Acknowledgements

From the charter of the United Nations University

ARTICLE I

Purposes and structure

1. The United Nations University shall be an international community of scholars, engaged in research, post-graduate training and dissemination of knowledge in furtherance of the purposes and principles of the Charter of the United Nations, In achieving its stated objectives, it shall function under the joint sponsorship of the United Nations and the United Nations Educational, Scientific and Cultural Organization (hereinafter referred to as UNESCO), through a central programming and co-ordinating body and a network of research and post-graduate training centres and programmes located in the developed and developing countries.

2. The University shall devote its work to research into the pressing global problems of human survival, development and welfare that are the concern of the United Nations and its agencies, with due attention to the social sciences and the humanities as well as natural sciences, pure and applied.

3. The research programmes of the institutions of the University shall include, among other subjects, coexistence between peoples having different cultures, languages and social systems; peaceful relations between States and the maintenance of peace and security; human rights; economic and social change and development; the environment and the proper use of resources; basic scientific research and the application of the results of science and technology in the interests of development; and universal human values related to the improvement of the quality of life.

4. The University shall disseminate the knowledge gained in its activities to the United Nations and its agencies, to scholars and to the public, in order to increase dynamic interaction in the world-wide community of learning and research.

5. The University and all those who work in it shall act in accordance with the spirit of the provisions of the Charter of the United Nations and the Constitution of UNESCO and with the fundamental principles of contemporary international law.

6 The University shall have as a central objective of its research and training centres and programmes the continuing growth of vigorous academic and scientific communities everywhere and particularly in the developing countries, devoted to their vital needs in the fields of learning and research within the framework of the aims assigned to those centres and programmes in the present Charter. It shall endeavour to alleviate the intellectual isolation of persons in such communities in the developing countries which might otherwise become a reason for their moving to developed countries.

7. In its post-graduate training the University shall assist scholars, especially young scholars, to participate in research in order to increase their capability to contribute to the extension, application and diffusion of knowledge. The University may also undertake the training of persons who will serve in international or national technical assistance programmes, particularly in regard to an interdisciplinary approach to the problems with which they will be called upon to deal.

ARTICLE II

Academic freedom and autonomy

1. The University shall enjoy autonomy within the framework of the United Nations. It shall also enjoy the academic freedom required for the achievement of its objectives, with particular reference to the choice of subjects and methods of research and training, the selection of persons and institutions to share in its tasks, and freedom of expression. The University shall decide freely on the use of the financial resources allocated for the execution of its functions ....

This book is published within the framework and as part of the Sub-programme on Human Nutritional Requirements and Their Fulfillment through Local Diets of the United Nations University's World Hunger Programme. The views expressed are those of the authors and not necessarily those of the United Nations University.

The United Nations University Toho Seimei Building, 15-1 Shibuya 2-chome, Shibuya-ku, Tokyo 150, Japan

Foreword

A working group convened by the United Nations University in San Jose, Costa Rica, in February 1977 discussed the issues surrounding protein energy under conditions prevailing in developing countries, summarized current knowledge, and indicated priority research needs. Its report was published in 1979 as a supplement to the Food and Nutrition Bulletin, but by that time the UN University had established an extensive research network and was already supporting many of the studies recommended.

The present report is the result of a workshop on protein-energy requirements convened in Cambridge, Massachusetts, USA, 19-23 May 1981, by Committee l/7 of the International Union of Nutritional Sciences (IUNS) under the chairmanship of Dr. Benjamin Torhe purpose of the workshop was to receive and review the first round reports of the UNU-sponsored research, evaluate the significance of the data presented, and make recommendations for the additional research most urgently required for an in-depth review of international recommendations for protein-energy requirements planned by the Food and Agriculture Organization, the World Health Organization, and the UN University for 5-17 October 1981 in Rome. Independent of the UNU research effort but in close co-ordination with it, the FAO and WHO jointly sponsored research in three developing countries-Guatemala, Thailand, and India- utilizing funds received from the Danish International Development Agency (DANIDA); some of the results of these studies are also presented and discussed.

The format of the workshop consisted of data presentations by the co-operating investigators, which are included in this report in the form of short papers. Protein requirements data from research groups in Berkeley, California; Cambridge, Massachusetts; Tokushima, Japan; and Taipei, Taiwan; and data from China on energy expenditures associated with various kinds of activity are also presented. These discussions and presentations of data are followed by a comparative tabulation and analysis of the nitrogen-balance data reported.

Following these plenary discussions, four working groups were established to answer a series of critical questions and to identify urgent research needs concerning (a) protein requirements for adults, (b) energy requirements for adults and the relationship of protein and energy, (c) protein requirements for children, and (d) energy requirements for children and the relationship of protein and energy. Each of the working groups' reports attempts to answer the questions posed in the light of the newly available data and indicates further research needs.

As indicated, this report describes research carried out on the basis of recommendations of the Costa Rica workshop referred to above to determine the amounts of protein in usual diets required for nitrogen balance and, in the case of children, for growth as well, as measured by short-term, multi-level nitrogen-balance studies. The Cambridge workshop, in turns, recommended not only obtaining additional data of this type but also other types of studies. One of these is to administer the amount of protein considered to represent the mean plus two standard deviations of intercept data in a large enough number of subjects to test the hypothesis that this is sufficient for nearly all other populations. The second type of additional study recommended is the administration of this level of protein intake to a small number of subjects for periods as long as two to three months. Both types of studies are being supported by the UN University, and the results will be made available in a workshop to be held in advance of the October 1981 FAO/WHO/UNU Consultative Group Meeting on Protein-Energy Requirements.

Comments, criticisms, and additional data relevant to the issues raised in this report should be addressed to the UNU World Hunger Programme, Cambridge office, 20A-201, MIT, Cambridge, Mass. 02139, USA, so that they can be taken into account in the planned future international meetings concerned with protein-energy requirements.

Nevin S. Scrimshaw
Senior Adviser
World Hunger Programme
The United Nations University

Statistical considerations in the estimation of protein requirements

Precise estimation of the amount of protein that human populations require is too complex and too general a problem to be defined, much less accomplished, with our present understanding of human nutrition. The more specific problem of how to estimate the needs of a specific fraction of a specific population for protein derived from a diet of known composition is, however, a problem that can be explored.

From a statistical point of view there are four distinct steps to reach such an estimate:

a. measurement of the nitrogen content of a sample;
b. determination of the nitrogen balance of an individual receiving a constant dietary intake;
c. estimation of the response of an individual to varying levels of a specific protein;
d. estimation of the fraction of a population that would be in positive nitrogen balance at various levels of a specific protein.

In order to interpret the data presented at this workshop, it is necessary to consider the uncertainties at each of these steps.

1. Measurement of Nitrogen Content

Determining the nitrogen content of a sample is a complex procedure, with various potential sources of error. The uncertainty of the resultant value of nitrogen content can be considered to be composed of two different types of error. The first is the error of the method itself as carried out by a specific laboratory. This can be estimated by the reproducibility of results within a laboratory; results run one time can be compared with results run at another time. Second, methods may differ, and laboratories need to be compared with each other to see whether different results indicate different phenomena or just different procedures.

2. Determination of Nitrogen Balance

Nitrogen balance is defined as the difference between nitrogen intake and output. Nitrogen output is, in turn, calculated as the sum of the nitrogen contained in urine and faeces and that of miscellaneous losses, primarily sweat, all usually expressed as per kilogram of body weight:

Nbal = I_N-(U_N + F_N + M_N)

Since the components of output appear to be uncorrelated, for a constant intake the variances of balance will be simply the sum of the variances of the individual quantities.

V (Nbal) = V (i_N) + V (U_N) + V (F_N) + V (M_N)

The observed variability of balance data can be usefully partitioned into measurement error (mentioned above) and inherent biological variability. The day-to-day variability of nitrogen balance has been explored by us using long-term studies of individuals fed a constant intake of protein. Most importantly, we found no pattern of variability over time. Other investigators have suggested the existence of longterm cycles in the data (i.e., that urinary nitrogen losses are serially correlated); however, we are unable to reproduce these findings.

Since many of the data regarding the response to constant levels of nitrogen intake are taken from experiments in which the individual changed from one level or pattern of nitrogen intake to another, the short-term adaptation period is very important. Our investigations, which are consistent with those of other workers in the field, suggest that within five to seven days individuals reach a steady state, or at least a state that cannot be discriminated from a new steady state.

3. Individual Response

Knowledge of how an individual responds to a single level of intake of a specific protein is only moderately useful-it tells us merely whether that level will or will not fulfil an individual's needs for nitrogen. If, however, an individual's balance is measured at several different levels of intake, these data can be used to describe the individual's general response to that particular protein and permit an estimation of the particular level necessary for that individual.

Fundamental to this is the concept of a function that relates nitrogen balance to nitrogen intake. Since balance directly includes intake, it is better to consider the relationship between output and intake. While we do not know the true form of this function, we can approximate it by a straight line for a limited range of intakes; it is this line that we seek to estimate.

O_N =a+bI_N

For experiments that follow the standard UNU protocol we have, for each individual, at each of the n levels of intake (I₁, ..., I_N), a single faecal determination and five urinary determinations.

(F_i, U_i1, U_i2, U _i3, U_i4, U_i5) i = 1, ..., u (no. of levels)

If faecal nitrogen does not vary over the ranges examined, faecal averages can be calculated:

For the urinary data the replicates at each level of intake can be used to calculate a mean and variance of urinary output at each level:

These values can then be used to estimate a urinary response curve by means of weighted linear regression, with each point being weighted by the reciprocal of its variance.

a_u = (1/n) S (I _i /S_j² ) - b_u(1/n)SU_i/S_i²

These estimates of urinary and faecal response are combined with an estimate of miscellaneous losses (usually 5 mg N/kg) to give a function representing nitrogen output for that individual.

O_N = (a_u+ F + 5) + b_u I_N

For an estimate of the amount of nitrogen that that individual would require (from that dietary source), the point at which intake would balance output is calculated. Note that this is our mean estimate for each specific individual.

I_R=(a_u+F+5)/(1 -b_u)

4. Population Requirement

The next step is to put individuals together to estimate a population response to a specific test protein. Here the direct approach is to use the estimates of the individual responses as a sample of the population response. This suggests use of average values and standard deviation, and this is what we do.

There are, however, two statistical constraints to this approach. The first is that use of the arithmetic mean to describe a population assumes a single population with no anomalous individuals, or outliers. If a population consists mainly of normal individuals but contains a few individuals who differ fundamentally, then more robust statistical techniques are necessary. Perhaps the simplest of these is the trimmed mean, where a fixed percentage of the high and low data is routinely discarded, resulting in a somewhat lower estimate of the variability. While this is a standard way of dealing with this sort of problem, we cannot really recommend it, since it requires the routine elimination of data that are expensive and difficult to obtain and usually are valid.

The alternative, followed by many investigators including ourselves, is to eliminate data for subjects with apparent clinical problems and use all the rest. This has the potential problem of introducing an investigator's subjective judgement into the analysis. One needs to be very careful on this point, and the general problem of deviant responses needs attention. Those subjects with anomalous responses need to be carefully examined and retested to determine the data's reproducibility; in addition, larger samples need to be examined to determine the actual extent of the outlier problem.

A second statistical constraint in the description of a population response comes up in the estimation of upper percentiles. Thus we are interested in determining the level of a test protein, or mixture of proteins, that would meet the requirements of a fixed fraction of population. While statistical methods do exist for estimating percentiles with confidence, such as calculating tolerance intervals, they have two problems. First, they require a Gaussian or normal distribution, or at least a distribution that is not too far from normal. Second, these techniques often result in unreasonably large values when based on data from small samples. The inherent problem is that we are trying to estimate the tail of a distribution when most of the data describe the central portion.

Practically, in relation to the assessment of protein quality, we use, as the best technique available, the estimated mean requirement plus an appropriate number of standard deviations. As a general caution, however, studies that involve relatively few subjects do not provide sufficient data to establish estimates of the extreme percentiles with confidence. We thus recommend that the mean of the requirement be used to estimate the mean population requirement and that two standard deviations above be considered as an estimate of the intake level that would suffice for 97.5 per cent of the population. Furthermore, as studies accumulate, consideration should be given to replacing the individual standard deviation by a pooled standard deviation.

A summary analysis of the nitrogen-balance data

Following the standard UNU protocol, six investigators conducted a total of 11 studies on adults. This presentation summarizes these studies.

Skin Losses

One investigator, P.C. Huang, measured skin losses of nitrogen in subjects consuming two separate diets. Analysis of his data (see table 1 ) shows that the two diets differed significantly in terms of the amount of nitrogen lost through the skin. Additionally, although skin losses did differ with different levels of intake, these differences were

TABLE 1. Skin Losses (mg N/kg/body weight)

Level	Diet
mg N/kg	Mixed (n=10)	Egg (n=7)
56	-	10.50
72	5.95	13.47
88	7.10	7.07
104	5.97	-
120	5.58	-
Mean:	6.15	10.35
subject S.D.	1.77	n.s.
measurement S.D.	0.75	2.75

S.D. = standard deviation.

TABLE 2. Faecal Losses (mg N/kg body weight)

Investigator	Diet	Levels of intake	N	Average faecal losses	Measurement S.D.
Inoue	Fish	56-104	6	12.06	1.03
	Mixed	56-104	8	13.2	0.82
	Soy	56-104	5	11.5	1.38
Huang	Egg	56-88	6	14.3	1.47
	Mixed	72-120	10	15.7	1.92
Bourges	Mixed	64-112	7	27.5	4.39
Fajardo	mixed (animal)	82-136	9	20.2	4.91
	mixed (vegetable)	72-152	5	-	5.90
Uauy	Egg	48-96	8	18.5	2.77
	Mixed	64-112	7	35.1	3.28

 

Faecal Losses

For nine of the ten studies for which faecal nitrogen values are available, there were no significant differences between those values for different levels of nitrogen intake (see table 2). The data of L. Fajardo obtained with a vegetable protein diet did indicate differences; this study examined six levels of intake from 72 to 152 mg N/kg. These results suggest that perhaps diets can be well characterized by faecal nitrogen losses. Furthermore, it appears that absorption measurements will vary with the level of intake.

Nitrogen Balance

Table 3 gives estimated means, standard deviations, and 97.5 per cent population requirements for the 11 studies. Note that the pooled coefficient of variation is 18 per cent. Table 4 shows the results of a series of standard protocol experiments conducted at MIT. Table 5 shows all these data arranged by diet.

 

TABLE 3. Nitrogen Balance (mg N/kg body weight)

Investigator	Diet	N	Mean	S.D.	C.V.1	'97.5 2,3	'97 5 4
Inoue	fish	8	101.0	15.7	15.6	132.5	137.6
	mixed	8	106.4	24.0	22.6	154.5	145.0
	soy isolate (Supro 620)	5	129.6	14.1	10.9	157.9	176.6
Huang	egg	7	93.5	15.8	19.0	129.0	127.4
	mixed	15	127.5	25.9	20.3	179.3	173.7
Tontisirin	egg	13	123.6	17.1	13.8	157.7	168.4
Bourges	mixed	8	121.4	27.2	22.4	175.8	165.4
Fajardo	vegetable	8	138.6	22.3	16.1	183.2	188.8
	animal	11	131.7	27.4	20.8	186.5	179.4
Uauy	egg	8	98.7	8.2	8.3	115.1	134.5
	mixed	7	133.4	27.5	20.6	188.4	182.4

 

Table 4 Summary of MIT Experiments

Investigator	Diet	No of subjects	Mean requirement (S.D.) (mg N/kg)	97.5% requirement (mg N/kg)
Fajardo	beef	7	83 (11)	113
	wheat	7	147 (58)	200
Puig	egg	7	92 (42)	125
	soy isolate1	8	111 (15)	151
Wayler	milk	7	93 (33)	126
	beef/soy isolate1	7	96 (35)	131
	beef	7	108 (20)	147
Wayler	milk	6	133 (49)	181
	soy isolate1	8	135 (28)	184
	soy isolate2	7	114 (36)	155
Uauy	wheat	6	135 (54)	184
Puig	beef	6	106 (19)	144

¹Supro 620 (Ralston-Purina).
²Supro 710 (Ralston-Purina).

TABLE 5. Experiments by Protein Source

		No of	Mean
Diet	Investigator	subjects	requirement
Egg	Huang	7	94 (16)
	Tontisirin	13	124 (26)
	Uauy	8	99 ( 8)
	Puig	7	92 (42)
Soy isolates	Inoue1	5	130 (14)
	Puig1	8	111 (15)
	Wayler1	8	135 (28)
	Wayler2	7	114 (36)
Milk	Wayler	7	93 (33)
	Wayler	6	133 (49)
Beef	Fajardo	7	83 (11)
	Puig	6	106 (19)
	Wayler	7	108 (20)
Wheat	Fajardo	7	147 (58)
	Uauy	6	135 (54)
'`Local"	Inoue	8	106 (24)
	Huang	15	128 (26)
	Bourges	8	121 (27)
	Uauy	7	133 (28)
	Fajardo	8	139 122)
	Fajardo	11	132 (27)
Other	Inoue (fish)	8	101 (16)
	Wayler (beef/soy isolate)	7	96 (35)

¹ Supro 620.
² Supro 710.

TABLE 6. Repeated Subjects

Investigator		Measurement	Standard deviations
			Between diets	Between subjects	Measurement
Inoue:	fish	faecal nitrogen
	vs.	(mg/kg)	1.6	1.9	0.9
	mixed	slope of
		response	n.s.	n.s.	0.18
		mean
		requirement
		(mg/kg)	n.s.	n.s.	16.5
Uauy:	egg	faecal nitrogen
	vs.	(mg/kg)	16.9	2.7	2.8
	mixed	slope of
		response	n.s.	n.s.	0.25
		mean
		requirement	34.1	n.s.	20.1

Paired Experiments

Two investigators, R. Uauy and G. Inoue, conducted experiments comparing two different diets using the same individuals. For these experiments, between-subject variability can be separated from withinsubject variability. The results are shown in table 6. These results suggest (a) that efficiency measures (slopes of response curves) are too variable to be of value, and (b) that the errors in determining mean requirement levels are so large (~ 20 mg/N) as to permit discrimination only between very different dietary proteins.

Protein requirements for adults

The working group on protein requirements for adults was asked to address the following major questions:

1. An important factor in the utilization of food proteins, especially of plant compared with animal sources, is their digestibility. What differences in faecal nitrogen exist between populations with low protein intakes corresponding to those used to measure obligatory losses at requirement levels, and populations with habitual levels of protein intake from different food sources?

2. What variation in nitrogen digestibility occurs with different diets and levels of dietary fibre in relation to various hygienic factors?

3 How should requirements, expressed as egg or milk protein, be adjusted for other protein sources and diets (i.e., of different nutritional quality)?

4 What is the significance of zero-balance intercepts for different diets and populations? What effect does inclusion of integumentary losses have on estimates of nitrogen balance and of protein requirements?

5. What are the effects of acute and chronic infections and of other stress situations?

6. What long-term indicators of dietary protein and energy adequacy are appropriate?

7. What reference protein source is appropriate for establishing requirements in adults?

The task force responded to these questions as follows.

1. Faecal Nitrogen

Mean values for endogenous faecal nitrogen loss reported by various groups range from 8 to 16 mg N/kg/day. It was thought that two factors might explain this variation.

First is the time at which measurements were made after initiation of the experimental, low-protein or protein-free diet, although the available data are not sufficiently extensive to assess the quantitative importance of this variable.

Second, the ingredient composition of the experimental diet may also influence the output of endogenous faecal nitrogen. Thus, in order to resolve the issue as to whether differences exist between specific population groups, a highly standardized diet would have to be used. However, a resolution of this problem would not be expected to contribute importantly to the further refinement of estimations of adult protein needs.

The limited data available do not suggest marked differences in faecal nitrogen between the groups of adults studied. There appears to be little need, therefore, to generate additional data on obligatory faecal nitrogen losses for the purpose of refining estimates of the protein requirement.

It has been customary to consider faecal nitrogen losses of individuals after brief adaptation to a nitrogen-free, but otherwise adequate, diet as representing obligatory losses; the latter are subtracted from faecal losses in calculating "true digestibility." There is no reason, however, for making such a correction when the object of the study is to determine the total amount of ingested nitrogen necessary to reach a defined level of body nitrogen balance. Moreover, there is no evidence to support the assumption that obligatory faecal losses measured on a nitrogen-free diet are representative of metabolic faecal nitrogen losses when the protein content of the diet approximates an intake in the requirement range. On the contrary, there are reasons to believe that this assumption is invalid.

Measuring the nitrogen balance at various levels of intake of usual diets avoids the problem. In other words, when attempts are made to determine the amount of dietary protein required for long-term nitrogen equilibrium, separate estimation of endogenous faecal nitrogen losses is unnecessary.

At requirement levels of intake with highly digestible protein sources and refined diets, faecal nitrogen output approximates 20 per cent of total nitrogen output and increases to about 35 per cent for diets containing predominantly vegetable protein sources. The available data from the current UNU sponsored studies, together with previous data from the Institute of Nutrition of Central America and Panama (INCAP) and other data from developed countries, suggest that there are minimal differences in mean faecal nitrogen losses between different groups of adults when highly digestible animal protein foods serve as the major source of protein intake. However, for diets based on vegetable protein sources, there appear to be larger differences in faecal nitrogen output both within and among the experimental populations comprising subjects from developed and developing regions. Among the possible dietary factors are kinds of protein (animal, mixed, or predominantly plant sources), levels of dietary fibre, presence of inhibitors of digestive enzyme activity, such as polyphenolic compounds, and the structural organization of the foods. In addition, the population differences observed may be due to the higher faecal nitrogen outputs arising from chronic, subclinical pathophysiological changes in gastrointestinal function and metabolism.

At habitual levels of protein and food intake that may provide protein at levels in excess of requirements, differences among populations may not be readily distinguishable, but there are no adequate data to evaluate this point.

2. Variation in Nitrogen Digestibility

As to variation in nitrogen digestibility with different types of diet, comparison among apparently healthy populations consuming the same diet indicates that differences in digestibility are due largely to differences in health, especially the reduced absorption due to what has been described as "tropical jejunitis" 11*). There is little evidence to indicate long-term and favourable adaptive responses of the gastrointestinal tract to poorly digestible diets by individuals or different ethnic groups. Furthermore, there is no good evidence that favourable adaptations in nitrogen digestion and absorption occur in response to conditions that initially result in poor nitrogen digestibility. Improvements over time can be achieved by appropriate health interventions or by upgrading the sanitary conditions of the environment.

3. Adjustment of Requirements for Various Protein Sources

The adjustment of requirements expressed as egg or milk protein for other protein sources and diets is necessary, according to data presented at this meeting. With diets based on mixed protein sources that include small amounts (10-25 per cent) of high quality protein, differences in protein requirements, relative to milk or egg as a reference, appear to be due mainly to differences in digestibility and absorption of ingested proteins. This observation underscores the importance of including digestibility, as proposed by the 1975 Consultative Group (2), in adjustments of protein quality based on a chemical score procedure.

The data presented at this meeting indicate that the mean requirement for a protein source of nutritional value equal to that of egg or milk protein is approximately 0.6 g/kg/day, or a value 30 per cent greater than that proposed in the 1973 FAD/WHO report (3).

An observation made in a number of these studies was that the efficiency of nitrogen utilization is low at very low protein intakes, in contrast to the experience reported in the literature (4) based on individual protein sources, such as egg or milk, and on studies in healthy Caucasian and Oriental subjects. This difference in the observations may reflect various factors: (a) an experimental design problem associated with rates of adaptation of nitrogen metabolism to different nitrogen intakes, and (b) biological and metabolic factors related to balance and utilization of amino acids. If this observation is biologically significant it will have important implications for protein nutritional status where people consume diets low in protein either on occasion or for more frequent or extended periods.

The problem of further exploring the rate of adjustment in nitrogen utilization to changes in source and level of nitrogen intake is not only a matter of significance in experimental design, but also possibly a problem that has importance in relation to the meal and daily pattern of protein intake to maintain an adequate protein nutritional status.

In reference to adults, an overview of the data at the meeting supports the appropriateness of correcting requirements for egg or milk to other protein sources of lower quality; this correction then applies to both adults and healthy, normally growing children. Protein quality adjustment factors must be developed and validated under test protein levels that approach maintenance requirements.

4. Zero-Balance Intercepts

For the measurement of zero nitrogen balance intercepts, three test levels are inadequate and four test levels represent a minimally acceptable design. In reference to zero nitrogen balance intercepts, estimates of balance should always include a specified level of integumentary loss, the level depending upon the particular environment under which the nitrogen balances were obtained. No data were presented at the meeting to substantiate the concept that there are quantitatively significant differences in protein requirements among genetically distinct groups of healthy individuals of comparable age and body composition.

5. Effects of Infections

Mild to moderate parasitic infestations of the gastrointestinal tract with helminths did not reveal unfavourable effects on nitrogen balance and utilization for generous protein intakes. The group felt that there is a continuing need to examine this issue in further studies involving levels of protein intakes that approximate requirements and to include other infestations, such as Giardia.

6. Long-term Indicators

The group agreed strongly that nitrogen-balance measurements do not provide a definitive evaluation of long-term protein requirements. There is an urgent need to examine the metabolic and health significance of nitrogen-balance data in critical detail.

In order to achieve this goal the most critical need is to define better the physiology of human protein and amino acid metabolism and its relationship to current measures of requirements, physiological function, and health status. This requires further basic research, specifically directed toward improving the definition of human protein and amino acid requirements and the factors that affect them.

This question must include exploration of the design of experimental protocols for assessing protein and amino acid requirements. The group considered that multiple approaches are necessary to arrive at a definite statement on these requirements, and that highly controlled metabolic studies involving both shorter- and longer-term diet periods play an important role in reaching this goal. However, the ultimate definition will require studies of populations living under natural environmental conditions, and these studies must incorporate improved measurements of food intake, as well as of the nutritional and health status of the members of the population.

