(introduction...)

Experimental details
Summary of main results
Conclusions
Acknowledgements

Enrique Yz, Ricardo Uauy, Digna Ballester, Gladys Barrera, Nelly Chavez, Ernesto Guzman, Maria T. Saitua, and Isabel Zacarias
Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile

Objective

To evaluate the capacity of the Chilean mixed diet to meet the protein-energy requirements of young adult men from a low socio-economic background, using the nitrogen-balance response to graded levels of dietary protein.

Experimental details

1. Subjects
Eight men, 20 to 31 years old, were selected. Their initial weights, heights, and energy intakes are described in table 1. All were chosen from among volunteers who answered a local advertisement. Their monthly incomes were lower than US$150, which corresponds to the lower tercile of the national income distribution. Their housing, sanitary environment, and educational background were consistent with their incomes. They were healthy, based on medical history, physical examination, and laboratory analysis of haematocrit, haemoglobin, total and differential white blood count, serum transaminase activities, and complete urinalysis.

2. Physical Activity
The men continued their normal daily routines, including their usual patterns of activity, but refrained from participating in competitive sports during the study. They slept in the Metabolic Unit of the Institute's Clinical Research Centre. All subjects remained under the supervision of a physician and a nurse throughout the

TABLE 1. Physical Characteristics and Energy Intakes of the Subjects Participating in the Study

					Energy intake
Subject	Age (years)	Weight (kg)	Height (cm)	W/H Index* (%)	(kcal/day)	(kcal/kg)
J.A.	26	55.0	161	89.5	2,800	51
H.F.	20	54.5	180	72.4	3,050	57
J.B.	25	75.0	177	104.7	3,150	42
O.G.	25	60.7	174	86.7	2,950	49
S.L.	28	59.0	166	91.1	3,000	51
M.L.	30	51.0	162	81.4	2,500	50
H.R.	25	60.5	171	88.6	2,800	47
E. R.	31	61.5	170	91.3	3,000	49
Mean	26.3	59.7	170.1	88.2	2,906	49.5
S.D.	3.4	7.2	6.8	9.2	202.6	4.2

* Based on D.B. Jelliffe, The Assessment of the Nutritional Status of the Community (World Health Organization, Geneva. 1966).

3. Diets
The subjects were fed a "Chilean mixed diet" designed according to available dietary survey information of a typical Chilean diet for the country's low-income groups. Its composition is shown in table 2. Protein was fed at 0.40, 0.55, and 0.70 g/kg of body weight per day. In addition, an egg diet that provided 0.30, 0.45, and 0.60 9 protein/kg/day was fed as a reference diet (table 2). Each subject's energy intake was calculated from his customary diet by the 24-hour dietary recall method for 15 consecutive days. All protein levels were fed with the same total dietary energy to a given individual. Vitamin and mineral supplements were provided daily to meet or exceed the 1974 NAS/NRC Food and Nutrition Board Recommended Dietary Allowances. The nitrogen content of dietary ingredients and preparations was analysed by the Kjeldahl method, using a macro digestion procedure followed by a semi-micro 45 distillation of the ammonia produced into 2 per cent boric acid containing a mixed indicator.

TABLE 2. Composition of Experimental Diets Used for Study of Protein Requirements in Young Men

Ingredient	Level of intake
	Mixed diet	Egg diet
Whole dried egg powder, g	-	34.3
Sucrose, g	106.4	114.0
Wheat flour, g	145.1	-
Margarine, g	54.1	67.1
Dried skim milk, g	27.9	-
Rice, g	61.4	-
Cornstarch, g	-	246.9
Bean soup powder, g	46.0	-
Vegetable oil, ml	32.7	70.5
Potato flakes, g	112.0	-
Soup flavouring, g	-	2.0
Apricot marmalade, g	15.0	-
Orange-flavoured beverage, g	23.6	37.5
Lemon-flavoured beverage, g	-	37.5
Water, ml	1,200	1,754
Vitamin/mineral supplement1

Food preparations:

Mixed diet: Wheat flour bread, rice/milk dessert, bean soup, potato flakes. Intake is given for a 61 kg subject.

Egg diet: Cornstarch bread, cornstarch soup, omelette, liquid egg formula, cornstarch dessert, protein free cookies. Intake is given for a 60 kg subject.

1 Multivitamin/mineral supplement, Laboratories Pfizer de Chile, Santiago, Chile. One tablet supplies: vitamin A, 5,000 I.U.; vitamin D2 1,000 I.U.; thiamin 1 mg; riboflavin 2 mg; pyridoxine 1 mg;vitamin Bl2 2 mcg; ascorbic acid 50 mg; niacinamide 12 mg; Ca pantothenate 2 mg; copper (as CuO) 70 mcg; iodine (Kl ) 50 mcg; iron 1 mg; potassium (Kl) 16 mcg; manganese (MnCO3) 28 mcg; magnesium (MgO) 108 mcg; zinc (ZnO) 71 mcg,

4. Experimental Design
Each experimental period started with a 1-day protein-free diet (NFD) followed by ten days on the experimental diet. A free-choice diet was eaten in the next three days, followed by one day on the nitrogen-free diet and ten days with another experimental level of dietary protein. The sequence of protein levels was randomly assigned to each individual. Three isoenergetic, isonitrogenous meals were provided, at 8 a.m., 1 p.m., and 7 p.m., and were consumed under the close supervision of a dietitian. The protein sources during the mixed diet period were distributed as equally as possible in the three daily meals.

5. Measurements
Complete 24-hour urine collections were obtained with HCI as a preservative throughout the study. An aliquot was analysed for total nitrogen, urea, and creatinine. Faeces were collected daily and pooled during the last eight days of each dietary period. Pools were separated by feeding autoclaved brilliant blue and carmine red markers. Nitrogen balance was calculated by subtracting the mean daily urine and faecal ( nitrogen excretion from the daily nitrogen intake. Integumental and miscellaneous nitrogen losses were estimated at 5 mg N/kg body weight/day. Body weights were measured on a 50g precision scale daily at 0800 hours, before breakfast, post-voiding, with subjects wearing minimal clothing. Fasting blood samples were drawn from an antecubital vein at 0800 hours at the beginning of the study and at the end of the lowest and highest dietary protein test periods. They were analysed for total serum protein, albumin, and urea concentrations; transaminase activities; and blood cell counts. Height; body weight; waist, gluteal, and mid-upper arm circumferences; and triceps and subscapular skin-fold thicknesses were measured at the beginning and end of each dietary level.

Summary of main results

Results of anthropometric changes are summarized in table 3. Tables 4 and 5 show the nitrogen-balance data. Urinary nitrogen excretion was greater with increasing nitrogen intakes from better diets. No correlation was found between faecal nitrogen, which included obligatory faecal nitrogen, and the level of protein intake for either protein. Faecal nitrogen excretion was significantly higher on the mixed diet (34 mg N/kg/day) than on the egg diet (18 mg N/kg/day) (p<0.001). This difference resulted in lower apparent nitrogen digestibility with the mixed diet (p < 0.001). Using 9 mg/kg/day as the obligatory faecal level, the mean true protein digestibilities of the two protein powders at three increasing levels of intake were 82.6, 89.1, and 92.8 per cent for egg and 71.4, 77.1, and 83.5 per cent for the mixed diet. Digestibility of the mixed diets was significantly lower (p < 0.001). Figure 1 shows the regression analyses of "true" nitrogen balance. The "pooled" regression equations are:

Egg diet y = -69.95 + 0.708 X, n = 24, r = 0.852
Mixed diet y = -91.95 + 0.737 X, n = 21, r = 0.807

TABLE 3. Anthropometric Changes in Subjects Participating in the Study

Subjects	Body weight (kg)	Waist (cm)	Gluteal circumference (cm)	Mid-upper left arm circumference (cm)	Left triceps skin-fold (mm)	Left subscapular skin-fold (mm)
J.A. Initial	55.0	72	85.0	26	5.6	8.4
Change	-1.22	-0.5	-1.0	-0.5	0.2	0
H.F. Initial	54.5	72	84.5	25	5.0	6.4
Change	+1.35	1.0	1.5	0	0.2	0
J.B. Initial	75.0	90	96	31	8.4	16.2
Change	-0.27	- 1.0	0	0	0	0.2
O.G. Initial	60.7	80	89.5	25.5	5.5	11.2
Change	+0.67	1.5	1.5	1.0	0.5	0.2
S.L. Initial	59.0	81	91	25.5	5.2	8.6
Change	-0.22	0	0	0	0	0.2
H.R. Initial	60.5	79	90	24.5	6.2	10.6
Change	+0.27	1.0	1.0	0.5	0.2	-0.2
E.R. Initial	61.5	80	88	27	5.6	7.4
Change	- 0.97	-2.0	0	0	-0.2	-0.4
Mean ± S.D.
Initial	60.9±6.8	79.1±6.1	89.1±3.9	26.4±2.2	5.9±1.2	9.8±3.3
Change	0.055±0.9	0.0±1.3	0.4±0.9	0.1±0.4	0.1±0.2	0.0±0.2

TABLE 4. Nitrogen Balance and Nitrogen Digestibility of Individual Subjects Given an Egg Diet et three Dietary Levels of Nitrogen Intake

Subjects (mg/kg/day)	Nitrogen intake	Urinary nitrogen (mg/kg/day)	Faecal nitrogen	Nitrogen balance 1,2 (%)	Apparent digestibility
J.A.	48	67.8	19.6	-44.4	59.2
H.F.	48	50.8	13.3	-21.1	72.3
J. B.	48	49.3	9.2	- 15.5	80.8
O.G.	48	53.4	18.5	- 28.9	61.5
S. L.	48	48.6	24.0	- 29.6	50.0
M.L.	48	77.0	21.8	-55.8	54.6
H. R.	48	70.1	25.0	- 52.1	47.9
E.R.	48	54.4	18.1	-29.5	62.3
Mean	48	58.9	18.7	-34.6	61.1
S.D.		11.0	5.3	14.6	11.1
J.A.	72	62.8	19.1	-14.9	73.5
H.F.	72	78.3	14.6	-25.9	79.7
J.B.	72	72.8	16.0	-21.8	77.8
O.G.	72	67.1	18.2	-18.3	74.4
S. L.	72	66.3	17.5	- 16.8	75.7
M.L.	72	62.5	20.0	- 1 5.5	72.2
H.R.	72	68.1	15.7	-16.8	78.8
E.R.	72	63.8	22.2	-19.0	69.2
Mean	72	67.7	17.9	- 18.6	75.2
S.D.		5.4	2.5	3.7	3.6
J.A.	96	72.0	22.6	- 3.6	76.5
H.F.	96	67.4	15.8	+ 7.8	83.5
J.B.	96	72.2	14.5	+ 4.3	84.9
O.G.	96	71.6	18.4	+ 1.0	80.8
S. L.	96	77.5	20.8	- 7.3	78.3
M.L.	96	72.6	20.9	- 2.5	78.2
H.R.	96	74.5	18.9	- 2.4	80.3
E. R.	96	75.4	19.4	- 3.8	79.8
Mean	96	72.9	18.9	- 0.8	80.2
S.D.		3.0	2.7	4.9	2.8

1 Estimated "true" balance, assuming 5 mg N/kg/day for integumental and miscellaneous losses.
2 Since individual aliquots from diet periods varied less than 5 per cent of calculated intake based on nitrogen analysis of ingredients, nitrogen balance was computed from the latter.

