(introduction...)

Objective
Experimental details
Summary of main results
Conclusions and comments

Ricardo Uauy, Enrique Yz, Digna Ballester, Gladys Barrera, Ernesto Guzman, MarT. Saitnd Isabel Zacarias
Institute of Nutrition and Food Technology (INTA), University of Chile, Santiago, Chile

Objective

To obtain information on obligatory urinary and faecal nitrogen losses of Chilean men from a low socioeconomic background.

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

Subject	Age (years)	Weight (kg)	Height (cm)	W/H* (%)	Energy intake
					Period 1			Period 2
					(kcal/ day)	(kcal/kg/ day)	(kcal/ day)		(kcal/kg/ day)
J.A.	26	55.0	161	90	2,800	51	3,500		64
J.B.	25	74.2	177	104	3,150	42	3,937		53
O.G.	25	61.7	174	89	2,950	48	3,687		60
S.L.	28	60.5	166	94	3,000	50	3,750		62
H.R.	25	61.8	171	91	2,800	45	3,500		57
E.R.	31	63.9	170	95	3,000	47	3,750		59
R.E.	31	69.9	169	104	3,000	43	3,750		54
N.A.	24	70.6	170	106	3,000	42	3,750		53
Mean	26.9	64.7	169.8	96	2,963	46	3,703		58
S.D.	2.8	6.3	4.8	6.9	116	4	145		4

* 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

Ingredient	Period 1	Period 2
Sugar, g	102	112
Honey, g	30	55
Cornstarch, g	224	230
Margarine, g	68	83
Vegetable oil, g	103	150
Orange-flavoured beverage, g	30	50
Soup flavouring, g	2	4
Baking powder, g	8	7
Carbonated beverage, ml	414	414
Apple sauce, g	98	97
Alphacel, g	6	6
Water, ml	1,522	1,722
Vitamin/mineral supplement* Dietary energy (%)
CHO	50.2	44.5
Fat	49.6	55.3
Energy intake (kcal)	3,000	3,750

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.

Summary of main results

1. Energy Intake
During period 1, the men received 46 i 4 kcal/kg/day, and during period 2, their energy intake was increased to 58 i 4 kcal/kg/day.

2. Anthropometry
Table 3 shows the anthropometric data obtained during the study. All subjects except one lost weight. The remaining anthropometric indices did not show significant changes.

3. Urinary Nitrogen Excretion
Figure 1 and tables 4A and 4B show the daily urinary nitrogen losses. It should be noted that subject E.R. on day 6 did not comply with the experimental protocol and exercised heavily. We eliminated the abnormally high value obtained that day and, for computational purposes, replaced it with the mean obtained from days 5 and 7. A kinetic evaluation of the data is presented in Table 4A. Subject S.L. did not reach stability by day 10 and his data were not included to calculate the mean parameters of the equations. The asymptotically derived urinary nitrogen loss (P3) after stability had been reached was 35.8 mg N/kg/day. The mean time to stabilitY (tS) was 6.5 days. Data from subject H.R. on day 15 and from E.R. on day 13 were not included in the pooled regression analyses and daily means because these abnormally elevated values corresponded to days on which the conditions of the experimental protocol were not fully met. The regression analysis of nitrogen loss versus days with excess energy showed a trend toward decline in nitrogen loss.

TABLE 3. Anthropometric Measurements of Subjects Fed a Nitrogen-Free Diet at Two Levels of Energy Intake

	Days
Variable	1	10	18
Height, cm	169.8 ±4.8*	169.8 ± 4.8	169.8 ± 4.8
Weight, kg	64.4 ± 6.4	63.9 ± 6.1	63.6 ± 6.4
Waist, cm	82.38±5.3	81.81 ±5.18	80.88±5.07
Gluteal circumference, cm	91.19 ±3.40	90.63 ± 3.68	89.88 ± 3.81
Mid-upper right arm circumference, cm	28.94 ±2.29	28.54 ± 2.34	28.19 ± 2.34
Mid-upper left arm circumference, cm	28.25 ±2.55	27.94 ± 2.44	27.69 ± 2.50
Right triceps skin-fold, mm	7.5 ± 2.5	7.5 ± 2.6	7.3 ± 2.6
Left triceps skin-fold, mm	7.6 ± 2.6	7.4 ± 2.6	7 3 ± 2.6
Right subscapular skin-fold, mm	11.9 ± 3.5	11.7 ± 3.4	11.5 ± 3.3
Left subscapular skin-fold, mm	11.8 ± 3.3	11.6 ± 3.2	11.4 ± 3.2

* Mean ± S.D.

4. Other Urinary Excretions
Table 5 shows the mean urinary creatinine, urea nitrogen, and total nitrogen lost daily during the last five days of each period.

