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Effect of nitrogen intake on nitrogen utilization (1, 2)
Concluding comment

Goro Inoue, Kyoichi Kishi, Yoshiaki Fujita, Shigeru Yamamoto, and Yukio Yoshimura
Department of Nutrition, School of Medicine, Tokushima University, Tokushima, Japan

Effect of nitrogen intake on nitrogen utilization (1, 2)

When nitrogen intake varied widely in the submaintenance range from minimal to marginal levels with maintenance energy intake in young men (see figure 1), the response of nitrogen balance to nitrogen intake was not linear. Only in the range of nitrogen intake greater than 30 mg/kg was the response linear and the difference in protein qualities indicated by the difference of the slopes. In the minimal nitrogen intake below this point of inflection, the slope through the endogenous nitrogen output of 46 mg/kg became very steep so that the quality difference disappeared.

As a whole, the biological value (BV) decreases with increasing protein intake, e.g., BV for wheat gluten is as high as 100 at a low nitrogen intake and decreases to 24 with increasing nitrogen intake (see table 1). This change of BV can be described by a general fractional equation for a limited range of linear response. BV curves based on this equation are illustrated in figure 2. The curves decrease exponentially with increasing nitrogen intake until maintenance nitrogen intake is reached.

Using the equations obtained for egg, rice, and wheat gluten, net protein utilization (NPU) corresponding to the respective nitrogen intakes was calculated and the NPU values relative to egg estimated for rice and wheat gluten (see table 2).

FIG. 1. Non-rectilinear Relationship between Absorbed Nitrogen and Nitrogen Balance in Young Men with Maintenance Energy Intake

Wheat gluten: above 0.2 g/kg, Y = 0.129X-26.16 (Inoue).
Egg protein: below 0.2 g/kg, Y = 1.028X-46.05 (Young);
above 0.2 g/kg, Y = 0.411 X-37.03 (Inoue).

The NPU value for egg at the maintenance intake of 0.56 9 protein/kg of body weight was 51, with the relative NPU values for rice and wheat gluten at their respective maintenance nitrogen intakes being 76 and 45, respectively. These are similar to those estimated from maintenance nitrogen intakes. On the other hand, using the slope ratio method, the relative efficiencies for rice and wheat gluten to egg were 65 and 31, based on slopes for egg, rice, and wheat gluten of 0.411, 0.268, and 0.129, respectively (see table 3). As a result, the figures estimated by the slope ratio method are considerably lower than those by the other estimations. The appropriateness of the slope ratio method requires critical review. It is clear that, even within the sub maintenance range of nitrogen intake, the nutritional efficiency of a protein may change inversely with the level of protein intake. The significance of nitrogen utilization at the minimal nitrogen intake should also be reconsidered.

TABLE 1. Body Weight, Nitrogen Balance, and Biological Value (3V) in Young Men Given Various Levels of Wheat Gluten


Nitrogen balance2

Protein (g/kg) No. of subjects BW 1 (kg) Intake (mg/kg) Urine (mg/kg) Faeces (mg/kg) Balance (mg/kg) BV
0 9 63.3±6.3 2 33.3±3.1 12.7 ± 1.5 - 46.0±73  
0.1 9 62.2±5.3 15 32.3±4.9 11 9 ± 2.2 - 29.2±5.7 106±2
0.2 10 56.6± 4.2 28 37.3±3.7 13 4 ± 2.0 - 23.2±2.4 85±9
0.4 3 58.5 ±4.4 60 66.0±1.4 1 1.8 ± 0.5 -18.4±1.6 45±3
0.6 3 63.7 ±5.4 100 94.8±0.5 16.2 ± 0.7 - 10.2±1.0 37±1
1.0 5 54.6 ±2.8 173 159.0±15 20.2 ± 2.5 - 5.9± 2.5 24 ± 2

1 Means ± S.D. for the last five days on standard diet.
2 Mean values of urinary nitrogen for the last five days and of faecal nitrogen for the entire period were used for estimating the nitrogen balance.
3 Figure indicates mean ± S.D. of endogenous nitrogen output.

TABLE 2. Changes of Nutritional Efficiency with Intake Level of Protein at Maintenance Energy Intake

Protein intake

Biological value

(g/kg) Egg Rice Wheat gluten
0.2 62    
0.3 59 (100)1 56 (95) 54 (92)
0.45 53 (100) 46 (87) 41 (77)
0.562 51 (100) 42 (82) 35 (69)
0.752   39 (76)3  
1.0     25
1 262     23 (45)2

FIG. 2. Inverse Curvilinear Relationship between BV and Protein Intake in Young Men Fed Egg Protein and Wheat Gluten.