TABLE 5. Nitrogen Balance and Nitrogen Digestibility of Individual Subjects Given a Chilean Mixed Diet at Three Dietary Levels of Nitrogen Intake

Subjects	Urinary nitrogen	Faecal nitrogen	Nitrogen balance1	Apparent digestibility
	(mg/kg/day)			(%)
Nitrogen intake 64 mg/kg/day
J. A.	71.8	36.7	-49.5	42.6
H.F.	58.6	29.1	-28.7	54.5
J.B.	55.7	31.2	-27.9	51.2
O.G.	84.7	30.8	-56.5	51.9
S. L.	69.4	44.9	- 55.3	29.8
H. R.	74.6	38.8	-46.4	39.4
E. R .	69.1	29.2	-39.3	54.5
Mean	69.1	33.2	-43.4	46.2
S.D.	9.8	5.7	11.8	9.3
Nitrogen intake 88 mg/kg/day
J. A.	75.2	40.0	-32.2	54 5
H.F.	69.5	38.8	-25.3	55.9
J. B.	65.4	30.5	- 12.9	57.5
O.G.	66.6	34.3	-17.9	61.0
S. L.	82.8	44.9	-44.7	43.1
H.R.	96.0	30.8	-43.8	65.0
E. R .	81.9	34.4	- 33.3	60.9
Mean	76.8	36.2	-30.0	56.8
S.D.	11.0	5.3	12.1	7.0
Nitrogen intake 112 mg/kg/day
J. A.	84.1	35.4	-12.5	68.4
H . F .	78.1	35.0	- 6.1	68.7
J.B.	64.2	32.5	+10.3	71.0
O.G.	83.4	32.6	- 9.0	70.9
S. L.	73.4	43.4	- 9.8	61.2
H. R.	99.9	28.7	- 21.6	74.4
E. R.	80.0	34.4	- 7.4	69.3
Mean	80.4	34.6	- 8.0	69.1
S.D.	11.0	4.5	9.6	4.0

¹ Estimated "true" balance, assuming 5 mg N/kg/day for integumental and miscellaneous losses.

Mean nitrogen requirements for equilibrium were estimated as 97 mg N/kg/day for the mixed diet. Based on the 95 per cent confidence bands about the regression, the safe levels of protein intakes would be 1.1 g/kg/day for egg and 1.5 g/kg/day for the mixed diet; assuming that the coefficient of variation would be 15 per cent, as suggested by the 1971 FAO/WHO Expert Committee, the safe levels of protein intakes would be 0.8 9 and 1.0 9 protein/kg/day for the egg and mixed diets, respectively.

Table 6 summarizes the results of total serum protein, albumin, blood urea nitrogen (BUN), serum glutamic oxaloacetic transaminase (SGOT), and serum glutamic pyruvate transaminase (SGPT). Significant changes relative to initial values were found

FIG. 1. Regression Analyses of "True" Nitrogen Balance in total serum protein and albumin values with the mixed diet (2-way ANOVA). Blood urea nitrogen decreased with both diets.

Conclusions

1. The mean nitrogen requirements with egg and the mixed Chilean diets correspond to 97 and 125 mg N (or 0.61 and 0.78 9 proteins)/kg/day, respectively,

2. The current FAD/WHO safe level of egg protein intake (0.6 g/kg/day) was adequate for only three of eight men. The safe level of intake for our subjects is 0.8 to 1.1 9 egg protein/kg/day, depending on the approach used to estimate inter individual variability.

3. Faecal nitrogen did not vary at the three levels of protein intake, although it was

TABLE 6. Plasma Constituents for Subjects Consuming Two Levels of Egg Protein and a Chilean Mixed Diet ¹

	Initial	Egg (g/kg)		Mixed diet (g/kg)		Diet effect 2 - way ANOVA
						F	P
		0.30	0.60	0.40	0.70	(4.33)
Total protein, g/dl	7.5 ± 0.4	7.6 ± 0.4	7.6 + 0.3	6.9 ± 0.4	7.1 ± 0.5	4.7	<0.05
Albumin, g/dl	5.2 ± 0.3	5.3 ± 0.3	5.3 + 0.4	4.8 ± 0.4	4.7 ± 0.3	5.5	<0.05
Urea nitrogen, mg/dl	13.7 ± 1.7	7.0 ± 1.4	8.0 ± 1.6	6.1 ± 2.1	8.2 ± 2.8	17.5	<0.001
SGOT,Karmen units/dl	18.7±10.1	14.0±5.6	16.2+5.2	23.9 ±12.7	18.1+7.0	1.5	n.S.
SGPT, Karmen units/dl	17.1 ± 9.6	10.1±3.4	10.3±3.5	13.8±10.7	10.2±4.4	1.5	N.S.

¹ Mean ± S. D.

4. The anthropometric indices suggest that our subjects are leaner than the normal standard, and hence a higher energy intake on a body-weight basis can be expected. There was a negative correlation (r = 0.89) between the weight-height (W/H) index and the energy intake required to maintain stable body weight. From this regression, the estimated energy intake for a subject with a W/H index of 100 per cent is 44.7 kcal/kg, which is similar to the energy requirements of healthy, normal Caucasian subjects. Our subjects consumed a mean of 49.5 kcal/kg. The minor changes observed in weight, body circumferences, and skin-fold measurements suggest that our subjects were close to equibrium and that their high energy intakes are accounted for by their body composition and activity pattern.

5. Based on the regression equations with the egg diet, the mean obligatory nitrogen losses were 69 mg N/kg/day, which are similar to those estimated from available data corrected by the 30 per cent factor for decreased efficiency of utilization within the maintenance range.

6. BUN decreased with both diets, suggesting a change in the urea pool. With the mixed diet there was also a decrease in serum protein and albumin.

7. It is necessary to re-evaluate dietary protein recommendations. Long-term evaluations with 0.8 or 1.0 9 protein/kg/day, respectively, of egg or a mixed diet of mainly vegetable origin should be undertaken.

Acknowledgements

This study was supported by a Research Grant from the United Nations University World Hunger Programme. The authors gratefully acknowledge Laboratories Pfizer de Chile for kindly supplying the vitamin/mineral supplement (Polyterra) used in these experiments.

(introduction...)

Objectives
Summary of the main results
Conclusions

Luis F. Fajardo, Oscar Bolanos, Giovanni Acciarri, Fanny Victoria, Jaime Restrepo, Ana B. Ramz, and Luz M. Angel
Escuela de Medicina, Universidad del Valle, Cali, Colombia

Objectives

These studies were designed to determine the level of protein intake necessary to maintain nitrogen balance in a population living under the conditions of a developing tropical country. Two different local diets were examined.

Experimental Details

1. Subjects
For the first study, 11 healthy individuals consumed a predominantly animal protein diet (table IA). Eight healthy males took part in the second study of a predominantly vegetable diet (table IB).

2. Study Environment
Location: Universidad del Valle, Colombia. Climate: tropical.

3. Physical Activity
All subjects were students.

4. Diets
For the first study a menu largely made up of animal protein typical of the upper socio-economic class of Colombia was designed, as shown in table 2A. For the second study, the diet was patterned after that in the Cauca Valle region; 80 per cent of this diet is of vegetable origin (table 2B).

TABLE 1. Characteristics of the Subjects

	1A. Animal Protein Diet
Subject	Sex	Age (years)	Weight (kg)	Height (cm)
1	M	26	61	167
2	M	21	53	177
3	M	23	62.5	178
4	M	22	90.5	165
5	M	22	68	174
6	M	23	71.5	170
7	M	21	56	175
8	M	22	61	172
9	F	23	53	166
10	F	22	74	175
11	F	22	64	158
X		22.4	64.9	170
S.D.		1.37	10.9	0.061
1B. Vegetable Protein Diet
O.A.01	M	25	55.7	174
A.A.02	M	23	61.0	172
R.E.03	M	22	57.5	163
J.A.04	M	22	64.0	172
J.G.05	M	25	66.5	166
F.P.06	M	24	57.2	170
W.V. 07	M	23	57.7	174
J.D. 08	M	25	63.2	170
X		23.6	60.3	170.1
S. D.		1.3	3.8	3.89

TABLE 2A. Animal Protein Diet

Breakfast:	cafe au fait bread margarine marmalade juice
Mid-morning:	Guava candy, sweetened carbonated beverage
Lunch:	soup fried chicken fried cassava salad: carrots cabbage tomatoes lettuce onion mayonnaise banana with milk, cream black coffee, sweetened carbonated beverage
Afternoon:	baked plantain, sweetened carbonated beverage
Supper:	fried meat baked potato salad fruit brown sugar water

Take-home snack: Guava candy and sweetened carbonated beverage

5. Experimental Design
For the animal protein study there were four periods; for the vegetable protein study there were seven. Each period consisted of a one-day protein-free diet, a five-day adaptation period, a five-day collection period, and a three-day break.

TABLE 2B. Predominantly Vegetable Protein Diet

Breakfast:	orange juice with sugar
	fried plantain and margarine
Mid-morning:	guava candy and sweetened carbonated beverage
Lunch:	sancocho:
	typical soup made
	of plantain, cassava,
	potatoes, meat, oil,
	tomatoes, onion,
	and parsley
	white rice
	beans and potatoes
Afternoon:	guava candy and sweetened carbonated beverage
Supper:	bean soup
	beans and potatoes
	peaches
	sweetened carbonated beverage

Take-home snack: guava candy and sweetened carbonated beverage

6. Nitrogen Balance
The nitrogen content of the diet, urine, and faeces was measured by the micro Kjeldahl technique. Miscellaneous losses were estimated at 5 mg/kg.

Summary of the main results

1. Animal Protein Data
Table 3 shows the nitrogen-balance data. Four of the 11 subjects were classified as behaving atypically and are listed separately in table 3A.

TABLE 3. Nitrogen Balance in Subjects Consuming Protein Mainly of Animal Origin

Subject	Nl	UNE	FNE	TNB	BV	D
Level #1
1	82	85	18	- 26	36	92
3	80	79	20	-24	41.6	90
6	88	70	18	- 5	59.7	93
7	93	91	23	-27	34	88
8	92	76	17	- 6	55	94
9	85	68	26	- 14	56	83
11	80	82	14	-16	42	97.5
X	85	78.7	19.4		46.3	91
S	5.44	8.16	4.0		10.3	4.6
Level #2
1	103	98	21	-15	41	91
2	108	91	21	- 9	45	91
3	104	86	21	- 8	48	91
6	105	105	18	-23	31	94
7	106	88	16	- 3	50	96
8	97	101	20	-29	28	91
9	105	71	22	+ 7	64	90
11	98	74	15	-19	48	96
X	103.2	91	19.25		43	92
S	3.8	10.2	2.60		11	2.4
Level #3
3	120	109	20	-14	35	93
3	122	79	21	+16	62	92
6	114	107	10	- 8	39	101
7	114	92	18	- 1	49	94
8	116	86	20	+ 5	54	93
9	126	89	16	+17	57	96
11	1 22	96	13	+ 7	51	99
X	119	94	16.8		49.5	95.4
S	4.6	11	4.1		9.6	34
Level #4
6	135	99	23	+ 8	50	91
7	138	106	28	- 2	43	88
8	136	106	16	+ 9	47	97
9	132	93	18	+11	51	95
11	136	72	11	+48	74	100
X	135	96.2	19.2		53	94.2
S	2.19	14.0	6.53		12.4	4.7

Key:

Nl = nitrogen intake
UNE = urinary nitrogen excretion
FNE = faecal nitrogen excretion
TNB = total nitrogen excretion (assuming 5 mg/kg miscellaneous losses)
BV = biological value
D = digestibility

2. Results

1. Liver function was evaluated by measuring serum transaminase levels (SOOT) at the end of each period; although it was evident that there was a slight increase in the mean level of SGOT with increased protein intake, the mean value of SGOT between groups was not significantly different (ANOVA: p < 0.05).
2. The regression equation relating the nitrogen intake (Nl) with the urinary nitrogen excretion (UNE) was as follows (values in g/day): UNE = 2.78 + 0.308 Nl; thus, the UNE for 0 intake of protein would be 2.78 9. Assuming a mean weight of 60.7 kg, this corresponds to an obligatory urinary nitrogen excretion of 45.7 mg/kg, which is 7.7 mg/kg higher than that reported by Scrimshaw and others.
3. There are significant differences in total faecal nitrogen excretion with increasing levels of protein intake (p < 0.001). The regression equation relating faecal nitrogen excretion (FNE) with nitrogen intake (Nl) was: FNE = 0.77 + 0.21 (Nl) (values in g/day); thus, the obligatory nitrogen faecal excretion for zero protein intake was estimated to be 12.4 mg/kg, higher than the figure reported by Young et al. (V.R. Young, M.A. Hussein, and N.S. Scrimshaw, Nature, 218: 568 [1968] ), and just above the 12 mg/kg accepted by the FAD/WHO Committee.
4. Table 4 shows the data on nitrogen intake, urinary nitrogen excretion, faecal nitrogen excretion, and nitrogen balance expressed in mg/kg/day. The nitrogen balance increases significantly with an increase in nitrogen intake. The regression equation of the nitrogen intake (Nl) expressed in mg/kg/day on the true nitrogen balance (NB) was: NB = -65.84 + 0.50 (Nl).

