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close this bookProtein-Energy Requirements of Developing Countries: Evaluation of New Data (UNU, 1981, 268 p.)
close this folderProtein requirements-children
close this folderProtein requirements of pre-school children: milk and soybean protein isolate
View the document(introduction...)
View the documentObjectives
View the documentExperimental details
View the documentSummary of main results
View the documentConclusions

(introduction...)

Objectives
Experimental details
Summary of main results
Conclusions

Benjamin Toraria 1. Cabrera Santiago, and Fernando E. Viteri
Institute of Nutrition of Central America and Panama (INCAP), Guatemala City, Guatemala

Objectives

1. To determine protein requirements using milk or a soybean isolate as the protein source and following traditional nitrogen balance techniques.
2. To evaluate the protein quality of the soybean isolate relative to milk.

Experimental details

(all values given as mean S.D.)

1. Subjects

  1. Ten children, all males, of mixed Maya and Caucasian descent (Lading).
  2. Chronological age: 23 4 months (range: 17 to 31). Height-age: 15 3 months (range: 9 to 23).
  3. All had been treated for severe, oedematous protein-energy malnutrition (kwashiorkor or marasmic kwashiorkor). They had recovered fully at least one month before beginning the studies, based on clinical, anthropometric, and biochemical criteria (plasma proteins, non-essential/essential amino acid ratio, haematological indices, urinary creatinine excretion, and creatinine-height index [CHI] ). Only one child had a weight-for-height below 93 per cent of the expected (88 per cent) and a CHI of 0.84.
  4. Weight: 10.13 1.00 kg (range: 8.82 to 11.961. Height: 77.6 4.4 cm (range: 71.3 to 86.4). Weight-forheight, percentage of expected: 97 4 (range: 88 to 104; see above). CHI: 0.95 0.08 (range: 0.84 to 108; see above).
  5. Intestinal parasites: Three children had Ascaris lumbricoides. One of them plus another child had Trichuris trichiura. These two children and two others had Giardia lamblia. All infestations were mild (few eggs and protozoa in stools), and they were not treated before the study.
  6. All children were healthy throughout the study, except for occasional episodes of upper respiratory infections with or without fever. When a child had fever, the study was interrupted and he received a diet that provided 2 to 2.5 9 protein and 100 kcal/kg/day for at least seven days after the fever and other symptoms had subsided. The study was then reinitiated with a protein intake at the level that preceded the level he was eating when he became ill. The only exceptions were when a child became ill during the final level of dietary protein intake. In that case, the study was terminated and his data were evaluated with only three levels of protein intake.

2. Study Environment
INCAP's Clinical Centre in Guatemala City; 1,500 m above sea level. Temperature: 18 to 24 C. Relative humidity: 40 to 50 per cent, except on rainy days.

3. Physical Activity
The children were encouraged to participate in games that involved walking, running, climbing stairs or ramps, and tossing balls between two and five hours every day, including the metabolic balance periods.

4. Duration of the Study

a. Four consecutive 9-day dietary periods, or 36 days in all, with a protein source.
b. Fourteen days with a diet that provided 2 to 3 9 protein and 100 kcal/kg/day.
c. Four consecutive 9-day dietary periods, or 36 days in all, with the other protein source.

5 . Diets
a. The components of the experimental diets are given in table 1. The amino acid compositions of the two protein sources are given in table 2.

TABLE 1. Constituents of Experimental Diets (g/kg/day)

 

Protein intake levels

  A B C D
Skimmed milk 3.55 2.85 2.13 1.43
(or soybean protein isolate.) (1.44) (1.15) (0.86) (0.57)
Cornstarch 1.50 1.50 1.80 2.20
Supar 13.25 13.80 14.12 14.28
Cottonseed oil 3.26 3.27 3.29 3.30
Mineral mixture 0.61 0.61 0.61 0.61
Water, to total of 80 80 80 80
Protein, g/kg/day 1.25 1.00 0.75 0.50
Energy, kcal/kg/day 100 100 100 100

* Purina Protein 220, Ralston-Purina Co., St. Louis, Mo., USA. These formulas contained more, cornstarch than the milk formulas to compensate for the higher enargy content of milk.
** Provides (in mEq): K 6; Na 1; Ca 1; Mg 0,4; Cl 6; PO4 1; CO3 1; SO4 0.4.

b. The liquid diets were cooked for 10 to 15 minutes and final weights were adjusted with water after cooling. Cinnamon flavour was added. Fibre content was extremely low, and fat provided 30 per cent of total energy.

c. Diets were fed as five isonitrogenous, isoenergetic meals at three-hour intervals, beginning at 8 a.m. Vitamin and mineral supplements were given each day to satisfy the children's requirements. Additional water was offered ad libitum. At the end of each meal the food containers were rinsed with water and the child drank it. Intake was measured weighing the containers immediately before and after each meal.

d. Dietary levels: The protein content of the diet was increased (ascending design) or decreased (descending design) by 0.25 g/kg/day at 9-day intervals. The changes were isoenergetic with carbohydrate replacement of protein and vice versa. Half the children began with 1.25 g/kg/day (descending design) and half with 0.5 g/kg/ day (ascending design ).