Choice of Reference Protein

The question was raised by the drafting group as to whether there was evidence indicating a more adequate reference protein than milk or egg. This question was addressed, in part, because various investigators at the meeting have observed a relatively large variation in nitrogen-balance data when using egg, compared with other sources of protein tested. Although the group did not re-examine the data in specific detail, it was their tentative conclusion that there is no justification for recommending high quality protein sources other than egg, milk, or lean beef as substitute reference proteins. A good quality soy-protein product might be considered as an alternative reference protein, but more comparative data from other investigators and laboratories would be required to examine this issue.

It was the recommendation of the group that a description of the reference protein used in metabolic studies should be given and based, in part, on results obtained with standard chemical and animal bioassay procedures. The importance of a reference protein in human metabolic studies is analagous to the use of a standard in analytical chemistry. However, this does not imply that concurrent studies with a reference protein are always necessary in each series of human metabolic experiments.

Energy requirements for adults and energy-protein relationships

The working group on energy requirements for adults and energy-protein relationships was asked to address the following major questions:

1. What is the effect of various factors on the digestion and absorption of dietary energy?
2. What is the consequence of energy deficits for population groups; of seasonal variation in energy requirements; and of adaptation to chronic or seasonal energy deficits?
3. What is the influence of dietary energy on protein metabolism and nitrogen balance?
4. What is the influence of dietary protein on energy metabolism and nitrogen balance?
5. What is the significance of protein-energy ratios of bulk, energy density, and the fat content of diets?

1. Host Effects

Genetic: It is impossible, in metabolic studies, to separate the effects of genetic and environmental factors on variation in nitrogen and energy absorption. It may be possible, however, by studying different populations under similar conditions, to obtain information on this point, although this is doubtful and has not been achieved to date. This comment applies to protein. When it comes to differences in digestible energy, there is little doubt that differences in lactose hydrolysis associated with differences in intestinal lactase activity have a genetic basis.

While the net effect on digestible energy is small, because lactase-deficient individuals are not likely to have milk as a major energy source in their diet, it would be prudent to be alert for other genetic differences in utilization of energy sources within or among different populations. The fact that low lactase levels can also be induced by environmental factors does not change the preceding argument. It does suggest, however, that, where milk is used as a sole source of protein in experimental studies, this factor should be considered in the interpretation of the data.

Intestinal parasites: Data were presented at the meeting (5) to suggest that mild-tomoderate infections with intestinal helminths have minimal or no detectable effects on nitrogen and energy digestion and absorption. Severe infections, however, do measurably reduce protein absorption, and in some populations where the burden of intestinal helminths is heavy this can have public health significance. A similar general statement cannot be made for intestinal protozoa. Those not associated with the production of diarrhea probably have little effect, although impaired absorption of nitrogen has been found with Giardia infections (M. Gupta, unpublished INCAP data).

The most important infections for most developing-country populations are those associated with acute and chronic diarrhoea. Since the effect is so variable, depending on the nature, frequency, and severity of the diarrhoea, it is difficult at present to give any quantitative estimates of the consequence of diarrhoea infections on nitrogen digestibility and absorption in actual "field" situations.

2. Energy Deficits in Population Groups

Appropriateness of dietary energy standards: It is not possible to make valid global recommendations for mean energy requirements. It must be assumed that, for adult populations who are maintaining body weight and composition, energy intake and energy expenditure are in balance in spite of wide variations in individual intakes. The consequences for pregnant and lactating women, however, may be lower birth weight babies that experience greater morbidity and mortality during infancy and reduced breastmilk output during lactation.

For adults and children an adaptation of great significance is a reduction in physical activity, although growth may also be affected. However, the observation that most populations in developing countries are consuming considerably less than current FAD/WHO estimated mean calorie requirements does not necessarily mean that they are deficient in calories. It may indicate that the current requirement estimations are too high, or that the individuals have been forced to reduce their physical exertion for work, recreation, or social organization.

The biological and social consequences of low energy intakes may be partly mitigated by metabolic alterations and by altering patterns of activity, both resulting in greater efficiency of utilization of dietary energy. To some extent, individuals or societies may have adapted to a lower availability of dietary energy, and a sudden increase in dietary energy would not necessarily be wholly beneficial. For example, it might lead to a greater prevalence of obesity.

Seasonal affects: Even though long-term adaptation to low dietary energy intakes may occur, as evidenced by survival of a population, this may conceal important seasonal effects. Under the conditions prevailing among lower socio-economic groups of a number of developing countries, individuals, particularly women, experience a significant loss of weight during the season of the year when food is most scarce and costly, and regain it when the harvest season arrives (6).

This has two particularly serious adverse consequences. First, women who are in late stages of pregnancy during the adverse season have infants with lower birth weights and higher infant morbidity and mortality. Second, there may also be an adverse effect on lactation performance. Also, periods of food shortage often come just at the time when there is a need for considerable energy expenditure in agricultural labour, hence a resulting weight loss. However, under experimental conditions, change in energy intake may have significant effects on nitrogen retention. It is clear that it is extremely important to adjust the energy intake of studies designed to determine protein requirements to one that is appropriate for the subjects and not associated with any long-range change in body weight or composition. Since the latter cannot be determined precisely in periods of less than a month, this means that definitive studies to establish protein requirements must be conducted over relatively long periods of time.

3. Effect of Dietary Protein on Energy Requirements

It is known that, with an energy intake that is borderline or adequate, an increase in protein intake can result in weight gain. The functional implications of this are not understood. It does appear, however, that energy requirements for maintenance of weight and body composition may be less when dietary protein is adequate.

4. Significance of Protein-Energy Ratios: Bulk, Energy Density, and Fat Content of Diets

The protein-energy ratio of a diet has been proposed as a means of evaluating the diet's ability to meet human protein needs when consumed at levels sufficient to meet energy requirements. Unfortunately, since energy requirements of individuals and populations depend on physical, biological, and social factors in the environment, no single set of protein-energy ratios can have general validity.

A further reason why use of protein-energy ratios in diets must be approached with great caution is the lack of any fixed association between relative protein and energy requirements. In fact, the environmental circumstances-physical, biological, and social-that require an adaptation to low dietary energy intakes are likely to be associated with factors such as infections, parasites, and low digestibility of diets that lead to increased protein requirements compared with those of more privileged populations. It is particularly hazardous to assume that protein-energy ratios calculated for populations have any significance for individuals because of this disassociation between energy and protein requirements.

It is still valuable to know the concentration of each in the diet relative to the amount consumed or consumable if the distribution of protein and energy requirements for a specific population is known. It will sometimes be evident that the percentage of protein relative to energy in a diet is grossly inadequate to meet protein needs of specified groups even if enough of the diet could be consumed to meet energy needs. This is particularly likely to occur for young children during periods of recovery from severe clinical malnutrition and/or infection, when the percentage of protein calories required is further increased (7). The low protein-energy ratios of diets consisting mainly of a cereal or cassava as the major energy source must be improved by either legumes or a source of animal protein.

5. Constraints Due to Dietary Bulk for Energy and/or Protein Density

It is not necessary to define or specify a protein-energy ratio in order to examine the adequacy to meet the needs of specific target groups of the protein and energy concentrations of diets relative to their bulk. However, it is necessary to determine whether enough of the diet can be consumed to meet energy needs and whether, if this is the case, protein needs are also met. It is not uncommon for underprivileged, vulnerable groups in the developing countries to be consuming habitual diets in which sheer bulk and energy density are constraints.

It is possible that when cassava, starch, or sugar becomes a major component of the diet, protein density is a constraint. Diets consisting almost entirely of cereals are likely to be inadequate in both protein and energy density. As indicated above, children recovering from severe malnutrition and/or infection are particularly vulnerable to such constraints, and it may often be necessary to add fat to the diet to provide sufficient energy density, or a more concentrated source of protein, or both. This means that an improvement in the quality and not just the quantity of the habitual diet is required.

Protein requirements for children

The working group on protein requirements for children was asked to address the following major questions:

1. An important factor in the utilization of food proteins, especially of plant compared with animal sources, is their digestibility. What differences in faecal nitrogen exist between populations with low protein intakes corresponding to those used to measure obligatory losses of requirement levels, and populations with habitual levels of protein intake from different food sources?

2. What is the significance of zero-balance intercepts; the use of nitrogen retention required for normal growth; and the use of integumentary losses in nitrogen-balance calculations?

3. What are the effects of acute and chronic infections and of other stress situations?

4. How do these affect catch-up growth?

Standardization of Methodology

The drafting group members were conscious of a number of limitations in the investigative approach used in the UNU-sponsored research and agreed that it should be supplemented by research following the guidelines for protein requirement studies on children devised by a 1977 FAO/WHO Expert Consultation (18).

Selection of Subjects

In principle, children should be representative of the general population from which they come, but this was rarely the case. At INCAP, they were all children who had previously been severely malnourished. The children who were studied in the Philippines were mostly mildly undernourished at the time of the study, the majority being below 90 per cent weight/height.

For future investigations it is recommended that the children selected for this type of study be representative of the segment of the population about whom there is concern. Children without obvious morbidity should be selected and, preferably, they should never have been severely malnourished. If, on initial selection, they are below 90 per cent weight/height, they should be rehabilitated for at least four weeks before the commencement of the study. Unpublished INCAP data show that it takes as long as 30 days for the creatinine-height indices to return to normal, even though weight/ height has achieved normality.

While frequently the investigators in the studies carried out so far had little option but to work with the subjects they did, in the future greater efforts might be made to obtain a more representative selection of the target population.

Energy Intake

Ideally, the customary energy intake of each individual child should be determined over a representative period of time, perhaps one month, and then the nitrogenbalance procedure should be carried out at that child's established intake. The standardization of energy intake levels used left much to be desired. In the studies carried out using the multiple protein level approach, either the energy intake was fixed at the 1973 WHO/FAD theoretical average requirement for that age or it was ad libitum based on fixed protein-to-energy ratios. In this latter situation, children who had low energy intakes also had low protein intakes.

We must recognize that these inconsistencies represent a shortcoming of experimental design. In the studies by Intengan, for example, the energy intakes were up to 10-20 per cent above the assumed physiological requirement because the ad libitum intakes were measured during recovery from mild to moderate malnutrition. The data must be interpreted in the light of these facts. In spite of this, however, it must be recognized that the INCAP team observed that a 10 per cent energy intake reduction at a fixed protein intake below the WHO/FAD safe level did not affect nitrogen balance in children. The same would appear to be true of the studies carried out by Tontisirin.

Energy Content of Food

Another potential source of error is the assumed energy content of food. It may be inadequate to use food composition tables to define the energy content of the diet, particularly for those based on traditional foods. What is important is the available energy content of food. It is recommended that in future studies the energy content of food and faeces should be measured directly by bomb calorimetry. If necessary, suitably prepared samples should be sent to a regional reference laboratory for this purpose.

General Health Status

The UNU collaborative study represents a mixture of children. Some were infested with intestinal parasites, although usually only mildly or moderately so. The experience of Ju, as described at the meeting, has clearly been that a low level of Ascaris and Trichuris infestation does not affect nitrogen balance. Even though infections with these worms are endemic to most developing countries, it appears unnecessary to deworm such subjects, since this would not significantly affect requirements. The influence of Giardia on nitrogen balance, however, deserves more detailed study. Apart from worm infestations, the UNU research subjects were apparently free of clinically manifest signs of infection that might have influenced the validity of the resuIts.

Protein Characteristics of the Dietary Proteins Tested

In some of the studies presented, information was not given on the amino acid composition of the diets used. Such information should be obtained, and this would facilitate comparisons among the studies from the different research centres. Food table values for amino acid compositions are rarely adequate, and it is recommended that in future studies of this type a reference laboratory should be available for the conduct of amino acid analyses. In the subsequent published accounts of the present studies a concerted attempt should be made to make amino acid data available for comparative purposes, in spite of the difficulties and limitations involved.

Physical Activity

Physical activity can also influence the efficiency of dietary nitrogen utilization. Energy output should therefore be standardized. Play facilities should be provided during the investigation and the child should be encouraged to use them. The use of "metabolic beds" should therefore be avoided whenever possible. One or two studies in the UNU project could have been influenced by a lack of attention to this detail.

Number of Levels of Protein to Be Fed for Balance Studies

The drafting group was concerned about the statistical procedures adopted for the analysis of the children's nitrogen-balance regressions. While the pooled regression analysis gives an adequate estimate of the mean nitrogen intake needed for a given level of balance, it does not provide a precise estimate of variation in requirements between individuals. We recommend that the present data be recalculated using regressions for individual children and that future studies be planned in such a way that they can be analysed on this basis.

The use of individual rather than pooled regression analysis necessitates at least four and preferably more levels of nitrogen intake. These should be fed around the estimated level for normal growth plus and minus 20 per cent. Lower or higher intakes would serve no practical purpose.

It was considered premature to make any comments on nitrogen requirements either on the apparent mean or on variance until the data are supplemented with those from additional studies under way and then reanalysed. However, there appears to be a remarkable similarity among most of the data from the different centres.

Integumental Nitrogen Losses

Such information on integumental nitrogen losses is essential for the true interpretation of nitrogenbalance data. The UNU data present, for the first time, nitrogen balance information on children between one and three years of age from two cultures, Taiwan and Guatemala. These studies were done under conditions where there was no overt sweating. With protein intakes of 1.4-3 g/kg/day, integumental losses were virtually the same (6-9 mg N/kg/day). Interestingly, and confirming previous adult findings, lower protein intakes yielded slightly lower integumental losses. With 0.5 9 protein/kg/day, it was 6-7 mg N, and with a nitrogen free diet it was 5 mg N/kg/day. It is recommended, in the interpretation of nitrogen-balance studies on children who are not sweating, that 8 mg N/kg/day be incorporated into the calculation of nitrogen balance. For studies carried out under circumstances where environmental temperatures are such that sweating is more profuse, the extra integumental nitrogen losses are unlikely to produce a significant error in the balance calculations.

Exclusion of Anomalous Data

The decision to use individual regression equations will ensure the detection of individuals producing anomalous patterns of nitrogen retention relative to intake. When the results are biologically unrealistic (for example, a negative regression coefficient) for the relationship between nitrogen intake and balance, the data for that individual should be discarded. In less clear-cut cases, it is recommended that objective decisions be made based on accepted statistical methods. If it is possible to repeat the nitrogen study on a given individual, this should be done.

While the exclusion of data always represents a difficult decision, it is essential to deal with this problem so that the calculation of requirements does not result in unrealistically high values. When data have been excluded in the calculation of nitrogen requirements for the pooled multi-centre studies, this should be clearly stated and the specific reasons given.

Zero-Balance Intercept and Growth

For the non-pregnant, non-lactating adult the zero-balance intercept (Bo), after taking into account the integumental losses, can be used as an indicator of the mean nitrogen requirements. In the case of the growing child, the adequate level of nitrogen intake should be one that allows the child to grow at an acceptable rate.

It was recommended that the present data be analysed by the individual regression approach to produce two values, Bo, the nitrogen for maintenance nitrogen balance, and Bg, the nitrogen intake to allow for daily growth corresponding to the mean annual growth rate at the 50th centile for a given size. Bo is largely of academic interest, but on a per kilogram basis the data would seem to indicate that it is the same for children as adults. For population groups, the relevant value for children is Bg.

At the level of the individual, however, growth, even in healthy children, is by no means a uniform process (7). Variations in growth rate up to five times the 50th centile are commonplace. At these times, more protein would be needed. It is impossible, at the present time, to predict exactly how much more is needed since changes in physiological efficiency are a distinct possibility. Furthermore, this variable growth rate limits the value of protein requirement estimates based on data observed from short-term nitrogen balance studies.

Valuable data would accrue if healthy children in different countries were followed longitudinally to determine precisely the natural fluctuations in growth and the extent to which they are paralleled by fluctuations in food intake. Special attention should also be placed on the voluntary selection of foods and the resulting different nutrient intakes to see whether or not there is a "hunger" for individual nutrients as well as for energy.

Determination of Obligatory Nitrogen Loss in Children

Three studies were presented at the meeting concerning obligatory nitrogen losses on diets containing very low amounts of protein. There was good agreement as to faecal nitrogen losses, but a discrepancy in the urinary nitrogen. The two INCAP studies suggested 34 mg N/kg/day as the obligatory loss, whereas the Taiwan data suggested 54 mg N/kg/day. A possible explanation lies in methodological differences; the diets in the Taiwan study provided 12-28 mg N/kg/day, whereas the INCAP diets were virtually nitrogen-free. Another possible explanation was that the Taiwan data were based on pooled collections covering days four to ten, whereas the daily analyses performed in the INCAP studies suggested that day four was too early to be included in the calculation of obligatory nitrogen losses.

Regardless of this unresolved problem, we do not recommend that new studies be initiated. Obligatory nitrogen losses are of value for calculating protein requirements using the factorial approach and for estimating true digestibility and BV data for protein foods, but are not needed for requirement estimates based on the zero-balance intercept approach. Moreover, ethical problems make further studies of this type undesirable, even though there were no ill effects to the subjects as a consequence of the low-protein intake in the studies presented.

Suggestions for Future Investigative Approaches

Laboratory and metabolic ward approaches are invaluable, but are inevitably of relatively short-term duration and can never accurately reproduce normal living circumstances. To be truly sure that a given level of energy or a nutrient is adequate, long-term studies are essential.

Two approaches suggest themselves. One would be on free-living children for whom food intake would be measured sequentially over an extended period of time in conjunction with measurement of growth, body composition, function, and basic well-being. The aim would be to determine, under prevailing circumstances, the level and quality of food that is compatible with health and with appropriate physiological and psychological performance. This approach cannot identify minimum requirements. In certain circumstances, food intake could be approached by relevant modifications of intake.

The second approach is a natural extension and would apply to circumstances where one had a more complete control over children's food intake, such as in child welfare institutions. Here apparent safe levels for short-term metabolic studies could be introduced and tested on a long-term basis. As an alternative to multilevel studies in a few individuals, a single level of dietary protein intake considered adequate could be given to a large number of individuals to observe the distribution of their nitrogenbalance response. If appropriate for most normal individuals, negative nitrogen balance responses should be rare but might occur occasionally.

The drafting group suggests that relevant indicators of overall health and well-being in studies of dietary protein adequacy should include:

(a) weight and height growth;
(b) assessments of lean body mass and adiposity, using techniques appropriate to technical facilities available; and
(c) the immune response.

Energy requirements for children and energy-protein relationships

The working group on energy requirements for children and energy-protein relationships was asked to address the following major questions:

1. What is the effect of various factors on the digestion and absorption of dietary energy?
2. What is the consequence of energy deficits for population groups; of seasonal variation in energy requirements; and of adaptation to chronic or seasonal energy deficits?
3. What is the influence of dietary energy on protein metabolism and nitrogen balance?
4. What is the influence of dietary protein on energy metabolism and nitrogen balance?
5. What is the significance of protein-energy ratios of bulk, energy density, and the fat content of diets?

The Energy Requirement of Children

1. The evidence from recent studies in Thailand and at INCAP shows that, by the classical criteria of weight gain and nitrogen balance, a net intake (measured by bomb calorimetry) of about 90 kcal/kg is adequate for children 2-3 years old.

It appears from these studies that net intake, determined by bomb calorimetry, is about 10 per cent lower than the calculated intake based on the Atwater factors (see table 1). If the FAD/WHO (8) estimated requirement of 101 kcal/kg at 1-3 years is reduced by 10 per cent, it becomes identical with the figure of 90 kcal/kg obtained by direct measurement. The consideration of net energy intake is essential if one is to compare energy intake with expenditure. When comparisons of energy intake are being made with requirement recommendation figures, or with other energy intake figures, both should be expressed in the same terms, that is, both based on Atwater factors. However, further work is needed on the comparison of conventional measurements of intake with those obtained by bomb calorimetry of food and faeces.

2. The criteria used for estimating energy requirements need to be critically reviewed. "Adequate" growth is usually assessed by reference to western standards. These may be inappropriate, not so much because of possible genetic variations in growth potential but because the average western child could be somewhat overweight.

Even in the most carefully conducted trials, the relation between nitrogen retention and weight gain appears to be quite variable. This can be explained partly by variations in the amount of fat and lean tissue deposited. It has been shown that children on the same diet do differ in the composition of tissue laid down (9). As a result, the expectation expressed at the 1977 FAD/WHO (8) meeting, that nitrogen balance might be the most sensitive criterion of the adequacy of energy intake, seems not to be substantiated by these findings.

TABLE 1. Energy Requirements and Nitrogen Balance: Mean Daily Protein and Energy Intakes

Protein (g/kg/day)
Theoretical intake: 1.75	Actual intake: 1.73	Absorbed:* 1.14
Energy (kcal/kg/day)
Theoretical intake		120	110	100	92	83
Gross intake**		118	106	99	91	81
Net intake***		106	96	90	82	71

Source: B. Tord F. Viteri, unpublished INCAP data, 1980.
0 Coefficients of variability between 3 and 5 per cent.
* "True" N digestibility = 66 9 per cent.
** Gross intake measure by bomb calorimetry.
*** Net intake = gross intake-faecal energy (bomb calorimetry).

There also seems to be little justification for the suggestion (3) that for nitrogen retention to be considered "adequate" in children 1-3 years old it should be at least 70 mg/kg/day. The expected retention for growth at this age would be more nearly in the range of 15-25 mg/kg/day. Substantial variation from day to day is to be expected; what matters is that the average retention over a period should reach the level indicated.

3. Almost important criterion of the adequacy of energy intake is that it should support a "satisfactory" level of physical activity. There is evidence from one sutdy (INCAP) that the initial response to a fall in energy intake is a decrease in expenditure rather than in growth or nitrogen retention. Physical activity promotes not only skeletal growth but also interaction with the environment and hence stimulates mental development. Estimates of physical activity are not easy when it is measured, and the definition of what is "satisfactory" must be subjective. Nevertheless, observations of physical activity should be regarded as essential in all future studies of this kind, and a decrease in physical activity below "normal" should be avoided.

4. The rate of linear growth is, for many purposes, a better measure of the nutritional state of young children than the rate of gain in body weight. This criterion should be used whenever possible in future studies of the adequacy of energy intakes, but a longer period of observation will be necessary and it must be certain that protein is not a limiting factor. Thought needs to be given to other possible criteria, particularly those that measure outcome over an extended period.

5. It is clear from measurements of food intake that throughout the third world the energy intakes of young children frequently fall below the estimated requirement. A common estimated intake would be 75 kcal/kg/day (calculated), representing a deficit of about 25 per cent. The most obvious manifestations of this deficit are reductions in weight for height and in physical activity.

A commonly observed pattern of change in developing-country populations is that growth in weight-forheight begins to fall at 3-6 months, reaches a minimum at 12-18 months, and then returns toward normal by three years, leaving a child who is stunted but not necessarily underweight.

Weight for height is usually assessed in relation to western standards, and some percentage of the median standard is commonly taken as a cut-off point separating those who require action ("malnourished") from those who do not require action ("normal" or mildly undernourished). A cut-off point of this kind needs to be related to measures of risk-of death, morbidity, or impairment of function, A start has been made in several centres on the assessment of risk, but much more needs to be done. When weight deficits are being related to morbidity, it is essential to separate children into age groups, because the risks associated with a given deficit vary with age.

6. In all parts of the world there appear to be seasonal variations in growth. In poor countries these may be attributed to variations in food intake and in disease transmission; in rich countries the cause is not clear. It may, perhaps, be a difference in the level of physical activity. These seasonal effects must obviously be taken into account in comparative studies.

7. One of the most important questions for the future is the significance of a child's adaptation to deficits in energy intake. Adaptation is difficult to define, and this term has been used with a number of different meanings. Nevertheless, the general concept is an essential component of modern thinking in biology and nutrition.

One form of adaptation to shortage in food supply is a decrease in growth. It has been suggested, for example, that stunting is an adaptive change. Another is a decrease in physical activity. A third is a change in the efficiency of the utilization of food for metabolic and mechanical work. In all these cases the balance of advantage and disadvantage of the adaptive change to the well-being of the individual should be considered.

Energy-Protein Relationship

Energy and protein metabolism are closely interrelated. Several short-term (10-day) studies have shown that the intake of protein for zero nitrogen retention decreases with increasing energy intake, not only when the starting level is at deficient energy intake levels but also when intake reaches excess levels. A long-term study in Thailand (40 days) failed to show a decrease of nitrogen retention with decreasing energy intakes to levels considered 10 per cent below requirements and associated with lower energy expenditure and rate of weight gain. It would appear from present evidence that long-term studies are warranted that are based on energy intake and activity level immediately preceding the study.

On the other hand, a reduction of protein intake while energy intake remains constant can reduce the rate of growth in terms of weight and height even when nitrogen balance is not negative. The mechanisms for this effect are not clear. Apart from the specific dynamic action of proteins, a high protein intake does not appear to affect energy requirements.

Effects of Energy Intake on Physical Activity and Growth

Apathy and low physical activity have been observed in children of populations where energy deficiency is prevalent. Recently, quantitative estimates of physical activity and energy expenditure of children on energy intakes below estimated requirements have shown proportional decrements in the activity component of energy expenditure.

Moreover, such decrement takes place rapidly (within a week) in one- to three-year" old children when energy intake is reduced experimentally to levels slightly below estimated energy requirements. Energy conservation by a reduction in activity thus appears to be a response to deficient energy intakes in children as well as in adults.

Recent studies in animals and children in rapid growth phases and receiving sub optimal energy intakes have shown that a programme of enforced physical activity is associated with increased linear and lean body mass growth when compared with pair-fed but less active animals and children (10). These findings suggest that physical activity is necessary for adequate growth and that it enhances the efficiency of energy and/or protein utilization. They also point out the desirability of the maintenance of physical activity in children in metabolic studies aimed at exploring dietary energy protein interrelations, particularly when growth is considered a dependent variable. This has not been considered in most past studies.