Curves were drawn using the following fractional equations: with wheat gluten ( --- ), BV = (19.8/X +0.13) x 100; with egg protein (--- ), BV = (12.5/X + 0.36) x 100. A general fractional equation is as follows: BV/100= (EN-b)/X +a where X: nitrogen intake in mg/kg, EN (endogenous nitrogen): a constant of 46.0 mg/kg, and a and b are the slope and Y-intercept, respectively, in the response equation.

TABLE 3. NPU Estimated by Slope Ratio Method


Regression equation

  Subj. no. Slope Y-intercept Relative NPU
Egg protein 11 0.411 37.03 (100)
Rice protein 14 0.268 31.98 65
Wheat gluten 21 0.129 26.16 31

FIG. 3. Regression Equations in Young Men Given Egg Protein Diet at Various Levels of Energy Intake

TABLE 4. Changes of Nitrogen Requirement and Nutritional Efficiency of Egg Protein in Young Men Given Various Levels of Energy Intake

  Energy intake (kcal/kg) Subj. no. Maintenance nitrogen intake (mg/kg) Equations for1 computing NPU NPU² (N: 90 mg/kg) NPU³
Slightly deficient 40 15 124 12.4/X+0.74 41 37
Maintenance 45 15 90 12.4/X + 0.34 51 51
Slightly excess 48 31 81 12.0/X + 0.24 55 57
Excess 57 6 67 9.8/X + 0.54 65 69

1 X is nitrogen intake (mg/kg).
2 Values are estimated as the figures corresponding to maintenance nitrogen intake of 90 mg/kg.
3 Values corresponding to respective maintenance nitrogen intakes

Effect of Energy Intake on Nitrogen Utilization
As shown in figure 3, four series of nitrogen balance studies were carried out with a total of 67 young Japanese men given an egg protein diet, with nitrogen intake varying from about 25 to 100 mg/kg. Energy intakes were about 40,45,48, and 57 kcal/kg, respectively. The intake of 45 kcal/kg met approximately the maintenance requirement; 40 was slightly deficient, 48 was slightly in excess, and 57 was greatly in excess by about 700 kcal/kg. As a result, the slope of the regression line became greater with increasing energy intake, being 0.27, 0.37, 0.42, and 0.54, respectively, in order of energy level. This means that the efficiency of nitrogen utilization was affected greatly by energy intake.

The changes in nitrogen requirement and NPU corresponding to the respective energy intakes are shown in table 4. At the maintenance intakes of both nitrogen and energy, 51 per cent of ingested egg protein may be utilized in the amino acid pool, whereas at the same nitrogen intake of 90 mg/kg, NPU decreases by about 20 per cent with slightly deficient energy and increases by about 30 per cent with excess energy.

Nitrogen requirement is also greatly affected by the level of energy intake. This is very important because the significance of energy intake on nitrogen balance has not been fully taken into account in numerous past reports (3). If dietary energy is supplied in excess, the egg protein requirement could be reduced to about 0.4 g/kg. From this point of view, the safe intake of 0.57 g/kg of egg protein that was proposed by the 1973 FAD/WHO report must be reconsidered, as Garza, Scrimshaw, and Young have pointed out (4).

FIG. 4. Interrelationship between Nitrogen Balance and Nitrogen and Energy Intakes.

NB = 0.01049N E-0.1049N + 0.02714E-35.39, where
NB: nitrogen balance (mg/kg/day); N: nitrogen intake (mg/kg/day); E: energy intake (kcal/kg/day)

Interactive Effect of Protein-Energy Intakes on Nitrogen Utilization (5)

Using the multiple regression analysis, the interaction between nitrogen and energy intakes on nitrogen balance and NPU can be expressed as the following equations:

I. NB = 0.01049N E-0.1049N + 0.0271E-35.39 (n = 67, R2 = 0 77)
II. NPU = 7.384/N + 884.9/N + 0.9672E-7.458 (n = 67, R2 = 0.66)

where NB: N balance in mg/kg; N: N intake ranged from 25 to 100 mg/kg; and E: energy intake ranged from 45 to 57 kcal/kg.

1. The interrelationship between nitrogen and energy intakes on the nitrogen balance obtained in equation I is illustrated in figure 4. The improvement in nitrogen balance is only 0.32 mg with a unit increase of nitrogen intake at the submaintenance energy intake of 40 kcal/kg, but it rose to 0.50 mg at an excess energy intake of 57 kcal/kg.

FIG. 5. Change in the Efficiency of Intake Nitrogen with Nitrogen and Energy Intakes Varied around Maintenance Levels, Which Are Approximately 93 mg N/kg and 45 kcal/kg.

As shown in figure 5, when both the nitrogen and energy intakes change around maintenance level (93 mg N/kg and 45 kcal/kg), the effect of energy intake on nitrogen balance is larger by about 2.7 times in unit change and by about 1.3 times in percentage change than that of nitrogen intake.