TABLE 2. Essential Amino Acids in Cow's Milk and Soybean Protein Isolate Used to Study Protein Requirements (mg of Amino Acid per Gram of Protein)*

Amino acid Milk Soy
Histidine 34.5 29.4
Isoleucine 56.4 50.2
Leucine 98.8 78.0
Lysine 85.8 62.1
Total sulphur amino acids 38.0 26.4
Methionine 27.6 13.0
Cystine 10.4 13.4
Total aromatic amino acids 93.0 88.7
Phenylalanine 52 .7 52 .5
Tyrosine 40.3 36.5
Threonine 44.1 36.5
Tryptophan 19.8 16.0
Valine 64.2 52.8

* Prom Tor al. (1980) and Cabrera-Santiago and Tor980). Amino acid analyses performed at the Ralston-Purina company's Research Laboratories, based on 24- and 88-hour hydrolysis.
** Purina-Protein 220, USA.

At the end of the fourth protein level, the children ate a diet that provided 2 to 3 9 of protein and 100 kcal/kg/day for 14 days and then followed once more the same experimental design with the other protein source. Half the children began the study with milk and half with soybean protein isolate.

6. Indicators and Measurements
a. Nitrogen balance was determined during the last four days of each nine-day period. Faeces were homogenized and dried at 80 C. Aliquots of diets, faeces, and urine were digested and analysed by a micro-Kjeldahl technique, and the results were corrected by the recovery factor of tryptophan standards that were digested and analysed simultaneously.

"Apparent" balance was calculated (i.e., dietary nitrogen-faecal nitrogen-urinary nitrogen). However, instead of using the zero-balance intercept to calculate nitrogen requirements, a retention of 24 mg N/kg/day was used to represent "balance," therefore allowing 9 and 15 mg N/kg/day to compensate for integumental losses and growth, respectively.

b. Protein digestibility was calculated both as "apparent" and as "true." For the latter, 20 mg N/kg/day was used as the mean obligatory (endogenous) faecal nitrogen.

c. Body weight was measured daily before breakfast. Height and other anthropometric measures were obtained at 14-day intervals.

d. Protein quality was calculated by computing the regression coefficients of nitrogen balance (Y) on intake (X) to determine the relative protein value (RPV) of the diet. Nitrogen intakes required to support a retention of 24 mg N/kg/day, as mentioned above, were also determined-that is, relative nitrogen requirement (RNR).

e. At the beginning of the study and at the end of each nine-day period, a blood sample was obtained to determine haematocrit (micro-centrifugation), plasma proteins (refractometry), serum albumin (dye binding with bromcresol purple), and serum aminotransferases (kinetic U.V. method, with and without addition of pyridoxal pyrophosphate). Urea (carbamido-diacetyl reaction) and creatinine (modified Folin and Wu) were determined in the urine collected during the balance periods.

Summary of main results

Tables 3 and 4 summarize the results derived from the nitrogen balance studies. Table 5 shows that total plasma proteins decreased after nine days with an intake of 0.5 9 milk protein/kg/day and also after nine days with 0.75 9 soybean protein/kg/day.

Except for a decrease in urinary urea with the decrease in nitrogen intake, there were no consistent diet-related changes in the other biochemical indicators explored.

Rates of weight gain during the nine-day periods were slower with milk protein intakes of 0.5 9 milk protein/kg/day than with 1.00 or 1.25 g/kg/day (ANOVA: F3 34 = 3.473; least significant difference 13.1 g/day).

Although the mean weight gain was also lower with the lowest levels of soybean protein intakes, these did not differ from the higher levels because of the large inter individual variability.

TABLE 3. Data Derived from Nitrogen Balances: Milk

 

Child

Digestibility
(% of intake)

Regression of apparent
Balance (Y) on intake (X)
(mg N/kg/day) b

Nitrogen intake required to retain
24 mg N/kg/day c

 

App.

"True"a

Y = a + bX

r

 
Descending design  
A.A,

81

96

- 68 + 0.690X

0.991

133

C.R.

76

91

- 74 + 0.922X

0.970

106

I.G.

88

104

- 16 + 0.652X

0.994

61

H.A.

78

92

- 24 + 0.546X

0.972

88

W.G.

76

91

- 49 + 0.765X

0.988

95

D.B.

78

92

- 42 + 0.718X

0.992

92

Mean

80

94

- 46 + 0.716X

 

96

S.D.

5

5

23 .125

 

24

Pooled data

(n=22) d

   

- 51 + 0.766X

0.914

98

Ascending design  
W.M.

83

97

- 34 + 0.725X

0.995

80

M.V.

75

91

- 72 + 0.642X

0.997

150

A.Z.

82

96

- 51 +0.816X

0.999

92

I.T.

78

93

- 16 + 0.552X

0.958

72

Mean

80

94

- 43 + 0.684X

 

99

S.D.

4

3

24 .113

 

35

Pooled data (n=16)    

- 48 + 0.718X

0.836

100

Both designs  
Mean

80

94

- 45 + 0.703X

 

97

S.D.