TABLE 3A. Atypical Subjects

Values in mg/kg/day
Nl	UNE	FNE	TNB	BV	D
Subject # 2
93	115	30	-57	- 4	80
99	122	32	- 60	- 7	80
121	122	27	-34	+19	87
136	115	26	- 10	+36	89
Subject #4
85	85	12	- 18	+43	100
101	108	21	-33	+22	91
112	107	20	-21	+32.6	92.8
11 5	108	24	- 22	+32	89
Subject # 5
79	92	16	-34	+26	94
96	111	24	-44	+12	87
122	110	30	- 23	+29	85
138	120	20	-10	+35	93
Subject #10
87	78	23	-20	+46	87
94	113	12	-36	+19	100
106	101	27	-27	+30	86
137	114	18	0	+41	96

A close look at the individual data values shows that subject 01 never attained a positive nitrogen balance, and the slope of the regression level relating nitrogen intake with nitrogen balance was 0.35. Although the data for this individual still met the criteria outlined by the WHO-UNU guideline, they were not typical, and it is perhaps not wise to include the data for this subject in the calculations. He had a history of renal stones, and during the study he had a severe attack of renal lithiasis.

TABLE 4. Nitrogen Data for Subjects Consuming the Predominantly Vegetable Protein Diet 1

Subject	Level I (0.45)*	Level II (0.55)	Level III (0.67)	Level IV (0.77)	Level V (0.86)	Level VI (1)
Nitrogen intake
2	68.21	96.21	118.7	120.7	131.5	155.36
3	76.53	89.96	109.8	128.9	137.8	152.0
4	72.11	96.57	106.9	120.1	138.7	161.2
5	69.56	89.98	103.9	127.3	140.6	164.1
6	-	-	107.9	129.9	145.4	163.9
7	66.92	88.64	114.3	135.7	148.9	164.9
8	-	83.48	102.3	114.3	-	150.4
X	70.66	90.80	109.1	125.27	140.48	158.8
S.D.	3.79	4.94	5.76	7.25	6.1	6.1
Urinary nitrogen excretion
2	69.2	84.4	88.45	91.71	77.60	99.37
3	76.53	83.7	85.53	95.80	93.10	87.25
4	72.11	67.9	75.23	79.61	78.90	85.10
5	59.6	70.4	81.53	87.17	86.80	109.50
6	-	-	77.51	86.83	76.72	95.14
7	75.10	73.5	89.67	80.11	84.13	96.20
8	-	58.7	65.29	84.85	-	92.21
X	70.308	73.1	80.458	86.58	82.87	94.82
S.D.	6.78	9.81	8.57	5.84	6.37	8.19
Faecal nitrogen excretion
2	14.27	22.2	27.0	39.18	47.00	37.4
3	27.30	33.3	26.5	23.0	41.30	44.2
4	37.22	38.3	48.19	46.30	52.37	38.95
5	30.49	36.3	35.84	21.72	45.55	48.09
6	-	-	22.7	30.21	53.71	32.00
7	25.32	29.32	35.9	34.10	42.57	43.17
8	-	33.93	42.49	39.49	-	54.57
X	28.92	32.22	34.08	33.428	47.08	42.62
S.D.	5.20	5.77	9.24	9.0	5.0	7.40
Nitrogen balance
2	-14.58	-10.38	+ 3.25	-19.19	+ 6.92	+18.52
3	-11.44	-27.12	- 2.2	+10.19	+ 3.39	+20.53
4	-37.22	- 9.76	-16.51	- 5.81	+ 7.41	+37.07
5	-20.53	-16.95	-13.97	+18.41	+ 8.33	+ 6.57
6	-	-	+ 7.66	+12.25	+15.02	+36.78
7	-33.5	-14.05	-11.25	+21.53	+22.49	+25.54
8	-	- 9.16	- 5.5	-10.04	-	+ 4.68
X	-23.45	-14.57	- 5.43	+ 5.19	+10.56	+21.39
S.D.	(±11.41)	(± 6.83)	(± 8.93)	(±13.5)	(± 6.88)	(±12.95)
ANOVA		SS	DF	F	P
Protein intake		8812.4	5	13.3426	0.0000
		12152.3

1 Excluding four ''atypical'' subjects.
* All values expressed in mg/kg/day.

The impression of the research team was that throughout the study the subject suffered from a chronic renal infection. This observation and other values considered by the team as errors in collection were deleted. The regression line relating the nitrogen balance (NB) with the nitrogen intake (Nl) after deleting the questionable data was found to be: NB = - 66.7 - 0.52 (Nl).

Conclusions

1. Mean requirement for animal protein was 114 mg N/kg; for vegetable protein it was 128 mg N/kg. (These correspond to 0.71 and 0.80 9 protein, respectively.)

2. With the vegetable protein diet, faecal nitrogen excretion increased as nitrogen intake increased.

(introduction...)

Objectives
Experimental details
Summary of main results

Mixed dietary protein and egg protein at usual levels of energy intake

P.C. Huang and C.P. Lin
Department of Biochemistry, College of Medicine, Taiwan University, Taipei, Taiwan

Objectives

1. To determine the protein requirements of young Chinese male adults eating typical local diets supplying the amount of energy needed for their usual daily lives.

2. To determine the requirements of young Chinese men for egg protein when they are consuming customary amounts of energy.

Experimental details

1. Subjects
Twenty-eight students 20 to 29 years old in the College of Medicine and in a junior college volunteered to participate. Twenty ate a mixed Chinese diet at one, three, or four different levels of protein intake. Thirteen of them also participated in the egg study at one, two, or three different protein levels. Eight other men participated only in the egg protein studies. All subjects remained essentially healthy throughout the experiment. Their characteristics are shown in table 1.

2. Study Environment
Subjects lived in the metabolic ward located on the College of Medicine campus throughout the experiment. Room temperature and relative humidity were 9.0 to 37.2° C and 70 to 90 per cent, respectively.

TABLE 1. Characteristics of 28 Young Chinese Men Participating in the Studies

Items	Mean1	S.D.1	Minimum	Maximum
Age (years)	24.2	2.57	20	29
Body weight (kg): initial	59.4	6.89	46.2	70.2
final	58.8	6.92	45.0	69.7
Height (cm)	168.2	4.95	160.0	178.5
Height/weight	2.89	0.31	2.46	3.46
Urinary creatinine (g/day)	1.242	0.2022	0.89	1.70
Skin-fold (mm): triceps	9.6	3.56	4.0	16.0
subscapular	12.8	3.15	8.5	19.5
Calculated energy intake (kcal/kg/day)	42	1.94	38	46

1 Values given are mean and standard deviation for 28 subjects.
2 Mean and standard deviation for 1,155 determinations.

TABLE 2. Ingredients and Nutrient Composition of the Chinese Mixed Diet, Planned According to the Food Balance Sheet (1976)1

Ingredients (gm)	Levels of protein intake (g/kg body wt/day)
	0.45	0.55	0.65	0.75
Rice	110	142	161	180
Cornstarch	240	225	210	196
Sugar	8	8	8	7
Soybean oil	46	40	34	31
Butter	16	15	16	16
Potassium phosphate	2.3	2.3	2.4	2.4
Calcium phosphate	2.3	2.3	2.4	2.4
Cellulose	4.5	4.5	4.8	4.8
Sodium chloride	10	10	10	10
Mung bean noodles	70	70	70	70
Wheat flour	20	24	29	33
Sweet potato	9	12	14	16
Soybean curd	56	68	81	92
Peanuts	3	4	5	5
Kale	11	17	23	25
Chinese cabbage	54	80	100	100
Carrots	30	40	53	56
Cabbage, dried	10	9.5	10	13
Bananas	7	8	10	11
Watermelon	40	50	50	50
Pork	19	23	24	37
Chicken	6	7	9	13
Egg	5	6	7	8
Fish	15	23	25	37
Whole milk	1	1.2	1.4	1.6
Skim milk	1	1.2	1.4	1.6
Vitamin and mineral supplements2
Nutrients: Protein (gm)	25.6	31.3	37.1	42.4
Fat (gm)	96.1	88.7	96.9	94.5
Carbohydrate (gm)	388.2	399.1	372.6	370.4
Calculated calories (kcal)	2,520	2,520	2,511	2,502

1 The amount is for a 60 kg subject.
2 Vitamins and minerals were supplemented each day to meet the National Research Council re commended allowances, using a preparation from the China Chemical and Pharmaceutical Co.

3. Physical Activity
All subjects maintained their usual school activities without unusual physical exercise.

4. Duration of the Study
On the first day of an experimental period, the subjects were given 0.1 9 of egg protein/kg followed by an experimental diet for ten days. Between the consecutive nitrogenbalance studies, the men ate their ordinary diets with more than 1.5 9 protein/kg/day for three or four days. The protein intakes with the Chinese test diets were fed in an order of 0.65, 0.45, 0.75, and 0.55 g/kg in the first and third series, and in reverse order (0.55, 0.75, 0.45, and 0.65 g/kg) in the second series. With the egg formulae, the order in which the protein levels were fed was 0.45, 0.35, and 0.55 g/kg in the first and third series; the order was reversed in the second series. Skin nitrogen losses were determined for two days during each balance period.

5. Diets
Food ingredients of the ordinary Chinese mixed diet were selected according to the Taiwan Food Balance Sheet of 1976. A part of the mixed diet was served as a liquid formula prepared by blending a mixture of milk, egg, sweet potato, methyl-cellulose, salt, soy bean oil, butter, and cornstarch with water in a proportion of 1:2 and steamed at about 95 C for at least 30 minutes. Details of the dietary ingredients and the nutrient composition are shown in tables 2 and 3. The test diet was provided in four meals a day, at 0730,1200, 1730, and 2200 hours.

