balance for the four Nitrogen intake levels for each protein source. Data processing followed the methods suggested by Rand (8) and Nitrogen Balance Index relating nitrogen inta ke or nitrogen absorbed to nitrogen retained was calculated (9). RESULTS The different preparations and formula containing SPF or milk were readily accepted and consumed 96-100% of what was offered. The children remained healthy during the investigation and did not show adverse reactions to either diet. Table 4 shows the observed anthropometric indices. No significant changes were noted. Two children (MA and JL) had elevated temperature due to an in tercurrent viral infection during the SPF diet and for this reason the data fo~ the corresponding level was not considered in the pooled data for the SPF evaluation. The c h i l d i d e n t i f i e d as AB was removed from the study a t the request o f his parents during the m i l k p r o t e i n e v a l u a t i o n . TABLE 4 Changes in Anthropometric Indices f o r Subjects Fed Soybean Protein Isolate Milk
Weight (kg) Height (cm)
SPF-200 (n=7) Initial Final 14.9" + 2.5 14.9 + 2.7 94.7 ~- 8.5 96.0 ~- 8.9
AMA cm2 AFA cm
12.2 5,6
WIH ~o.*~
I 0~-. 7
AMA: Arm Muscle Area *Mean + S,D.
+ 2.1 T].4
I OE. 4 11.6 + 1.6 6.1 T 2 , 4
M i l k (n=6) Initial Final 14.4 + 2.3 14.8 + 2.5 93.7 T 7.1 93.9 ~ 7.0
100-.2 10.8 + 0.9 6.4 T 2 . 7
I 0E. 3 10.5 + 1,1 7.1 ~-3.3
AFA: Arm Fat Area **of NCHS Standard (I)
and
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199
Table 5 shows apparent N digestibility N balance and Net Protein utiliza tion for both proteins determined at the various N intakes. The individual results of N digestibility ranged between 86 to 70% for SPF and 83 to 75~ for milk. No significant differences in digestibility were observed between the protein sources at the same N intake. However SPF showed a significantly decrease apparent digestibility value for the lowest level of N intake (p(.01). The results of nitrogen balance at the different N intake levels show that N retentions for SPF were significantly lower than milk and ranged between 63 to 0 mg N/kg/day with SPF and for milk between 90 to 28 mg N/kg/day at the highest and lowest N intake levels, respectively. Apparent utilization data derived from the different levels of N intake suggests a better retention for milk protein. The higher apparent N utilization at larger intakes reflects the smaller contribution of endogenous N losse~ TABLE 5 Mean Apparent Nitrogen D i g e s t i b i l i t y , N Balance and Net Protein U t i l i z a t i o n in Preschool Children fed Soybean Protein Isolate and Milk N intake m9 N k 9 day
Digestibility % SPF-200 Milk
A P P A R E N T N Balance mg/kg/day SPF-200 Milk
Net P r o t e i n U t i l i z a t i o n SPF-200 Milk
240
86.21 +--3.2 84.5+--5.2
63+_23b
90+_18a
26.4+--9.6
37.9~ 8.5
200
83.21 +-3.2 78.7+--6.8
43+18 b_
65+19 a
21.4+--8.6
32.7~ 9.4
160
80.91 +-6.0 76.4~7.5
29+11 b
50+_16a
18.3+--7.1
31.0+_10.3
120
71.62 +-9.2 74.7~7.6
O~ 8 b
28~18 a
0.1+-7.0
%
23.8~14.0
I ; 2 = d i f f e r from each o t h e r (anova p ( . 0 1 ) a;b -- d i f f e r from each other (student's paired " t " test (p<.05) I n d i v i d u a l regressions based on d a i l y 24 hr values and pooled regression f o r N intake versus N r e t e n t i o n f o r both p r o t e i n s are shown in Table 6. Compar i s o n of the slopes showed no s i g n i f i c a n t d i f f e r e n c e s between the p r o t e i n s ( p > . 0 5 ) although m i l k had s l i g h t l y h i g h e r values. Nitrogen balance index based on values of N r e t e n t i o n over N absorption is also shown in Table 6. No s i g n i f i c a n t d i f f e r e n c e between the regression c o e f f i c i e n t s of the p r o t e i n sources was observed. TABLE 6 Mean Regression Equations of Apparent Nitrogen Retention over Nitrogen Intake and Nitrogen Absorbed (mg N/kg/day) Mean o f I n d i v i d u a l Regressions a
b
Pooled data a
b
r
Nitrogen Intake SPF-200 Milk
-54.0 -33.6
0,49 0.51
-57,2 -37.6
0,51 0.53
0.77 0.77
Nitrogen Absorbed SPF-200 Milk
-40.3 -20.6
0.51 0.56
-40.3 -24,8
0.51 0.59
0.79 0.85
Biochemical i n d i c a t o r s in plasma as shown in Table 7, demonstrated t h a t both d i e t s induced s i m i l a r non s i g n i f i c a n t changes, A s i g n i f i c a n t l y h i g h e r serum transaminase c o n c e n t r a t i o n was observed on the l a s t day o f study which co-
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J . l . Egana et al.