TABLE 4A. Kinetic Analysis of Daily Nitrogen Excretion in Subjects Fed a Nitrogen-Free Diet for Ten Days. Dietary energy intake: 46 kcal/kg/day.

Day	J.A.	J.B.	O.G.	S.L.*	H.R.	E.R.	R.E.	N.A.	Mean ± S.D.
(mg N/kg/day)
1	115.7	134.4	100.8	92.2	117.7	161.0	82.5	94.6	115.2± 26.3
2	76.3	68.4	69.7	97.6	81.6	92.9	56.2	67.4	73.2 ± 11.7
3	53.2	54.6	44.5	57.9	69.3	74.3	44.4	54.4	56.4 ± 11.5
4	44.9	57.3	42.9	54.9	65.0	52.1	42.4	41.5	49.4 ± 9.0
5	41.6	47.3	31.0	56.3	43.9	77.3	37.8	44.1	46.1 ± 14.7
6	36.3	37.2	31.0	51.6	43.5	65.1*	32.5	34.0	40.0 ± 11.8
7	39.9	36.3	26.7	53.4	43.9	54.1	32.7	28.7	37.5 ± 9.5
8	29.8	32.4	32.7	54 4	45 7	54.1	27.4	33.9	36.6 ± 9.7
9	34.8	29.0	24.2	21.4	43.5	35.2	27.4	30.0	32.0 ± 6.4
10	31.6	33.8	25.2		65.0	33.9	27.4	27.4	34.9± 13.7
P1	159.0	229.5	135.8	92.0	136.8	244.4	93.3	106.9	158.0 ± 58.2
P2	0.6591	0.8488	0.5996	0.1500	0.6668	0.7885	0.5454	0.4819	0.6557±0.1295
P3	33.46	36.19	26.27	13.29	47.50	49.91	28.44		35.77 ± 9.47
S.D.	1.369	3.188	1.731	49.72	4.109	5.867	1.507	1.920
ts	7.2	5.0	7.3	*	5.3	4.7	7.6	8.3	6.5 ± 1.4

 

* Subject S,L did not reach a stable nitrogen loss during this period. His data were not included in the means. P1 and P3 are expressed in mg N/kg/day; P2 is given in days—1; ts stands for days needed for stability.

TABLE 4B, Daily Nitrogen Excretion in Subjects Fed a Nitrogen-Free Diet with Excess Energy. Dietary energy intake: 58 ± 4 kcal/kg/day.

Day	J.A.	J.B.	O.G.	S.L.	H.R.	E.R.	R.E.	N.A.	Mean ± S.D.
(mg N/kg/day)
11	36.6	35.1	31.1	42.7	48.3	51.8	27.6	32.7	38.2 ± 8.5
12	36.6	*	26.7	43.9	48.8	30.0	27.7	30.9	34.9 ± 8.5
13	35.1	32.6	23.7	57.8	42.3	90.7**	27.5	28.9	35.4 ± 11.5
14	33.5	30.3	29.5	46.4	50.3	46.1	31.6	28.9	37.1 ± 8.9
15	30.2	31.5	26.6	44.8	69.7**	46.0	30.3	31.6	34.4 ± 7.7
16	29.2	27.6	25.1	49.4	36.3	44.6	27 6	32.9	34.1 ± 8.8
17	35.4	26.3	20.8	31.0	25.6	29.7	26.3	31.5	28.3 ± 4.5
18	37.3	22.7	17.9	***	27.2	***	19.7	27.6	25.4 ± 7.0

* Sample was lost.
** Data not included in the calculation because they deviated from expected values. Regression equations for (a) the complete set of data, and (b) for days 15 through 18: (a) v = - 1.50x + 55.4, r = - 0.39, p < 0.01; (b) - 3.31 x +8.52, r = - 0.46, p < 0.05.
*** Subjects did not complete study

FIG. 1. Obligatory Nitrogen Excretion in Chilean Young Adult Males Fed Two Levels of Dietary Energy Intake

5. Faecal Nitrogen Excretion
Table 5 also shows the mean daily faecal nitrogen excretion for both experimental periods.

6. Measurements in Serum
Table 6 summarizes the biochemical measurements obtained initially and at the end of each period.

7. Factorial Calculation
Table 7 shows the factorial calculation of mean protein requirements. Adding 30 per cent for individual variability as suggested in 1973 by FAD/WHO, the safe level of protein intake would be 0.62 g/kg/day.