6. Indicators and Measurements
Regression analyses of nitrogen balance on nitrogen intakes were performed to obtain the mean protein requirements. The 97.5 per cent confidence limits were calculated using the pooled data regressions. The nitrogen content of all specimens and diets was determined by a semi-micro-Kjeldahl method. Biological value (BV), net protein utilization (NPU), and apparent and true digestibilities were calculated. The obligatory urinary and faecal nitrogen losses used for the calculations were those from our previous study: 33.4 and 13.1 mg N/kg, respectively.

Summary of main results

Table 4 and figures 1 and 2 show the nitrogen-balance data. All men were in negative nitrogen balance at an intake of 0.45 and 0.55 9 protein/kg in the mixed diets, and also at the 0.35 g level of egg protein/kg. At higher protein intakes, some subjects achieved positive nitrogen balance (figures 1 and 2).

The mean protein requirements for the mixed Chinese diet and the egg diet were 0.79 and 0.61 g/kg/day, respectively. The 97.5 per cent confidence limits for the requirements were calculated as 1.18 and 0.89 g/kg/day, respectively. The efficiency of utilization of the Chinese mixed dietary protein was 77 per cent that of the egg protein, based on relative nitrogen requirements.

TABLE 3. Composition of the Experimental Egg Diet

Ingredients (gm)	Levels of protein intake (g/kg body wt/day)
	0.35	0.45	0.55
Whole egg	158	196	246
Cornstarch	296	270	260
Sugar	20	20	20
Soybean oil	41	36	30
Butter	30	30	30
Potassium phosphate	3	3	3
Calcium phosphate	3	3	3
Methyl-cellulose	6	6	6
Sodium chloride	10	10	10
Watermel on	200	200	200
Chinese cabbage	100	100	100
Mung bean noodles	200	200	200
Vitamin and mineral supplements2
Nutrients: Protein (gm)	21	27	33
Fat (gm)	81.2	84	78.4
Carbohydrate (gm)	444	423	420.6
Calculated calories (kcal)	2,591	2,556	2,520

1 See footnote 1 in table 2.
2 See footnote 2 in table 2.

The actual energy intakes of the subjects in the two series of studies ranged from 38 to 46 kcal/kg. Most of the subjects spent a large part of their time in academic studies and their energy expenditure was light to moderate. When a body-weight increase of more than 0.2 kg or a decrease of more than 0.7 kg occurred, an adjustment in energy intake was made by subtraction or addition of soybean oil. Changes in body weight over the 56-day period ranged from -2.5 to +0.6 kg.

Conclusions and Comments

1. The results obtained suggest that the 1973 FAD/WHO recommendation for young men of 0.57 9 egg protein/kg/day is inadequate. The recommended dietary allowance of protein estimated in 1972 for adults in Taiwan was 1.1 g/kg/day. That figure coincides with the present results using mixed Chinese diets.
2. The NPU of a similar mixed Chinese diet was found to be 62 when weanling rats were used as the experimental animal. In contrast, the present study with young Chinese men showed the NPU of the mixed Chinese dietary protein to be only 43. The NPU of egg protein was 55 to 58 in the present study as compared with a value of 70 obtained in our earlier studies with infants.
3. The mean nitrogen requirements of young Chinese men, based on regression analysis of the pooled data, are 127 mg (0.79 9) protein/kg/day with the mixed Chinese diet and 98 mg (0.61 9) protein/kg/day with the egg diet.

 

TABLE 4. Daily Nitrogen Balance Data1 with Chinese Mixed Diets and Egg Diets

Nitrogen intake	Urinary nitrogen	Faecal nitrogen	Skin nitrogen	Total nitrogen loss	Nitrogen balance
(mg/kg body weight/day)
Chinese mixed diets
72.4±0.79	70.9±9.641	15.2+2.26	7.2+2.97	93.3±10.56	-20.9+10.30
87.9±1.03	77.9±10.47	16.9+2.20	8.7±7.37	103.6±11.81	-15.8+11.10
103.3±0.53	91.3±10.35	17.0+3.79	7.5+2.88	116.4±10.08	-12.5±10.14
121.0±0.87	99.0± 9.12	16.7±325	5.5±1.46	121.2± 8.16	- 0.2± 7.97
Egg diets
56.3±0.74	54.8+ 8.41	13.9±2.35	8.4±3.10	77.1± 9.50	-20.8± 9.11
71.7±0.78	63.9± 9.76	13.1+2.39	10.5±4.28	87.4± 8.56	-15.7± 8.95
89.0±1.37	71.5± 7.53	14.5+2.38	6.8+2.44	92.7± 8.41	- 3.7± 7.94

¹ Mean ± S.D. of 15 subjects.

FIG.1 Daily Nitrogen Balance with Chinese Mixed Diet

FIG. 2. Daily Nitrogen Balance with Egg Diet

TABLE 5. Calculated Biological Value (BV), Net Protein Utilization (NPU), and True and Apparent Digestibility of the Test Dietary Proteins at Different Levels of Intake in Young Men

Level of protein intake	BV1	NPU1	Digestibility
(g/kg/day)			Apparent	True
Mixed diet
0.45	47±13a,A	45±13a,A	7g±3c	97±3
0.55	47±12a,A	45±11a,A	81±3d,e	96±3
0.65	42±10a,A	40±10a,A	84±4c,d	96±3
Egg diet
0.35	sg±17b	58±18b	75±4f	98±4
0.45	57±11b	56±11b	82±3	98±2
0.55	56±10b	55±10b	84±3f	98±2

¹ Mean +S.D. (n = 15).
a,b Same letters within a column are not significantly different.
A F ratio obtained from ANOVA test is significantly lower (P<0.01) than those in egg diet series.
c-f Group means within a column followed by the same letter are significantly different: c,e, p<0,01; d,f, p<0,05.

(introduction...)

Objective
Experimental details
Summary of the main results
Conclusions

Hector Bourges R. and Blanca Rosa Lopez-Castro
Division of Nutrition, National Institute of Nutrition, Tlalpan, Mexico D.F., Mexico

Objective

To determine the amount of protein needed by normal young male adults fed a rural Mexican diet.

Experimental details

1. Subjects
Eight healthy young males participated in the study in which the multiple-level nitrogenbalance technique was used while they consumed a "rural" diet. In order to have reference data, three of these subjects (A.D.F., S.R., U.R.) participated twice, first on the rural diet and later on a milk diet.

All the subjects were born in and are still living in the Malinalco State of Mexico. Their town is 120 km from Mexico City and 1,700 m above sea level, and its main economic activity is agriculture. Table 1 shows the characteristics of the subjects.

2. Physical Activity
During the study period the subjects were sedentary throughout the day with a halfhour period of exercise on a stationary bicycle, except for A.D.F. who, during both the rural and milk diet studies, exercised for 45 minutes per day.

TABLE 1. Characteristics of the Eight Subjects

Age (x ± S.D.)	21.5 ± 2.92
Sex	male
Racial origin	mixed Indian-Spanish
Physiological status	young adult
Nutritional status
(ideal weight ± S.D.)	95.15 ± 6.26
Health status	normal

3. Duration of the Study
For each subject there was a stabilization period of approximately 15 days, in which the energy intake was adapted to individual requirements as judged by body-weight changes.

After the stabilization period, four different nitrogen balance studies were conducted on each subject. Each test took the following sequence:

1. One day on a nitrogen-free diet.
2. A 6-day period of adaptation to the test level of protein intake (no collections).
3. A 4 day period for nitrogen balance (collection).
4. A 3 day rest period (same diet but protein intake at 1 g/kg body weight). For the whole study each subject remained in the metabolic unit for approximately 71 days.

4. Diet
Rural Diet: A standard diet was given, consisting of corn, beans, and wheat pasta supplying, respectively, 52, 31.5, and 63 per cent of the total protein at each level.

The typical menu consisted of boiled beans, corn tortillas, pasta soup, fruit, vegetables, and lemonade (fruits and vegetables provided 10.2 per cent of the total protein). The basis for the design of this diet was the information obtained from dietetic surveys in the rural areas of the Mexican plateau. Levels of 0.4, 0.5, 0.6, and 0.7 9 of protein/kg were administered to the subjects.

Milk diet: The milk diet consisted of whole milk, cheddar cheese, and cream supplying 25, 70, and 5 per cent of milk protein, respectively. Fruit, vegetables, cornstarch, corn oil, sugar, candies, and protein free desserts (jelly, preserved peaches, and jam) were added. Fruits and vegetables provided a maximum of 16 per cent of the total protein in the diet.

TABLE 2. Amount and Source of Dietary Energy

A. Rural Mexican Diet: 41.36 2.79 kcal/kg*
Protein intake level (g/kg body weight)
% of calories	0.4	0.5	0.6	0.7
Protein	3.87	4.86	5.71	6.73
	±0.24	±0.28	±0.37	±0.41
Fat	27.20	29.38	31.34	26.03
	± 8.82	± 6.52	± 7 07	± 7.21
Cabohydrates	68.92	66.13	62.78	66.97
	± 8.94	± 6.84	± 7.30	± 7.12
B. Milk Diet: 42.03 ± 2.91 kcal/kg*
Protein intake level (g/kg body weight)
% of calories	0.3	0.4	0.5	0.6
Protein	2.93	2.83	4.78	5.92
	±0.17	±0.28	±0.28	±0.17
Fat	25.69	31.66	32.35	31.49
	± 2.92	± 0.62	± 3.07	± 6.10
Carbohydrates	71.40	64.50	62.86	62.57
	± 2.88	± 10.54	± 3.19	±6.27

* Mean ± S.D.

The menu essentially consisted of milk with coffee, sweet cornstarch, vegetable soup, cooked vegetables with cheese and cream, vegetable salad, fruit, and jelly. Each subject ate this diet at a level of 0.3, 0.4, 0.5, and 0.6 9 protein/kg.

Fruits, vegetables, and the non-protein ingredients were used to dilute both diets in order to obtain the different protein levels. Each daily ration per subject was prepared individually, carefully weighing the ingredients and using individual containers. The beans and tortillas were prepared in the typical way and the soup was made with pasta, onion, tomato, and chicken broth. Energy values of the diet are given in table 2.

Liquid intake was maintained constant throughout the study, and two capsules of Unicap T were given daily in order to meet vitamin and mineral requirements. A dietitian supervised all the procedures.

5. Measurements
The composition of the diets was determined by analysing the nitrogen content of the main components individually (corn, beans, and wheat pasta) plus a pool of the fruits and vegetables, using the macro-Kjeldahl method. The energy content of the diet was calculated with the aid of tables.

Nitrogen balance: The urinary nitrogen excretion for each intake level corresponds to the average urinary nitrogen excretion for the last four days of each balance period. The faecal nitrogen was determined, by the macro-Kjeldahl method, in each of the corresponding pools for the last four days of the faecal collection in each balance period.

Integumental nitrogen losses were taken as 5 mg N/kg (Calloway et al., J. Nutr., 101: 775 [1971] ). Since environmental temperature in the metabolic unit was around 20° C, no appreciable sweating occurred.

Summary of the main results

Table 3 shows the nitrogen-balance data for all subjects.