Significance of Protein-Energy Ratios: Bulk, Energy Density, and Fat Contents of Diets

From all available evidence, a protein concentration above 7.5 per cent of calories when corrected for quality appears not to bring additional benefits to healthy children. For children recovering from protein energy malnutrition, infection, or other stress, this proportion should be higher, but probably need not be beyond 12 per cent. The lower limit for this value of corrected protein-energy intake should be above 5 per cent for healthy pre-school children. This should be kept in mind in attempts to increase the energy density of diets based on cereal-legume mixtures.

Studies on pre-school children fed five times a day on a free choice of corn and bean preparations common in their habitual diets have proven that these foods can be consumed in amounts that fulfil safe protein intakes, but that energy intake was inadequate and resulted in poor growth (weight) gains. An increase in fat calories from 8 per cent to near 20 per cent, and energy density from 4.5 to 4.9 kcals/g food (9 per cent increase), overcame both the energy intake deficit and the poor weight gains. Further addition of fat brought no additional improvement.

Addition of fat to infant and pre-school-child food preparations not only increases the energy density but also facilitates the swallowing of the solids and porridges that otherwise may be too gelatinous [agglutinated) for easy consumption by young children. This, and probably bulk, impair the capacity of cereal-legume mixtures, without fat added in appropriate amounts, to fulfil energy requirements.

Bulk-energy density-fat interrelationships in cereal-legume diets require very active research, including consideration of population beliefs and practices about infant feeding of such foods.

Infection and Catch-up Growth

Infection leads on the one hand to anorexia and decreased food intake, and on the other to losses of energy, nitrogen, and other body components. The extent of these changes is highly variable according to the nutritional status of the host and the severity and type of disease. In order to determine the effect of infections on the requirement for energy and nutrients, the magnitude of these losses must be known. In view of the range of variation, it will be difficult to draw realistic general conclusions from direct measurements on small numbers of children under closely controlled metabolic ward conditions.

A promising approach, which needs to be more widely pursued, is to establish the relationship under field conditions, and on a population basis, among the three variables: frequency, severity, and duration of the infection; food intake; and growth. It should then be possible to estimate the extent to which, on average, infections contribute to growth deficit, and hence to calculate the extra intake of energy and protein needed to make good the deficits, i.e., for catch-up. A beginning has been made with this kind of study in the Gambia, Peru, and Guatemala.

Catch-up

When one attempts to calculate the requirements for catch-up 17) two points emerge. First, relatively more protein is needed for weight gain (assuming normal balanced tissue) than for maintenance; therefore the protein-energy ratio in the diet needs to be somewhat higher than normal (the exact value will depend on the rate of catch-up aimed at). Second, catch-up growth obviously requires an increased intake of both protein and energy, but the increase is probably four to five times greater for protein than for energy. Quantitative estimates of the intakes needed for various rates of catch-up growth were tabulated in a previous UNU report 17). These estimates were based on the assumption that tissue of balanced composition is being laid down, and on observed values for the energy cost of weight gain 111). Studies on children recovering from malnutrition in hospital have shown that these estimates are realistic. For example, a net intake of 150 kcal and 3.5 9 protein/kg/day (P:E ratio about 10 per cent) will, support rates of weight gain of up to 10 g/kg/day.

In practice, the main difficulty in securing intakes adequate for catch-up growth is that even the child whose appetite is good may be physic ally unable to eat the necessary quantity of food. In this situation, the nutrient density of the food and the extent to which it is glutinous or easily swallowed become matters of great importance.

Research under metabolic ward conditions has been, and continues to be, essential in the efforts to define as accurately as possible protein and energy requirements and the mechanisms involved. Upon seclusion in metabolic wards, however, the inevitable changes that take place in living habits and very often in levels and type of energy and protein intakes, as well as the limits of time of study imposed by these conditions, make it very difficult to translate the data into "real-life, natural" conditions and into "safe levels of intake" for different population groups.

Research should be directed to develop techniques and evaluate methodology that would allow investigators to conduct field research in protein and energy nutrition as closely as possible to the accuracy and precision of metabolic ward conditions. This should allow the scientific community to obtain quantitative information on an adequate number of subjects under free-living conditions.

A series of population groups in different food and ambient ecological settings whose intakes are considered low could then be selected. A series of measurements could be made, aimed at defining their characteristics in terms of protein and energy nutrition. if the indicators used for this purpose indicate "normal population behaviour," one could try to identify populations with even greater intake deficits, if available.

In any case, the proof that energy and/or protein intakes are not limiting should be ascertained by means of nutrition interventions. The choice of indicators to detect the normal or abnormal behaviour of population groups is critical. Measurements of good health and adequate performance were proposed, such as rate of linear growth for age, lean body mass and muscle mass for age, low morbidity, milk production, birth weight, physical work capacity and fitness, and immune responses.

(introduction...)

References

The chemical energy in foodstuffs, manifested by the liberation of heat during combustion, is used primarily for doing work (internal and external work). External work is that performed by the body in its environment; internal work is mechanical and chemical Synthesis of compounds in reactions that would not proceed spontaneously, transport of ion against electrochemical gradients, etc.) In the steady state, i.e., when there is no change in chemical composition and body mass, all the internal work performed is dissipated as heat as prescribed by the first law of thermodynamics.

Energy utilization should be taken to mean the amount of work (external and internal) performed in or by the body by a unit change (decrease) in body energy. The latter change is equal to the heat output in thermal steady state. The relation between the two is subject to the restriction imposed by the second law of thermodynamics, which can be expressed as DH = G-TDS, where the entropy term, TDS, is almost always so small that it can be neglected. The change in Gibbs free energy, DG, is equal to the maximum work that can be harvested in the process. This amount of work can never be realized lit is the theoretical case where all processes proceed reversibly). The actual work performed is always less than the maximum work and depends on the degree of coupling that the body can make between the spontaneous chemical reactions and the work Synthesis. ion pumping, muscular contraction, etc.). The efficiency of the process is then given by the ratio of the actual work done to the maximum possible work.

Although the energy input to the body can always be measured, the total amount of work performed by the body cannot. Therefore, the efficiency of energy utilization cannot be measured. However, it is possible to imagine an experiment in which changes in efficiency can be measured. If measurements of energy expenditure are made on the same individual, during two situations identical in total work (i.e., a given "regime" or "programme"), then any change in energy expenditure directly reflects a change in efficiency of energy utilization.

References

1. "Tropical Jejunitis (Tropical Enteropathy): Morphology, Specificity," session 11 in l.H, Rosenberg and N.S. Scrimshaw, eds., "Malabsorption and Malnutrition," A.J. Clin. Nutr., 25: 1080-1102 (Oct.-Nov. 1972).

2. Consultative Group Meeting on Energy and Protein Requirements, "Recommendations by a Joint FAO/WHO Informal Gathering of Experts," Food and Nutrition (FAO), 1 (2): 11-19 (1975).

3. Energy and Protein Requirements: Report of a Joint FAO/WHO Ad Hoc Expert Committee, WHO Tech. Rep. Ser. 522 (World Health Organization, Geneva, 1973).

4. V.R. Young and N.S. Scrimshaw, "Nutrition Evaluation of Proteins and Protein Requirements," in M. Milner, N.S. Scrimshaw, and D.l.C. Wang, eds., Protein Resources and Technology (Avi Publishing Company,Westport, Conn., USA, 1978), pp. 136-173.

5. Jin Soon Ju, W.l. Hwang, T.G. Ryu, and S.H. Oh, "Protein Absorption of Adult Men with Intestinal Helminthic Parasites," pp.131-138 below.

6. R.G. Whitehead, "Infant Feeding Practices and the Development of Malnutrition in Rural Gambia," Food and Nutrition Bulletin, 1 (4): 36 41 (Aug. 1979).

7. F. E. Viteri, R.G. Whitehead, and V.R. Young, eds., Protein-Energy Requirements under Conditions Prevailing in Developing Countries: Current Knowledge and Research Needs (WHTR-1/UNUP-18, The United Nations University, Tokyo, 1979).

8. "Protein and Energy Requirements: A Joint FAO/WHO Memorandum-An Informal Consultation Held at the Food and Agriculture Organization of the United Nations, Rome, 1977,"Bul/. Wld. Hlth. Org., 57 (1): 65-79 (1979).

9. G.G. Graham, A. Cordano, R.M. Blizzard, and D.B. Cheek, "Infantile Malnutrition: Changes in Body Composition during Rehabilitation," Rediat Res., 3: 579-589 (1969).

10. B. Tor. Schutz, R. Bradfield, and F.E. Viteri, "Effect of Physical Activity upon Growth of Children Recovering from Protein-Calorie Malnutrition," in H. Koishi, Y. Yasumoto, K. Iwai, M. Kanamori, Y. Muto, and T. Oanaka, eds., The Tenth International Nutrition Congress (Victory-sha Press, Kyoto, Japan, 1976), pp. 247-249.

11. D.W. Spady, P.R. Payne, D. Picou, and J.C. Wateriow, "Energy Balance during Recovery from Mainutrition," Am. J. Clin. Nutr., 29: 1073-1088 (1976).

(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.

(introduction...)

Objectives
Experimental details
Conclusions and comments

E. Vargas and R. Bressani
Institute of Nutrition of Central America and Panama (INCAP), Guatemala City, Guatemala

Objectives

To evaluate the protein quality of rice-and bean diets.
To determine the effect of supplementary animal protein and energy density on the quality of rice-andbean diets.
To estimate the protein and amino acid needs of normal adult individuals fed such diets.

Experimental details

1. Subjects Number: ten per experimental run.
Experimental runs: four.
Age: 20 to 31 years.
Sex: male.
Racial origin: Maya Indian and Spanish.

TABLE 1. Ascending Protein Intake Sequence

Protein intake	Calorie intake
level (g/kg/day)	level (g/kg/day)	No. of days	Diet fed
0.6	45 & 50	6	Regular diet
0.0	45 & 50	3	Nitrogen-free diet
0.2	45 & 50	2	Rice and bean*
0.4	45 & 50	2	Rice and bean
0.6	45 & 50	2	Rice and bean

*Rice and bean (60:40 protein distribution) in study I with 45 kcal/kg/day; in study 2 with 50 kcal/kg/days; in study 3 with 10 per cent milk protein substitution with 45 kcal/kg/day and in study 4 with 10 per cent milk protein substitution with 50 kcal/kg/day.

Physiological status: normal.

Nutritional status: acceptable-weight 49.1 to 65.0 kg; height 157 to 172 cm.

Health status: free of chronic infections and free of intestinal parasites.

2. Study Environment Location:
Metabolic Unit of the Division of Food Science, INCAP. Climatic characteristics: temperature 21 to 25 C (day); relative humidity 77 to 85 per cent; altitude 1,470 m above sea level; months of September to December 1979.

3. Physical Activity Normal
(laboratory technicians and institution maintenance crew).

4. Duration of Study
Total time per Study nine days.

Procedure: short-term, multiple-point nitrogen-balance assay.

Protein intake changes: every two days, as indicated in table 1.

TABLE 2. Nitrogen-Free Diet Composition per Assay

Food	1	2	3	4	Calories from each ingredient (kcal)
Instant coffee, g	3	3	3	3	3
White sugar, g	25	25	25	25	100
Apple or pineapple marmalade, g	30	30	30	30	78
Wheat starch bread,1 g	300	300	300	300	801
Margarine, g	80	80	80	80	576
Cornstarch soup²	480	480	480	480	144
Vegetable (Guisquil), g	100	100	100	100	52
Apple (with peel), g	200	200	200	200	116
Artifical fruit-flavoured drink (glasses)	3	3	3	3	228
Cookies (units)³	2	1	1	1	100/unit
Carbonated drinks (units)	1	-	1	-	136
Vitamin/mineral supplement (pills) 4	1	1	1	1	-
Total calories	2,434	2,198	2,334	2,198

1,3 Prepared from wheat starch (Jolly Joan, Ener-G Foods, Inc., P.O. Box 24723, Seattle, Washington 98124, USA).
2 Prepared from cornstarch and margarine, and seasoned with aromatic herbs. Herbs were not consumed
4 UNIT TMR.

5. Diets Table 2 describes the ingredient composition of the basal diet fed (nitrogen-free diet), and table 3 shows the protein content of the rice and beans used in the four studies. Table 3 also describes the vitamin and mineral composition.

The protein and energy intake from rice and beans was calculated every two days. It was given to the subjects in three equal portions, or during lunch or dinner. Large batches of raw rice and beans were purchased to reduce the variability that could be caused by differences in quality. The beans were cooked in large lots by soaking for 14 to 16 hours followed by cooking at 151bs/inch2 (1.05 kg/cm2) for 30 minutes. The material was then stored frozen. Rice was steamed.

TABLE 3. Protein Content of Protein Sources

Food	Moisture	Dry matter	Protein (N x 6.25)
	(%)	(%)	(%)*
Rice	68.5	31.5	2.68
Beans	76.1	23.9	7.38
Basal diet (nitrogen-free diet)	76.4	23.6	0.35

*Fresh basis.

6. Measurements and indicators
Composition of diets: AOAC methods of proximate chemical analysis.

Digestibility: apparent and true.

Nitrogen balance: apparent-does not include other losses.

Indicators of protein quality and utilization: (a) linear regression analysis (y = a + bx) of nitrogen intake (Nl) to nitrogen retention (NR) and of nitrogen absorption (NA) to NR; N i for N R = 0; (b) quadratic regression analysis (y = a + bx + cx²) of Nl to NR and of NA to NR, used primarily to estimate recommended protein intake as obtained by calculating the first derivative dy/dx = b + cx, where x (Nl or NA) is equal to-(b/2c).

Energy: digestibility and metabolizable energy at 0.6 9 protein intake and by measuring energy in food, faeces, and urine.

Other determinations and measurements: none.

Summary of Main Results Protein digestibility (apparent) of the rice and bean diet (60:40) was not affected by a difference in energy intake of 45 to 50 kcal (59.1 vs. 59.6 per cent). It was increased by the 10 per cent milk replacement of the protein in the rice and bean mixture without being affected by energy intake (65.3 and 64.6 per cent). The protein digestibility of milk was 75.6 per cent. Energy digestibility of the diets varied from 93.3 to 94.6 per cent, while metabolizable energy varied from 91.9 to 92.9 per cent.

The linear coefficient of regression between Nl and NR was not affected by energy intake without milk (0.75 and 0.79) or with milk (0.95 and 0.86), but milk supplementation improved it significantly, and was no different from milk alone (0.91). The Nl for NR = 0 followed the same trend. For 45 and 50 kcal/kg/day the values without milk were 95.6 and 92.9 mg/kg/day, and with milk, 78.4 and 80.8 mg/kg/day. The milk reference value used was 86.6 mg/kg/day. The relative coefficients of regression to milk equal to 100 per cent were 82.4, 86.8, 104.4, and 94.5 per cent.

The effect of milk was attributed to an improvement in protein digestibility rather than to an improvement in essential amino acid balances, as judged by amino acid content and by the linear regression coefficients of NA to NR, which were statistically alike (0.99, 1.02, 1.10, and 1.04 for diets, and 0.91 for milk). Using the quadratic regression equations, the protein intakes for maximum nitrogen retention were 0.79, 0.79, 0.69, and 0.74, with diets of better quality giving lower values. Correcting for differences in digestibility, ail values were similar, with an average of 74.8 mg N/kg/ day. This value was interpreted to represent the amount needed for all of the population to be in positive and maximal balance.

Conclusions and comments

1. The short term assay yields data calculated and interpreted in the same way as that from the longterm, conventional method. It is sensitive to differences in quality and reduces problems that may develop in subjects.

2. Rice and bean diets (60:40 protein ratio) are not improved by energy intake; however, replacement of 10 per cent of the protein by milk protein increases quality through an improvement in digestibility.

3. Although present interpretation of results from linear regression analysis provides useful data for estimating protein digestibility, protein quality, and intakes for maintenance, the quadratic regression analysis should be included as part of the whole analysis. This should be tested further.

(introduction...)

Objective
Experimental details
Summary of main results
Conclusions and comments

Ricardo Bressani, Delia A. Navarrete, Emilio Vargas, and Olivia Gutierrez
Division of Agricultural and Food Sciences, Institute of Nutrition of Central America and Panama (INCAP), Guatemala City, Guatemala

Objective

The staple foods for large population groups in Latin America are common beans (Phaseolus vulgaris) and maize or rice. The cereal grains are sometimes replaced by tubers (e.g.. plantain! or roots (e.g., cassava or yam). These studies were carried out to evaluate the protein nutritional quality of common beans mixed with starch, a cereal, or plantain, using a short-term, multi-level nitrogen-balance assay.

Experimental details

1. Subjects
Four sets of experiments were carried out. A total of 32 healthy young men volunteered for the study. They were laboratory technicians and maintenance employees. They were Spanish or mixed Spanish-Mayan Indian, from 23 to 35 years old, with weight ranges from 46.5 to 65.0 kg and heights from 157 to 172 cm. They did not have chronic infections or intestinal parasites.

2. Study Environment
The men lived in their homes in Guatemala City and worked at INCAP. All of their meals were eaten in the Division of Food Sciences Metabolic Unit. The daily ambient temperature ranged from 21° to 25° C in some studies and from 27° to 32° C during others. Relative humidity ranged from 72 to 85 per cent. Guatemala City is 1,510 m above sea level.

TABLE 1. Basal Diet Used in Human Metabolic Studies

Food	Amount (g)	kcal
Instant coffee	3	3
Sugar	25	100
Apple jelly	30	78
Toasted bread*	300	801
Margarine	80	576
Soup * *	480	144
Chayote	100	52
Apple	200	116
Artificially flavoured drink, glass	3	228
Starch cookies,*units	2	200
Carbonated beverage, bottle	1	136
Total energy	-	2,434

* Made from wheat starch only
** Made from cornstarch.

3. Physical Activity
All men performed their usual chores.

4. Duration of the Study
After subjects had consumed the standard protein diet for several days, a nineday experiment was carried out. During the first three days the men ate the diet described in table 1, which provided only between 20 and 30 mg N/kg/day. After that they ate the experimental diet in which protein content was changed every two days. The three two day periods of protein intake provided 0.2, 0.4, and 0.6 9 protein/kg/day. The details of this short-term, multiple nitrogen-balance assay have been described elsewhere (Bressani et al., in H.L. Wilcke, D.T. Hopkins, and D.H. Waggle, eds., Soy Protein and Human Nutrition [Academic Press, New York, 1979] ).

5. Experimental Diets
The four experimental diets were as follows:

TABLE 2. Regression Equation between Nitrogen Intake and Nitrogen Retained of Subjects on a Diet of Wheat Starch and Black Beans (Phaseolus vulgaris)

Subject	a 1 b1	Nitrogen intake for equilibrium (mg/kg/day)	r
1F-HM	-39.7 +0.32	124.1	0.56
2F-AF	-100.9+0.86	117.3	0.93
3F-GM	-50.6 + 0.47	107.6	0.71
4F-WH	-85.9 + 0.80	107.4	0.83
5F-SY	-35.9 + 0.30	119.7	0.99
6F-AS	-63.1 +0.62	101.8	0.96
Average	-62.6 + 0.55	113.8	0.75

1 NR=a+b(NI).

TABLE 3. Regression Equation between Nitrogen Intake and Nitrogen Retained of Subjects on a Diet of Tortilla and Black Beans (Phaseolus vulgaris)

Subjects	a1 b1	Nitrogen intake for equilibrium (mg/kg/day)	r
7T- FS	-89.5 + 0.94	95.2	0 96
8T-VO	-105.9 + 0.89	118.9	0.82
10T-AL	-83.5 + 0.91	91.7	0 96
11T-WS	-88.4 + 0.91	97.1	0 94
12T-EM	-80.6 + 0.95	84.8	0 99
Average	-86.9 + 0.89	97.6	0.89

1. Common beans with wheat starch were the main energy sources (6 men participated).
2. Corn tortillas and black beans. The maize-bean mixture was provided in proportions of 70:30 on a dry-weight basis (6 men participated).
3. Rice and common beans fed in a 60:40 ratio, based on protein content (10 men participated). d. Common beans and cooked, mature plantain, with black beans providing between 30 and 32 per cent of total energy (12 men participated).

1 NR=a+b(NI).

The initial low-nitrogen diet provided between 2,400 and 2,500 kcal/day. Energy intake was adjusted to 45 to 50 kcal/kg/day to meet each individual's energy needs and to allow him to maintain body weight throughout the experiment. A multivitamin and mineral tablet was provided each day. Water intake was maintained at a constant level for this study.

6. Indicators and Measurements
a. The composition of the diets was calculated with AOAC methods of proximate chemical analysis. b. Urine and faeces were collected as 24 hour pools. Faecal markers were used to separate faecal collections. c. The nitrogen contents of diets, urine, and faeces were determined using the macro Kjeldahl technique. Apparent nitrogen balance was calculated by subtracting urinary and faecal nitrogen from dietary nitrogen. Apparent digestibilties were calculated, not including obligatory faecal nitrogen losses,

Summary of main results

Tables 2 to 5 show the individual regression equations of apparent nitrogen balance on nitrogen intake. Table 6 shows the nitrogen intake required to obtain equilibrium and the amounts of foods in the diet that provided such nutrients. Table 7 shows the apparent digestibility of the proteins fed in various experiments.

Conclusions and comments

1. Larger amounts of beans are needed to obtain nitrogen equilibrium when they are eaten with starch or plantains than when they are eaten with cereal grains (see table 7). This is due both to the nitrogen contribution of the cereal grains and to the sulphur amino acids that they provide. The difference in the protein quality of the mixtures is demonstrated by the higher regression coefficients of the bean-andmaize or beanand-rice mixtures.

TABLE 4. Regression Equations between Nitrogen Intake and Nitrogen Retention of Subjects Fed Rice and Black Beans (Phaseolus vulgaris) in a 60:40 Protein Ratio

Subject	a1 b1	Nitrogen intake for equilibrium (mg/kg/day)	r
M.R.	- 92.7 + 0.76	121.5	0.92
R.A.	- 60.6 + 0.63	96.9	0.96
L.J.	- 65.9 + 0.81	81.8	0.96
F.M.	- 81.8 + 0.78	105.1	0.97
A.G.	- 76.8 + 0.79	96.9	0.95
C.E.	- 54.8 + 0.79	69.5	0.99
J.L.	-54.5 + 0.55	98.8	0.98
G.P.	- 70.1 +0.70	100.4	0.95
O.B.	- 84.8 + 0.92	92.3	0.96
R.C.	- 86.1 +0.87	98.6	0.92
Average	-71.6 +0.75	95.6	0.87

¹ NR-a+B(NI),

Protein digestibilities were low in the four studies, more so when the bean-and-plantain diet was used. The polyphenolic compounds in beans increase faecal nitrogen output.

Plantain also contains polyphenolic compounds that may add to the faecal nitrogen excretion.

2. More sulphur-containing amino acids are needed in the bean-paste diets eaten by populations who also consume starchy foods such as plantain or cassava. This does not seem to be so important in the diet of populations who consume cereal grains in addition to beans.

TABLE 5. Regression Equation between Nitrogen Intake and Nitrogen Retained of Subjects on a Diet of Plantains and Black Beans (Phaseolus vulgaris)

Subjects	a1 b1	Nitrogen intake for equilibrium (mg/kg/day)	r
11P-F.M.	- 121.7 + 1.20	101.4	0.91
12P-O.B.	- 54.0 + 0.47	114.9	0.82
13P-R.A.	- 75.6 + 0.70	108.0	0.99
14P-S.P.	- 66.9 + 0.53	126.2	0.76
15P-R.S.	- 53.4 + 0.34	1 57.0	0.73
16P-O. B.	--69.7 + 0.63	110.6	0.99
17P-H. R.	- 109.8 + 0.92	119.3	0.92
18P-M.R.	- 63.6 +0.29	219.3	0.87
19P-O.H.	- 126.3 + 1.51	83.6	0.85
20P-N. Ro.	- 75.3 + 0.64	117.6	0.96
Average	-81.7 + 0.73	111.9	0.77

¹ NR=a+b(NI).

TABLE 6. Nitrogen Intake for Nitrogen Equilibrium and Amounts of Foods Needed

Diet	Nitrogen intake for nitrogen equilibrium (mg/kg/day)	Beans/day		Other food/day
		dried wt. (g)	cooked wt. (g)	dried wt (g)	cooked wt. (g)
Beans/starch	114	186	638	-	-
Beans/plantain	112	185	636	-	855
Beans/maize	98	82	170	193	495
Beans/rice	95	52	194	281	802

TABLE 7. Apparent Protein Digestibility of Black Beans (Phaseolus vulgaris) Fed with Starchy Foods and Cereal Grains

Diet	Nitrogen intake (mg/kg/day)	Apparent protein digestibility (%)
Beans/starch	115.6 ± 0.9	60.0 ± 2.2
Beans/plantain	117.4 ± 0.6	52.5 ± 4.0
Beans/maize	127.6 ± 0.5	61.0 ± 9.0
Beans/rice	102.5 ± 1.1	59.1 ± 7.4

(introduction...)

Objective
Experimental details
Summary of main results
Conclusions and comments

Ricardo Uauy, Enrique Yz, Digna Ballester, Gladys Barrera, Ernesto Guzman, MarT. Saitnd Isabel Zacarias
Institute of Nutrition and Food Technology (INTA), University of Chile, Santiago, Chile

Objective

To obtain information on obligatory urinary and faecal nitrogen losses of Chilean men from a low socioeconomic background.

Experimental details

1. Subjects
Eight young, healthy men, 24 to 31 years old, who belonged to the low socio economic class in Chile. Their physical characteristics are given in table 1.

2. Study Environment
They slept in INTA's metabolic unit for the entire duration of the experiment, and they were asked to maintain their usual daily activities, refraining from unusual exercise. During the entire study the men were under the supervision of a physician and a nurse.

