(introduction...)

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

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.

References

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),

(introduction...)

Objective
Experimental details
Summary of main results
Conclusions and comments

R G. Whitehead, A.A. Paul, A.E. Black, and S.J. Wiles
Medical Research Council Dunn Nutrition Unit, Cambridge, England

Objective

To obtain information on the dietary energy intakes of British women during pregnancy and lactation, since estimates of the dietary intakes of individuals living in the United Kingdom have shown a consistent downward trend in recent years.

Experimental details

1. Subjects
Twenty-five women were recruited near the beginning of the second trimester of pregnancy through the antenatal clinic of the Cambridge Maternity Hospital. They were 21 to 35 years old (mean 29) and belonged to social grades I, II, and III. Twelve were primiparous and worked during most of their pregnancy, mainly in a clerical capacity. None of the multiparous mothers had outside jobs. Their mean height was 161.7 cm (147.5 to 172.5), and the stated pre-pregnant weight was 56.2 (43.5 to 71.7). The validity of the latter measurement was verified by comparison with the initial weight found on recruitment.

2. Dietary Intake
Energy and nutrient intakes were measured over four consecutive days each month throughout pregnancy and lactation by the mother herself, after instruction, weighing the food and drink she consumed. The food intake measurements were interpreted using food composition tables.

3. Weight Changes and Stored Energy
A number of anthropometric measurements were made, including weight, at monthly intervals throughout pregnancy, at two weeks after delivery, and then once again at monthly intervals. Energy stored as fat during pregnancy was estimated from the difference in body weight between two weeks postpartum and the pre-pregnant weight, making the assumption that adipose tissue provides, during lactation, 6.5 kcal/g body-weight change (Thomson et al., Brit,J. Nutr., 24: 565 [1970] ).

4. Duration of Pregnancy and Birth Weights
Birth weights, which were all over 2.6 kg, were obtained by the maternity hospital staff. Mean gestational age was 39 completed weeks, range 36 to 43 weeks.

5. Breast-milk Production
Breast-milk intake was also measured by the mother on four consecutive days each month by test weighing, using Salter Baby weigher Model 40 Scales. The test weighing measurements in a number of subjects were checked by the recently developed deuterium oxide method (Coward et al., Lancet, ii: 13 [1979], the milk intakes showing good agreement between the two procedures.

Summary of main results

1. Pregnancy
a. The principal pregnancy data are given in table 1. There was no significant difference in energy intake between the second and third trimesters of pregnancy, and both values agreed closely with those for the first trimester reported by

Smithells in Leeds (Brit. J. Nutr., 38:497 11977] ).

TABLE 1. Energy Intakes and Body-Weight Changes of 25 Mothers during the Second and Third Trimesters of Pregnancy (Mean ± S.D.)

Energy intake, 2nd trimester	(kcal/day)	1,950 ± 380
3rd trimester	(kcal/day)	2,005 ± 345
2nd & 3rd trimester	(kcal/day)	1,978 ± 350
Weight gain during pregnancy*	(kg)	12.6 ± 4 .0
Estimated maternal energy store	(kcal)	38,662 ± 28,570
Birth weight	(kg)	3.31 ± 0.35

* To the 36th week.

TABLE 2. Energy Intake and Milk Output of 17 Mothers at Different Stages of Lactation (Mean + S.D.)

Month of lactation	Energy intake (kcal/day)	Breast-milk output (9/24 hr)
2	2,278 ± 458	715 ± 148
3	2,300 ± 470	773 ± 140
4	2,380 ± 408	755 ± 136

b. Among the Cambridge mothers, there was no correlation between energy intake in the last trimester of pregnancy and birth weight (r = 0.01).

2. Lactation
a. Of the 25 mothers, 4 breast-fed for only 2 to 14 days, and another 4 for less than three months. Seventeen breast-fed at least up to the beginning of the fifth month, 11 exclusively, but the remaining 6 had by then introduced small amounts of other foods, which supplied only an average of 18 per cent of the babies' total energy intake.

b. The basic lactation data are summarized in table 2. Lactation was associated with an increase in food intake, but daily energy consumption was still 450 kcal less than the United Kingdom's DHSS Recommended Daily Amount. In the fourth month, there was little difference in energy intake among the 11 mothers who were exclusively breast-feeding (2,278 ± 431 kcal [mean ± S.D.] ) and the six who were not (2,363 ± 402 kcal), although the mean milk output of the former, 791 ± 93 g/day, was higher than that of the mothers who were using mixed feeding, 688 ± 186 g/day.