TABLE 3. Mean Ingested, Urinary, and Faecal Nitrogen and Nitrogen Balance for Each Subject and Level with the Rural (R) and Milk (M) Diets (mg N/kg BOOT)

Subject	Ingested	Urinary	Faecal	Balance
A.D.F. (R)	65.21	48.61	32.03	- 20.44
	81.78	59.88	34.50	- 1 7.62
	113,00	83.53	32.72	- 8.24
A.C. (R)	64.61	56.30	32.49	- 28.18
	81.34	78.32	25.22	- 27.18
	96.82	88.60	25.21	- 21.99
	112.63	88.90	28.30	- 9.58
J.S. (R)	64.21	74.73	23.51	- 39.04
	80,85	84.11	23.66	- 31.92
	113.69	87.44	13.82	- 7.54
I.G. (R)	64.87	70.42	21.90	- 32.45
	82.11	70.66	30.71	- 24.25
	96.87	75.81	25.25	- 9.19
	112.63	67.94	20.16	+19.52
A.R. (R)	65.49	68,49	30.72	- 38.72
	81.06	84.09	16.67	- 24.68
	97.40	79.28	1 7.09	- 3,97
	113.08	80.15	22.07	+ 5.86
S.R. (R)	64.55	54.76	34.41	- 29.62
	81.64	64.70	27.57	- 15.64
	98.05	71.71	28.26	- 6.91
	112.79	66.74	32.10	+ 8.97
U.R. (R)	65.12	66.59	28.49	- 34.96
	81.29	62.11	34.75	- 20.56
	96.27	62.78	33.25	- 4.75
	112.61	72.32	26.53	+ 8.76
E.R. (R)	65.40	63.50	22.71	- 25.80
	81.21	65.10	29.79	- 18.69
	97.43	72,84	31.18	- 11.59
	113.74	83.03	29.68	- 3.96
S.R. (M)	48.43	54.46	15.38	- 26.07
	63.01	55.99	23.35	- 21.55
	79.17	61.57	23.87	- 11.26
	93.91	68.85	24.98	- 4.92
A.O.F. (M)	48.42	54.73	18,51	- 23.83
	63.57	53.27	1 7.82	- 1 2.51
	79.63	74.47	12.18	- 10.23
U.R. (M)	48.38	53.49	14.69	- 24.78
	62,89	53.43	22.02	- 1 7.25
	78.28	67.22	21.69	- 15.23
	95.86	76.72	14.87	- 0.78

Conclusions

1. Mean protein requirements for milk and rural diets were very close: 103 and 112 mg N/kg body weight, respectively, equivalent to 0.66 and 0.70 9 of protein/kg body weight. Even the mean requirement obtained for the milk diet (0.66 g/kg) was notably above the recommendation of FAD/WHO 1973. The safe levels of intake for 97.5 per cent of the population (PR_0.975) were, respectively, 121.6 and 136.4 mg N/kg body weight.
2. Since the chemical score for the rural and milk diets was 71 per cent (limiting amino acid: Iysine) and 94 per cent (limiting in sulphur amino acids), respectively, theoretically the protein requirement mean (PRm) for the rural diet should have been about 136.5 mg N/kg, and PRo 975 should have been 161 mg N/kg. The values found experimentally were much lower, suggesting that the lysine value in the 1973 FAO/ WHO provisional pattern may be too high
3. Neither the different clinical laboratory parameters measured nor the indices of body composition obtained from somatometric measurements showed any changes or abnormalities.
4. Subject A.D.F. exhibited a distinct increase in protein requirement when on the rural diet. Although a native of Malinalco, he had moved to Mexico City for several months, where be became accustomed to a daily intake of meat, eggs, and milk. Considering his dietary pattern, it would seem as though he had "lost" his ability to utilize the protein in a rural diet.

(introduction...)

Objective
Experimental details
Summary of main results
Conclusions

G. Inoue, T. Takahashi,* K. Kishi, T. Komatsu, and Y. Nilyama
Department of Nutrition, School of Medicine, Tokushima University, Tokushima, Japan, and *Institute of Health and Sport Science, Tsukuba University, Ibaraki-ken, Japan

Objective

The nutritive values of soy protein isolate (Supro 620, Ralston-Purina Co., St. Louis, Mo, USA) alone and mixed with fish were compared with that of fish protein.

Experimental details

1. Subjects
Twenty-one male university students served as the subjects in these three series of experiments. They lived in a metabolic ward of our laboratory throughout the experiment. During the study, they continued their daily routine activities but were not allowed to do any hard physical work. Characteristics of the students are shown in table 1.

2. Diets
Each subject was given, successively, four levels of low-protein diets. Protein sources were cod fish (as a standard) for eight subjects, a soybean protein isolate (Supro) for five subjects, and a 50:50 mixture of both for eight subjects. Among these, five men received both the cod and mixed protein diets (see table 1). The fish and Supro were prepared as paste products (kamaboko) and fed in equal amounts three times a day. An example of the diet composition is shown in tables 2 and 3.

Each experimental period consisted of one day on a protein-free diet and ten days

TABLE 1. Characteristics of the Subjects

	Subject	Age (years)	Ht. (cm)	Wt (kg)	Chest circumference (cm)	Subscapular skin-fold (mm)	BMR (kcal/kg)	Blood pressure (mmHg)
Fish	A	21	171	63.3	86.1	13.5	22.7	125/75
	B	20	164	56.5	84.6	7.5	25.6	135/82
	C	19	171	58.1	84.8	8.2	27.4	145/90
	D	24	171	69.8	97.6	15.2	21.2	120/75
	E	22	179	64.3	90.0	9.5	22.4	115/75
	F	22	172	63.2	90.8	11.8	22.5	130/85
	G	20	168	57.6	83.4	11.0	24.5	120/70
	K	22	166	71.6	91.8	18.8	25.6	125/84
	Mean	21	170	63.1	88.6	11.9	24.0	127 80
	S.D.	2	5	5.6	4.8	3.8	2.1	10 7
Supro	M	21	173	68.2	92.0	7.0
	N	21	161	53.9	87.4	8.0
	O	28	164	54.6	89.8	7.0
	P	22	164	74.0	92.7	12.0
	Q	26	174	77.5	99.8	14.0
	Mean	24	167	65.6	92.3	9.6
	S.D.	3	6	10.9	4.7	3.2
Mixed	A	21	171	63.4	84.9	15.3	21.7	105/70
	B	21	164	59.2	86.7	7.5	21.9	133/92
	C	20	170	59.4	85.1	7.8	24.2	148/93
	D	24	171	68.3	98.3	15.0	24.8	118/75
	E	22	178	62.3	88.5	8.5	25.9	122/83
	H	25	170	60.3	87.8	8.5	23.2	95/58
	I	21	166	52.0	83.4	6.0	27.3	108/72
	J	21	168	61.0	85.7	10.5	25.9	117/83
	Mean	22	170	60.7	87.6	9.9	24.4	118 77
	S.D.	2	4	4.6	4.6	3.5	2.0	17 12

 

TABLE 2. Diet Composition (Example). Subject: 65 kg; mixed protein 0.55 g/kg; energy 45 kcal/kg.

Materials	g/day
Cod kamaboko *	155
Supro kamaboko*	107.5
Sugar	323
Cornstarch	300
Margarine	32
Corn oil	30
Agar	3
Baking powder	5
Salt	2.5
Mineral mixture	6
Vitamin mixture	3

* For composition of the kamaboko, see table 3. The nitrogen contents are as follows: Dried cod 89.6 mg/g; dried Supro 147.2 mg/g; cod kamabako 18.45 mg/g; Supro kamaboko 26.6 mg/g. Nitrogen intake was 2.86 g/day equally in cod and Supro.

TABLE 3. Composition of Kamaboko Products

	Cod		Supro
	Parts	%	Parts	%
Cod paste	100	77.2	-	-
Supro 620	-	-	20	17.5
NaCI	3	2.3	3	2.6
Potato starch	5	3 9	5	4 4
Sugar	1.5	1.2	1.5	1.3
Water	20	15.4	85	74.2

Total 129.5 100.0 114.5 100.0 on the experimental diet, followed by three days on a free choice (ad libitum) diet. Four periods were included in the study, with 0.35, 0.45, 0.55, and 0.65 9 protein/ kg/day fed to each man in a random order. Energy intake was constant for each individual to maintain body weight (mean + S.D. = 44.6 + 2.4 kcal/kg; range = 38.4 to 49.8).

3. Measurements and Indicators

a. Anthropometry: body weight, height, arm and leg circumferences, skin-fold thickness.
b. Blood analyses: RBC and WBC count, haemoglobin, haematocrit.
c. Plasma analyses: total proteins (Biuret), albumin (dye-binding, HABCA), urea (indophenol colour reaction), glucose (Somogyi-Nelson), triglyceride (Van Handel), total cholesterol (Zak-Hanly), Na and K (atomic absorption), SGOT and SGPT (Reitman-Frankel). d. Urine analyses: urea (urease-indophenol), ammonia (phenol-hypochlorite), creatinine (Folin-Wu), uric acid (phosphotungstic acid), Na and K (atomic absorption).
e. Nitrogen balance: total nitrogen in diets, faeces, and urine was measured with a semi-micro-Kjeldahl technique during the last five days of each ten-day period on the experimental diets.

Summary of main results

1. Nitrogen Balance
See tables 4, 5, and 6 for individual data. Table 7 shows the individual and pooled regression equations of apparent nitrogen balance (i.e., without allowances for integumental and other insensible nitrogen losses) and nitrogen intake.

Figure 1 shows the individual variability and the upper 95 per cent confidence band for the pooled data of the fish-Supro mixture.

2. Relative Protein Quality of the Soybean Isolate
Based on the regression coefficients ("slope ratio"), nitrogen requirements for nitrogen balance ("maintenance intake"), and net protein utilization (NPU) shown in table 7, the quality of Supro 620 relative to cod fish was 82, 73, and 74 per cent, respectively (mean: 76 per cent). The fish-Supro mixture gave values of 115, 96, and 96 per cent, respectively, compared with fish alone.

3. Other Measurements
Anthropometric, blood, and urine indicators were not significantly affected by changes in dietary protein source.

TABLE 4. Summary of Nitrogen Balance Data for Young Men Given Different Levels of Fish Protein ¹

Protein level	Subject	Body wt. (kg)	Energy intake (kcal/kg)	Nitrogen balance (mg/kg)				Digestibility4 (%)
				Intake nitrogen	Urinary nitrogen	Faecal nitrogen	Nitrogen balance
0.35 g/kg/day	A	64.0	44 3	55.2	48.6	10.2	- 8.6
	B	57.8	46.2	56.2	67.0	11.4	- 27.2
	C	58.0	42.0	56.0	54.1	12.1	- 15.2
	D5	_	_	_	_	_	_
	E	64.1	44.9	55.9	51.5	12.5	- 13.1
	F	63.7	47.5	55.4	62.2	14.9	- 26.7
	G	57.3	45.5	56.7	56.2	11.9	- 16.4
	K6	70.8	47.1	56.2	58,9	8.1	- 15.8
	Mean	60.8	45.1	55.9	56.6	12.2	- 17.9	100.0
	S. D.	3 4	1.9	0.5	6.9	1.6	7.5
0,45 g/kg/day	A	63.7	45.5	71.3	61.2	10.5	- 5.4
	B	57.1	44.1	70.6	64.1	11.2	- 9.7
	C	57.8	42.1	72.3	64.9	13.7	- 11.3
	D	71.1	38.8	69.9	60.2	9.7	- 5.0
	E	65.1	44.2	70.8	61.9	12.1	- 8.2
	F	63.0	47.0	72.1	74.0	13.3	- 20.2
	G	57.9	45.1	72.2	64.8	11.2	- 8.8
	K6	71.3	47.8	71.8	86.6	8.8	- 28.6
	Mean	62.2	43.8	71.3	64.4	11,7	- 9.8	100.0
	S.D.	5.1	2.7	0.9	4.6	1.5	5.1
0,55 g/kg/day	A	63.5	44.6	87.4	64.6	11.6	+ 6.2
	B	57.5	46.6	87.3	72.2	11.1	- 1.0
	C5	-	-	-	-	-	-
	D	70.3	39.3	86.3	73.5	8,7	- 0.9
	E	64.7	44.5	87.0	74.5	16.8	- 9.3
	F	63.2	46.9	87.7	78.2	15.2	- 10.7
	G	58.7	44.5	86.9	70.9	13.3	- 2.3
	K6	70.7	48.2	88.4	109,1	9.1	- 34.8
	Mean	63.0	44.4	87.1	72.3	12.9	- 3.0	99.4
	S.D.	4.6	2.7	0.5	4.5	2.9	6.2
0.65 g/kg/day	A	64.5	43.7	103.1	80.6	12,9	+ 4.6
	B	57.9	45.3	102.4	92.7	1 2.4	- 7.7
	C	58.6	41.6	102.9	84.8	13.0	+ 0.1
	D	70.1	39.4	102.4	77.3	10.1	+10.0
	E	64.2	44.9	103.7	90.2	12.8	- 4.3
	F	63.6	44.6	103.0	89.5	14.9	- 6.4
	G	58.4	44.9	101.5	82.7	14.4	- 0.6
	K6	70.9	46.1	104.2	118.7	8.9	- 28.4
	Mean	62.3	43.5	102.7	84.5	1 2.9	- 0.6	99.5
	S.D.	4.4	2.2	0.7	5.6	1.5	6.3