3. Diet
The usual protein intake of the subjects before the study was estimated to be about 1 g/kg/day, based on a 15-day dietary history and a prospective observation period.

The daily energy intake of each subject was calculated in the same way, also based on his caloric expenditure according to the pattern of his usual energy intake and activity.

 

TABLE 1. Physical Characteristics of the Subjects

Subject	Age (years)	Weight (kg)	Height (cm)	W/H* (%)	Energy intake
					Period 1			Period 2
					(kcal/ day)	(kcal/kg/ day)	(kcal/ day)		(kcal/kg/ day)
J.A.	26	55.0	161	90	2,800	51	3,500		64
J.B.	25	74.2	177	104	3,150	42	3,937		53
O.G.	25	61.7	174	89	2,950	48	3,687		60
S.L.	28	60.5	166	94	3,000	50	3,750		62
H.R.	25	61.8	171	91	2,800	45	3,500		57
E.R.	31	63.9	170	95	3,000	47	3,750		59
R.E.	31	69.9	169	104	3,000	43	3,750		54
N.A.	24	70.6	170	106	3,000	42	3,750		53
Mean	26.9	64.7	169.8	96	2,963	46	3,703		58
S.D.	2.8	6.3	4.8	6.9	116	4	145		4

* Relative to standards suggested by D.B. Jelliffe, The Assessment of the Nutritional Status of the Community (World Health Organization, Geneva, 1966).

Table 2 lists the components of the experimental diet, which provided less than 2 mg N/kg body weight/day. Two successive experimental periods were conducted, as shown in table 1. From day 1 through day 10 the subjects were fed the nitrogen-free diet at their estimated energy requirement level (period 1), and from day 11 through day 18 dietary energy was raised by 25 per cent. Three isoenergetic meals were provided at 8 a.m., 1 p.m., and 7 p.m. and consumed under the supervision of a dietitian. A vitamin and mineral supplement was given each day at lunch to meet or exceed the 1974 NAS/NRS Food and Nutrition Board Recommended Dietary Allowances. Supplements of calcium and zinc were also given.

4. Duration of the Study
Eighteen days on a nitrogen-free diet.

5. Indicators and Measurements
The men were weighed before breakfast each day after voiding and wearing minimal clothing. Complete 24-hour urine collections were made throughout the study.

TABLE 2. Composition of Protein-Free Diets Used for Study of Obligatory Nitrogen Losses

Ingredient	Period 1	Period 2
Sugar, g	102	112
Honey, g	30	55
Cornstarch, g	224	230
Margarine, g	68	83
Vegetable oil, g	103	150
Orange-flavoured beverage, g	30	50
Soup flavouring, g	2	4
Baking powder, g	8	7
Carbonated beverage, ml	414	414
Apple sauce, g	98	97
Alphacel, g	6	6
Water, ml	1,522	1,722
Vitamin/mineral supplement* Dietary energy (%)
CHO	50.2	44.5
Fat	49.6	55.3
Energy intake (kcal)	3,000	3,750

Food preparations: protein-free cookies; cornstarch soup; cornstarch bread; cornstarch dessert; apple sauce. The intake is given for a 63 kg subject.

* Vitamin/mineral supplement (Polyterra), Laboratories Pfizer de Chile, Santiago, Chile. One tablet supplies: vitamin A 5,000 I.U.; vitamin D 1,000 l.U.; thiamine 1 mg; riboflavin 2 ma; pyridoxine I mg; vitamin B_l2 2 mcg; ascorbic acid 50 mg; niacinamide 12 mg; Ca pantothenate 2 mg; copper (as CuO) 70 mg; iodine (Kl) 50 mcg; iron 1 mg; potassium- (Kl) 16 mcg; manganese (MnCO₃) 29 mcg; magnesium (MgO) 108 mcg; zinc (ZnO) 71 mcg. In addition, each subject received dairy a 15 mg zinc supplement, as zinc chloride, and a table of Calcium Sandoz Forte providing 500 mg of calcium.

Samples were collected in plastic bottles with 10 ml of 10 per cent (v/v) sulphuric acid. Each 24-hour collection was made up to 3,000 ml with distilled water and thoroughly mixed. Aliquots were analyzed immediately for total nitrogen, urea, and creatinine. Another sample was frozen for subsequent analysis. Faeces were collected daily in plastic containers and kept in a freezer until analysed. Composites were made for each subject from the faecal pools for the entire duration of each experimental period. Blood samples were drawn from an antecubital vein after an overnight fast of 12 hours on days 1, 10, and 18 and analysed for total serum protein, albumin, urea, aminotransferases, and complete blood count. Anthropometric measurements (height; body weight; waist, gluteal, and mid-upper arm circumferences; triceps skin fold and subscapular skin-fold) were obtained on days 1, 10, and 18.

6. Statistical Analyses The analysis of urinary obligatory nitrogen was made as suggested by Rand et al. (Am. J. Clin Nutr., 29: 639 [1976] ). A single exponential model was used:

y = P₁e-P₂t + P₃

where

y = urinary nitrogen excretion;
P₁ = difference between y at time 0 and at P₃;
P₂ = rate of change in nitrogen excretion;
P₃ = value at the asymptote of the curve; and
t = time in days.

In addition to P₁, P₂, and P₃, an additional index used was the time required for stabilization of P₃ (t _s). The time to stability is defined as the time taken for y to achieve a value not significantly different from P₃ ±1 S.D. The mathematical calculations were carried out at the INTA Biometrics Unit with the aid of a non-linear least squares fits programme, using the Marquardt algorithm from the Public Library of IBM System 370, APL language.

Summary of main results

1. Energy Intake
During period 1, the men received 46 i 4 kcal/kg/day, and during period 2, their energy intake was increased to 58 i 4 kcal/kg/day.

2. Anthropometry
Table 3 shows the anthropometric data obtained during the study. All subjects except one lost weight. The remaining anthropometric indices did not show significant changes.

3. Urinary Nitrogen Excretion
Figure 1 and tables 4A and 4B show the daily urinary nitrogen losses. It should be noted that subject E.R. on day 6 did not comply with the experimental protocol and exercised heavily. We eliminated the abnormally high value obtained that day and, for computational purposes, replaced it with the mean obtained from days 5 and 7. A kinetic evaluation of the data is presented in Table 4A. Subject S.L. did not reach stability by day 10 and his data were not included to calculate the mean parameters of the equations. The asymptotically derived urinary nitrogen loss (P3) after stability had been reached was 35.8 mg N/kg/day. The mean time to stabilitY (tS) was 6.5 days. Data from subject H.R. on day 15 and from E.R. on day 13 were not included in the pooled regression analyses and daily means because these abnormally elevated values corresponded to days on which the conditions of the experimental protocol were not fully met. The regression analysis of nitrogen loss versus days with excess energy showed a trend toward decline in nitrogen loss.

TABLE 3. Anthropometric Measurements of Subjects Fed a Nitrogen-Free Diet at Two Levels of Energy Intake

	Days
Variable	1	10	18
Height, cm	169.8 ±4.8*	169.8 ± 4.8	169.8 ± 4.8
Weight, kg	64.4 ± 6.4	63.9 ± 6.1	63.6 ± 6.4
Waist, cm	82.38±5.3	81.81 ±5.18	80.88±5.07
Gluteal circumference, cm	91.19 ±3.40	90.63 ± 3.68	89.88 ± 3.81
Mid-upper right arm circumference, cm	28.94 ±2.29	28.54 ± 2.34	28.19 ± 2.34
Mid-upper left arm circumference, cm	28.25 ±2.55	27.94 ± 2.44	27.69 ± 2.50
Right triceps skin-fold, mm	7.5 ± 2.5	7.5 ± 2.6	7.3 ± 2.6
Left triceps skin-fold, mm	7.6 ± 2.6	7.4 ± 2.6	7 3 ± 2.6
Right subscapular skin-fold, mm	11.9 ± 3.5	11.7 ± 3.4	11.5 ± 3.3
Left subscapular skin-fold, mm	11.8 ± 3.3	11.6 ± 3.2	11.4 ± 3.2

* Mean ± S.D.

4. Other Urinary Excretions
Table 5 shows the mean urinary creatinine, urea nitrogen, and total nitrogen lost daily during the last five days of each period.

TABLE 4A. Kinetic Analysis of Daily Nitrogen Excretion in Subjects Fed a Nitrogen-Free Diet for Ten Days. Dietary energy intake: 46 kcal/kg/day.

Day	J.A.	J.B.	O.G.	S.L.*	H.R.	E.R.	R.E.	N.A.	Mean ± S.D.
(mg N/kg/day)
1	115.7	134.4	100.8	92.2	117.7	161.0	82.5	94.6	115.2± 26.3
2	76.3	68.4	69.7	97.6	81.6	92.9	56.2	67.4	73.2 ± 11.7
3	53.2	54.6	44.5	57.9	69.3	74.3	44.4	54.4	56.4 ± 11.5
4	44.9	57.3	42.9	54.9	65.0	52.1	42.4	41.5	49.4 ± 9.0
5	41.6	47.3	31.0	56.3	43.9	77.3	37.8	44.1	46.1 ± 14.7
6	36.3	37.2	31.0	51.6	43.5	65.1*	32.5	34.0	40.0 ± 11.8
7	39.9	36.3	26.7	53.4	43.9	54.1	32.7	28.7	37.5 ± 9.5
8	29.8	32.4	32.7	54 4	45 7	54.1	27.4	33.9	36.6 ± 9.7
9	34.8	29.0	24.2	21.4	43.5	35.2	27.4	30.0	32.0 ± 6.4
10	31.6	33.8	25.2		65.0	33.9	27.4	27.4	34.9± 13.7
P1	159.0	229.5	135.8	92.0	136.8	244.4	93.3	106.9	158.0 ± 58.2
P2	0.6591	0.8488	0.5996	0.1500	0.6668	0.7885	0.5454	0.4819	0.6557±0.1295
P3	33.46	36.19	26.27	13.29	47.50	49.91	28.44		35.77 ± 9.47
S.D.	1.369	3.188	1.731	49.72	4.109	5.867	1.507	1.920
ts	7.2	5.0	7.3	*	5.3	4.7	7.6	8.3	6.5 ± 1.4

 

* Subject S,L did not reach a stable nitrogen loss during this period. His data were not included in the means. P1 and P3 are expressed in mg N/kg/day; P2 is given in days—1; ts stands for days needed for stability.

TABLE 4B, Daily Nitrogen Excretion in Subjects Fed a Nitrogen-Free Diet with Excess Energy. Dietary energy intake: 58 ± 4 kcal/kg/day.

Day	J.A.	J.B.	O.G.	S.L.	H.R.	E.R.	R.E.	N.A.	Mean ± S.D.
(mg N/kg/day)
11	36.6	35.1	31.1	42.7	48.3	51.8	27.6	32.7	38.2 ± 8.5
12	36.6	*	26.7	43.9	48.8	30.0	27.7	30.9	34.9 ± 8.5
13	35.1	32.6	23.7	57.8	42.3	90.7**	27.5	28.9	35.4 ± 11.5
14	33.5	30.3	29.5	46.4	50.3	46.1	31.6	28.9	37.1 ± 8.9
15	30.2	31.5	26.6	44.8	69.7**	46.0	30.3	31.6	34.4 ± 7.7
16	29.2	27.6	25.1	49.4	36.3	44.6	27 6	32.9	34.1 ± 8.8
17	35.4	26.3	20.8	31.0	25.6	29.7	26.3	31.5	28.3 ± 4.5
18	37.3	22.7	17.9	***	27.2	***	19.7	27.6	25.4 ± 7.0

* Sample was lost.
** Data not included in the calculation because they deviated from expected values. Regression equations for (a) the complete set of data, and (b) for days 15 through 18: (a) v = - 1.50x + 55.4, r = - 0.39, p < 0.01; (b) - 3.31 x +8.52, r = - 0.46, p < 0.05.
*** Subjects did not complete study

FIG. 1. Obligatory Nitrogen Excretion in Chilean Young Adult Males Fed Two Levels of Dietary Energy Intake

5. Faecal Nitrogen Excretion
Table 5 also shows the mean daily faecal nitrogen excretion for both experimental periods.

6. Measurements in Serum
Table 6 summarizes the biochemical measurements obtained initially and at the end of each period.

7. Factorial Calculation
Table 7 shows the factorial calculation of mean protein requirements. Adding 30 per cent for individual variability as suggested in 1973 by FAD/WHO, the safe level of protein intake would be 0.62 g/kg/day.

TABLE 5. Obligatory Nitrogen Losses in Young Male Subjects Fed a Protein-Free Diet at Two Levels of Energy Intake

	Days 6 to 10 46 ± 4 kcal/kg/day ( g/day )	Days 14 to 18 58 ± 4 kcal/kg/day (g/day )	Paired t test p<
Creatinine	1.33 + 0.25*	1.22 + 0.29	N.S.
Urea nitrogen	1.41 ± 0.68	1.54 ± 0.46	N.S.
Total urinary nitrogen	2.365 ± 0.477	2.107 ± 0.388	0.01
Faecal nitrogen	1.029 + 0.194**	0.562 + 0.141 ***	0.001

* Mean ± S.D.
** Faecal nitrogen losses for days 1 to 10.
*** Faecal nitrogen losses for days 11 to 18.

TABLE 6. Plasma Biochemical Measurements for Subjects Consuming a Nitrogen-Free Diet at Two Levels of Energy Intake

	Day of study			ANOVA
	1	10	18	P	L.S.D.*
Protein (g/dl)	7.4 ± 0.5	6.9 ± 0.5	6.9 ± 0.6	N.S.	-
Albumin (g/dl)	5.0 ± 0.3	4.6 ± 0.3	4.2 ± 0.3	<.001	0.61
Glucose (mg/dl)	81.4 ± 8.7	78.3 ± 8.5	86.6 ± 6.4	N.S.	-
Urea (mg/dl)	24.9 ± 5.2	10 2 ± 2.6	12.5 ± 3.9	< 001	7.7
Urea N (mg/dl)	11.6 ±3.4	4.7 ± 1.2	5.8 ± 1.8	< 001	3.6
SGOT (Karmen units/dl)	28.8 ± 11.0	23.1 ± 10.9	38.6 ± 11.0	<.05	11.4
SGPT (Karmen units/dl)	17.1 ± 10.2	13.9 ± 9.6	15.4 ± 14.5	N.S.	-
Creatinine (mg/dl)	0.65 ± 0.1	0.79 ± 0.1	0.72 ± 0.1	<.05	0.01
Triglycerides(mg/dl)	52.8±25.1	44.9±11.5	49.1 ±12.5	N.S.	-
Cholesterol (mg/dl)	174.9 ± 38.1	160.8 ± 42.2	134.6 ± 14.3	N.S.	-

* Least significant difference to the indicated p value.

TABLE 7. Factorial Nitrogen Losses for Young Males Consuming a Nitrogen-Free Diet

Subject	P3a	Urinary nitrogeb	Faecal nitrogenc	Total obligatory nitrogen lossesd	Correction factor 1.3
(mg N/kg/day)
J.A.	33.5	34.5	13.9	53.4	69.4
J.B.	36.2	33.7	16.0	54.7	71.1
O.G.	26.3	28.0	18.9	51.9	67.5
S.L.	13.3	45.0	14.0	64.0	83.2
H.R.	47.5	48.3	18.0	71.3	92.7
E.R.	49.9	48.5	20.2	73.7	95.8
R.E.	28.4	29.5	13.7	48.2	62.6
N.A.	28.6	30.8	13.7	49.5	64.4
Mean	35.8	36.2	16.1	58.4	75.8
S.D.	9.5	8.6	2.6	9.9	12.9
FAO/WHO (1973)		37	12	54	70

1. Urinary nitrogen loss (mp N/kg/day) after asymptotic stabilization,
2. Obligatory urinary nitrogen losses for days 6 to 10.
3. Obligatory faecal nitrogen losses for days 1 to 10.
4. Assuming 5 mg N/kg/day for integumental and miscellaneous losses.

Conclusions and comments

1. Our results coincide with those obtained in the US and Taiwan, indicating that obligatory nitrogen losses are independent of ethnic and environmental conditions.

2. The correction factor of 1.3 used in factorial calculations underestimates protein requirements, as indicated by our studies using multi-level nitrogen balance techniques (see Young et al., this volume)

3. The relatively higher obligatory faecal nitrogen losses suggest that endogenous nitrogen excretion may be increased in subjects from developing regions with poor environmental sanitation and chronic, subclinical intestinal mucosal damage. Prolongation of the nitrogen-free diet showed a decrease in faecal nitrogen of about 50 per cent. That decrease might have been due to a further decline in labile nitrogen or to changes in the gut flora that contributed to faecal nitrogen losses.

4. Our subjects had higher energy intakes than are customary in this type of study. The men with low weight/height indices had higher energy intakes per unit of body weight.

5. Weight losses may have been due to losses in lean body tissue, mainly muscle, as suggested by the drop in mean daily urinary creatinine excretion.

6. The excessive energy intake decreased the urinary nitrogen losses, especially during the last four days. The men had stable physical energy patterns. The effect of energy balance deficit can be anticipated to raise obligatory nitrogen losses. Nevertheless, the biological significance of excess energy appears to be minor.

Acknowledgements

The authors gratefully acknowledge Laboratories Pfizer de Chile for supplying the vitamin/mineral supplement (Polyterra) used in this experiment.

(introduction...)

Objective
Experimental details
Summary of main result
Conclusions and comments

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

Objective

To determine the obligatory nitrogen losses in healthy adult Thai males.

Experimental details

1. Subjects
Four male adult Thais 21 to 25 years old. Their body weights were 45 to 67.5 kg, and their heights were 164.5 to 169 cm. 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. The climatic characteristics were those of tropical countries.

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

4. Duration of the Study
The study of obligatory nitrogen losses was carried out over a 14-day period.

TABLE 1. Composition of the Protein-Free Diet

Ingredients	g/day
Mung bean starch	100.4
Mung bean noodles	100.0
Sugar	1 08.6
Margarine	48.0
Oil	30.0
Onion leaf	30.0
Wax gourd	27.0
Salt	5.1
Lemon juice	2.4
Soy sauce	2.0
Garlic	2.0
Carbonated beverage	variable

5. Diets
The composition of the protein-free meal is shown in table 1. The subjects were given four meals per day at 7.30 a.m., 12 noon, 5.00 p.m., and 9.30 p.m. Daily energy intakes were constant, at about 45 kcal/kg/day. Fat provided approximately 30 per cent of the daily energy intake. Multivitamin and mineral tablets were given twice each day. Water was offered ad libitum but the volume was recorded daily.

6. Indicators and Measurements

1. Total nitrogen in urine and faeces was measured by a calorimetric semi-automated procedure (Munro and Fleck, Mammalian Protein Metabolism, vol. 3 [1969]). Nitrogen balance was calculated from the last five and ten days of urinary and faecal nitrogen losses, respectively.
2. Basal metabolic rate (BMR) was measured at seven-day intervals with a respirometer (closed circuit).
3. Body weight was recorded daily.
4. Serum concentrations of total protein, albumin, urea nitrogen, and some amino acids and amino transferase activities were measured at seven-day intervals.

TABLE 2. Obligatory Nitrogen Losses in Four Adult Thai Males

Subject	U	F	S	Total
M.P.	38.6	15.1	5	58.7
V.D.	32.5	12.4	5	49 9
S. R.	33.4	12.4	5	50.9
S.S.	35.0	10.6	5	50.6
Mean ± S.D.	34.9 ± 2.7	12.6 ± 1.9	5 ± 10	52.5 ± 4.2

U = urinary nitrogen losses, average from the last 5 days of 14-day dietary period.
F = faecal nitrogen losses, average from the last 10 days.
S = skin and other minor route nitrogen losses, taken from FAD/WHO.

Summary of main result

1. Daily Obligatory Nitrogen Losses in Four Adult Thai Males
Table 2 shows the data for obligatory nitrogen losses of the subjects. The means of urinary and faecal nitrogen losses were similar to most studies done in other countries. Total obligatory nitrogen losses were calculated after allowing 5 mg N/kg/day for skin and other minor losses.

2. Plasma Amino Acids
Table 3 shows that the concentration of valine decreased, while concentrations of alanine and glycine increased on days 7 and 14. The ratios of non-essential/essential amino acids (NEA/EA ratio) and of glycine/valine increased.

3. Other Measurements
Table 3 also shows that BMR and blood urea nitrogen (BUN) were lower and ALT was higher by day 7. By day 14, all indicators shown in the table differed from initial values.

TABLE 3. Blood Chemistry and Basal Metabolic Rates of Four Adult Thai Males Eating a Protein-Free Diet for 14 Days

		Protein-free diet
Measurement	Initial value	day 7	day 14
BMR (kcal/m²/day)	1,134 ± 94	927 ± 261*	906 ± 129*
Total protein (g/dl)	7.5 ± 0.5	6.9 ± 0.9	6.9 ± 0.3*
Albumin (g/dl)	5.3 ± 0.1	5.2± 0.3	4.8 ± 0.3*
BUN (mg/dl)	10.3 ± 3.1	3.3 ± 1.0*	2.7 ± 0.7*
AST (sigma unit)	21.0 ± 3.4	24.0 ± 4.6	24.5 ± 3.8*
ALT (sigma unit)	17.6 ± 0.9	23.3 ± 3.0*	24.0 ± 0.9*
Valine (um/L)	258.5 ± 7.7	175.1 ± 30.8*	165.9 ± 18.6*
Alanine (um/L)	219.8 ± 61.4	696.9 ± 225.4*	846.0 ± 153.8*
Glycine (um/L)	299.7 ± 39.7	468.3 ± 15.0*	486.9 ± 31.8*
NEA/EA ratio	2.2 ± 0.1	3.4 ± 0.5*	3.3 ± 0.5*
Gly/Val ratio	1.2 ± 0.1	2.7 ± 0.4*	3.0 ± 0.5*

* Differs from initial value p < 0.05.

Conclusions and comments

1. The results of obligatory nitrogen losses in this study of adult Thai males were quite similar to those that have been reported on 4 Indian men, on 13 men studied at the University of California, on 9 Nigerian men, and in 83 studies on MIT students and 50 Chinese studies on males. It seems, therefore, that the obligatory urinary and faecal nitrogen excretion of young men, expressed as mg/kg/day, does not differ among ethnic groups.

2. Factorial calculation of safe levels of protein intake, using a correction factor of 1.3 and adding another 30 per cent for individual variability, as suggested in 1973 by FAO/WHO, results in 0.56 g/kg/day, which is almost identical to the safe level of intake recommended by FAD/WHO. That recommendation has recently been questioned for populations at large or for long-term studies. Our own studies using multi-level nitrogen balance techniques indicate that 0.56 9 protein/kg/day is too low (see Tontisirin et al., this volume).

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 the main results
Conclusions and comments

Jin Soon Ju, W.l. Hwang, T.G. Ryu, and S.H. Oh
Department of Nutrition and Biochemistry, Korea University College of Medicine, Seoul, Republic of Korea

Objective

The digestion and absorption of protein and energy may be affected by intestinal parasite infestation. The purpose of this study is to observe the effects of intestinal helminths on the protein absorption of adult men before and after deworming.

Experimental details

1. Subjects
Twenty men were selected after a faecal screening test on 305 farmers from an agricultural village in southwestern South Korea, about 240 km from Seoul. They were 20 to 45 years old, weighed 51.8 to 72.0 kg, and measured 155.8 to 176.6 cm in height. They were healthy and had no abnormalities except for the intestinal parasites, based on medical history, physical examination, and laboratory tests (blood cells, haemoglobin, haematocrit, serum transaminases, alkaline phosphatase, and urinalysis).

2. Study Environment
The study was performed during winter in a metabolic unit. Outdoor temperature:

-2° to-10°C; indoor 18° to 22 C.

3. Physical Activity
Before the study: heavy agricultural work; during the study: light exercise.

4. Duration of the Study
Twenty-eight days divided into three periods:

a. Experimental diet before deworming: 7 days.
b. Deworming and break, free choice of diet at home: 14 days.
c. Experimental diet after deworming: 7 days.

5. Diets
Daily dietary allowances were 50 to 60 kcal/kg and 1.2 to 1.5 9 protein/kg. The subjects were given an adequate level of calories based on individual diet history. Three different daily menus, based on local diets, were offered on alternate days (see table 1). All covered daily dietary requirements. Sometimes additional food intake, mainly rice, was allowed according to individual requests, and all food intake was recorded.

Although the total energy intake varied among the subjects, based on individual dietary history (50 to 60 kcal/kg/day), it was relatively constant for each man throughout the experimental feeding periods.

Mineral and vitamin supplements were given once a day to meet recommended dietary allowances.

Three isoenergetic, isonitrogenous meals were offered at 8.30 a.m., 1.00 p.m., and 5.30 p.m. and eaten under a dietitian's supervision. Table 2 shows the essential amino acid composition of the experimental diet.

6. Anti-holminthic Treatment
Combantrin (10 mg/kg) was used to treat those without hookworms, and Amidantol (Bayer Co.) (6 mg/kg) was given to those who had hookworms with or without ascaris. The drugs were given as a single dose on the first morning of the second experimental period. (See "Duration of the Study," above.)

7. Indicators and Measurements
a. Complete urine and faecal collections were made daily throughout the experimental feeding periods before and after deworming. Faecal samples were pooled the last four days of each experimental feeding period.

b. Total nitrogen (micro-Kjeldahl), urea nitrogen (Folin-Wu), and creatinine (diacetyl monoxime) were determined in urine. Nitrogen was also measured in faeces and food.

c. Fats (Soxhlet) and ash (combustion) in food and faeces were measured. Carbohydrates were calculated by subtracting protein, fat, ash, and moisture from total weight of food and faeces. The energy contents of diets and faeces were calculated using 4, 4, and 9 kcal per gram of carbohydrate, protein, and fat, respectively.

d. True nitrogen balance was calculated assuming integumental and miscellaneous losses of 5 mg N/kg/day. The true digestibility of protein was calculated using 12 mg N/kg/day for obligatory faecal losses.