2. NPU curves corresponding to the respective energy intakes are shown graphically in figure 6. Assuming that NPU decreases linearly with an increase of nitrogen intake in the range above 50 mg/kg, equation 11 may be reformulated as follows: NPU = 0.119N + 1.367E-0.200. Thus, NPU decreases 0.12 units with an increase of 1 mg/kg of nitrogen and increases 1.37 units with an increase of 1 kcal/kg of energy (see figure 7).

Figure 5b

This result was reconfirmed by rat studies using carcass analysis (6). Two-hundred adult male rats were fed daily an allocated amount of food for three weeks at each of five levels of nitrogen (lactalbumin) and energy intakes (64 to 192 mg/rat/day and 40 to 72 kcal/rat/day). When N and energy varied with percentage change around weight maintenance intakes (139 mg N/day and 55.4 kcal/day), the slopes for energy and nitrogen changed accordingly, as summarized in table 5. It was found that the effect of energy intake on body nitrogen retention was 1.6 times larger than that of nitrogen intake, while that on body weight was 5.4 times larger. An increase in nitrogen intake led to a loss of body fat in contrast to gains in water and nitrogen.

FIG 6. Relationship between NPU and Nitrogen Intake with Several Levels of Energy Intake. Equations relating to NPU vs. nitrogen intake:

E = 40, NPU = 1182/N + 31.42; E = 45, NPU = 1217/N + 36.07; E = 48, NPU = 1241/N + 39.16; E = 57, NPU = 1306/N + 47.67.

FIG. 7. Change in NPU

Concluding comment

Energy intake has a proportionately greater effect on nitrogen utilization than does nitrogen intake in young men when energy and protein intakes vary around the maintenance level. Recently, Tor al. (7), studying the effect of physical exercise on protein requirements, suggested that not only are energy requirements influenced by the level of protein intake but also protein requirements are affected by energy intake. Clearly, the interaction of protein and energy must be taken into account in defining the dietary requirements for both protein and energy and also in treating persons with proteinenergy malnutrition.

It should be also be emphasized that evaluation of food protein involves many complex factors. These include not only dietary variables, such as the level of protein

TABLE 5. Increment Rate of Body Composition with Increasing Energy and Nitrogen Intakes


Body composition

Body weight

Water Fat N x6.25
Slope for Et (mg/day) 23.9 23.4 5.4 (52.7) 53 7
Slope for N2(mg/day) 10.3 - 4.6 3.3 ( 9.0) 10.0
E/N ratio 2.4 - 5.1 1.6   5.4

E (energy intake) varied with the percentage of the change from weight maintenance of 55.4 kcal/day i 100 per cent) and nitrogen intake was constant at maintenance of 139 mg/day. N (nitrogen intake) varied with the percentage of the change from weight maintenance of 139 mg/day (100 per cent), and E intake was constant at maintenance of 55,4 kcal/day. and energy intake, but also physiological status, i.e., growth, nutritional status, the presence of infections and other diseases, etc.


1. G. Inoue, Y. Fujita, K. Kishi, S. Yamamoto, and Y. Niiyama, "Nutritive Values of Egg Protein and Wheat Gluten in Young Men," Nutr. Rep. Int., 10: 201 (1974).

2. V. R. Young, Y. S. M. Taylor, W. M. Rand, and N. S. Scrimshaw, "Protein Requirements of Man: Efficiency of Egg Protein Utilization at Maintenance and Submaintenance Levels in Young Men," 1 Nutr., 103: 1164 (1973).

3, N. S, Scrimshaw, "Shattuck Lecture: Strengths and Weaknesses of the Committee Approach," New Engl. J. Med,, 294: 1 36, 198 11976).

4. C. Garza, N. S. Scrimshaw, and V. R. Young, "Human Protein Requirements: A Long-term Metabolic Nitrogen Balance Study in Young Men to Evaluate the 1973 FAO/WHO Safe Level of Egg Protein Intake," l Nutr., 107: 335 11977).

5. K. Kishi, G. Inoue, Y. Yoshimura, Y. Fujita, and S. Miyatani, "Quantitative Effects of Energy and Nitrogen Intakes at Near Maintenance Level on Egg Protein Utilization in Young Men" (submitted to Am. l C/in. Nutr.).

6. Y. Yoshimura, G. Inoue, K. Kishi, and Y. Matsumoto, "Quantitative Relationship between Effects of Energy and Protein Intakes on N Utilization in Adult Rats" [submitted to Am. J. C/in. Nutr.).

7. B. Tor. S. Scrimshaw, and V. R. Young, "Effect of Isometric Exercises on Body Potassium and Dietary Protein Requirements of Young Men," Am. J. Clin. Nutr,, 30: 1983 (1977),