1 Values are means for the last five days at each protein level.
2 Mean faecal nitrogen loss for the last eight days at each protein level,
3 True nitrogen balance was calculated using 5.0 mg N/kg as an estimate of miscellaneous losses.
4 True digestibility was calculated using 1 2.4 mg N/kg as faecal metabolic nitrogen,
5 The experiments of subject D in low-protein diet 1 and Subject C in low-protein diet 3 were interrupted because of febrile upper respiratory infections.
6 All data of subject K were omitted from further analyses because the nitrogen balance was inversely related to nitrogen intake.

TABLE 5. Summary of Nitrogen Balance Data for Young Men Given Different Levels of Supro 620¹

Protein level	Subject	wt.² (kg)	Nitrogen balance (mg/kg)				Digestibility4 (%)
			Intake nitrogen	Urinary nitrogen	Faecal nitrogen	Nitrogen balance
0.35 g/kg/day	M	68.2	56.0	67.7	7.0	- 23.8
	N	53.9	56.2	60.3	10.2	- 19.3
	O	54.6	55.9	68.1	11.4	- 28.6
	P	74.0	56.0	62.2	13.4	- 24.6
	Q	77 5	55.9	59.7	11.2	- 20.1
	Mean	65.6	56.0	63.6	10.6	-23.3	100.0
	S.D.	10.9	0.1	4.0	2.3	3.8
0.45 g/kg/day	M	68.2	72.0	76.5	6.5	- 16.0
	N	53,9	72.0	73.5	8.7	- 15.2
	O	54.6	72.0	81.1	14.7	- 28.8
	P	74.0	72.2	69.7	10.4	- 13.0
	Q	77.0	72.0	74.2	12.5	- 19.7
	Mean	65.6	72.0	75.0	10.6	- 18.5	100.0
	S.D.	10.9	0.1	4.2	3.2	6.2
0.55 g/kg/day	M	68.2	88.0	82.7	8.5	- 8.5
	N	53.9	88.1	84.2	11.9	- 13.0
	O	54.6	88.1	89.7	16.3	- 23.0
	P	74.0	88.0	85.4	11.9	- 14.3
	Q	77.5	88.1	88.0	12.1	- 17.0
	Mean	65.6	88.1	86.0	12.1	- 15.1	100.0
	S.D.	10.9	0.1	2.8	2.8	5.4
0.65 g/kg/day	M	68.2	104.0	95.6	8.7	- 5.3
	N	53 9	103.9	97.2	10.6	- 8.9
	O	54.6	104.0	98.4	14.1	- 13.4
	P	74.0	104.1	90.5	14.3 -	5.8
	Q	77,5	104.1	93.3	15.1 -	9,3
	Mean	65.6	104.0	95.0	12.6 -	8.5	99.8
	S.D.	10.9	0.1	3.2	2.8	3.3

1 Energy intake: 45 ± 1 kcal/kg
2 Values ate means for the last five days at each protein level.
3 Mean faecal nitrogen loss for the last eight days at each protein level.
See footnotes for table 4.

TABLE 6. Summary of Nitrogen Balance Data for Young Men Given Different Levels of a Mixture Containing 50 per cent Fish Protein and 50 per cent Supro 620¹

Protein level²	Subject	Body wt. (kg)	Energy intake (kcal/kg)	Nitrogen balance (mg/kg)				Digestibility4 (%)
				Intake nitrogen	Urinary nitrogen	Faecal nitrogen	Nitrogen balance
0.35 g/kg/day	A	62.9	44.1	56.1	57.2	14.1	- 20.2
	B	60.4	47.9	54.6	55.0	13.2	- 18.6
	C	69.5	44.6	55.5	69.2	12.9	- 31.6
	D	69.0	39A	55.2	46.8	9.3	- 5.9
	E	62.9	44.4	55.2	55.3	1 7.0	- 22.1
	H	60.2	44.9	55.8	56.0	13.6	- 18.8
	I	52.2	45.8	55.7	54.8	12.1	- 16.2
	J	60.4	46.7	56.6	56.0	10.8	- 15.2
	Mean	60.9	44.7	55.6	56.3	12.9	- 18.6	99.1
	S.D.	4.7	2.5	0.6	6.1	2.3	7.2
0.45 g/kg/day	A	62.8	44.1	72.3	70.0	12.9	- 17.6
	B	58.7	48.2	72.4	73.3	13.8	- 19.7
	C	59.8	44.4	71.1	63.5	14.0	- 11,4
	D	68.6	39.7	71.4	61.7	9.6	- 4.9
	E	63.4	44.7	71.6	71.3	15.9	- 20.6
	H	60.6	44.6	71.3	65.0	14.4	- 13.1
	I	52.5	45.6	71.2	62.3	11.6	- 7.7
	J	60.9	46.1	70.9	71.3	11.3	- 16.7
	Mean	60.9	44.7	71.5	67.6	12.9	- 14,0	99.3
	S.D.	4.6	2.4	0.5	4.0	2.0	5.7
0,55 g/kg/day	A	63.1	44,9	87.8	80.2	13.9	- 11,3
	B	59.2	49.8	87.7	89.2	14.9	- 21.4
	C	59.7	40.5	86.9	67.5	14.6	- 0.2
	D	69.0	38.4	86.7	69.3	8.7	+ 3.7
	E	63.1	44.2	86.5	70.5	17.0	- 6.0
	H	60.8	44.4	86.8	70.4	16.3	- 4.9
	I	52.6	45.5	87.1	81.7	11.8	- 11.4
	J	61.2	45.1	86.3	85.6	12.4	- 16.7
	Mean	61.1	44.1	87.0	76.8	13.7	- 8.5	98.5
	S.D.	4.6	3.4	0.5	8.4	2.7	8.3
0.65 g/kg/day	A	63.8	44.4	10.7	77.7	1 2.2	+ 7.8-
	B	60.1	48.1	102.2	87.5	14.0	- 4.3
	C	59.7	44.5	102.8	91,1	14.2	- 7.5
	D	69.0	39.4	102.5	75.9	9.4	+ 12.2
	E	62.8	45,4	102.7	76.6	1 5.4 +	5,7
	H	62.8	45.4	103.5	81.9	16.7 -	0.1
	I	52.7	45.4	102.6	76.5	13.3 +	7.8
	J	60.8	45.4	102.6	92.3	12.0 -	6.7
	Mean	61.2	44.7	102.7	82.4	13.4 +	1.9	99.0
	S.D.	4.6	2.4	0.4	6.9	2.2	7.5

1 Values are means for the lest five days at each protein level,
2 Each period consists of one day on protein-free diet and ten days on experimental diet,
3 Mean faacal nitrogen loss for the last eight days at each protein level,
4 True digestibility was calculated using 12,4 mg N/kg as faecal metabolic nitrogen.

TABLE 7. Summary of Maintenance Nitrogen and Equations Relating Nitrogen Balance to Nitrogen Intake

Individual data for apparent balance
Fish				Supro				Mixture
Subject	Regression equation¹	Main- tenance²	NPU	Subject	Regression equation	Main- tenance	NPU	Subject	Regression equation	Main- tenance	NPU
A	Y= 0.320X-21.2	66.3	69.4	M	Y= 0.396X-40.0	101.0	45.5	A	Y = 0.577X-51.3	88.9	51.7
B	Y=0.430X-40.4	94.0	49.0	N	Y=0.210X-25.9	123.3	373	B	Y=0.249X-30.8	123.7	37.2
C	Y = 0.322X-29.4	88.6	50.9	O	Y= 0.320X-44.1	137.8	33.4	C	Y = 0.528X-49.4	93.6	49.2
D	Y =0.416X-33.4	72.5	63.5	P	Y= 0.345X-37.0	107.2	42.9	D	Y= 0.399X-25.2	63.2	72.8
E	Y = 0.158X-16.3	103.2	44.6	Q	Y= 0.218X-29.0	133.0	34.6	E	Y = 0.620X-54.7	88.2	52.1
F	Y = 0.445X-46.4	104.3	44.1					H	Y = 0.405X-36.3	89.6	51.3
G	Y = 0.363X-30.8	84.8	54.2					I	Y = 0.435X-36.3	83.4	55.1
K¦	Y=0.274X +0.03	-	-					J	Y=0.171X-22.3	130.4	35.3
Mean	0.357X	87.7	53.7	Mean	0.298X	120.5	38.7	Mean	0.423X	95.1	50.6
S.D.	0.105	14.5	9.6	S.D.	0.081	16.0	5.3	S.D.	0.155	21.8	11.5
C.V.	29.4	12.7	5.2	C.V.	27.3	19.3	2.0	C.V.	36.6	20.7	5.8

¹Y: Nitrogen balance (mg/kg), X: Nitrogen intake (mg/kg).
² Maintenance: Nitrogen intake for the maintenance of nitrogen equilibrium (mg/kg).
³ K: emitted from group analyses (see table 4).

Pooled data

Y=0.365X-31.8	Y=0.298X-35.2	Y=0.423X-38.3
Maintenance: 87.1±17.2	Maintenance: 118.1±15.4	Maintenance: 90.5±17.1
N = 26, r = +0.727	n = 20, r = +0.774	n = 32, r = +0.727
NPU = 52.8	NPU = 38.9	NPU = 50.8

Statistical analysis
Fish vs. Supro: t = 6.337, P < 0.01, significant
Fish vs. Mixture: t = 0.751, P > 0.10, not significant
Mixture vs. Supro:t = 5.877, P < 0.01, significant

FIG. 1. Relationship between Nitrogen Intake and Nitrogen Balance for the Mixed Protein of Fish and Supro 620

TABLE 8. Amino Acid Pattern Codfish and Soybean (Amino Acid Content of Foods, FAO, 1970)

	Nitrogen (g/100 9)	Isoleu- cine	Leucine	Lysine	Methionine	Methionine - Cystine	Phenyl-alanine	PhenyI-alanine + Tyrosine	Threonine	Tryptophan	Valine	Total EAA
FAOIWHO reference (1973)		250	440	340	-	(220)*	-	(380)	250	60	310	2,250
Codfish	2.72	293	533	626	211	(284)	316	(561)	323	70	327	3,017
Soybean	6.65	284	486	399	79	(162)	309	(505)	241	80	300	2,457
50% soy + 50%cod	4.68	289	510	513	145	(223)	313	(533)	282	75	314	2,737

*Numbers in parentheses are totals.