TABLE 1. Foods Offered Each Day, in Grams

	Diets
Food	A*	B*	C*
Rice, 70% polished	750	750	750
Potato	-	-	50
Vegetables	500	580	520
Soybean curd	30	30	-
Soybean paste	25	40	-
Soy sauce	-	-	30
Fruits	90	90	-
Candy	27	-	27
Crackers	-	30	-
Egg	45	45	45
Beef	20	-	45
Pork	-	20	-
Fish	60	75	40
Vegetable oil	8	3	3
Sodium glutamate	3	3	3
Energy, kcal**	3,150	3,200	3,100
Protein, g, total***	88	93	83
animal	20	22	18
Fat, % of energy	25	25	20

* A: days 1.4,7; 8: days 2,5; C: days 3,6.
** 50 to 60 kcal/kg/day.
*** 1.2 to 1.5 g protein/kg/day.

TABLE 2. Essential Amino Acid Composition of Experimental Diet A (mg/g N)*

Amino Acid	FAD/WHO mg (S)	Diet mg (D)	D/S %
Isoleucine	40	41.2	103.0
Leucine	70	70.9	101.3
Lysine	55	52.1	94.7
Methionine + Cystine	35	31.6	90.3
Phenylalanine + Tyrosine	60	69.0	115.0
Threonine	40	35.3	88.3
Tryptophan	10	10.5	105.0
Valine	50	48.9	97.8
Total	360	359.5

* Total essential amino acid intake was about 18.7 g/day.

e. The apparent biological value (by) of protein was calculated as:

BV = [N intake-(N faecal + N urinary )]/N intake

and net protein utilization (NPU) was calculated as:

NPU = digestibility x biological value

f. Subjects were weighed each morning without clothes after voiding and before breakfast.

g. Venous blood samples were drawn before breakfast on the first and last days of each experimental dietary period. Haemoglobin, haematocrit, urea, creatinine, SOOT, SGPT, thymol turbidity, alkaline phosphatase, total proteins, albumin, and total and differential leukocyte counts were determined.

TABLE 3. Parasitological Observations

Group		Egg count per gram of faeces before treatment*	Worms expelled after treatment
A	Ascaris
	lumbricoides	2,600-12,500	2
	(n = 7)	(6,800)**	(6)
B	Ascaris +	Al: 0-1,800	1-4
	hookworm	(200)	(2)
	(n = 6)	Hw; 300-12,000	1-72
		(3,000)	(28)
C	Hookworm	300-4,400	5-21
	(n = 7)	(1,200)	(11)

* All men in group A, four men in group B, and four men in group C also had Trichuris trichiura (100 to 700 eggs/g faeces) One of the men in group C also had Taenia saginata.
** Mean.

h. Quantitative parasitological stool examinations were performed daily on the first three days of each experimental feeding period. The type and number of parasites expelled in the first three days after vermicidal treatment were recorded.

Summary of the main results

1. Parasitological Observations
Table 3 shows the ova counts before treatment and the number of helminths collected during the three days after treatment. Some men did not have ova in the initial stool examination, but expelled parasites after treatment. Fifteen men also had Trichuris trichiura and one of them had Taenia saginata.

After treatment all analyses were negative for Ascaris lumbricoides and hookworm, but 13 men still had T. trichiura (100 to 300 eggs/g of faeces).

2. Body Weight
Changes before and after deworming were small and inconsistent.

3. Nitrogen Digestibility and Balance
Group results are summarized in table 4. The men in group C (without ascaris) had lower faecal nitrogen and higher apparent nitrogen digestibility after treatment. Although group B showed higher urinary nitrogen excretion after treatment, nitrogen balance, biological value, and NPU did not change in any group.

4. Dietary Energy Absorption
Before treatment, the apparent absorption was (mean ± S.D.) 95.1 ± 2.0, 94.6 ± 2.8, and 94.3 ± 2.8 per cent in groups A, B, and C, respectively. It did not change with treatment.

5. Other Observations
Only one man with hookworm was anaemic, as shown in table 5 (11.5 9 haemoglobin/100 ml). All men with hookworm showed slight increases in haemoglobin after treat ment. All other biochemical indicators remained constant, within normal ranges.

Conclusions and comments

1. The men had light intestinal loads of helminths.
2. Apparent protein digestibility improved after treatment in some but not all men. Energy absorption was not affected by the parasites.
3. Nitrogen balance was not affected by the parasites.
4. Unusually heavy infestations with an intestinal helminth can probably interfere with the intake, absorption, and retention of proteins, but the mere presence of a parasite does not justify an assumption of metabolic or clinical significance. The contribution of these parasites to protein malnutrition is often overemphasized.

TABLE 4. Nitrogen-Balance Data

Nitrogen	Group A Ascaris (n = 7)	Group B Ascaris+ hookworm (n = 6)	Group C Hookworm (n = 7)
Intake (mg N/kg/day)
Before deworming	204.9 ± 11.1	241.1 ± 9.3	2218. ± 11.3
After deworming	209.7 ± 13.8	245.6 ± 8.1	226.9 ± 6.8
Urinary nitrogen output (mg N/kg/day)
Before	135.8 ± 6.3	147.4	123.6 ± 7.6
After	136.3 ± 7.0	163.1 ± 5.9*	142.7± 9.0
Faecal nitrogen output(mg N/kg/day)
Before	44.1 ± 4.3	58.1 ± 3.3	53.1 ± 4.4
After	42.6 ± 4.3	53.6 ± 4.4	43.5 ± 4.1
Nitrogen Balance(mg N/kg/day)
Before	20.0 ± 4.3	30.6 ± 10.0	40.7 ± 6.5
After	25.9 ± 7.4	24.1 ± 9.1	35.6 ± 9.2
True digestibility (%)
Before	84.6 ± 1.0	81.0 ± 0.8	81.7 ± 1.4
After	86.0 ± 1.4	83.2 ± 1.4	86.2 ± 1.7
Biological value (%)
Before	36.3 ± 1.7	34.8 ± 3.2	43.0 ± 3.3
After	38.2 ± 2.5	31.6 ± 3.0	39.5 ± 3.9
NPU (%)
Before	30.7 ± 1.6	28.1 ± 2.1	35.4 ± 3.3
After	32.8 ± 2.3	26.3 ± 2.7	34.1 ± 3.5

Changes with deworming: * p < 0.05; ** p < 0.01.

TABLE 5. Haematological Observations

Group	Parasite	Subject no.	Haemoglobin			Haematocrit
			Initial	Final	Difference	Initial	Final	Difference
A	Ascaris Lumbricoides	4	14.5	15.5	1.0	41	45	4
		6	14.0	15.4	1.4	40	45	5
		8	14.2	14.8	0.6	40	42	2
		10	13.8	13.7	- 0.1	41	41	0
		12	13.7	13.7	0	41	40	- 1
		22	14.5	14.2	- 0 3	45	40	5
		31	14.8	15.0	0.2	43	43	0
B	Ascaris + hookworm	3	13.3	14.9	1.6	37	43	6
		7	14.5	15.7	1.2	41	45	4
		9	14.4	15.4	1.0	46	41	- 5
		21	15.0	15.6	0.6	43	45	3
		28	14.2	14.7	0.5	41	41	0
		30	14.6	15.3	0.7	43	44	1
C	Hookworm	1	16.1	17.1	1.0	48	50	2
		3	11.5	12.0	0.5	33	36	3
		11	14.0	14.6	0.6	41	43	2
		23	14.4	16.2	1.8	43	46	3
		24	13.8	14.0	0.2	40	41	1
		25	13.6	14.4	0.8	39	42	3
		29	14.0	14.6	0.6	41	42	1

 

(introduction...)

Objectives
Experimental details
Summary of main results
Conclusions

R.E. Schneider, B. Tor. Shiffman, C. Anderson, and R. Helms
Institute of Nutrition of Central America and Panama (INCAP), Guatemala City, Guatemala, and Institute of Nutrition, University of North Carolina, Chapel Hill, North Carolina, USA

This brief report summarizes the results of two studies carried out with healthy adult males from the rural Pacific lowlands of Guatemala consuming a diet qualitatively similar to that habitually eaten in that region. Some of these men lived for certain periods of time in environments with improved sanitary conditions, as described below.

Objectives

1. To determine the apparent absorption of total energy, protein, and fat from the customary rural diet in: (a) military conscript males (soldiers) who were born and had lived all their life in rural Guatemala except for the two years before being studied, during which they lived in military installations with better sanitary conditions than those prevailing in their rural homes; and (b) men living in two rural communities.

2. To evaluate the effect of sanitation measures, such as the introduction of an intra-domiciliary water supply system and a sanitary education programme, on the absorptive capacity of men from one of the rural communities studied.

Hypothesis
Environmental sanitary conditions influence the capacity of Guatemalan adults to absorb the major food nutrients present in their habitual diet. if this is true, the soldiers should absorb the nutrients better than men living in their rural homes.

Furthermore, the absorptive capacity of the rural men in the community where sanitary measures were introduced should also improve.

Experimental details

1. Absorption Studies in Soldiers
One hundred soldiers who were born and had always lived in the lowlands of Guatemala and who had been for two years at an army station (MZ) near Guatemala City were interviewed and evaluated clinically. Besides being exposed during this period to improved environmental sanitation, these men had been eating a better diet than the one commonly eaten in rural areas. From this group 13 volunteers, 18 to 22 years old, who fulfilled the following criteria were selected: (a) there was no history of acute or chronic gastrointestinal diseases; (b) the result of the physical check-up was normal; (c) there was normal urinary excretion of d-xylose five hours after an oral dose of 25 9; and (d) two direct examinations of fresh stools for ova and parasites proved negative. Table 1 gives the volunteers' pertinent characteristics.

The soldiers lived for 21 days in a metabolic unit set up at the military post infirmary in Guatemala City (altitude 1,500 metres above sea level; temperature 19 to 22 C; low humidity). The experimental protocol followed was: Days 1 and 2: adaptation to the typical rural diet. Days 3 to 18: five consecutive three-day balance periods (Balances 1 to 5). Days 19 and 20 were used to complete faecal collections. A final physical check-up was done on day 21 before discharging the subjects from the unit.

2. Absorption Studies in Men from Two Rural Communities
In 1973, two villages, Guanagazapa (GU) and Florida Aceituno (FA), located in the lowlands near the Pacific coast of Guatemala, were chosen in order to carry out a study to evaluate the effect of introducing sanitation measures on the absorptive capacity of their inhabitants. The villages were within one hour's drive from Guatemala City, with a distance of 32 km between the two villages. Their altitudes were 200 and 235 m above sea level, with an annual rainfall of 2,000 mm. Temperature was 20° C during the day and cooler at night. Their populations of 973 for GU and 923 for FA were approximately 20 per cent Maya Indian and 80 per cent Ladino (mixed Maya and Caucasian descent). Both communities had water supplies of poor quality consisting of private wells and some communal faucets.

Studies were carried out for four years in both villages, divided into three stages: (a) two years of basal studies were made (1973-1974); (b) sanitary measures were implemented in GU (test village). In December 1974 an intra-domiciliary water supply system became operative, and a sanitary education programme was started in early 1975-neither measure was implemented in FA (control village); (c) two years were spent evaluating the impact of the sanitary interventions (1975-1976).

TABLE 1. General Characteristics of the 13 Soldiers Studied

Age, years	20.7 ± 1.2*
Body weight, kg	60.1 ± 4.5
Height, cm	164.0 ± 4.6
Body surface, m2	1.66 ± 0.39
Weight/height (kg/m)	37.0 ± 0.04
D -xylose, % excreted	28.3 ± 4.6
Plasma proteins, g/dl	8.1 ± 0.8
Haemoglobin, g/dl	16.4 ± 1.5
Haematocrit, %	49.0 ± 2.4
Urine analysis	Normal
Two direct stool examinations	Negative for parasites
Ethnic background	Maya Indian or Ladino (mixed Maya/Caucasian descent)

* Mean ± S.D.

In 1973, 60 male volunteers aged 14 to 45 years were randomly chosen in each community among those men who had lived there at least ten years. By 1974 some had emigrated and were replaced by others of the same ages, also chosen at random, in order to study 120 men each year. The same procedure was followed in 1975 and 1976. Therefore, at the end of the four years there was a "longitudinal" group formed of men who participated one, two, or three times in the study. Table 2 gives the number of subjects in both groups each year. All were healthy at the time of the studies. Table 3 gives their characteristics. All men had mild or moderate infestations with one or more of the following intestinal parasites: Ascaris lumbricoides, Trichuris trichiura, hookworms.

Absorption studies were carried out between May and November in four consecutive years beginning in 1973. The men were housed in groups of 8 to 10 in a rural metabolic unit built adjoining the hospital of a nearby city (Escuintla) with the same climate as that of the study villages.

TABLE 2. Number of Subjects Included in the Statistical Analyses

	Balance I				Balance II
	1973	1974	1975	1976	1974	1975	1976
Guanagazapa
Longitudinal	34	34	34	34	34	34	34
Non-longitudinal	12	13	14	15	13	14	15
Whole sample	46	47	48	49	47	48	49
Florida Aceituno
Longitudinal	28	28	28	27*	28	28	28
Non-longitudinal	18	23	23	26	23	23	26
Whole sample	46	51	51	53*	51	51	54

* One of the longitudinal subjects from FA was excluded from Balance I in 1976 due to diarrhoea.

TABLE 3. General Characteristics of the Men from Florida Aceituno (FA) and Guanagazapa (GU), 1973

	FA	GU
Number of men	46	46
Body weight, kg	50.2 ± 6.8*	57.1 ± 9.5
Height, cm	155.6 ± 6.8	162.5 ± 7.9
Body surface, m²	1.47 ± 0.13	1.62 ± 0.42
Weight/height, kg/m	32.0 ± 3.5	32.2 ± 3.1
D-xylose, % excreted	18.5 ± 6.4	19.9 ± 5.8
Plasma proteins, g/dl	6.8 ± 1.2	6.9 ±1.6
Haematocrit, %	40.6 ± 7.7	44.0 ± 4.9

* Mean ± S.D.

The groups alternated between men of each village and they lived in the metabolic unit for five days in 1973 and for eight days in each of the following years. Metabolic-balance studies began on the day after admission; in 1973 only one three-day metabolic-balance study was performed, and in each of the following years two consecutive three-day balance studies were done (hereafter referred to as Balance I and Balance II). During the last two days, faecal collections were completed and d-xylose absorption tests were carried out.

3. Rural Diet Study
The same diets were used in the absorption studies with soldiers and with men from GU and FA. The diet was prepared with the foods and recipes used by the population from which these men came, except it included certain amounts of commercial canned black beans and more animal protein, since the men did not eat meat every day at home. Table 4 gives the amounts of food offered each day, divided into three meals. These amounts provided 2,800 kcal (28 per cent of animal origin), 95 g protein (34 per cent animal protein), and 35 g fat (22 per cent animal fat). The men were encouraged, but not forced, to eat all the food served in the metabolic unit.

The maximum amount of food offered to each man from GU and FA in 1973, 1975, and 1976 provided 2,800 kcal/day, based on the mean intakes of 75 men from each village surveyed in 1972. In 1974, diets that provided 2,000, 2,400, or 2,800 kcal/day were offered during the first three days (Balance I ) to each man, depending on his personal dietary history; during the following five days (which included Balance II), food amounts equivalent to 2,800 kcal/day were offered to all men. This was done in an effort to assess the effect of the usual dietary intakes preceding admission to the metabolic unit. The proportions of nutrients offered were constant at all levels of energy intake, since the changes were achieved through proportional variations in the amounts of each food served.

4. Measurements
The amounts of each food eaten by each man were weighed at every meal, and the nutrient intake was calculated from the analyses of representative food aliquots. Complete urine and faecal collections were also obtained, using carmine red as the faecal marker. Aliquots of the foods and of the three-day stool collections from each balance period were analysed, and their contents of total energy (bomb calorimetry), nitrogen (macro-Kjeldahl), and fat (Van de Kamer) were used to calculate apparent absorptions. Urinary nitrogen was also determined (macro-Kjeldahl) to calculate apparent nitrogen balance.

Summary of main results

1. Soldiers
Table 5 summarizes the results of the five consecutive three-day balance periods. One man's data were excluded from Balances ill and IV because he had diarrhoea.

TABLE 4. Amount of Food Prepared and Offered Daily in the Metabolic Unit, in Grams

Cooked beans*	307
Fried beans*	40
Corn tortilla	570
Rice	200
Bread	45
Sweet rolls	66
Meat	1 07
Cheese	1 09
Chayote**	100
Squash	68
Carrots	66
Sugar	37

Total energy 2,800 kcal
Total protein 95 9 = 380 kcal (13.6% energy)
Total protein 95 9 = 380 kcal (13.6% energy)
Total fat 35 9 = 315 kcal (11.2 % energy)

* Black beans: Phaseolus vulgaris.
** Chayote: Sechium edule.

1. There was a clear tendency to gain weight through tout the 15 days. This suggests that energy intake was in excess of expenditure.
2. Stool weight was higher in the first balance period. This might be due to faecal residues of the food ingested before coming into the metabolic unit and suggests that two days are not enough for "adaptation" to the new diets.
3. Apparent absorption of nitrogen and of total energy was also lower during the first balance period. This is probably related to the larger faecal excretion in that balance period.
4. Urinary nitrogen excretion was constant throughout the five balance periods.
5. Nitrogen balance was lower in Balance I than in Balances II and IV. It did not differ from the overall mean balance of Balances II to V.

TABLE 5. Guatemalan Soldiers: Results of Metabolic-Balance Studies in Five Consecutive Three-Day Periods (Mean + S.D.)

Measurement	Three-day balance periods					Average of balance periods II-V	Least significant difference**
	I (13)*	II (13)	III (12)	IV (12)	V (13)
Body weight, kga	62.27 ± 3.08	62.77 ± 3.02	63.30 ± 2.91	62.47 ± 2.98	63.21 ± 2.94	62.94 ± 2.89	0.17
Stool weight, g/3 days	789 ± 303c	627 ± 291b	633 ± 255b	660 ± 273b	630 ± 261b	636 ± 261b	60
Nitrogen, mg/kg/day
Intake	289 ± 14	285 ± 14	286 ± 13	286 ± 14	285 ± 13	285 ± 13
Faecal	60 ± 16C	40 ± 20b	43 ± 20b	46 ± 20b	37 ± 14b	41 ± 18b	11
Urinary	200 ± 27	193 ± 21	213 ± 33	194 ± 29	216 ± 23	204 ± 28
Apparent	30 ± 20b	52 ± 22c	30 ± 31b	47 ± 24c	32 ± 25b	40 ± 27	15
Apparent absorption, %	79 ± 5b	86 ± 6c	85 ± 6c	84 ± 6c	88 ± 5c	86 ± 6c	3.8
Energy, kcal/kg/day
Intake	50 ± 3	50 ± 2	49 ± 2	50 ± 2	49 ± 2	49 ± 3
Faecal	6 ± 3c	4 ± 2b	4 ± 2	4 ± 1b	4 ± 2b	4 ± 2b	1.4
Apparent absorption, %	89 ± 5b	93 ± 4c	92 ± 3	92 ± 2c	93 ± 5c	92 ± 4c	3.0
Fat, mg/kg/day
Intake	556 ± 27	583 ± 28	515 ± 23	586 ± 28	547 ± 25	558 ± 38
Faecal	87 ± 32	88 ± 34	80 ± 28	71 ± 27	68 ± 31	70 ± 32
Apparent absorption,%	84 ± 6	85± 3	84 ± 5	88 ± 5	88 ± 6	86 ± 6

* Number of men in parentheses. ** L.S.D. shown only when groups differed by analysis of variance, p < 0.05.
a Linear tendency to gain weight with time. b Lower than values with superscript c, P <0.05..

2. Rural Men

1. There were some differences between Balances I and II but they were not consistent throughout the four years of the investigation. Based on these findings, on the studies in the soldiers, and on the fact that some men were offered less than 2,800 kcal of diet in Balance I of 1974, the data from the second Balance period were used for comparisons.
2. There were no consistent differences between 1974, 1975, and 1976 in FA or between 1974 and 1975 in GU. Absorption of nutrients was higher in GU in 1976 than in other years (see tables 6 and 7).
3. There were no differences between the "longitudinal" and "non-longitudinal" groups.
4. Table 6 summarizes the results of the second Balance periods in 1974 and 1976. Table 7 shows the differences in absorption of nutrients and in nitrogen balance between the two communities and between them and the soldiers.
5. The differences in nitrogen balance seem to be mainly due to the high urinary nitrogen excretion in GU in 1976. f. The men from FA had a tendency for larger stool volumes throughout the four years of the study than those of men from GU and MZ, although the differences were not always statistically significant.
6. The men from FA were thinner and weighed less. Consequently, dietary intakes per unit of body weight tended to be higher in them than in GU and MZ men.
7. The apparent absorption of nutrients was lower in 1974 and 1975 in both villages than in MZ. In 1976, however, the men from GU absorbed as well or better than those from MZ men.
8. All men from GU and FA had between one and seven intestinal parasite species at the metabolicbalance studies (A. lumbricoides, T. trichiura, hookworm, Enterobius vermicularis, Giardia tamblia, Enterococcus coli, Entamoeba histolytica). All were asymptomatic, and ova counts suggested mild-tomoderate infections. There was no correlation between apparent absorption of nitrogen total energy and the number of parasite species in the host's intestine.

TABLE 6. Guatemalan Rural Men: Results of Metabolic-Balance Studies after Three Days in the Metabolic Unit (Balance I I, Longitudinal Group) and Comparison with Soldiers^a

Measurement	FA—1974 n=28b	GU—1974 n=34	FA—1976 n=28	GU—1976 n=34	MZ—11 n=13
Body weight, kg	50.35 ± 5.67c	58.86 ± 10.39	51.02 ± 5.51	60.69 ±10.53	62.77 ± 3.02
Stool weight, g/3 days	1,056 ± 442	776 ± 238	928 ± 442	759 ± 229	627 ± 291
Nitrogen, mg/kg/day
Intake	324 ± 46	300 ± 62	329 ± 42	303 ± 49	285 ± 14
Faecal	83 ± 32	70 ± 29	71 ± 24	44 ± 20	40 ± 20
Urinary	194 ± 40	171 ± 51	220 ± 37	225 ± 46	193 ± 21
Apparent balance	59± 62	79 ± 25	34 ± 39	31 ± 26	52 ± 22
Apparent absorption, %	75 ± 8	77 ± 8	78 ± 9	84 ± 6	86 ± 6
Energy, kcal/kg/day
Intake	57 ± 8	53 ± 10	56 ± 6	49 ± 8	50 ± 2
Faecal	7± 3	5± 2	6± 3	3± 1	4± 2
Apparent absorption, %	88 ± 5	90 ± 3	89 ± 6	95 ± 2	93 ± 4
Fat, mg/kg/day
Intake	702 ± 78	589 ± 106	574 ± 60	573 ± 89	583 ± 28
Faecal	150 ± 51	115 ± 45	96 ± 46	54 ± 26	87 ± 34
Apparent absorption, %	79± 6	81± 6	84± 7	91± 7	85±3

1. Men from villages FA and GU in 1974 and 1976, and soldiers from MZ (Balance 11).
2. Number of men.
3. Means ± S.D. Results of comparisons betueen groups shown in table 7.

TABLE 7. Comparisons between the Second Metabolic-Balance Periods Rural Men (FA and GU,1974 and 1976) and Soldiers (Mz)a

	FA-1974 Compared with				GU-1974 Compared with			GU-1976 Compared with
	GU		FA	GU		GU	FA		FA
	1974	MZ	1976	1976	MZ	1976	1976	MZ	1976
Stool weight, 9/3 days	­ **b	­ **	-	­ **	-	-	-	-	-
Apparent absorption
Nitrogen	-	¯ **	-	¯ **	¯ **	¯ **	-	-	­ **
Total energy	-	¯ **	-	¯ **	¯ **	¯ **	-	-	­ **
Fat	-	¯ **	¯ **	¯ **	¯ **	¯ **	-	­ ***	­ **
Nitrogen balance, mg/kg/day
Intake	-	­ **	-	-	-	-	¯ *	-	¯ *
Faecal	-	­ **	-	­ **	­ **	­ **	-	-	¯ *
Urinary	-	-	¯ **	¯ **	-	¯ **	¯ **	­ *	-
Balance	-	-	-	­ *	­ **	­ **	­ **	¯ *	-

a From data shown in table 6.
b Mean values were higher ( ­ ) or lower ( ¯ ) based on student's grouped ''t" test with p < 0.05 (*) or 0.01 (**).

Conclusions

The results indicate that adult men from rural areas of a developing country who live under conditions of poor sanitation, without appropriate use of potable water, and who eat diets largely based on corn and black beans with some animal protein and low fat (about 11 per cent of total energy intake) have apparent absorptions of the order of 90 per cent of total energy, 75 to 80 per cent of protein, and 75 to 85 per cent of fat. It is also evident that these men's absorptive capacity improves after they have lived for two years in environments with better sanitation and have modified their hygiene habits through education. Their apparent absorptions become about 93 per cent of total energy, 85 per cent of protein, and 90 per cent of fat.

If we assume faecal obligatory losses of 12 to 14 mg N/kg/day, the "true" nitrogen digestibilities would be about 5 per cent higher than apparent digestibilities.

The diets used in this study were typical of the region, although their protein content (P% about 13.6 ) and the contribution made by protein of animal origin (about 34 per cent of total protein) were higher than those in the diets of most men of similar ethnic, cultural, and socioeconomic conditions in other parts of the country. It is conceivable that protein absorption may be somewhat lower in the latter, whose diets have a P% closer to 10 than 13, with animal proteins contributing only 20 to 25 per cent to the total.

The results obtained also indicate that future research studies involving measurements of absorptive capacity in rural subjects should allow about five days of dietary adaptation before starting the metabolic-balance studies.

