(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)