4

4

22 .115

 

27

Pooled data (n=38)    

- 49 + 0.740X

0.877

98

a "True" digestibility assumes obligatory faecal nitrogen loss of 20 mg/kg/day.
b Apparent balance = intake—urinary nitrogen—faecal nitrogen.
c Allowing 15 mg N/kg/day for growth and 9 mg N/kg/day for insensible losses.
d Using all data for the corresponding design; n = number of determinations.

TABLE 4. Data Derived from Nitrogen Balances: Soybean Protein Isolate

Child

Digestibility
(% of intake)

Regression of apparent Balance (Y) on intake (X)
(mg N/kg/day) b

Nitrogen intake required to retain 24 mg N/kg/day c

 

App.

"True"a

Y = a + bX

r

 
Descending design  
A.A. 81 96 - 34 + 0.522X 0.986 111
C.R. 67 83 - 52 + 0.558X 0.985 136
I.G. 82 95 - 36 + 0.563X 0.993 107
H.A. 76 96 - 27 + 0.473X 0.992 108
W.G. 73 89 - 52 + 0.543X 0.981 140
Mean 76 91 - 40 + 0.532X   120
S.D. 6 5 11 .036   16
Pooled data in

(n = 18) d

    - 44 + 0.553 0.936 123
Ascending design  
W.M. 83 98 - 26 + 0.545X 0.998 92
W.M.A. 80 95 - 53 + 0.743X 0.998 104
M.V. 80 96 - 68 + 0.654X 0.965 141
A.Z. 76 91 - 63 + 0.698X 0.975 125
I.T. 77 92 - 46 + 0.528X 0.989 132
Mean 79 94 - 51 + 0.634X   119
S.D. 3 3 16 .094   20
Pooled data

(n = 18)

    - 55 + 0.662X 0.921 119
Both designs  
Mean 78 94 - 46 + 0.583X   120
S.D. 5 4 15 .086   17
Pooled data

(n = 36)

    - 50 + 0.610X 0.920 121

a, b, c, d See footnotes in table 3.

TABLE 5. Plasma Protein Concentrations with Different Levels of Protein Intake (g/dl)

Protein intakes (g/kg/day)

 

Descending design

Ascending design

 

2.00

1.25

1.00

0.75

0.50

1.50*

0.50

0.75

1.00

1.25

Milk

mean

6.90

6 80

6.90

6.62

6.37 a

6.90

6.50 b

6.75

6.58

6.73

S.D.

0.20

0.29

0.54

0.31

0.38

0.29

0.29

0.96

0.67

0.25

   

4/5 c

3/6

4/6

6/6

 

4/4

2/4

3/4

2/3

Soybean protein

mean

6.94

6.76

6.64

6.28 b

6.20

7.03

6.25 b

6.22

6.30

6.78

S.D.

0.23

0.43

0.21

0.16

0.57

0.36

0.44

0.43

0.64

0.53

   

3/5

315

5/5

2/4

 

4/4

3/5

3/5

2/5

a,b Differs from preceding protein intake (student's paired "t" test): a: p< 0.01; b: p<0.05.
c Proportion of children with decreased concentrations after nine days on the corresponding dietary protein intake.
* Basal period.

 

The RPV and RNR of the soybean protein isolate, compared with milk, were 83 per cent and 81 per cent, respectively.

Conclusions

1. The mean nitrogen requirements, allowing for integumental losses and nitrogen retention for growth, were 98 mg (0.61 9 protein) of milk/kg/day and 120 mg (0.75 9 protein) of soybean isolate/kg/day. The milk requirement is 33 per cent lower than the estimate made by the FAD/WHO Committee of Experts on Energy and Protein Requirements (WHO Tech. Rep. Ser. No. 522, 1973).

2. The coefficients of variation between individuals of the regression coefficients and of the nitrogen requirements for the two protein sources were between 14 and 16 per cent, except for the mean nitrogen requirement with milk, which was 28 per cent. If two children with high requirements were excluded, variability would be reduced to 16 per cent. The safe levels of intake of the two proteins can be calculated by adding 30 per cent to the mean requirements (FAO/WHO, 1973), or by using the higher limit of the 95 per cent confidence bands (Rand et al., Am. J. Clin. Nutr., 30: 1129 [1977] ). With the former approach, they would be 127 mg nitrogen or 0.79 9 protein/ kg/day for milk, and 156 mg nitrogen or 0.98 9 protein/kg/day for soybean protein isolate. Using the confidence bands, they are 151 mg nitrogen or 0.94 9 protein/kg/day for milk, and 162 mg nitrogen or 1.01 9 protein/kg/day for the soybean isolate. All of these values are lower than the 1.19 9 milk protein/kg/day suggested by FAD/WHO

3. The nutritive value of the soybean protein isolate was 82 per cent relative to milk.

Acknowledgements

The Ralston-Purina Company provided us with the milk, soybean protein isolate, and other materials used in these studies. Ms. Cabrera-Santiago participated in the investigation under a Fellowship from the United Nations University World Hunger Programme.