rresponded to the lowest level o f N intake f o r both p r o t e i n s . The c h o l e s t e r o l lowering e f f e c t was more marked f o r the SPF. No s i g n i f i c a n t changes were noted in hematological indices between p r o t e i n s f o r high and low l e v e l s o f N int ak e . TABLE 7 Biochemical Indices f o r Subjects on SPF-200 and M i l k SPF-200 Initial
MILK Final*
Albumin (g/dl) 4,5 +--0.41 4.2 +__0.3 Globulin (g/dl) 2.4+-0.5 2.5 +--0.5 B.U.N. (mg/dl) 10.1 +-3.8 8.8 +_ 1.7 Creatinine (mg/dl) 0..3+-0.1 0.5+-0.2 Total L i p i d s (mg/dl) 507.1 +--40 472.4 +j06.8 Triglycerides (mg/dl) 74.8 +--35.8 61.7 + 9.6 a Cholesterol (mg/dl) 144.1 +14.1 115.9 +20.3 a SGOT IU/I 20.4 + 7.4 122.9 +__132.5 SGPT IU/] 6.4 +- 2.7 68.6 +83.0 a
Initial
4.5 +--0.4 2.4+-0.6 10.4+-4.1 0.3 +__0.1 509.7 +-43.2 75.0 +--39.2 146.6 +15.2 21.5+- 9.6 7.7 +_2.8
Final*
4.4+__0.3 2.4 +__0.4 6.6+-0.8 0.5 +__0.1 469.5 +-71.9 61.8 +-18.5 132.1 +27.6 116.9 +67.9 a 38.4 +_24.0a
1Mean + standard d e v i a t i o n ~ S i g n i T i c a n t l y d i f f e r e n t from i n i t i a l value ( s t u d e n t ' s p a i r e d " t " ( p < . 0 5 ) sample taken on the l a s t day o f 0.75 g kg day p e r i o d f o r each p r o t e i n DISCUSSION Our r e s u l t s i n d i c a t e t h a t the d i g e s t i b i l i t y o f the i s o l a t e d soy p r o t e i n studied is s i m i l a r to t h a t o b t a i n e d w i t h m i l k . A lower apparent d i g e s t i b i l i t y was found a t the lowest l e v e l s o f N intake due to the r e l a t i v e l y l a r g e r c o n t r i b u t i o n o f endogenous f e c a l N. This e f f e c t was more marked f o r SPF and is s i m i l a r to r e s u l t s o b t a i n e d by o t h e r authors using comparable experimental designs
(I0). Nitrogen balance was s i g n i f i c a n t l y h i g h e r f o r m i l k w i t h i n the range o f i ~ takes s t u d i e d . This was determined by the lower u r i n a r y N e x c r e t e d w h i l e the c h i l d r e n consumed m i l k . I t is important to note t h a t our e x p e r i m e n t a l design included N intakes much lower than the normal f o r preschool c h i l d r e n . D i f f e r e n c e s in p r o t e i n qual i t y are maximized at lower intakes and decrease at higher N intakes. Few s t u dies o f p r o t e i n q u a l i t y e v a l u a t i o n are done in h e a l t h y c h i l d r e n . Most have been done in malnourished c h i l d r e n a f t e r being subjected to n u t r i t i o n a l recovery (10,11). The s l i g h t y lower N r e t a i n e d by our subjects may be e x p l a i n e d by this fact. The l i m i t a t i o n s o f an e i g h t day d i e t a r y p e r i o d were r e a d i l y apparent to the i n v e s t i g a t o r s at the time t h i s study was designed indeed f u l l a d a p t a t i o n t o a given p r o t e i n intake may take several weeks to occur. Nevertheless f o r pract i c a l reasons a compromise between the ideal and the f e a s i b l e must be reached. Children have f a s t e r N t u r n o v e r rates and e a r l y a d a p t a t i o n t o a given p r o t e i n intake occurs in three to f o u r days, t h e r e f o r e most studies use a seven to ten day m e t a b o l i c p e r i o d (12,13). The v a l i d i t y o f t h i s approach can o n l y be e s t a b l i s h e d by long term studies w i t h p e r i o d i c e v a l u a t i o n o f N balance and o t h e r i ~ dices o f p r o t e i n s t a t u s .
