Experimental details

1. Subjects
Eight young, healthy men, 24 to 31 years old, who belonged to the low socio economic class in Chile. Their physical characteristics are given in table 1.

2. Study Environment
They slept in INTA's metabolic unit for the entire duration of the experiment, and they were asked to maintain their usual daily activities, refraining from unusual exercise. During the entire study the men were under the supervision of a physician and a nurse.

3. Diet
The usual protein intake of the subjects before the study was estimated to be about 1 g/kg/day, based on a 15-day dietary history and a prospective observation period.

The daily energy intake of each subject was calculated in the same way, also based on his caloric expenditure according to the pattern of his usual energy intake and activity.

TABLE 1. Physical Characteristics of the Subjects

* Relative to standards suggested by D.B. Jelliffe, The Assessment of the Nutritional Status of the Community (World Health Organization, Geneva, 1966).

Table 2 lists the components of the experimental diet, which provided less than 2 mg N/kg body weight/day. Two successive experimental periods were conducted, as shown in table 1. From day 1 through day 10 the subjects were fed the nitrogen-free diet at their estimated energy requirement level (period 1), and from day 11 through day 18 dietary energy was raised by 25 per cent. Three isoenergetic meals were provided at 8 a.m., 1 p.m., and 7 p.m. and consumed under the supervision of a dietitian. A vitamin and mineral supplement was given each day at lunch to meet or exceed the 1974 NAS/NRS Food and Nutrition Board Recommended Dietary Allowances. Supplements of calcium and zinc were also given.

4. Duration of the Study
Eighteen days on a nitrogen-free diet.

5. Indicators and Measurements
The men were weighed before breakfast each day after voiding and wearing minimal clothing. Complete 24-hour urine collections were made throughout the study.

TABLE 2. Composition of Protein-Free Diets Used for Study of Obligatory Nitrogen Losses

Food preparations: protein-free cookies; cornstarch soup; cornstarch bread; cornstarch dessert; apple sauce. The intake is given for a 63 kg subject.

* Vitamin/mineral supplement (Polyterra), Laboratories Pfizer de Chile, Santiago, Chile. One tablet supplies: vitamin A 5,000 I.U.; vitamin D 1,000 l.U.; thiamine 1 mg; riboflavin 2 ma; pyridoxine I mg; vitamin B_l2 2 mcg; ascorbic acid 50 mg; niacinamide 12 mg; Ca pantothenate 2 mg; copper (as CuO) 70 mg; iodine (Kl) 50 mcg; iron 1 mg; potassium- (Kl) 16 mcg; manganese (MnCO₃) 29 mcg; magnesium (MgO) 108 mcg; zinc (ZnO) 71 mcg. In addition, each subject received dairy a 15 mg zinc supplement, as zinc chloride, and a table of Calcium Sandoz Forte providing 500 mg of calcium.

Samples were collected in plastic bottles with 10 ml of 10 per cent (v/v) sulphuric acid. Each 24-hour collection was made up to 3,000 ml with distilled water and thoroughly mixed. Aliquots were analyzed immediately for total nitrogen, urea, and creatinine. Another sample was frozen for subsequent analysis. Faeces were collected daily in plastic containers and kept in a freezer until analysed. Composites were made for each subject from the faecal pools for the entire duration of each experimental period. Blood samples were drawn from an antecubital vein after an overnight fast of 12 hours on days 1, 10, and 18 and analysed for total serum protein, albumin, urea, aminotransferases, and complete blood count. Anthropometric measurements (height; body weight; waist, gluteal, and mid-upper arm circumferences; triceps skin fold and subscapular skin-fold) were obtained on days 1, 10, and 18.

6. Statistical Analyses The analysis of urinary obligatory nitrogen was made as suggested by Rand et al. (Am. J. Clin Nutr., 29: 639 [1976] ). A single exponential model was used:

y = P₁e-P₂t + P₃

where

y = urinary nitrogen excretion;
P₁ = difference between y at time 0 and at P₃;
P₂ = rate of change in nitrogen excretion;
P₃ = value at the asymptote of the curve; and
t = time in days.

In addition to P₁, P₂, and P₃, an additional index used was the time required for stabilization of P₃ (t _s). The time to stability is defined as the time taken for y to achieve a value not significantly different from P₃ ±1 S.D. The mathematical calculations were carried out at the INTA Biometrics Unit with the aid of a non-linear least squares fits programme, using the Marquardt algorithm from the Public Library of IBM System 370, APL language.