Acknowledgements

These studies were done as part of a project carried out jointly by the University of North Carolina and INCAP with the economic support of the US Agency for International Development. The United Nations University contributed support for the last stages of data analysis.

(introduction...)

Objective
Experimental details
Summary of main results

Chen Hsue-cun
Institute of Health, Chinese Academy of Medical Sciences, Beijing, China

Objective

Several studies have been conducted by Chinese nutritionists in the last 30 years to assess energy intake, expenditure, and requirements of Chinese men and women engaged in various working activities. A summary of the main results will be presented.

Experimental details

1. Energy Intakes
Energy intakes were calculated from individual dietary surveys.

2. Energy Costs of Activities
Energy costs of activities were calculated by indirect calorimetry using Douglas bags and analysing the O2 and CO2 contents of exhaled air. Measurements were obtained under basal conditions, at rest and at work.

3. Energy Expenditures
Energy expenditures were calculated using time-motion techniques and the average energy cost of work.

Summary of main results

1. Basal Energy Expenditure of Young Men
The mean value obtained from 19 college students 20 to 30 years old was 0.63 kcal/m2 of body surface/minute.

2. Energy Expenditure of College Students
Table 1 shows the energy cost of different activities of college students. The mean total energy expenditures of 16 men and 6 women were 2,420 and 2,170 kcal/day, respectively.

The make-up of the dietary energy absorbed by 9 men was 12.0 per cent from proteins, 27.9 per cent from fats, and 60.1 per cent from carbohydrates. Based on their non-protein respiratory quotient and urinary nitrogen, the different sources of expended energy were 12.3 per cent from proteins, 28.8 per cent from fats, and 58.9 per cent from carbohydrates.

3. Energy Expenditures of Iron and Steel Workers and of Coal Miners
Tables 2 and 3 give the mean energy expenditure of men engaged in these physically demanding jobs. The figures include the pauses and resting periods during the eight-hour shift. The average cost (kcal/min/m2) of the specific activities performed by the men varied widely. For example, among iron and steel workers, energy costs ranged from 1.67 while putting core into moulds and 1.71 when walking in the furnace area, to 4.93 during heavy hand-rolling. Among coal miners, the energy costs ranged from 1.60 while packing holes with explosives and 1.75 while drilling rocks, to 4.43 when climbing in the wind tunnels, and 5.09 carrying trestles in such tunnels.

4. Dietary Intakes of Workers
Table 4 gives the mean energy and protein intakes of labourers engaged in different jobs in several provinces of China.

5. Energy Expenditures of Peasants
Tables 5 and 6 give the energy costs of work-related activities for male and female peasants. Total energy expenditure varies cyclically, being greater during the summer harvest and spring digging. Table 7 gives time allocations in the various seasons, and table 8 gives some of the corresponding total daily energy expenditures of men and women engaged in different agricultural chores.

TABLE 1. Energy Cost of Different Activities of College Students

Sedentary	Activity	Energy expenditure
		(kcal/m /mm)
	Lying at ease	0.65 (0.58 - 0.74)*
	Sleeping	0.73 (0.69 - 0.82)
	Snooze; resting between classes;
	reading Iying down	0.79 (0.65 - 0.89)
	Sitting (watching movie; watching demonstration;
	writing; reading; studying; attending meeting)	0.82 (0.71 - 1.08)
	Taking examinations	0.92 (0.73 - 1.02)
	Writing on blackboard; standing and listening	0.98 (0.88 - 1.09)
	Working in laboratory	1.00 (0.71 - 1.19)
	Cleaning window	1.98 ( - )
	Dressing or undressing	2.20 (2.06 - 2.23)
	Making bed	2.26 (2.20 - 2.32)
	Washing clothes	2.36 (2.17 - 2.56)
Moderate	Standing and conducting singing	2.64 (2.37 - 2.92)
	Ordinary morning drill	2.65 (2.22 - 3.80)
	Walking	2.70 (2.33 - 3.30)
	Cleaning floor	2.72 (2.62 - 2.81)
	Broadcast drill	2.77 (2.56 - 2.98)
	Scrubbing floor	2.82 ( - )
	Dancing	4.03 (3.31-4.80)
	Playing baseball	4.03 (3.91 - .1 5)
Vigorous	Playing volleyball	4.07 (3.82 - 4.32)
	Running	5.30 (3.85 - 6.75)
	Playing basketball	5.78 (5.00 - 7.92)
	Playing football	5.96 (5.33 - 6.60)

* Mean, with range between parentheses.

TABLE 2. Energy Expenditure of Iron and Steel Workers in an Eight-Hour Shift

Type of work	Working time (%)*	Energy cost of work		Total energy expenditure (kcal/man/8 hrs)
		(kcal/m²/min)	(kcal/min)
Furnaceman	55	1.86	2.92	1402
Ore-carrying worker	72	3.06	5.28	2534
Ore-sieving worker	60	2.71	4.97	2386
Blast furnaceman	72	2.97	4.82	2314
Sintering (non-mechanized)	80	2.69	4.42	2122
Open-hearth furnace	42	1.77	2.84	1363
Electric open-hearth	40	1.78	2.86	1373
Coke-oven man (mechanized)	70	1.95	2.95	1416
Coke-oven man (semi-mechanized)	60	1.60	2.41	1157
Rolling worker	55	1.66	2.71	1301
Rolling worker	46	1.52	2.36	1138
Tube-casting foundry work
Moulder	75	1.60	2.58	1238
Core layer	46	1.40	2.25	1080
Fettling work	59	1.92	2.89	1387
Fettling work	63	1.88	3.21	1541
Chassis-making	74	2.12	3.40	1637
Core removal	82	1.93	3.10	1488

* The remaining time was spent in pauses and rest periods.

TABLE 3. Energy Expenditure during Coal Mining in an Eight-Hour Shift

Type of work	Working time (%)	Energy cost of work		Total energy expenditure (kcal/man/8 furs)
		(kcal/m²/min)	(kcal/min)
Coal miner	56	2.21	3.45	1655
Coal-timbering worker	56	2.28	3.55	1702
Coal-drilling worker	64	1.93	3.01	1444
Blasting worker	82	2.35	3.66	1756
Tunnelling worker	58	1.81	2.92	1403
Rock-transferring worker	48	1.95	3.63	1513
Timbering worker	63	2.09	3.35	1607

* The remaining time was spent in pauses and rests.

TABLE 4. Dietary Energy and Protein Intakes of Workers

Type of work	Location	Energy (kcal)	Protein (g)
Iron and steel work	Wuhan	4028	125
Iron end steer work	Hunan	3368	110
Iron and steel work	Anshan	3921	132
Electric power industry	Chonguing	3112	105
Coal mining	Anhui	3713	96
Iron factory	Hangzhou	3347	80
Machine factory	Guangxi	3168	86
Silk plant	Hangzhou	2885	89
Building worker	Beijing	3614	108
Mining	Guangdong	3950	86

TABLE 5. Energy Cost of Activities Performed by Male Peasants during Work

Activity	kcal/m2 /min
Resting	1.02
Loading corn onto carts	1.30
Pulling radishes by hand	1.54
Winnowing beans	1.54
Picking corn	1.57
Mowing beans	1.82
Binding corn stalks	1.89
Picking potatoes with hoe	1.97
Weeding with hoe	2.03
Walking	2.13 ± 0.39
Transplanting rice seedlings	2.28 ± 0.31
Spreading manure	2.30
Picking corn roots with hoe	2.51
Raking the soil	2.71
Weeding seedling fields	2.72 ± 0.33
Mowing wheat	2.79 ± 0.20
Picking radishes with hoe	2.93
Light ploughing	2.94 ± 0.18
Carrying water in pails	3.19 ± 0.14
Weeding rice fields (shallow)	3 33 ± 0 54
Carrying manure in holder on shoulder	3 50
Shovelling earth	3.51 ± 0.08
Planting fields	3.91 ± 0.35
Weeding rice fields (deep)	4.12 ± 0.42

TABLE 6. Energy Cost of Activities Performed by Female Peasant during Work

Activity	kcal/m2 /min
Resting	0.88 ± 0.28
Spreading fertilizer (standing)	1.62 ± 0.22
Mowing wheat	1.79 ± 0.06
Weeding rice field	1.91 ± 0.16
Walking	2.00 ± 0.29
Spreading manure	2.95 ± 0.14
Spreading fertilizer (stooping)	3.04 ± 0.21
Thinning young shoots	3.16 ± 0.33
Pulling up wheat	3.30 ± 0.21
Shovelling earth	3.44 ± 0.14
Planting fields	3.86 ± 0.63

TABLE 7. Distribution of Daily Time in Hours

	Spring and autumn		Summer		Winter		Summer harvest		Deep digging
Type of work	Part- time labour	Full- time labour	Full- time labour	Part- time labour	Part- time labour	Full- time labour	Part- time labour	Full- time labour	Part- time labour	Full- time labour
Productive labour	7.5	8.25	6.0	7.0	6.0	7.0	9.0	11.5	8.2	10.75
Sleeping	8.5	8.5	9.0	9.0	10.0	10.0	8.5	6.5	9.0	7.5
Taking meal	1.5	1.5	1.5	1.5	1.0	1.0	2.0	2.0	2.0	2.0
Sewing	0.5	0.0	1.0	0.0	1.5	0.0	0.0	0.0	0.0	0.0
Cleaning street	0.25	0.0	0.25	0.25	0.25	0.25	0.25	0.0	0.25	0.0
Rest	1.0	1.0	1.0	1.0	0.5	0.5	1.0	1.0	1.5	1.5
Reading and attending meeting	1.5	1.5	1.0	1.0	2.0	2.0	0.0	0.0	0.0	0.0
Toilet	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
Dressing and undressing	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
Chatting	0.5	0.5	1.0	1.5	0.75	1.0	0.25	0.25	0.5	0.0
Washing clothes	0.5	0.0	1.0	0.0	0.25	0.0	0.25	0.0	0.25	0.0
Carrying water	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25
Walking	1.0	1.5	1.0	1.5	0.5	1.0	1.5	1.5	1.0	1.0

TABLE 8. Average Daily Energy Expenditure (kcal) of Male and Female Peasants in Spring, Fall, and Summer

Type of work	Average daily energy expenditure in spring and fall			Average daily energy expenditure in summer
	Male	Female		Male	Female
	Full-time labour	Full-time labour	Part-time labour	Full-time labour	Full-time labour	Part-time labour
Light ploughing	3,898	-	-	3,691	-	-
Spreading	-	3,408	3,310	-	3,310	3,055
Thinning young shoots	-	3,647	3,445	-	3,365	3,102
Spreading fertilizer (stooping)	-	3,563	3,369	-	3,437	3,163
Planting field	4,681	4,189	3,938	4,356	3,897	3,558
Shovelling earth	4,343	3,886	3,663	4,069	3,640	3,337
Mowing	-	-	-	4,538	3,003	2,740
Pulling up wheat	-	-	-	-	4,568	4,008
Deep digging	4,954	4,435	3,812	-	-	-

(introduction...)

Objectives
Experimental details
Summary of the main results
Conclusions

BenjamTord Fernando E. Viteri
Institute of Nutrition of Central America and Panama (INCAP), Guatemala City, Guatemala

Objectives

1. Measurement of obligatory faecal and urinary nitrogen excretion in children two years old or a little older.
2. Calculation of such children's protein requirements following the factorial nitrogen approach.

Experimental details

1. Subjects

a. Five children, all males, of mixed Maya and Caucasian descent (Lading).
b. Chronological age: 24 ± 5 months (range: 17 to 31). Height-age: 16 ± 5 months (range: 10 to 23).
c. All had been treated for severe, oedematous protein-energy malnutrition (kwashiorkor and marasmickwashiorkorl. They had recovered fully at least one month before beginning the studies, based on clinical, anthropometric, and biochemical criteria (plasma proteins, non-essential/essential amino acid ratio, haematological indices, urinary creatinine excretion, and creatinine-height index [CHI] ).
d. Weight: 10.66 ± 1.14 kg (range: 8.82 to 11.96). Height: 79.8 ± 4.9 cm (range: 72.9 to 86.4). Weight-for-height, percentage of expected: 98 ± 1 per cent (range: 96 to 100 per cent). CHI: 0.95 ± 0.07 (range: 0.89 to 1.04).
e. Intestinal parasites: Two children had asymptomatic giardiasis. One of them also had a light infestation with Trichuris trichiura (one or two eggs per microscopic slide preparation). They were not treated before the study.
f. All children were healthy throughout the study.

2. Study Environment
INCAP's Clinical Centre in Guatemala City, 1,500 m above sea level. Temperature: 18 to 24° C. Relative humidity: 40 to 50 per cent.

3. Physical Activity
Since no child had diarrhoea and defecation habits were known by the nurses, the children were confined to metabolic beds only part of the day. During most of the day they moved freely in the Clinical Centre and outdoor playing grounds wearing urine-collection bags, except for those children who were toilet-trained. They participated in games that involved climbing ramps, walking uphill, and tossing balls.

4. Duration of the Study
Four children were studied simultaneously for nine days. A fifth child was studied five months later for seven days.

5. Diet

1. A nitrogen-free, liquid formula was prepared with the following ingredients (g/kg/day): cornstarch 2.5; sugar 15.2; cottonseed oil 3.3; mineral mixture (NaCI, KCI, Na₂HPO₄, CaCO₃, mg SO₄) 0.6; water to make a final weight of 80 g/kg/day.
2. The liquid formulas were cooked for 10 to 20 minutes. They were free of fibre, and vegetable oil provided 30 per cent of the energy.
3. The diet, which provided 100 kcal/kg/day, was divided into five isoenergetic meals and fed at threehour intervals. It was supplemented with vitamins, iron, iodine, zinc, and manganese. Additional water was offered ad libitum.
4. Prior to the study, the children consumed milk-based liquid formulas for several days.

6. Indicators and Measurements

1. Urine was collected at 23- to 25-hour intervals, and the volume was corrected mathematically to correspond to a 24-hour period. Faeces were not collected during the first day of the study. Beginning on the second day, carmine red and brilliant blue were given alternately as markers with breakfast every two days. Faeces were collected between markers as 48-hour pools. They were dried at 80° C and homogenized.
2. Urinary and faecal aliquots were digested, and their nitrogen contents were determined by a microKjeldahl technique using selenium (Se) as a catalyst. Diet aliquots were analysed in the same way to ensure the absence of nitrogen. Tryptophan standards were analysed simultaneously.
3. Body weight was measured daily before breakfast.

Summary of the main results

1. Obligatory Nitrogen Losses
The results are summarized in table 1 and figure 1. The mean and S.D. for the combined data of days five to nine were 34.0 5.3, 19.5 6.9, and 53.7 8.1 mg N/ kg/day for urinary, faecal, and both urinary and faecal nitrogen, respectively.

If the study had been done in only six days and the mean values of days five and six used, the corresponding results would have been 33.2 5.9, 19.9 6.8, and 53.0 7.7 mg N/kg/day for urinary, faecal, and both urinary and faecal nitrogen, respectively.

2. Factorial Calculations
Assuming that integumental nitrogen losses were of the order of 5 mg N/kg/day on a protein-free diet, total obligatory nitrogen losses would be 59 mg/kg/day, or 40 per cent less than the current FAD/WHO estimates. Adding 15 mg N/kg/day for growth of children of the same height-age and multiplying by 1.3, as suggested by FAD/WHO (WHO Tech. Rep. Ser. No. 522, 1973), results in an estimated mean requirement of 96.2 mg N/kg/day, equivalent to 0.60 9 of milk or egg protein/kg/day. This value coincides with the mean requirement of 0.61 g/kg/day calculated by us using multiple-level nitrogen balance techniques (see summary of study by Torabrera Santiago, and Viteri, this volume).

Conclusions

1. The obligatory faecal and urinary nitrogen excretion on a protein-free, low-residue diet can be assessed during the last two of six experimental days.

2. Urinary and faecal nitrogen are 34 ± 5 and 20 ± 7 mg N/kg/day, respectively.

Child	Days on a nitrogen - free diet
	1	2	3	4	5	6	7	8	9
Urinary nitrogen	58.9	28,9	41.0	37 4	25 5	33.6	27,4	31.3	44.2
C.R.	58.9	28.9	41.0	37.4	25,5	33.6	27,4	31,3	30 2
W.M.	81.3	52.4	29.8	39.0	31.2	33.4	35.2	34.0	30.2
I.G.	85.4	39.0	37.1	33.6	31.1	39.8	34.3	39.4	33.3
H.A.	89.4	68.6	60.8	43.4	40.8	38.4	39.8	31.1	40.3
A.A.	-	58.2	36.9	39.9	22.8	.5.5	29.2	-	-
Mean	78.8	49.4	41.1	38.6	30.2	36.1	33.2	33.9	37.0
S.D.	11.8	15.8	11.8	3.6	6.9	2.8	4.9	3.4	6.0
Faecal nitrogen
C R.a		19.8		26,3		27,9		28.2
W.M. a,b		19.0		23.1		20.1		13,9
I.G.		9.7		7.4		15.2		9.7
H.A.		29.7		12.1		27.2		22.1
A.A.		12.9		22.0		17.9		-
Mean		18.2		18.1		21.6		18.5
S.D.		7.7		8.0		5.7		8.3
Total (urinary + faecal nitrogen)
Mean -		67.6	59.3	56.7	48.4	57.7	54.8	52.4	55.5
S.D. -		20.5	18.4	9.4	6.8	5.8	7.3	5.2	14.4

a Giardia lamblia in stools.
b Trichuris trichiura in stools. Both parasites diagnosed in faecal specimens collected four to nine weeks before the study.

FIG. 1. Urinary and Faecal Nitrogen Excretion on a Nitrogen-Free Diet

3. The mild parasitic infestations in two of the five children did not increase faecal nitrogen.

4. Factorial calculations using the empirical correction factor of 30 per cent support the conclusions of our studies with milk protein.

(introduction...)

Objectives
Experimental details
Summary of main results
Conclusions

Fernando E. Viteri and Cristina Martinez
Institute of Nutrition of Central America and Panama (INCAP), Guatemala City, Guatemala

Objectives

1. To determine the nitrogen losses by different routes in pre-school children ingesting different nitrogen levels.
2. To determine whether the nitrogen source (whole egg or rice-soy-milk) affects nitrogen losses by different routes.

Experimental details

1. Subjects

1. Four boys of mixed Maya Indian and Caucasian descent (Lading).
2. Chronological age: 18 ± 3 monthts (range: 14 to 24). Height-age: 14 ± 2 months (range: 11 to 18).
3. All had been treated for severe, oedematous protein-energy malnutrition. They had recovered fully at least two months before beginning the studies, based on clinical, anthropometric, and biochemical criteria (plasma proteins, non-essential/essential amino acid ratio, haematological indices, and creatinine height index).
4. Weight: 10.5 ± 0.8 kg (range: 9.7 to 12.1). Height: 71 ± 2 cm (range: 69 to 75). Weight for height: 101 ± 3 per cent (range: 97 to 105).
5. All children were healthy throughout the study, except for one child who developed a mild upper respiratory infection without fever.
6. No other characteristics of the subjects are pertinent to the study.

2 Study Environment
INCAP's Clinical Centre in Guatemala City; 1,500 m above sea level; mean temperature 24.6 C with a maximum of 28.5 and a minimum of 19.0 C; mean relative humidity 66.6 per cent, range from 46 to 96 per cent. All the children remained in the metabolic ward during the study.

3. Physical Activity
The study lasted 40 days with each source of protein and 10 days at the end of a nitrogen-free diet. The children were off the study for three weeks in between nitrogen sources. During the study periods the children were allowed to exercise freely for the first 4 days of each 10 consecutive days. The last 6 days of each 10-day period were nitrogen-balance days, during which the children remained confined to bed but not necessarily Iying down. Energy expenditure was measured by insensible water loss determinations during nitrogen balance periods and by using the Newburgh's factor of 2.2125 kcal/g IWL/day.

4. Duration of the Study
The four children were in the study a total of 111 days, distributed as follows: a. 40 days on a rice-soy-milk formula. b. 21 days on the rice-soy-milk formula and whole egg. c. 40 days on the whole egg diet. d. 10 days on a nitrogen-free diet.

5. Diet
a. Rice-soy-milk: 40 per cent rice flour; 38 per cent full-fat soy flour; 5 per cent skim milk powder; 14.85 per cent sugar; 1.9 per cent mineral mix; 0.1 per cent vitamin mix; and 0.15 per cent artificial flavour. b. Whole egg protein: Iyophilized whole egg homogenate, mineral, and vitamin mix. c. Nitrogen-free diet: purified corn starch, vegetable oil, sugar, minerals, vitamins, artificial flavour, and water. Twenty per cent of calories came from fat.

The three diets were prepared as liquid formulas that provided 90 kcal/kg body weight/day, 20 per cent of which came from fat. Each protein source (a and b) was fed on four consecutive levels, each for 10 days' duration, starting with a nitrogen intake of 320 mg/kg/day and decreasing to 240, 160, and 80 mg/kg/day (equivalent to 2, 1.5, 1.0, and 0.5 g protein/kg/day). Protein was replaced by cornstarch sugar to maintain constant energy intake.

6. Indicators and Measurements
a. Nitrogen Macro-Kjeldahl for food, urine, and faeces. MicroKjeldahl for integumentary losses. Food nitrogen was measured for each ten-day period. Faecal and urinary nitrogen were measured in three-day pools for each child.

Integumentary losses were measured as follows: Before nitrogen balance was started the child was bathed with a non-ionic detergent (nitrogen-free) and dried with nitrogen-free towels by blotting. The bedding, pyjamas, and bibs were all nitrogen-free (pre-washed with 0.5 per cent acetic acid) and were analysed for nitrogen at the end of three days of contact with the children. To this, nitrogen from bath water at the end of three days was added.

Hair and nails were cut to the same length every ten days and analysed with the rest of integumentary nitrogen for that level of intake.

Residual nitrogen in food utensils was 9 mg/day (less than 1 mg/kg/day). Recovery of integumentary nitrogen in pyjamas, bedding, and bibs, tested in "dummy children" with bedding, etc., on which diluted urine was sprinkled repeatedly throughout three days, was 97.3 i 1.8 per cent (N = 6) (range: 95.8 to 100 per cent).

b. Serum protein and albumin, urea, and ammonia were measured at the end of each ten-day period.

c. Basal oxygen consumption was also measured at the end of ten days.

Summary of main results

1. Total serum proteins, albumin, haemoglobin, and creatinine-height index remained constant, with averages of 7.0, 4.2,11.9, and 0.94, respectively.

2. Basal oxygen (VO2 ) consumption did not change. The averages for different nitrogen intakes, regardless of the source, were 107, 96, 104, and 97 per cent of expected. For nitrogen-free diets, the mean was also 97 per cent (mean ± S.D.: 48.5 ± 6.1 kcal/kg/ day). The respiratory quotient was also constant, the means being 0.88, 0.88, 0.82, 0.81, and 0.82 for the different nitrogen intakes. All variation was random.

3. Energy expenditure by insensible water loss determinations came to a mean of 74 kcal/kg/day for diets containing 0.5 g protein/kg/day or higher. For the nitrogen-free diet, it was 58 kcal/kg/day.

4. Weight changes are presented in figure 1 (mean and range). Weight loss in all subjects occurred only with the nitrogen-free diet. Prior to this period there was a consistent increase in skin-fold thickness.

FIG. 1. Mean and Range of Weight Changes in Four Pre-school Children Who Received Different Levels of Protein and 90 kcal/kg/day

5. Obligatory nitrogen losses are:

		mg N/kg/day	mg N/kcal/day
Integumentary	(mean ± S.D.) N = 8	5 ± 1	0.12 ± 0.02
Faecal	(mean ± S.D.) N = 8	19 ± 2	0.38 ± 0.03
Urinary	(mean ± S.D.) N = 8	36 ± 7	0.77 ± 0.17
Total	(mean ± S.D.) N = 8	58 ± 8	1.19 ± 0.21

6. Faecal nitrogen is consistently lower with egg than with the rice-soy-milk (RSM) mixture, by a mean of 4 mg/kg/day. Mean true digestibility for egg was 94 ± 5; for RSM, it was 91 ± 2. Urinary nitrogen was higher for egg protein than for RSM at intakes of 2.0 and 1.5 9 protein/kg/day, but at lower values the opposite occurred. Total integumentary nitrogen/kg/day obtained was as follows:

Nitrogen Intake	Egg Protein	RSM
320	10.4 ± 1.3 (9.6)*	8.0 ± 2.4 (7.3)
240	10.2 ± 1.2 (9.3)	6.6 ± 1.5 (5.7)
160	6.1 ± 0.8 (5.3)	6.5 ± 1.7 (5.6)
80	6.1 ± 1.3 t5.4)	5.9 ± 1.3 (5.1)
0	4.7 ± 0.6 (3.8)	4.7 ± 0.6 (3.8)

* (sweat and skin mean)

True nitrogen retention (mg/kg/day) obtained was as follows:

Nitrogen Intake	Egg Protein	RSM
320	58 ±16	94 ± 12
240	39 ± 15	52 ± 14
160	33 ± 18	22 ± 20
80	+2 ± 11	- 20 ± 15
0	-58 ± 8	-58 ± 8

7. Integumentary nitrogen correlated significantly with serum urea nitrogen (r = 0.56). Egg protein at different levels of intake, however, by producing greater changes in serum urea than RSM, also produced higher sweat and skin nitrogen losses.

Nitrogen in skin and sweat = 39.9 + 6.65 urea nitrogen (egg) r = 0.80
Nitrogen in skin and sweat = 51.3 ± 2.34 urea nitrogen (RSM) r = 0.41

Conclusions

1. The nitrogen losses by different routes have been determined in four pre-school children ingesting different nitrogen levels, at constant energy intake. Faecal, urinary, and integumental nitrogen changed proportionally to nitrogen intake. Obligatory faecal and urinary nitrogen agreed with accepted values. Integumental obligatory nitrogen is 4.7 ± 0.6 mg N/kg/day and increases up to 2.5 times at an egg protein intake of 2 g/kg/day.