c. Figure 1 shows the relationship between dietary energy intake and milk output. The line of best fit (r = 0.76, p < 0.001) was significant(y curvilinear (p < 0.01). Mean milk outputs were not significantly different in mothers with energy intakes of 2,000 to 2,400 kcal/day and in those with intakes over 2,400 kcal-768 ± 63 to 780 ± 148 g/day, respectively. Energy intakes below 2,000 kcal were, however, associated with significantly lower milk outputs: 455 ± 227 g/day (t-4.2, p < 0.001). Three of the mothers who could not breast-feed for more than two months had intakes below 1,720 kcal/day, and they had the three lowest milk outputs. Data for the fourth mother were omitted because she had been complying with advice to eat beyond her appetite-3,338 kcal/day-in an unsuccessful attempt to boost her milk output.

FIG 1. Relation between Maternal Energy Intake and Breast-Milk Output.

Most points are the mean of dietary energy and milk output measurements over 12 days during the second, third, and fourth months of lactation.

d. There was no important correlation between the average amount of milk that the individual mother produced during the first four months and her corresponding loss of weight (r = 0.20 NS). There was a significant correlation between weight loss and overall energy intake during lactation (r = 0.56, p < 0.02), and the relationship was even stronger (r = 0.78, p < 0.001) when weight change was related to the increase in energy intake that occurred when the mothers passed from pregnancy into lactation.

FIG. 2. Relation between Lactation Dietary Energy Increment and Weight Loss over the First Four Months Post Partum

Figure 2 predicts that at the DHSS recommended daily energy increment for lactation-600 kcal-mothers would lose none of their excess fat, while at the mean increment for the group studied, 281 kcal, the mothers lost weight at an average rate of 570 g/month. e. Figure 3 partly explains why some mothers ate so little extra during lactation. There was a high(y significant negative relationship (r = 0.73, p. < 0.001) between the extra food energy consumed during lactation and the amount of weight a mother had retained after her pregnancy.

FIG. 2. Relation between Weight retainrd after Pregnancy and Lactation Dietary Energy Increment

Conclusions and comments

1. The energy intake of women did not increase during pregnancy, and it was comparable to that of nonpregnant and non-(actating Cambridge women of similar age and social background: 2,029 ± 392 kcal/day (Nelson et al., unpublished).
2. In spite of these low intakes, mean weight gain during pregnancy coincided with the conventional standard of 12.5 kg. Mean birth weight was also normal, as was the weight retained by the mothers after parturition and hence the estimation of their energy stores for lactation.
3. Since the pregnant women were not demonstrably less active, it would appear that they must have been able to satisfy at least part of the additional needs of pregnancy by subtle changes in activity or by an enhanced efficiency of metabolism.
4. Milk outputs did not increase with dietary intakes above 2,000 kcal per day, but they decreased with lower levels of energy intake.
5. The maintenance of substantial amounts of milk production at relatively low levels of energy intake cannot be explained just on the basis of an increased utilization of the subcutaneous fat stored during pregnancy. At the mean dietary energy increment of 281 kcal/day observed during lactation in the present study, it can be calculated from figure 2 that only 124 additional kcal/day would be made available from the body weight changes (a loss of 19 9 body fat/day), resulting in a net gain of 405 kcal/day. Yet the mean volume of milk produced, 747 g/day, would contain an average 515 kcal. Conventional estimates of the efficiency of conversion of dietary energy for human milk production have clearly been misleading. The present results show that calculated efficiency factors vary with the level of energy intake. At a dietary increment of 800 kcal/day during lactation, the calculated efficiency of conversion would be the generally accepted value of 80 per cent, but at the mean measured increment of 281 kcal/day, as just calculated, the apparent efficiency is 127 per cent -at first sight a nonsense factor. The anomaly can be resolved, however, if it is accepted that there have been substantial compensatory alterations in the non lactational component of the mother's physiological efficiency.
6. We have produced circumstantial evidence for energy-conserving adaptations during lactation as well as pregnancy that ensure foetal development and subsequent milk production without the need for excessively high energy intakes or, alternatively, drastic changes in the body composition of the mother. The data presented indicate that, for the type of population studied, current recommended daily amounts of dietary energy during pregnancy and lactation are unnecessarily high: lower mean values of 2,000 kcal and 2,400 kcal, respectively, could be safely adopted. The suggested value for lactation would also enable the mother to attain her pre-pregnancy weight within a more acceptable time.

Acknowledgements

We thank Professor C. Douglas, Mr. R.E. Robinson, and Dr. N.R.C. Roberton and the staff of the Cambridge Maternity Hospital for their collaboration, and Miss M.J. Whichelow for her help in recruiting the subjects. This study was financially supported by the Department of Health and Social Security.