Experimental details

1. Subjects

1. Four boys of mixed Maya Indian and Caucasian descent (Lading).
2. Chronological age: 18 ± 3 monthts (range: 14 to 24). Height-age: 14 ± 2 months (range: 11 to 18).
3. All had been treated for severe, oedematous protein-energy malnutrition. They had recovered fully at least two months before beginning the studies, based on clinical, anthropometric, and biochemical criteria (plasma proteins, non-essential/essential amino acid ratio, haematological indices, and creatinine height index).
4. Weight: 10.5 ± 0.8 kg (range: 9.7 to 12.1). Height: 71 ± 2 cm (range: 69 to 75). Weight for height: 101 ± 3 per cent (range: 97 to 105).
5. All children were healthy throughout the study, except for one child who developed a mild upper respiratory infection without fever.
6. No other characteristics of the subjects are pertinent to the study.

2 Study Environment
INCAP's Clinical Centre in Guatemala City; 1,500 m above sea level; mean temperature 24.6 C with a maximum of 28.5 and a minimum of 19.0 C; mean relative humidity 66.6 per cent, range from 46 to 96 per cent. All the children remained in the metabolic ward during the study.

3. Physical Activity
The study lasted 40 days with each source of protein and 10 days at the end of a nitrogen-free diet. The children were off the study for three weeks in between nitrogen sources. During the study periods the children were allowed to exercise freely for the first 4 days of each 10 consecutive days. The last 6 days of each 10-day period were nitrogen-balance days, during which the children remained confined to bed but not necessarily Iying down. Energy expenditure was measured by insensible water loss determinations during nitrogen balance periods and by using the Newburgh's factor of 2.2125 kcal/g IWL/day.

4. Duration of the Study
The four children were in the study a total of 111 days, distributed as follows: a. 40 days on a rice-soy-milk formula. b. 21 days on the rice-soy-milk formula and whole egg. c. 40 days on the whole egg diet. d. 10 days on a nitrogen-free diet.

5. Diet
a. Rice-soy-milk: 40 per cent rice flour; 38 per cent full-fat soy flour; 5 per cent skim milk powder; 14.85 per cent sugar; 1.9 per cent mineral mix; 0.1 per cent vitamin mix; and 0.15 per cent artificial flavour. b. Whole egg protein: Iyophilized whole egg homogenate, mineral, and vitamin mix. c. Nitrogen-free diet: purified corn starch, vegetable oil, sugar, minerals, vitamins, artificial flavour, and water. Twenty per cent of calories came from fat.

The three diets were prepared as liquid formulas that provided 90 kcal/kg body weight/day, 20 per cent of which came from fat. Each protein source (a and b) was fed on four consecutive levels, each for 10 days' duration, starting with a nitrogen intake of 320 mg/kg/day and decreasing to 240, 160, and 80 mg/kg/day (equivalent to 2, 1.5, 1.0, and 0.5 g protein/kg/day). Protein was replaced by cornstarch sugar to maintain constant energy intake.

6. Indicators and Measurements
a. Nitrogen Macro-Kjeldahl for food, urine, and faeces. MicroKjeldahl for integumentary losses. Food nitrogen was measured for each ten-day period. Faecal and urinary nitrogen were measured in three-day pools for each child.

Integumentary losses were measured as follows: Before nitrogen balance was started the child was bathed with a non-ionic detergent (nitrogen-free) and dried with nitrogen-free towels by blotting. The bedding, pyjamas, and bibs were all nitrogen-free (pre-washed with 0.5 per cent acetic acid) and were analysed for nitrogen at the end of three days of contact with the children. To this, nitrogen from bath water at the end of three days was added.

Hair and nails were cut to the same length every ten days and analysed with the rest of integumentary nitrogen for that level of intake.

Residual nitrogen in food utensils was 9 mg/day (less than 1 mg/kg/day). Recovery of integumentary nitrogen in pyjamas, bedding, and bibs, tested in "dummy children" with bedding, etc., on which diluted urine was sprinkled repeatedly throughout three days, was 97.3 i 1.8 per cent (N = 6) (range: 95.8 to 100 per cent).

b. Serum protein and albumin, urea, and ammonia were measured at the end of each ten-day period.

c. Basal oxygen consumption was also measured at the end of ten days.