2. Protein source influences integumental losses as well as faecal nitrogen losses.

Acknowledgements

We are grateful to the staff of the Clinical Centre at INCAP, to Mr. R.D. Mendoza, and to Mrs. J. de Melgar. This study was partially supported by a grant from the World Health Organization.

The protein requirements of normal infants at the age of about one year: maintenance nitrogen requirements and obligatory nitrogen losses

P.C. Huang, C.P. Lin, and J.Y. Hsu
College of Medicine, Taiwan University, Taipei, Taiwan

Abstract from the Journal of Nutrition*

With 34 normal, healthy male infants aged 9 to 1 7 months, a total of 61 nitrogen (N) balance studies were conducted with N intake between 12 and 180 mg/kg/day. By regression analysis, the crude N maintenance requirements, either with whole egg or cow's milk protein, were estimated to be about the same, 106 and 103 mg/kg/day, respectively. The 97.5 per cent confidence limits for the requirements were 128 (egg) and 142 (milk) mg/kg, respectively. Sums of the obligatory urinary and faecal N for the egg and milk protein series were 75 and 71 mg/kg compared with 76 mg/kg of the actually measured figure. Ratio of the maintenance N requirement to the obligatory N loss was 1.4. In another 15 N balance studies, for which N intake from milk formulae ranged between 220 and 320 mg/kg/day, the mean apparent N retention was 25% of the intake. Total integumental N losses (skin + hair + nail) of infants fed 217 to 522 mg N/kg/day amounted to 7.9 ± 2.9 mg/kg daily. Egg protein had somewhat higher digestibility than cow's milk protein, 92 versus 87%, but a lower biological value, 76 versus 82. Net protein utilization (NPU) estimated from the regression line was about the same for both proteins, 71 and 69, respectively.

FIG. 1. Nitrogen Retention by Male infants 9 to 17 Months of Age at Various Levels of Egg Protein Intake.

The regression lines were drawn as follows: The regression equation for line UU' (-- ), for those whose nitrogen intake was less than 63 mg/kg/day, is Y = 0.578X - 71.85 (n = 11, r = 0.86, p < 0.01) where X = nitrogen intake and Y = nitrogen retention in mg/kg/day. The regression equation for line MM' (- - ), for those whose nitrogen intake was more than 70 mg/kg/day, is Y = 0.664X-69.40 (n = 18, r = 0.85, p < 0.01). Line R R' ( ) was plotted against all information; its regression equation is Y = 0.706X 75.11 (n = 29, r = 0.97, p <0.01).

FIG. 2. Nitrogen Retention by Male Infants 9 to 17 Months of Age at Various Levels of Milk Protein Intake

(introduction...)

Objective
Experimental details
Summary of main results
Conclusions and comments

Carmen LI. Intengan, Benigna V. Roxas, Anacleta Loyola, and Estelita Carlos
Food and Nutrition Research Center, Manila, Philippines

Objective

To determine the protein requirements of young children eating a diet based mainly on rice gruel and fish, habitually consumed in the Philippines, when dietary energy is not a limiting factor.

Experimental details

1. Subjects
Two sets of, respectively, three and four Filipino boys, 18 to 26 months old, were recruited from welfare institutions. During the first month of confinement in the metabolic unit of the Food and Nutrition Research Institute in Manila, their weights were adjusted to at least less than 10 per cent of their normal weight for height. Their characteristics are given in table 1. Five boys were mildly undernourished, i.e., they were 10 per cent below the ideal weight for their age, based on Philippine standards. They also had low weight for their heights. All children were 2.7 to 14.4 cm below the ideal height for their age. Two boys had light infestations of Trichuris trichiura (800 to 900 ova/al and very light infestations of Ascaris lumbricoides (2,200 to 2,400 ova/g).

2. Study Environment
The children lived in a metabolic unit in Manila throughout the study.

TABLE 1. Characteristics of the Subjects

Subjects	Age (months)	Body wt. (kg)	Ht. (cm)	Hgb. g/100 mg	Hct. vol. %	Serum albumin g/100 ml	Vit. A mcg/ 100 ml	Classification by wt.
R.A.*	26	10.3	82.5					1°1
A.C.	18	8.9	77.4					1°
M.C.	20	8.7	73.6					1°
G.L.	26	11.6	83.1	10.2	32	-	12	N²
J.M	20	8.9	77	12.1	36	2.89	39	1°
C.S.	23	12.3	86.5	14.5	42	3.01	16	N
R.A., Jr.**	24	10.3	82.3	11.5	36	3.23	24	1°

* Ascaris lumbricoides 2,200/g; Trichuris trichiura 900/g.
** Ascaris lumbricoides 2,400/g; Trichuris trichiura 800/g.
1 Degree of undernourishment: 1° = - 10 per cent underweight.
2 N - normal.

TABLE 2. Diet Composition per 100 kcal

Food	Grams	Food	Grams
Rice	14.0	Squash	2.5
Spanish makerel	3.1	Banana or	3.0
Sweet potato	5.0	Papaya	6.0
Bottle gourd	5.0	Coconut-corn oil	31.0
Sponge gourd	2.5	Sugar	1.7

3. Duration of the Study
Fifty-seven days for three children who ate six levels of dietary protein, and 47 days for four children who ate five levels of protein.

4. Diets
Diets were based on rice, fish, vegetables, and fruits habitually consumed in the Philippines (see table 2). The diets were calculated to provide 110 keel/kg body weight/day, based on the observed amount of energy consumed ad libitum during the first month of confinement in the metabolic unit. A blend of 50:50 coconut oil and corn oil provided 30 per cent of the energy intake. Three meals and two snacks were served during the day.

Vitamin and mineral supplements were provided each day: Vitamin A (3,000 I.U.) 0.9 mg; vitamin D2 (400 I.U.) 10 mcg; vitamin C 50 mg; thiamine 1.5 mg; riboflavin 1.2 mg; pyridoxine 1 mg; vitamin B 12 3 mcg; nixinamide 10 mg; iron 3 mg; iodine 75 mcg; calcium 40 mg; phosphorus 43 mg; magnesium 3 mg; manganese 0.5 mg; zinc 3.071 mg; choline 5 mg; dexpanthenol 5 mg; and inositol 5 mg. In addition, two teaspoons of Cetrin were given daily. Each 30 ml contains 250 mg vitamin C. Water was maintained at 80 ml/kg body weight/day.

Three children were fed six levels of protein, starting with 2.0 g/kg body weight/day and gradually decreasing to 1.75, 1.5, 1.25, 1, and 0.75 9. The other four children were given five protein levels starting at 1.75 g/kg/day. One-third of the dietary protein was provided by fish. Adjustment in protein level was done by replacing protein calories with starchy roots, fruits, or rice noodles (mung bean starch). Each dietary protein level was fed for seven days with a three-day break of ad libitum feeding between two consecutive experimental levels.

5. Indicators and Measurements
a. Nitrogen balance: Apparent nitrogen balance was calculated during the last four days of each seven-day period. Daily urines, two-day pooled faecal specimens, and dietary aliquots were analysed using a macro-Kjeldahl method. Apparent nitrogen absorption was calculated from dietary and faecal nitrogen. Biological value was calculated by dividing the percentage of nitrogen retained by the apparent absorption.

b. Protein requirements: An allowance of 10 mg N/kg/day was made for integumental and miscellaneous nitrogen losses to calculate the true nitrogen balance. The group's mean nitrogen requirement was calculated as the zero-balance intercept from the regression equation of true nitrogen balance (Y) on nitrogen intake (X), pooling all data points. The safe level of protein intake was calculated from the upper 95 per cent confidence band.

c. Urinary creatinine and urea nitrogen: These were determined by Folin's method and by a modified Van Slyke and Cullen method, respectively.

d. Anthropometry: Body weights were taken daily every morning before breakfast, post-voiding, with minimal clothing. The following measurements were obtained on day 0 and day 10 of each dietary period: height; arm, waist, chest, and head circumferences; and tricipital and subscapular skin-fold thicknesses.

Summary of main results

1. Nitrogen Balance and Absorption Summaries of individual mean nitrogen and energy intakes, apparent nitrogen balances, apparent nitrogen digestibility, and weight changes for each experimental period are presented in table 3. Significant drops in nitrogen retention, nitrogen absorption, and biological values were observed with the lowest level of nitrogen intake 1120 mg/kg/ day), and in some cases with intakes of 160 mg/kg/day.

2. Protein Requirements Figure 1 shows the regression analysis of true nitrogen balance on nitrogen intake (Y = - 60.3 + 0.537 X, Se = 12.6, n = 38). The mean nitrogen requirement was calculated as 112 mg, or a PRm of 0.70 9 protein/kg/day. The safe level of intake for 97.5 per cent of the population was calculated as 161 mg nitrogen, or 1.00 9 protein/ kg/day.

3. Body Weight Table 4 indicates that three of the five undernourished children reached normal weights at the end of the study. They included the two children with intestinal parasites.

TABLE 3. Individual Nitrogen and Energy Intakes, Apparent Nitrogen Balance, Apparent Nitrogen Digestibility, Protein Biological Value, and Changes in Body Weight at Each Level of Dietary Protein Intake

Subject	Mean daily nitrogen Biological intake (mg/kg)	Mean daily energy intake		Mean daily nitrogen excretion		Balance, apparent (mg/kg)	Absorption apparent (% of intake)	Retention, apparent (% of intake)	Biological value		Changes in body wt. (g/kg)
		(kcal/kg)	(%PE*)	urine (mg/kg)	faeces (mg/kg)
R.A.	346	109	7.9	115	96	135	72.2	39.0	54.0		6.1
	282	115	6.1	92	76	114	73.0	40.4	55.3		2.5
	260	112	5.8	76	87	107	66.5	41.2	56.1		0.5
	227	110	5.1	66	81	79	64.2	35.0	54.5		0.9
	153	123	4.1	53	72	78	64.5	38.4	59.5		3.8
	122	107	2.8	45	54	23	55.7	1 8.9	33.8		0
A.C.	358	113	7.9	113	119	126	66.8	35.2	52.7		9.9
	321	118	6.8	94	99	128	66.2	39.9	57.7		- 1.2
	275	121	5.7	86	88	100	67.9	36.5	53.8		5.9
	244	127	4.8	66	91	87	62.7	35.6	56.9		1.1
	209	129	4.0	56	63	90	69.9	43.1	61.6		1.8
	133	108	3.1	45	61	27	54.1	20.3	37.5		2.4
M.C.	338	119	7.1	106	86	146	74.6	43.2	57.9		9.6
	310	121	6.4	117	94	98	69.6	31.6	45.6		4.3
	271	116	5.9	80	88	103	67.5	38.0	56.3		3.7
	242	125	4.8	71	76	46	68.6	19.0	57.2		1.1
	158	109	3.6	62	76	20	51.9	12.8	24.4		0
	127	111	2.9	49	67	11	47.2	8.7	18.3		0.6
G.L.	277	98	7.0	130	79	68	71.5	24.6	34.3		2.9
	234	100	5.9	100	69	65	70.5	27.8	39.4		2.4
	204	100	5.1	79	62	63	69.6	30.9	44.4		0
	170	100	4.3	57	64	49	62.4	28.8	46.2		- 1.0
	125	99	3.2	53	55	17	56.0	13.6	24.3		1.9
J.M.	284	109	6.3	111	66	107	76.8	37.7	49.1		6.6
	240**	113	5.3	92	82	66	65.8	27.5	41.8		0.5
	200	100	5.0	57	51	62	74.5	31.0	41.6		- 1.7
	154	100	3.9	59	48	47	68.8	30.5	44.3		1.8
	120	99	3.0	65	48	7	60.0	5.8	9.7		-0.5
C.S.	263	107	6.4	99	78	86	70.3	32.7	76.5		- 2.3
	255	108	5.9	88	75	92	70.6	36.1	51.1		2.2
	190	107	4.4	68	76	46	60.0	24.2	40.4		1.1
	171	109	3.9	61	79	31	53.8	18.1	33.7		2.7
	112	110	2.5	47	60	5	46.4	4.5	9.6		- 0.8
R.A., Jr.	266	108	6.2	98	96	72	63.9	27.1	42.4		- 5.9
	245	107	5.5	86	80	79	67.4	32.2	45.2		- 2.1
	204	110	4.6	72	79	53	61.3	26.0	42.4		0.5
	160	110	3.7	68	65	27	59.4	16.9	28.4		- 0.5
	123	111	2.8	60	59	4	52.0	3.3	6.3		- 0.5

* Percentage of energy derived from dietary proteins.
** Child was given an antibiotic.

FIG. 1. Regression Analysis of "True" Nitrogen Balance on Nitrogen Intake (Pooled data, n = 38)

As table 3 shows, there was a trend towards increased rates of weight gain with protein-energy above 4 per cent. Appreciable weight gains were observed when nitrogen intakes were 270 mg/kg/day or higher.

4. Urinary Urea Nitrogen
Table 5 shows the results. There was a significant correlation between urinary urea nitrogen and nitrogen intake (r = 0.78, p < 0.001). The ratio of urinary urea nitrogen to urinary total nitrogen also correlated with nitrogen intake (r = 0.57, p < 0.001).

Conclusions and comments

1. The mean protein requirement with this Philippine diet was 0.70 g/kg/day and the safe level of intake was 1.0 g/kg/day. All children had good nitrogen retentions with intakes of 150 mg nitrogen, or 0.94 9 protein/kg/day. All these values are lower than current FAD/WHO estimates and recommendations.
2. Nitrogen absorption was low, probably due to indigestible proteins in rice.
3. When protein intake is too low, dietary energy is not used very efficiently and weight gains are reduced
4. Light infestations with A. Iumbricoides and T. trichiura did not affect dietary nitrogen utilization.

TABLE 4. Classification of the Subjects by Weight at Various Phases of the Study

Subjects	At start of study			After rehabilitation			At end of study
	Age (months)	Wt. (kg)	Degree of nourishment	Age (months)	Wt. (kg)	Degree of Nourishment	Age (months)	Wt. (kg)	Degree of nourishment
R.A.	26	10.3	1°1	29	12.1	N²	31	13.2	N
A.C.	18	8.9	1	20	9.9	1	23	10.5	1°
M.C.	20	8.7	1	23	10.1	1	26	10.1	1°
G.L.	26	11.6	N	28	11.7	N	30	13.2	N
C.S.	23	12.3	N	25	13.8	03	26	14.1	O
J.M.	20	8.9	1	22	10.4	1	24	11.2	N
R.A., Jr.	24	10.3	1	26	11.9	N	27	11.9	N

1 Degree of undernourishment.
2 N = normal.
3 0 - overweight.

TABLE 5. Summary of Individual Average Urinary Total Nitrogen (Utn) and Urea Nitrogen (Uun) for Each Protein Level

Subtract	Nitrogen intake (mg/kg)	Utn	Uun	UUn/Utn	Uun/g nitrogen Intake
R.A.	346	1.41	1.22	0.87	0.29
	282	1.14	0.78	0.68	0.22
	260	0.97	0.58	0.60	0.18
	227	0.86	0.68	0.79	0.23
	203	0,70	0.58	0.83	0.22
	122	0.60	0.43	0.72	0.27
A.C.	358	1.14	1.01	0.89	0.28
	321	0.96	0.84	0.88	0.26
	275	0.89	0.49	0.55	0.17
	244	0.71	0.57	0.80	0.22
	209	0.60	0.43	0.72	0.19
	133	0.47	0.34	0.72	0.24
M.C.	338	1.04	0.73	0.70	0.22
	310	1.18	0.72	0.61	0.26
	271	0.82	0.68	0.83	0.24
	242	0.74	0.68	0.92	0.27
	158	0.63	0.37	0.59	0.23
	127	0.50	0.26	0.52	0.20
G.L.	277	1.65	1.23	0.74	0.35
	234	1.28	0.93	0.73	0.31
	204	1.02	0.77	0.75	0.29
	170	0.75	0.56	0.75	0.25
	125	0.70	0.35	0.50	0.21
J.M.	284	1.17	0.71	0.61	0.24
	240-	1.03	0.44	0.43	0.16
	200	0.97	0.58	0.60	0.26
	154	0.65	0.40	0.62	0.23
	120	0.73	0.36	0.49	0.27
C.S.	263	1.35	0.94	0.70	0.26
	255	1.21	0.79	0.65	0.22
	190	0.95	0.49	0.52	0.19
	171	0.86	0.36	0.42	0.15
	112	0.67	0.18	0.27	0.11
R.A., Jr.	266	1.14	0.58	0.51	0.19
	245	1.01	0.38	0.38	0.14
	204	0.84	0.24	0.29	0.10
	160	0.81	0.25	0.31	0.13
	123	0.72	0.19	0.26	0.13

* Subject war given antibiotic.

(introduction...)

Objectives
Experimental details
Summary of main results
Conclusions

Benjamin Toraria 1. Cabrera Santiago, and Fernando E. Viteri
Institute of Nutrition of Central America and Panama (INCAP), Guatemala City, Guatemala

Objectives

1. To determine protein requirements using milk or a soybean isolate as the protein source and following traditional nitrogen balance techniques.
2. To evaluate the protein quality of the soybean isolate relative to milk.

Experimental details

(all values given as mean S.D.)

1. Subjects

1. Ten children, all males, of mixed Maya and Caucasian descent (Lading).
2. Chronological age: 23 4 months (range: 17 to 31). Height-age: 15 3 months (range: 9 to 23).
3. All had been treated for severe, oedematous protein-energy malnutrition (kwashiorkor or marasmic kwashiorkor). They had recovered fully at least one month before beginning the studies, based on clinical, anthropometric, and biochemical criteria (plasma proteins, non-essential/essential amino acid ratio, haematological indices, urinary creatinine excretion, and creatinine-height index [CHI] ). Only one child had a weight-for-height below 93 per cent of the expected (88 per cent) and a CHI of 0.84.
4. Weight: 10.13 1.00 kg (range: 8.82 to 11.961. Height: 77.6 4.4 cm (range: 71.3 to 86.4). Weight-forheight, percentage of expected: 97 4 (range: 88 to 104; see above). CHI: 0.95 0.08 (range: 0.84 to 108; see above).
5. Intestinal parasites: Three children had Ascaris lumbricoides. One of them plus another child had Trichuris trichiura. These two children and two others had Giardia lamblia. All infestations were mild (few eggs and protozoa in stools), and they were not treated before the study.
6. All children were healthy throughout the study, except for occasional episodes of upper respiratory infections with or without fever. When a child had fever, the study was interrupted and he received a diet that provided 2 to 2.5 9 protein and 100 kcal/kg/day for at least seven days after the fever and other symptoms had subsided. The study was then reinitiated with a protein intake at the level that preceded the level he was eating when he became ill. The only exceptions were when a child became ill during the final level of dietary protein intake. In that case, the study was terminated and his data were evaluated with only three levels of protein intake.

2. Study Environment
INCAP's Clinical Centre in Guatemala City; 1,500 m above sea level. Temperature: 18 to 24 C. Relative humidity: 40 to 50 per cent, except on rainy days.

3. Physical Activity
The children were encouraged to participate in games that involved walking, running, climbing stairs or ramps, and tossing balls between two and five hours every day, including the metabolic balance periods.

4. Duration of the Study

a. Four consecutive 9-day dietary periods, or 36 days in all, with a protein source.
b. Fourteen days with a diet that provided 2 to 3 9 protein and 100 kcal/kg/day.
c. Four consecutive 9-day dietary periods, or 36 days in all, with the other protein source.

5 . Diets
a. The components of the experimental diets are given in table 1. The amino acid compositions of the two protein sources are given in table 2.

TABLE 1. Constituents of Experimental Diets (g/kg/day)

	Protein intake levels
	A	B	C	D
Skimmed milk	3.55	2.85	2.13	1.43
(or soybean protein isolate.)	(1.44)	(1.15)	(0.86)	(0.57)
Cornstarch	1.50	1.50	1.80	2.20
Supar	13.25	13.80	14.12	14.28
Cottonseed oil	3.26	3.27	3.29	3.30
Mineral mixture	0.61	0.61	0.61	0.61
Water, to total of	80	80	80	80
Protein, g/kg/day	1.25	1.00	0.75	0.50
Energy, kcal/kg/day	100	100	100	100

* Purina Protein 220, Ralston-Purina Co., St. Louis, Mo., USA. These formulas contained more, cornstarch than the milk formulas to compensate for the higher enargy content of milk.
** Provides (in mEq): K 6; Na 1; Ca 1; Mg 0,4; Cl 6; PO₄ 1; CO₃ 1; SO₄ 0.4.

b. The liquid diets were cooked for 10 to 15 minutes and final weights were adjusted with water after cooling. Cinnamon flavour was added. Fibre content was extremely low, and fat provided 30 per cent of total energy.

c. Diets were fed as five isonitrogenous, isoenergetic meals at three-hour intervals, beginning at 8 a.m. Vitamin and mineral supplements were given each day to satisfy the children's requirements. Additional water was offered ad libitum. At the end of each meal the food containers were rinsed with water and the child drank it. Intake was measured weighing the containers immediately before and after each meal.

d. Dietary levels: The protein content of the diet was increased (ascending design) or decreased (descending design) by 0.25 g/kg/day at 9-day intervals. The changes were isoenergetic with carbohydrate replacement of protein and vice versa. Half the children began with 1.25 g/kg/day (descending design) and half with 0.5 g/kg/ day (ascending design ).

TABLE 2. Essential Amino Acids in Cow's Milk and Soybean Protein Isolate Used to Study Protein Requirements (mg of Amino Acid per Gram of Protein)*

Amino acid	Milk	Soy
Histidine	34.5	29.4
Isoleucine	56.4	50.2
Leucine	98.8	78.0
Lysine	85.8	62.1
Total sulphur amino acids	38.0	26.4
Methionine	27.6	13.0
Cystine	10.4	13.4
Total aromatic amino acids	93.0	88.7
Phenylalanine	52 .7	52 .5
Tyrosine	40.3	36.5
Threonine	44.1	36.5
Tryptophan	19.8	16.0
Valine	64.2	52.8

* Prom Tor al. (1980) and Cabrera-Santiago and Tor980). Amino acid analyses performed at the Ralston-Purina company's Research Laboratories, based on 24- and 88-hour hydrolysis.
** Purina-Protein 220, USA.

At the end of the fourth protein level, the children ate a diet that provided 2 to 3 9 of protein and 100 kcal/kg/day for 14 days and then followed once more the same experimental design with the other protein source. Half the children began the study with milk and half with soybean protein isolate.

6. Indicators and Measurements
a. Nitrogen balance was determined during the last four days of each nine-day period. Faeces were homogenized and dried at 80 C. Aliquots of diets, faeces, and urine were digested and analysed by a micro-Kjeldahl technique, and the results were corrected by the recovery factor of tryptophan standards that were digested and analysed simultaneously.

"Apparent" balance was calculated (i.e., dietary nitrogen-faecal nitrogen-urinary nitrogen). However, instead of using the zero-balance intercept to calculate nitrogen requirements, a retention of 24 mg N/kg/day was used to represent "balance," therefore allowing 9 and 15 mg N/kg/day to compensate for integumental losses and growth, respectively.

b. Protein digestibility was calculated both as "apparent" and as "true." For the latter, 20 mg N/kg/day was used as the mean obligatory (endogenous) faecal nitrogen.

c. Body weight was measured daily before breakfast. Height and other anthropometric measures were obtained at 14-day intervals.

d. Protein quality was calculated by computing the regression coefficients of nitrogen balance (Y) on intake (X) to determine the relative protein value (RPV) of the diet. Nitrogen intakes required to support a retention of 24 mg N/kg/day, as mentioned above, were also determined-that is, relative nitrogen requirement (RNR).

e. At the beginning of the study and at the end of each nine-day period, a blood sample was obtained to determine haematocrit (micro-centrifugation), plasma proteins (refractometry), serum albumin (dye binding with bromcresol purple), and serum aminotransferases (kinetic U.V. method, with and without addition of pyridoxal pyrophosphate). Urea (carbamido-diacetyl reaction) and creatinine (modified Folin and Wu) were determined in the urine collected during the balance periods.

Summary of main results

Tables 3 and 4 summarize the results derived from the nitrogen balance studies. Table 5 shows that total plasma proteins decreased after nine days with an intake of 0.5 9 milk protein/kg/day and also after nine days with 0.75 9 soybean protein/kg/day.

Except for a decrease in urinary urea with the decrease in nitrogen intake, there were no consistent diet-related changes in the other biochemical indicators explored.

Rates of weight gain during the nine-day periods were slower with milk protein intakes of 0.5 9 milk protein/kg/day than with 1.00 or 1.25 g/kg/day (ANOVA: F_{3 34} = 3.473; least significant difference 13.1 g/day).

Although the mean weight gain was also lower with the lowest levels of soybean protein intakes, these did not differ from the higher levels because of the large inter individual variability.

TABLE 3. Data Derived from Nitrogen Balances: Milk

 

Child	Digestibility (% of intake)		Regression of apparent Balance (Y) on intake (X) (mg N/kg/day) b		Nitrogen intake required to retain 24 mg N/kg/day c
	App.	"True"a	Y = a + bX	r
Descending design
A.A,	81	96	- 68 + 0.690X	0.991	133
C.R.	76	91	- 74 + 0.922X	0.970	106
I.G.	88	104	- 16 + 0.652X	0.994	61
H.A.	78	92	- 24 + 0.546X	0.972	88
W.G.	76	91	- 49 + 0.765X	0.988	95
D.B.	78	92	- 42 + 0.718X	0.992	92
Mean	80	94	- 46 + 0.716X		96
S.D.	5	5	23 .125		24
Pooled data (n=22) d			- 51 + 0.766X	0.914	98
Ascending design
W.M.	83	97	- 34 + 0.725X	0.995	80
M.V.	75	91	- 72 + 0.642X	0.997	150
A.Z.	82	96	- 51 +0.816X	0.999	92
I.T.	78	93	- 16 + 0.552X	0.958	72
Mean	80	94	- 43 + 0.684X		99
S.D.	4	3	24 .113		35
Pooled data (n=16)			- 48 + 0.718X	0.836	100
Both designs
Mean	80	94	- 45 + 0.703X		97
S.D.	4	4	22 .115		27
Pooled data (n=38)			- 49 + 0.740X	0.877	98

a "True" digestibility assumes obligatory faecal nitrogen loss of 20 mg/kg/day.
b Apparent balance = intake—urinary nitrogen—faecal nitrogen.
c Allowing 15 mg N/kg/day for growth and 9 mg N/kg/day for insensible losses.
d Using all data for the corresponding design; n = number of determinations.