Soya Pr0tei n Quali ty
20]
The ideal design in the e v a l u a t i o n o f p r o t e i n q u a l i t y is a randomized assignment o f subjects to the d i f f e r e n t d i e t a r y l e v e l s . We were l i m i t e d by our clinical facilities to p r o v i d e f o u r d i f f e r e n t d i e t s s i m u l t a n e o u s l y . We t h e r e f ~ re chose a descending p a t t e r n of p r o t e i n intakes to decrease the r e s i d u a l e f f e c t o f improved N u t i l i z a t i o n observed a t lower N intakes (14). D if f e r e n c es in apparent N u t i l i z a t i o n are confounded by endogenous losses. The i n t e r p r e t a t i o n o f these data can o n l y be done i f r e s u l t s are c o r r e c t e d , but we lack a p p r o p i a t e values for these losses [n Chilean preschool children. We chose to approach the comparison of protein quality by analyzing the regression equation data. The relationship between N intake and N retention was linear and s i g n i f i cantly positive in a11 subjects. The mean obtained from the individual regression equations was similar to that derived from the pooled regression data. The slope of the pooled regression line of N intake on N retention is related to Net Protein U t i l i z a t i o n (NPU) (6,7). Basedon these considerations and comparing the slopes SPF protein has a value of 96% r e l a t i v e to milk protein. The Nitrogen Balance Index was similar for both proteins. No differences between the slopes were observed. The regression equations from pooled data for each protein were also similar to that obtained from the mean individual re gressions. Nitrogen Balance Index is related to Biological Value (BV). In t h ~ case SPF-200 had value of 86% r e l a t i v e to milk protein. For chJildten the recommendedprotein allowances should consider the mainte nance needs and the additional growth requirements. This implies that a positT ve N Balance should be established. Considering lO mg N kg day as skin and mis celIaneous losses, and the mean daily N accretion for growth of preschool chil ~ dren plus two standard deviations, 39 mg N kg day appears to be a satisfactory growth N allowance . T h i s should account for day to day v a r i a b i l i t y in growth rate. I f the given N intake required daily to retain 39 mg N kg day is computed from the regression of balance on intake the allowance to cover 97.5% of the population can be calculated (15,16). Basedon this approach using a coeff i c i e n t of variation of 12.5% to account for interindividual variance a milk protein intake of 0,91 per kg and for soy 1.27 g per kg day was computed (16). This higher requirement for the SPF would be more than compensatedat normal protein intakes which may double that figure and by the complementationof aminoacid patterns obtained by consuming a mixed diet. The results of anthropometric indices showed a s l i g h t weight gain while children consumedmilk and a stable weight on the SPF-200. This finding should be interpreted with due caution since the f i n a l periods correspond to the Ibwest N intake probably associated with some weight loss. The design used does not allow a v a l i d interpretation of weight change as an index of protein energy ad~ quacy because of the short duration of dietary periods. The biochemical changes were not s i g n i f i c a n t except for the lower lipids and cholesterol while on SPF-200 and the elevations in serum transaminases observed at the low N intakes for both proteins. As previously shown these enzymes are very sensitive indicators of protein inadecuancy. Garzaet al (17) have shown rises in aminotransferases in long term N balance studies which occ~ rred while subjects consumed 0.6 g egg protein per kg and reverted to normal with added protein. T h i s normalization occurred over the one week ad l i b diet between the two experimental periods. We conclude that the isolated soy protein we evaluated is well accepted by preschool children. Its d i g e s t i b i l i t y is similar to that of milk protein but at the range of intakes studied the biologica| value of milk appears to be greater. Basedon the individual responses the minimun safe intake recommended for e i t h e r protein is significant1:y lower than normal protein intake for most
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children. SPF-200 can be considered a protein of good q u a l i t y and compares f a vorably to other animal or vegetable protein. REFERENCES 1. National Center f o r Health S t a t i s t i c s : Growth Curves f o r Children Birth 18 years. DHEWPublication N~ 78-1650. Series 11-N, 1978. 2. National Research Council - National Academy of Sciences. tary Allowances. Ninth Edition. Washington, 1980.