Conclusions

1. The mean intakes of fish protein, alone (87 mg N/kg/day) or mixed 50 per cent with Supro (91 mg N/kg/day), that were required to attain apparent nitrogen balance were similar to those previously reported by us for egg protein (90 mg N/kg/day) (Inoue et al., J. Nutr., 103: 1673 [1973] ±. However, the coefficient of variation for the mixture (22.9 per cent) was larger than that for fish (16.5 per cent) or egg (15.6 per cent).

2. The protein quality of Supro 620 was increased by mixing it with fish, probably because of complementation of sulphur-containing amino acids that resulted in a higher amino acid score (table 8). in Codfish and Soybean Foods, FAO, 1970)

(introduction...)

Objective
Experimental details
Summary of main results
Acknowledgements

Kraisid Tontisirin, Prapaisri P. Sirichakawal, and Aree Valyasevi
Faculty of Medicine, Ramathibodi Hospital, and Institute of Nutrition, Mahidol University, Bangkok, Thailand

Objective

To determine, by the nitrogen balance response method, the physiological needs for high-quality protein (hen's egg) in healthy adult Thai male subjects.

Experimental details

1. Subjects
Thirteen adult male Thai students and laboratory assistants participated in the study. Table 1 shows their characteristics. They were healthy and well-nourished, based on medical history, physical examination, urinalysis, stool examination, chest x-ray, and a routine complete blood count.

2. Study Environment
The entire study was conducted at the clinical research ward (a metabolic unit) in Ramathibodi Hospital. Temperature and humidity were those typical of tropical countries.

3. Physical Activity
The subjects were allowed to continue their usual activities but not to participate in competitive, heavy sports.

TABLE 1. Initial Characteristics of 13 Adult Thai Male Subjects

Subject	Age (years)	Weight (kg)	Height (cm)
M.P.*	24	45.0	166.0
V.D.*	25	55.0	166.5
S.R.*	23	56.5	169.0
S.S.*	21	67.5	164.5
M.K.	24	55.5	165.4
T.P.	21	51.9	161.0
G.P.	24	49.7	163.0
S.K.	27	50.5	171.0
S.R.	23	57.5	169.0
C.N.	23	59.0	163.0
A.P.	19	47.0	169.0
S.S.	21	69.0	164.0
M.P.	23	46.8	166.0
Mean	22.9	54.7	165.9
S.D.	2.1	7.4	2.9

* Also participated in study on obligatory nitrogen losses.

4. Duration of the Study
Five men were studied for 55 days with protein intakes of 0.20, 0.35, 0.50, and 0.65 g/kg/day. Eight men were studied for 41 days with protein intakes of 0.55, 0.70, and 0.85 g/kg/day, during three experimental periods, respectively.

Each experimental period was of 10 days, duration, preceded by 1 day on a protein free diet and followed by 3 days on a free-choice diet between experimental periods. The sequence of protein administration was assigned randomly.

TABLE 2. Multivitamin and Mineral Supplements (Tablets)*

Vitamins		Minerals
Vitamin A	2,500±.U.	Calcium	25 mg
Vitamin D2	250±.U.	Phosphorus	19.3 mg
Thiamine mononitrate	2.5 mg	Iron	5 mg
Ribafiavin	2.5 mg	Copper	0.75 mg
Nicocotinamide	10 mg	iodine	0.05 mg
Pyridoxine hydrochloride	2.5 mg	Manganese	0.5 mg
Folic acid	0.25 mg	Magnesium	0.5 mg
Ca.pantothenate	5 mg	Potassium	1 mg
Cyanocobalamin	0.001 mg	Zinc	0.5 mg
Ascorbic acid	37.5 mg
Vitamin E	1 mg
Vitamin K	0.2 mg

* Panvitan-M, manufactured by Takeda (Thailand, Ltd., Bangkok.

5. Diets
Hen's egg was the protein source, fed scrambled and mixed with mung bean noodles at lunch and supper. The daily energy intake was kept constant at about 45 kcal/kg/day. Fat provided approximately 30 per cent of the daily energy intake. Vitamin and mineral tables (see table 2) were given twice each day. Water intake was provided ad libitum, but the volume was recorded daily.

6. Indicators and Measurements
a. The total nitrogen in diet, urine, and faeces was measured by a calorimetric semi-automated procedure (Munro and Fleck, Mammalian Protein Metabolism, vol. 3,1969). True nitrogen balance was calculated for urinary nitrogen during the last five days and for faecal nitrogen during the last eight days of each period. Fat in food and faecal samples was measured by Van de Kamer's method. Food and faecal energy were measured by bomb calorimetry (Parr Adiabatic Calorimeter). b. Basal metabolic rate (BMR) was measured daily and at the end of each dietary period with a respirometer (closed circuit). c. Body weight was recorded daily. d. Analyses of serum: Total proteins and albumin; urea nitrogen; and aspartate and alanine aminotransferases (AST and ALT) were determined at the beginning and at the end of each dietary period.

Summary of main results

1. Nitrogen Balance
Tables 3 and 4 show the nitrogen balance data. "True" balance was calculated assuming miscellaneous nitrogen losses of 5 mg/kg/day. Most men were in negative nitrogen balance with protein intakes up to 0.7 g/kg/day. At the highest level of intake (0.85 g/kg/day), five men were within + 2 mg N/kg/day of equilibrium and the other three had a positive nitrogen balance.

The individual regression equations of nitrogen balance on intake are shown in table 5. The intercept at zero balance of individuals ranged from 83.4 to 140.4 mg/N/kg/day, with the mean of the group 123.6 + 17.1 mg N/kg/day, or 0.77 + 0.11 9 protein/kg/day.

2. Other Measurements
Tables 6A and 6B show various parameters measured before the beginning of the study and at the end of each dietary period,

During the first study, BMR showed no significant changes, while total serum protein and albumin levels were increased at the low levels of protein intake. In addition, blood urea nitrogen (BUN) decreased significantly during the two low levels of protein intake of 0.20 and 0.35 g/kg/day, and there were also significant increases in AST and ALT activities, the non-essential/essential amino acid ratio, and the glycine/valine ratio when the protein intakes were decreased from 0.65 to 0.20 g/kg/day. For the second study, BMR was significantly lower at the highest intake, while changes occurred in faecal energy losses, fat absorption, and serum albumin at the end of each dietary period compared with the initial values. BUN decreased significantly at the end of all dietary periods. AST and ALT activities increased significantly only at the end of the dietary period in which protein intake was 0.55 g/kg/day.

TABLE 3. True Nitrogen Balance (mg N/kg/day) of Five Adult Thai Males Given Four Levels of Hen's Egg Protein.

Subject	Nl	UN	FN	TN	Nitrogen balance
M.P.	37.3	43.5	23.1	71.6	-34.3
V.D.	38.9	33.7	18.9	57.6	-18.7
S.R.	38.2	55.2	18.0	78.2	-40.0
S.S.	36.4	39.1	26.3	70.4	-34.0
M.K.	36.2	42.2	20.7	67.9	-31.7
x ±S.D.	37.4±1.2	42.74±7.9	21.4+3.4	69.14+7.5	-31.7±7.9
M.P.	53.8	47.2	17.2	69.5	-15.7
V.D.	54.8	48.0	21.3	74.3	-19.5
S.R.	55.9	38.1	18.0	61.1	- 5.2
S.S.	57.0	50.3	18.8	74.1	-17.0
M.K.	57.1	58.4	24.4	87.8	-30.7
x +S.D.	55.72±1.4	48.4±7.3	20.0±7.3	73.4±9.7	-17.7±9.1
M.P.	86.7	56.4	24.7	86.1	+ 0.6
V.D.	80.7	54.4	22.5	81.9	- 1.2
S.R.	80.9	69.6	23.5	98.1	-17.2
S.S.	80.6	61.5	16.7	83.2	- 2.6
M.K.	80.0	70.3	19.3	96.6	-14.61
x ±S.D.	81.8±2.8	62.4±7.3	21.3±3.3	89.2±3.3	- 7.0±8.3
M.P.	110.4	87.1	25.3	117.4	- 7.0
V.D.	235.7	79.0	22.9	106.9	+28.8
S.R.	105.7	93.2	20.7	118.9	-13.3
S.S.	91.1	70.1	25.9	101.0	- 9.9
M.K.	109.5	89.1	24.0	118.1	- 8.7
x ±S.D.	110.5±16.1	83.7±9.2	23.8±2.1	112.5±8.1	-2.0±17.4

* Nl, UN, FN, and TN represent daily nitrogen intake, urinary nitrogen, faecal nitrogen, and total nitrogen excretion, which included 5 mg N/kg/day for cutaneous losses.

TABLE 4. True Nitrogen Balance (mg N/kg/day) in Eight Adult Thai Males Given Three Levels of Hen's Egg Protein*

Subject	IN	UN	FN	TN	Nitrogen balance
T.P.	89.7	72.9	24.8	102.7	- 13.0
G.P.	86.9	73.0	22.9	100.9	- 14.0
S.K.	89.5	84.5	23.4	112.9	- 23.4
S.R.	84.3	83.4	23.7	112.1	- 27.8
C.N.	83.2	80.4	30.3	115.7	- 32.5
A.P.	76.2	86.3	19.1	110.4	- 34.1
S.S.	98.3	73.3	20.5	98.8	- 0.6
M.P.	118.3	84.2	28.7	117.9	+ 0.4
x±S.D.	90.8+12.8	79.8±5.8	24.2+3.8	108.9±7.2	- 18.1±13.5
T.P.	107.3	78.5	22.9	106.5	+ 0.9
G.P.	105.0	92.8	24.6	122.4	- 17.4
S.K.	107.0	99.7	16.8	121.5	- 14.5
S.R.	124.2	107.9	21.4	134.3	- 10.1
C.N.	113.3	89.9	27.8	122.7	- 9.4
A.P.	125.4	102.1	21.7	128.8	- 3.4
S.S.	106.8	83.2	25.0	113.2	- 9.5
M.P.	105.4	89.8	26.8	121.7	- 16.3
x±S.D.	118.8±8.4	93.0±9.8	23.4±3.5	121.4+8.6	- 10.0+6.3
T.P.	140.5	110.5	23.7	139.1	+ 1.4
G.P.	137.9	104.5	26.2	135.7	+ 2.2
S.K.	133.3	108.6	19.3	132.9	+ 0.4
S.R.	135.4	113.3	18.4	136.7	- 1.3
C.N.	138.3	101.4	23.9	130.3	+ 8.0
A.P.	139.4	99.8	33.6	138.4	+ 1.1
S.S.	134.3	98.2	24.2	127.4	+ 6.9
M.P.	149.1	106.0	15.4	126.3	+22.8
x±S. D.	138.5±5.0	105.3+5.3	23.1±5.6	133.4±4.9	5.2±7.8

* See note to table 3.