TABLE 5. Obligatory Nitrogen Losses in Young Male Subjects Fed a Protein-Free Diet at Two Levels of Energy Intake

	Days 6 to 10 46 ± 4 kcal/kg/day ( g/day )	Days 14 to 18 58 ± 4 kcal/kg/day (g/day )	Paired t test p<
Creatinine	1.33 + 0.25*	1.22 + 0.29	N.S.
Urea nitrogen	1.41 ± 0.68	1.54 ± 0.46	N.S.
Total urinary nitrogen	2.365 ± 0.477	2.107 ± 0.388	0.01
Faecal nitrogen	1.029 + 0.194**	0.562 + 0.141 ***	0.001

* Mean ± S.D.
** Faecal nitrogen losses for days 1 to 10.
*** Faecal nitrogen losses for days 11 to 18.

TABLE 6. Plasma Biochemical Measurements for Subjects Consuming a Nitrogen-Free Diet at Two Levels of Energy Intake

	Day of study			ANOVA
	1	10	18	P	L.S.D.*
Protein (g/dl)	7.4 ± 0.5	6.9 ± 0.5	6.9 ± 0.6	N.S.	-
Albumin (g/dl)	5.0 ± 0.3	4.6 ± 0.3	4.2 ± 0.3	<.001	0.61
Glucose (mg/dl)	81.4 ± 8.7	78.3 ± 8.5	86.6 ± 6.4	N.S.	-
Urea (mg/dl)	24.9 ± 5.2	10 2 ± 2.6	12.5 ± 3.9	< 001	7.7
Urea N (mg/dl)	11.6 ±3.4	4.7 ± 1.2	5.8 ± 1.8	< 001	3.6
SGOT (Karmen units/dl)	28.8 ± 11.0	23.1 ± 10.9	38.6 ± 11.0	<.05	11.4
SGPT (Karmen units/dl)	17.1 ± 10.2	13.9 ± 9.6	15.4 ± 14.5	N.S.	-
Creatinine (mg/dl)	0.65 ± 0.1	0.79 ± 0.1	0.72 ± 0.1	<.05	0.01
Triglycerides(mg/dl)	52.8±25.1	44.9±11.5	49.1 ±12.5	N.S.	-
Cholesterol (mg/dl)	174.9 ± 38.1	160.8 ± 42.2	134.6 ± 14.3	N.S.	-

* Least significant difference to the indicated p value.

TABLE 7. Factorial Nitrogen Losses for Young Males Consuming a Nitrogen-Free Diet

Subject	P3a	Urinary nitrogeb	Faecal nitrogenc	Total obligatory nitrogen lossesd	Correction factor 1.3
(mg N/kg/day)
J.A.	33.5	34.5	13.9	53.4	69.4
J.B.	36.2	33.7	16.0	54.7	71.1
O.G.	26.3	28.0	18.9	51.9	67.5
S.L.	13.3	45.0	14.0	64.0	83.2
H.R.	47.5	48.3	18.0	71.3	92.7
E.R.	49.9	48.5	20.2	73.7	95.8
R.E.	28.4	29.5	13.7	48.2	62.6
N.A.	28.6	30.8	13.7	49.5	64.4
Mean	35.8	36.2	16.1	58.4	75.8
S.D.	9.5	8.6	2.6	9.9	12.9
FAO/WHO (1973)		37	12	54	70

1. Urinary nitrogen loss (mp N/kg/day) after asymptotic stabilization,
2. Obligatory urinary nitrogen losses for days 6 to 10.
3. Obligatory faecal nitrogen losses for days 1 to 10.
4. Assuming 5 mg N/kg/day for integumental and miscellaneous losses.

Conclusions and comments

1. Our results coincide with those obtained in the US and Taiwan, indicating that obligatory nitrogen losses are independent of ethnic and environmental conditions.

2. The correction factor of 1.3 used in factorial calculations underestimates protein requirements, as indicated by our studies using multi-level nitrogen balance techniques (see Young et al., this volume)

3. The relatively higher obligatory faecal nitrogen losses suggest that endogenous nitrogen excretion may be increased in subjects from developing regions with poor environmental sanitation and chronic, subclinical intestinal mucosal damage. Prolongation of the nitrogen-free diet showed a decrease in faecal nitrogen of about 50 per cent. That decrease might have been due to a further decline in labile nitrogen or to changes in the gut flora that contributed to faecal nitrogen losses.

4. Our subjects had higher energy intakes than are customary in this type of study. The men with low weight/height indices had higher energy intakes per unit of body weight.

5. Weight losses may have been due to losses in lean body tissue, mainly muscle, as suggested by the drop in mean daily urinary creatinine excretion.

6. The excessive energy intake decreased the urinary nitrogen losses, especially during the last four days. The men had stable physical energy patterns. The effect of energy balance deficit can be anticipated to raise obligatory nitrogen losses. Nevertheless, the biological significance of excess energy appears to be minor.

Acknowledgements

The authors gratefully acknowledge Laboratories Pfizer de Chile for supplying the vitamin/mineral supplement (Polyterra) used in this experiment.