Recommended Die-
3. J e l l i f e , D.B. The Assesment of the N u t r i t i o n a l Status of the Community.Geneva. WHO Monograph 53, 1966. 4. Frisancho, A.R. Triceps skin f o l d an upper arm muscle size norms f o r asse~ ment of n u t r i t i o n a l status. Am.J.Clin. Nutr. 27:1052, 1974. 5. Association of O f f i c i a l A n a l y t i c a l Chemist.Official Methods of Analysis. 13th. Ed. Washington, 1980. 6. Bressani, R. Evaluaci6n biol6gica de las prote~nas. En: Recursos prote~nicos en Am@rica Latina. Ed. M. Behar y R. Bressani. Guatema. INCAP L-1 ,
1971. 7. P e l l e t , P.L. and Young, V.R.
N u t r i t i o n a l Evaluation of Protein Foods. The United Nations U n i v e r s i t y World Hunger Programme. Food and N u t r i t i o n Buile t i n Supplement 4 pp. 154, 1980.
8. Rand, W.M.; Scrimshaw, N.S. and Young, V.R. Determination of protein a l l o wances in human adults from nitrogen balance data. Amer.J.Clin.Nutr. 30: 1129, 1977. 9. A l l i s o n , J.B. and Anderson, J.A. The r e l a t i o n between absorbed nitrogen, nitrogen and b i o l o g i c a l value of proteins in adult dogs. J.Nutr. 29: 413, 1945. 10. Torun, B, N u t r i t i o n a l q u a l i t y of soybean protein i s o l a t e s : Studies in chi~ dren of preschool age. In: Soy protein and human n u t r i t i o n . H. Wilke, D.T. Hopkins and D.H. Waggle. Ed. pp. 101-119. Academic Press, 1979. 11. Bressani, R,, V i t e r i , F., E l i a s , L.G., Saghi, S,, Alvarado, J. and Odeil, A.D. protein q u a l i t y of a soy bean protein textured food in experimental animals and children. J.Nutr. 93:349, 1967. 12. United Nations U n i v e r s i t y World Hunger Programme. Protein Energy Requirements under conditions p r e v a i l i n g in developing countries. Current knowledge and research need. Food and Nutr. Bull. 1, 1979. 13. Young, V,R., Steffe, W.P., Pencharz, P.B., Winterer, J.C. and Scrimshaw, N. S. Total Human Body Protein Synthesis in Relation to Protein Requirements at Various Ages, Nature 253:192, 1975. 14. Inoue, G., Fuj ira, Y., Kishl, K., Yamamoto, S. and Niiyama, Y. N u t r i t i v e values of egg protein and wheat gluten in young men, Nutr. Rep. Int,: 10: 201, 1974. 15. Torun, B., Young, V.R. and Rand, W.M. Protein-Energy Requirements of Developing Countries: Evaluation of New Data the United Nations U n i v e r s i t y World Hunger Programme. Food and N u t r i t i o n B u l l e t i n . Supplement 5, PP.
268. 16. FAO/WHO/UNU. Report of Energy and Protein Requirements. Expert Consultation. Rome, October 1981, To be published, 17. Garza, C.G., Scrimshaw, N.S. and Young, V.R. Human protein requirements: A long-term metabolic nitrogen balance study in young men to evaluate the 1973, FAO/WHO Safe Level of Protein Intake. J,Nutr, 107:335, 1977.