TABLE 5. Linear Regression Line of Nitrogen Balance Response (mg N/kg/day) of 13 Adult Thai Males Given Four Levels of Hen's Egg Protein

Subject	Individual linear regression line	Intercept, y = 0
M.P.	y = - 41.19±0.37x	111.3
V.D.	y = - 43.35± 0.52 x	83.4
S.R.	V = - 37.74 ± 0.27 x	139.8
S.S.	y = - 48.67 ± 0.49 x	99.3
M.K.	y = - 46.20 ± 0.35 x	132.0
T.P.	y= - 31.53 ± 0.25x	126.1
G.P.	y = - 48.35 ± 0.35 x	138.1
S.K.	y = - 72.33 ± 0.54 x	133.9
S.R.	y = - 70.18 ± 0.50 x	140.4
C.N	Y = - 93.44 ± 0.74 x	126.3
A.P.	y = - 77.49 ± 0.57 x	136.0
S.S.	y = - 35.25 ± 0.30 x	117.5
M.P.	y = - 104.96 ± 0.86 x	122.1
	Mean ± S.D.	123.6 ± 17.1*

Conclusions Nitrogen balance data indicated the adequacy of protein intake at 0.85 g/kg/day in seven of eight subjects, while only one of the eight reached balance on an intake of 0.70 g/kg/day. The enzyme activities of AST and ALT also showed no changes during this dietary period. Since there were technical problems with the amino acid autoanalyser, serum amino acid levels are not available. The mean protein requirement of these subjects, based on the linear regression analysis of nitrogen balance response of individuals, was 123.6 + 17.1 mg N/kg/day, or 0.77 + 0.11 9 protein/kg/day. if 97.5 per cent of the population is expected to be covered, the safe level of intake would be mean + 2 standard deviations, or equivalent to 157.8 mg N/kg/day, or 0.99 9 protein/kg/day. This safe level of protein intake is clearly higher than the 0.57 9 protein/kg/day recommended by the FAD/WHO Expert Committee.

TABLE 6A. Basal Metabolic Rate, Blood Chemistry Values, and Amino Acid Ratio (Mean ± S.D.) of Five Adult Thai Males Given Four Levels of Hen's Egg Protein

			Protein intake (g/kg/day)
Measurement	Initial value	0.20	0.35	0.50	0.65
BMR (kcal/m²/day)	987 ± 236	991 ± 103	972 ± 83	838 ± 77	919 ± 150
Total protein (g/dl)	6.7 ± 0.9	7.6 ± 0.6	6.7 ± 0.6	6.6 ± 0.6	7.2 ± 0.4
Albumin (g/dl)	4.3 ± 0.2	4.9 ± 0.7*	4.6 ± 0.1 *	4.5 ± 0.2*	4.8 ± 0.3
BUN (mg/dl)	8.7 ± 4.2	2.6 ± 2.2*	3.0 ± 0.9*	4.4 ± 2.0	5.3 ± 1.0
AST (sigma unit)	15.9 ± 2.4	33.8 ± 9.4*	26.0 ± 6.1 *	24.8 ± 6.3*	20.9 ± 3.3*
ALT (sigma unit)	12.8 ± 3.1	26.6 ± 3.8*	30.8 ± 10.9*	32.2 ± 5.6*	22.9 ± 3.1*
NEA/EA ratio	2.2 ± 0.3	3.3 ± 0.1 *	3.1 ± 0.3*	2.7 ± 0.2*	2.5 ± 0.1*
Glycine/valine ratio	1.2 ± 0.9	2.0 ± 0.2	1.9 ± 0.3	1.5 ± 0.1	1.2 ± 0.2*

* Comparing with initial value p < 0.05.

TABLE 6B. Basal Metabolic Rate and Other Biochemical Values in Eight Adult Thai Males Given Three Levels of Hen's Egg Protein

		Protein intake (g/kg/day)
Measurement	Initial value	0.55	0.70	0.85
BMR (kcal/m² /day)	935 ± 125	821 ± 128	821 ± 170	771 ± 129*
Faecal energy loss (% of intake)		5.3 ± 1.0	5.1 ± 2.0	5.7 ± 0.9
Fat absorption (% of intake)		96.0 ± 1.4	95.8 ± 1.2	96.6 ± 1.1
Total protein (g/dl)	7.0 ± 0.6	7.6 ± 0.6	7.2 ± 0.6*	6.9 ± 0.5
Albumin (g/dl)	4.5 ± 0.5	4.2 ± 0.7	4.8 ± 0.3	4.7 ± 0.6
BUN (mg/dl)	10.8± 1.5	6.0 ± 1.7*	7.4 ± 1.7*	8.1 ± 1.0*
AST (sigma unit)	14.2 ± 5.3	18.4 ± 4.9*	20.4 ± 10.1	15.5 ± 7.8
ALT (sigma unit)	14.4 ± 7.6	18.8 ± 9.5*	21.8 ± 14.1	16.9 ± 11.0

* Comparing with initial value p < 0.05.

Acknowledgements

This study was supported by the research fund of the World Hunger Programme of the United Nations University. We would like to thank Dr. V. Tanphaichitr for medical care of the subjects, the staffs of the clinical research ward and food analysis laboratory, Ramathibodi Hospital, for their assistance, and the subjects who cooperated throughout the study.

(introduction...)

Objective
Experimental details
Summary of main results
Conclusions and comments

J.E. Dutra de Oliveira, Helio Vannucchi, and Rosa M.F. Duarta B.
Division of Nutrition, Metabolic Unit, University Hospital, Ribeirao Preto, Sao Paulo, Brazil

Objective

This study was carried out to investigate the composition and nutritive value of the diet, largely based on rice and beans, habitually consumed by agricultural migrant workers in Brazil.

Experimental details

1. Subjects
Fourteen healthy migrant workers, 17 to 26 years old, were selected. Their characteristics are shown in table 1.

2. Study Environment
All men were admitted to our metabolic unit for the duration of the experiment, and the study was carried out during the summer months (average temperature 23.1 to 24.0° C).

3. Physical Activity
The men were ambulatory; they could walk in the metabolic unit, play card games, and watch television. In addition, they pedalled two to three times per day on a bicycle ergometer, with an energy expenditure of about 850 kcal/day.

TABLE 1. Characteristics of Subjects

Subject no.	Age (years)	Height (cm)	Weight (kg)	Mid-arm muscle circumference (% of standard, according to Jelliffe)	Albumin (g/dl)	Haemoglobin (g/dl)
1	21	172	66.8	98.6	4.6	13.5
2	21	180	70.0	71.5	4.8	14.3
3	22	157	52.0	109.5	4.3	12.8
4	21	179	66.8	92.8	4.4	15.2
5	26	169	60.9	88.9	4.2	15.6
6	20	171	63.0	-	4.2	1 5.6
7	20	172	66.6	92.5	4.6	13.9
8	20	171	59.3	-	4.6	11.7
9	17	159	51.8	-	4.4	13.9
10	24	157	52.6	92.5	4.4	14.2
11	24	173	66.6	107.1	4.4	14.9
12	23	172	63.5	95.2	4.6	1 5.8
13	21	167	61.2	103.1	4.3	15.0
14	22	151	50.6	100.3	4 3	14.7
Mean	21.6	168.3	60.8	95.5	4.4	14.4
S.D.	2.2	81.0	66	10.3	0.2	1.1

4. Duration of the Study
The experiment was divided into one adaptation period of two to three days and one five-day metabolic balance study.

TABLE 2. Intakes of the Rice-and Bean Diet

Food	Amount per day (g)
	Range	X
Rice	422.4 - 960.0	764.7
Beans	307.2 - 768.0	546.6
Meat	19.2 - 96.0	55.5
Eggs	48.0	17.1 5
Vegetables	28.8 - 192.0	142.1
Coffee and sugar	170.0 - 600.0	508.4

TABLE 3. Protein and Energy Characteristics of the Experimental Diet (Mean Amounts Consumed by 14 Men)

Protein and energy intake	Mean ± S.D.
Total energy (kcal/kg/day)	41.4 ± 6.20
Total protein (g/kg/day)	1.14 ± 0.14
Energy from rice and beans (kcal/day)	1,714 ± 308
Energy from other foods (k cal/day)	789 ± 144
Protein from rice and beans (g/day)	45.6 ± 9.2
Protein from other foods (g/day)	23.6 ± 2.3
Dietary energy density (kcal/g)	1.55 ± 0.08
Rice/bean protein ratio (9/9)	0.72 ± 0.06
Energy/protein ratio (kcal/g)	36.2 ± 2.5

5. Diet
After an initial dietary survey of each individual's food intake, diets were individually prepared. Rice and beans were the main sources of protein, and bread, coffee, small amounts of meat, eggs, and vegetables were also included in the meals. Table 2 shows the range of daily intakes by each of the 14 men. The food was always offered as a bread, with coffee and sugar at breakfast, lunch at noon, a mid-afternoon snack of coffee, sugar, and bread, supper in the early evening, and coffee with sugar at night.

6. Indicators and Meassurements
a. Urine was collected on a timed 24-hour basis. Faeces were collected over the five-day balance period, between administration of carmine and charcoal faecal markers. Urinary creatinine was measured daily (picrate method). Nitrogen in urine, faeces, and food was measured by the Kjeldahl method. Each dietary component was analysed separately, and the nitrogen intake was calculated from the amount of each food consumed. b, Apparent nitrogen balances were calculated from the dietary intakes and urinary and faecal excretions over the five-day period.

Other Studies

A medical examination was carried out on each subject before admission, blood was taken for biochemical profile analyses, and stool and urine samples were obtained for routine laboratory examination.

Summary of main results

Table 3 shows that rice and beans were the main source of energy and protein in the diet. This table also shows the total daily energy and protein intakes, the contributions made by rice and beans, and the dietary energy density.

The results of apparent nitrogen balance and apparent digestibility of the diet are shown in table 4.

Body weight and urinary creatinine excretion did not change during the five-day period.

Conclusions and comments

The rice-and-bean diet satisfied the men's energy requirements, at least under the conditions of this short-term metabolic study.

TABLE 4. Nitrogen Balance and Apparent Digestibility of Dietary Nitrogen(Means of Five Days)

Subject	Nitrogen intake (mg/kg/day)	Total urinary nitrogen (mg/kg/day)	Faecal nitrogen (mg/kg/day)	Apparent nitrogen balance (mg/kg/day)	"True" nitrogen balance* (mg/kg/day)	Apparent digestibility (%)
1	162.9	109.9	43.5	9.5	4.5	73.3
2	210.6	130.0	54.4	26.2	21.2	74.2
3	176.2	140.4	46.2	- 10.5	- 15.5	73.8
4	170.8	138.5	38.3	- 6.0	- 11.0	77.6
5	160.6	98.4	59.9	2.4	- 2.6	62.8
6	204.3	108.1	81.1	15.0	10.0	60.3
7	165.3	88.4	59.9	17.0	12.0	63.8
8	204.7	146.0	63.4	- 4.7	- 9.7	69.0
9	222.2	137.0	75.3	9.8	4.8	73.4
10	168.6	128.1	44.6	- 4.2	- 9.2	67.5
11	1653	92.6	53.8	18.9	13.9	65.6
12	166.6	92.6	57.3	16.7	11.7	69.9
13	207.5	107.2	62.4	37.9	32.9	73.7
14	173.5	120.5	45.6	7.4	2.4	69.3
Mean	182.8	117.0	56.1	9.7	4.7	69.3
S.D.	21.7	16.9	12.2	13.6	-	5.2

* Assuming miscellaneous losses of 5 mg/kg/day.

Based on these results, the energy and protein needs of these workers could be met if sufficient amounts of the rice-and bean diet were available. This is not always the case at the community level, since preliminary surveys showed that there were inadequate intakes of energy, protein, and other nutrients.