(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.