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)

* 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)*

* 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.