TABLE 4. Data Derived from Nitrogen Balances: Soybean Protein Isolate

Child	Digestibility (% of intake)		Regression of apparent Balance (Y) on intake (X) (mg N/kg/day) b		Nitrogen intake required to retain 24 mg N/kg/day c
	App.	"True"a	Y = a + bX	r
Descending design
A.A.	81	96	- 34 + 0.522X	0.986	111
C.R.	67	83	- 52 + 0.558X	0.985	136
I.G.	82	95	- 36 + 0.563X	0.993	107
H.A.	76	96	- 27 + 0.473X	0.992	108
W.G.	73	89	- 52 + 0.543X	0.981	140
Mean	76	91	- 40 + 0.532X		120
S.D.	6	5	11 .036		16
Pooled data in (n = 18) d			- 44 + 0.553	0.936	123
Ascending design
W.M.	83	98	- 26 + 0.545X	0.998	92
W.M.A.	80	95	- 53 + 0.743X	0.998	104
M.V.	80	96	- 68 + 0.654X	0.965	141
A.Z.	76	91	- 63 + 0.698X	0.975	125
I.T.	77	92	- 46 + 0.528X	0.989	132
Mean	79	94	- 51 + 0.634X		119
S.D.	3	3	16 .094		20
Pooled data (n = 18)			- 55 + 0.662X	0.921	119
Both designs
Mean	78	94	- 46 + 0.583X		120
S.D.	5	4	15 .086		17
Pooled data (n = 36)			- 50 + 0.610X	0.920	121

a, b, c, d See footnotes in table 3.

TABLE 5. Plasma Protein Concentrations with Different Levels of Protein Intake (g/dl)

Protein intakes (g/kg/day)
	Descending design					Ascending design
	2.00	1.25	1.00	0.75	0.50	1.50*	0.50	0.75	1.00	1.25
Milk
mean	6.90	6 80	6.90	6.62	6.37 a	6.90	6.50 b	6.75	6.58	6.73
S.D.	0.20	0.29	0.54	0.31	0.38	0.29	0.29	0.96	0.67	0.25
		4/5 c	3/6	4/6	6/6		4/4	2/4	3/4	2/3
Soybean protein
mean	6.94	6.76	6.64	6.28 b	6.20	7.03	6.25 b	6.22	6.30	6.78
S.D.	0.23	0.43	0.21	0.16	0.57	0.36	0.44	0.43	0.64	0.53
		3/5	315	5/5	2/4		4/4	3/5	3/5	2/5

a,b Differs from preceding protein intake (student's paired "t" test): a: p< 0.01; b: p<0.05.
c Proportion of children with decreased concentrations after nine days on the corresponding dietary protein intake.
* Basal period.

 

The RPV and RNR of the soybean protein isolate, compared with milk, were 83 per cent and 81 per cent, respectively.

Conclusions

1. The mean nitrogen requirements, allowing for integumental losses and nitrogen retention for growth, were 98 mg (0.61 9 protein) of milk/kg/day and 120 mg (0.75 9 protein) of soybean isolate/kg/day. The milk requirement is 33 per cent lower than the estimate made by the FAD/WHO Committee of Experts on Energy and Protein Requirements (WHO Tech. Rep. Ser. No. 522, 1973).

2. The coefficients of variation between individuals of the regression coefficients and of the nitrogen requirements for the two protein sources were between 14 and 16 per cent, except for the mean nitrogen requirement with milk, which was 28 per cent. If two children with high requirements were excluded, variability would be reduced to 16 per cent. The safe levels of intake of the two proteins can be calculated by adding 30 per cent to the mean requirements (FAO/WHO, 1973), or by using the higher limit of the 95 per cent confidence bands (Rand et al., Am. J. Clin. Nutr., 30: 1129 [1977] ). With the former approach, they would be 127 mg nitrogen or 0.79 9 protein/ kg/day for milk, and 156 mg nitrogen or 0.98 9 protein/kg/day for soybean protein isolate. Using the confidence bands, they are 151 mg nitrogen or 0.94 9 protein/kg/day for milk, and 162 mg nitrogen or 1.01 9 protein/kg/day for the soybean isolate. All of these values are lower than the 1.19 9 milk protein/kg/day suggested by FAD/WHO

3. The nutritive value of the soybean protein isolate was 82 per cent relative to milk.

Acknowledgements

The Ralston-Purina Company provided us with the milk, soybean protein isolate, and other materials used in these studies. Ms. Cabrera-Santiago participated in the investigation under a Fellowship from the United Nations University World Hunger Programme.

(introduction...)

Objective
Experimental details
Summary of main results
Conclusions and comments

Jin Soon Ju, W. I. Hwang, and T.G. Ryu
Department of Nutrition and Biochemistry, Korea University, College of Medicine, Seoul, Republic of Korea

Objective

The purpose of this study was to define the changes in protein digestibility and protein requirements that occur in pre-school children infested with intestinal parasites, mainly Ascaris lumbricoides.

Experimental details

1. Subjects
Six boys and four girls, 52 to 70 months old, were selected among pre-schoolers from agricultural villages in the northwestern part of South Korea, about 48 km from Seoul. Their characteristics are given in table 1. They were healthy and normal, except for the intestinal parasites, based on medical history, physical examination, and laboratory analyses of blood and urine specimens, including transaminase and alkaline phosphatase activities.

2. Study Environrnent
The tests were done in a field metabolic unit during the fall. Outdoor temperature ranged from 3 to 25 C, and indoor temperature from 18 to 24 C.

3. Physical Activity
The activities of these children were normal for their age.

4. Duration of the Study
The study iasted ten weeks, divided into three periods: a. The first feeding period (BfV) was before treatment with a vermicide and lasted four weeks. b. The second period lasted two weeks, with a free-choice diet comparable to what the subjects ate at home. Ten mg/kg of Combantrin was given on the first day of this period. c. The third period (AV), after the vermicide treatment, lasted four weeks.

TABLE 1. Characteristics of Subjects

							Energy
							Intake		Approximate requimments ***
Subject	Sex	Age (years- months)	Height (cm) m³/hr)	Weight (kg)	SA* (m² )	BMR * (kcal/ m³/hr)	BfV **	AV	Estimated	FAO/ WHO
							(kcal/kg/day)
1	M	5-10	114.6	22.92	0.83	54.8	91.5	102.7	81.0	70.1
2	M	5- 9	11 5.6	20.5	0.80	55.4	86.9	102.7	88.2	74.2
3	M	5 - 9	107.5	1 7.67	0.72	59.9	101.8	100.0	102.5	80.3
Subjects 4 and 5 dropped
6	M	4- 4	107.3	18.64	0.73	60.7	92.9	102.5	96.9	75.7
7	M	4- 7	97.3	15.08	0.63	63.2	100.4	104.3	107.7	81 7
8	M	4- 7	96.3	14.83	0.62	64.2	101.2	106.8	109.5	82.6
9	F	5- 9	108.8	17.79	0.72	60.7	100.7	993	100,2	76.0
10	F	4- 8	95.8	15.92	0.63	61.2	103,7	99.6	98.8	80.1
11	F	4- 7	108.4	18.74	0.74	58.5	98.6	100.2	94,3	75,7
12	F	4 7	98.1	14.44	0.62	56.4	99.4	98.9	98.8	83.6

* Surface area (SA) and BMR were measured on the last day of the eighth experimental week. SA w0.425 x H0.725 x 71.84.
** BfV - before vermicide; AV ** after vermicide.
*** Approximate energy requirements: estimeted - BMR (kcal/day) x 1.7/body weight (BW); FAD/WHO = 1973 recommendations.

5. Dietary Intakes
Because of a generally low intake of animal protein, a daily supplemental feeding consisting of 150 ml of whole cow's milk was added to each subject's ordinary daily diet for one month before initiation of the study. The experimental diets were devised to provide approximately 100 kcal/kg/day and four levels of dietary protein (1.50, 1.75, 2.00, and 2.25 g/kg/day: diets A, B, C, and D, respectively), based on the local diet (see table 2). The four levels of dietary protein were fed in four consecutive weeks, both before and after treatment with the vermicide (BfV and AV, respectively). Three isoenergetic and isonitrogenous meals and light snacks were provided each day. Additional food intake, mainly rice, was allowed on request, especially in the AV period. A record was kept of each subject's food intake. Vitamin and mineral supplements were given once a day to meet requirements. Table 3 gives the estimated essential amino acid content and the chemical score of the experimental diets.

6. Indicators and Measurements

1. Complete urine and faecal collections were made daily throughout the two experimental feeding periods. Total nitrogen (micro-Kjeldahl), urea nitrogen (Folin-Wu), and creatinine (diacetyl-monoxime) were measured in the urine collected during the last four days of each week with a given diet. Nitrogen was also measured in faeces pooled during the same four days and in aliquots of the diets.
2. Fats (Soxhlet) and ash (combustion) in food and faeces were measured. Carbohydrates were calculated by subtracting protein, fat, ash, and moisture from total weights of food and faeces. Their energy contents were calculated using 4, 4, and 9 kcal per gram of carbohydrate, protein, and fat, respectively.
3. Nude body weight was recorded daily after the subject had voided the first morning urine before breakfast.
4. Venous blood samples were drawn before breakfast on the first and last days of the study. Haemoglobin, haematocrit, serum glucose, urea nitrogen, creatinine, SOOT, alkaline phosphatase, total proteins, albumin, and globulins were measured.
5. Stool examinations for the quantitative determination of helminth eggs were performed each of the first three days in both experimental periods (BfV and AV). The parasites expelled on the first three days after treatment were identified and counted.
6. The basal metabolic rate (BMR) was measured on day 14 of the AV period by the Douglas bag spirometer method.
7. The faecal obligatory nitrogen losses were estimated from the Y intercept of the regression equation of faecal nitrogen (Y) on nitrogen intake (X) (table 4). Digestibilities of protein and energy were expressed as the percentage of intake that was absorbed.

TABLE 2. Food Ingredients in the Daily Diet of a 17-kg Child (g/day)

Foods	Dietary Periods
	A	B	C	D
Rice	270	270	270	270
Wheat flour	-	13	-	-
Biscuits	-	30	-	20
Potato	80	80	80	30
Soybean curd	-	22	20	30
Soybean paste	-	25	-	9
Soy sauce	6	2.5	5	1
Radish	160	80	140	110
Spinach	-	25	-	30
Onion	5	10	10	10
Carrots	5	10	10	10
Seaweed	-	-	-	3
Candy	60
Sugar	-	12	70	46
Jam	50	30	-	10
Apple	-	-	80	100
Pear	75	100	100	100
Orange juice	100	100	100	100
Oil	1	2	1	1
Beef	10	2.5	-	15
Sausage	-	-	15
Dried small fish	-	2	1	1
Egg	-	-	-	30
Energy, kcal	1,660-	1,660	1,660	1,660
Protein, total g	26.2	34.1	29.5	38.3
g/kg	(1.5)	12.0)	(1.75)	12.25)
animal	3.6	4.3	2.6	7.5
Fibre,g	3.1	5.0	3.7	4.2

TABLE 3. Essential Amino Acid Composition of Experimental Diets (mg/g Protein)

		Diet A		Diet B		Diet C		Diet D
Amino acid	FAO/WHO (S)	mg	(A/S) %	mg	(B/S) %	mg	(C/S) %	mg	(D/S) %
Isoleucine	40	45.5	114	45.3	113	47.2	118	46.0	115
Leucine	70	83.8	120	82.1	118	82.1	118	81.5	116
Lysine	55	49.1 *	89	50.8	92	49.9 *	91	51.9	94
Methionine +Cystine	35	34.2	98	32.0 *	91	33.9	97	32.5*	93
Phenylalanine +Tyrosine	60	82.0	137	80.4	134	79.7	133	79.2	132
Threonine	40	39.8	99	38.9	97	39.5	99	39.4	99
Tryptophan	10	11.6	116	12.1	121	11.7	117	12.4	124
Valine	50	57.2	114	54.9	110	55.9	112	55.8	112
Total	360		428.6		421.4		423.9		422.6

* First limiting amino acid.

An obligatory faecal nitrogen loss of 32 mg/kg/day was used to calculate "true" protein digestibility. True nitrogen balance was calculated assuming 5 mg N/kg/day for integumental and miscellaneous losses. The biological value (BV) of protein was estimated as:

BV = (intake-faecal-urinary + faecal and urinary obligatory losses) X 100/(intake-faecal + faecal obligatory loss)

where the obligatory faecal and urinary losses were assumed to be 32 and 48 mg N/kg/day, respectively. The net protein utilization (NPU) was calculated from the biological value and digestibility.

Summary of main results

1. Body Weight
Most children gained weight at a rate three times greater than the Korean standard for the corresponding age during the experimental period (an average gain of 0.99 kg in 11 weeks). This suggests that energy intakes were above requirements. There were no differences in weight gains before and after treatment.

2. Height
Most children gained 1.8 to 2.2 cm in ten weeks, which is more than the Korean standard of 1.2 cm for the corresponding age group. There were no differences before and after treatment.

3. Parasitological Observations
Table 5 gives the ova counts on each of the three days before and after treatment and the number of helminths identified in faeces during the three days after treatment.

TABLE 4. Estimation of Faecal Obligatory Nitrogen Losses

No. of subjects	Levels of nitrogen intake		Faecal nitrogen output (mg/kg/day)
	Range (mg/kg/day)	Mean (mg/kg/day)
Before vermicide
8	228 - 269	250.0 ± 4.53*	79.46 ± 5.21
9	270 - 300	286.8 ± 3.47	84.86 ± 7.03
10	308- 340	322.1 ± 3.54	94.03± 5.77
10	344 - 371	353.5 ± 2.61	98.53 + 6.13
Y = 31.64 + 0.19x (for X = 0, Y = 32)
After vermicide
9	262 - 276	269.8 ± 1.70	83.90 ± 4.23
8	282 - 311	299.3 ± 3.44	92.66 ± 4.16
9	315 - 362	346.9 ± 4.70	96.61 ± 5.65
9	356 - 397	381.1 ± 3.32	111.90 ± 6.17
Y=21.69+0.23x (for X=0, Y=22)

* Mean ± S.E.

4. Nitrogen Digestibility and Balance
Table 6 gives the "true" nitrogen digestibility and "true" nitrogen balance data, as well as the biological values and NPU. There were no differences before and after treatment. There was a poor correlation between nitrogen balance and nitrogen intake.

5. Urinary Urea and Creatinine Excretions
Table 7 gives the results obtained.

6. Absorption of Nutrients
Table 8 gives the apparent absorption of carbohydrates and fat, the "true" absorption of protein, and the estimated absorption of total dietary energy.

TABLE 5. Parasitological Findings in Stools

Subject		EPG* before vermicide			Worms excreted in 3 days after vermicide			EPG after vermicide
		Day 3	Day 2	Day 1				Day 1	Day 2	Day 3
1	Al **	8,100	11,900	15,300	Al	8	Al	Neg.***	Neg.	Neg.
	Tt **	100	100		Tt	2	Tt	Neg.	Neg.	Neg.
2	Al	28,700	11,600	18,000	Al	8	Al	Neg.	Neg.	Neg.
	Tt	1,400	400	1,200	Tt	1	Tt	100	Neg.	Neg.
3	Al	22,600	32,000	35,100	Al	14	Al	Neg.	Neg.	Neg.
	Tt	500	500	300	Tt	2	Tt	100	Neg.	Neg.
Subjects 4 and 5 dropped
6	Al	28,300	44,900	4,300	AL	8	Al	Neg.	Neg.	Neg.
	Tt	400					Tt	Neg.	Neg.	Neg.
7	Al	6,500	10,200	19,200	Al	14	Al	Neg.	Neg.	Neg.
	Tt	3					Tt	Neg.	Neg.	Neg.
8	AL	6,100	9,800	2,700	Al	2	Al	Neg.	Neg.	Neg.
9	Al	4,200	13,700	18,100	Al	6	Al	Neg.	Neg.	Neg.
10	Al	11,000	10,900	5,000	Al	2	Al	Neg.	Neg.	Neg.
	Tt	200	400		Tt	2	Tt	Neg.	Neg.	Neg.
11	Al	300	600	4,300	Al	10	Al	Neg.	Neg.	Neg.
12	Al	200	400	200	Al	1	Al	Neg.	Neg.	Neg.

* EPG = number of eggs per gram of stool.
** Al = Ascaris lumbricoides; T = Trichuris trichiura.
*** Neg. = negative.

TABLE 6. Nitrogen Balance, BV, and NPU before and after Vermicidal Treatment

Diet	No of subjects	Nitrogen intake	Faecal nitrogen (mg/kg/day)	Urinary nitrogen	Balance	True digestibility (%)	BV	NPU (kcal/kg/ day)	Energy intake
Before vermicide
A	9	259.94 *	92.80	10.8.37	53.79	76.49	69.68	53.24	103.50
		±8.21	±6.70	±6.48	±9.44	±2.67	±3.05	±2.91	±2.74
C	10	276.36	92.48	130.67	48.26	78.17	61.50	48.05	98.0
		±9.12	±6.39	±4.85	±7.95	±2.15	±1.99	±1.89	±3.13
B	10	323.60	89.44	161.98	67.18	82.49	56.97	47.04	97.56
		±7.72	±6.97	±3.84	±7.05	±1.91	±1.73	±1.98	±2.36
D	11	340.53	91.95	191.09	52.49	82.43	49.05	40.55	92.15
		±7.14	±5.78	±6.30	±6.30	±1.60	±1.82	±1.89	±1.86
Average		302.12	91.65	150.10	55.39	79.90	59.30	47.22	98
			±3.11	±3.68	±3.84	±2.08	±2.15	±2.19	± 1.4
After vermicide
A	10	270.88	8522	117.28	63.38	80.36	68.17	54.76	102.49
		±1.94	±4.01	±5.11	±5.80	±1.50	±2.28	±1.99	±0.91
C	8	303.30	97.20	140.20	60.90	78.54	61.08	48.11	101.60
		±2.94	±6.60	±4.15	±7.15	±2.09	±1.96	±2.37	±0.89
B	9	353.49	92.93	178.79	76.77	82.77	55.28	45.73	101.98
		±3.52	±3.41	±4.68	±5.33	±0.97	±1.53	±1.37	±1.06
D	8	383.13	113.35	210.58	52.97	78.75	46.06	36.19	102.39
		±4.00	±6.97	±6.98	±2.99	±1.69	±1.36	±0.91	±0.90
Average		324.90	96.37	159.66	63.85	80.11	57.65	46.20	102
			±3.03	±6.65	±3.03	±1.56	±1.78	±1.67	± 0.5

* Mean ± S.E.

TABLE 7. Daily Urinary Excretion of Urea Nitrogen and Creatinine (mg/kg/day)

Item	Diets
	A	C	B	D
Before vermicide
Ures N (UuN)	77.97 + 5.50 *	91.15 ± 4.05	112.38 ± 2.96	139.57 ± 5.37
Creatinine (UCr)	15.65 ± 0.57	15.15 ± 0.57	16.12 ± 0.54	15.20 ± 0.48
UtN **/nitrogen intake	9.42	0.47	0.50	0.56
UuN/UCr	4.98	6.00	6.98	9.18
UuN/nitrogen intake	0.39	0.33	0 35	0.41
UuN/UtN	0.72	0.70	0.70	0.73
After vermicide
Urea N (UuN)	89.55 ± 3.91	109.93 ± 3.69	152.50±5.05	169.48±6.82
Creatinine (UCr)	15.13 ± 0.43	15.71 ± 0.46	15.90 ± 0.56	16.72 ± 0.69
UtN/nitrogen intake	0.43	0.46	0.51	0.55
Uun/UCr	5.93	7.00	9.59	10.15
UuN/nitrogen intake	0.33	0.36	0.43	0.44
UuN/UtN	0.76	0.78	0.85	0.80

* Mean ± S.E,
** UtN = total urinary nitrogen.

There were no differences at the various levels of protein intake or as a result of vermicidal treatment.

7. Analyses of Blood and Serum Samples
There were no significant changes during the study and all results were within normal ranges.

Conclusions and comments

1. There were no changes in weight or height gains as a result of deworming.
2. Deworming did not change the absorption of nitrogen, cabohydrates, and fats, or the total dietary energy.
3. Nitrogen balance and the protein biological value and NPU were not affected by the parasites.
4. The results coincide with our studies in adults, indicating that ascariasis and trichuriasis of the severity reported in our investigations do not interfere with the intake, absorption, and retention of protein.

TABLE 8. Absorption of Nutrients (Percentage of Intake)

Items
Energy,	BfV	94.7 ± 0.40*	95.5 ± 0.28	95.4 ± 0.45	93.8 ± 1.50
	AV	95.0 ± 0.40	94.8 ± 0.56	95.0 ± 0.36	94.3 ± 0.54
CHO,	BfV	98.9 ± 0.23	99.3 ± 0.23	99.0 ± 0.28	97.0 ± 1.74
	AV	98.9 ± 0.27	99.6 ± 0.29	99.7 ± 0.35	99.7 ± 0.21
Fat,	BfV	93.3 ± 1.02	94.5 ± 0.40	95.4 ± 0.55	92.9 ± 0.74
	AV	96.5 ± 0.25	93.5 ± 0.48	96.6 ± 0.39	93.6 ± 0.55
Protein,**	BfV	76.5 ± 2.67	78.2 ± 2.15	82.5 ± 1.91	82.4 ± 1.60
	AV	80.4 ± 1.50	78.5 ± 2.09	82.8 ± 2.09	78.8 ± 1.69

* Mean ± S.E.
** "True" absorption after accounting for estimates of obligatory faecal nitrogen.

(introduction...)

Objectives
Experimental details
Summary of the main results

F.E. Viteri, B. Tor. Arroyave, and O. Pineda
Institute of Nutrition of Central America and Panama (INCAP), Guatemala City, Guatemala

Objectives

1. To determine whether pre-school children can fulfil their protein requirements with a diet based on corn and beans.
2. To determine which proportions of corn-and-bean-protein mixtures are better, in the sense that they lead to lower protein requirements and are the proportions spontaneously selected by pre-school children.
3. To determine the energy density of corn-bean diets necessary to fulfil both the protein and energy requirements of pre-school children eating ad libitum. (Is bulk a limiting element in corn-bean diets in preschool children?)

Experimental details

1. Subjects
Healthy pre-school children, fully recovered from previous oedematous protein energy malnutrition. Chronological age: 2 to 3 years old; height-age: 1 to 3 years old.

2. Study Environment
Clinical Centre at INCAP.

3. Physical Activity
The children were free to play in the Clinical Centre and surrounding grounds when not undergoing urine and faecal collections. However, they were not encouraged to be active.

4. Duration of the Study and Diets Used
The duration and diet of each study are presented below in "Summary of the Main Results."

5. Indicators and Measurements

a. Anthropometry: body weight, height, and skin-fold thickness.
b. Serum proteins and albumin; creatinine-height index.
c. Nitrogen absorption and retention, as described in other INCAP studies.

Summary of the main results

1. A study to test the hypothesis that, with a mixture of corn and beans in the proportions usually eaten, one can obtain sufficiently high concentration and quality of protein (ND p cal %) to satisfy calorie and protein requirements of pre-school children.

Four children between 21 and 28 months of age, with height-ages between 14 and 16 months, were fed a diet in which protein was provided by corn and black beans only, in a proportion of 76 and 24 per cent, respectively. Energy intake was fixed at 100 kcal/kg/day, 20 per cent of which came from vegetable fat. The children received a vitamin supplement. Protein intake was constant for two weeks at each of the following levels (g/kg/day): 1.00, 1.25, 1.50,1.75, 2.00, and 2.25. Two children followed an ascending and two a descending order of protein intake. Nitrogen balance was measured the last three days of each protein level. The children gained adequate weight and no clear tendency in height changes was detected.

Nitrogen balance is presented in figure 1. Nitrogen retained has been corrected for skin and other losses amounting to 14 mg/kg/day. Intakes of corn-bean protein greater than 1.3 g/kg/day resulted in positive balance in all children. However, two children retained less nitrogen than ideal for maintenance and growth (calculated for children of their height-age to be 20.7 mg/kg/dayl, although they were very close to that figure (19 and 16 mg/kg/day).

If 1.4 g of protein/kg/day is taken as the safe level of intake and is compared with 1.25 obtained with egg protein, this corn-and-bean mixture has a relative value of 89 per cent protein quality in relation to egg. This is very close to the relative urea:creatinine ratio in urine at different levels of protein intake obtained with the two proteins.

FIG. 1. Nitrogen Retention Necessary for Normal Growth of Children One to Two Years Old (Height-Age of Subjects)

The 95 per cent confidence interval from the nitrogen balance data yields 1.7 9 of corn-bean protein/kg/day as needed for all children studied under the above conditions.

2. Based on the results of the previous study ( 1, above), the following study, consisting of three phases, each of two months' duration, was conducted in four pre-school-age children. The four children participated in three phases aimed at testing the following hypotheses:

Phase 1: A corn-bean diet that provides 76:24 protein from the respective sources is adequate for pre-school children when fed for two months at a level providing 1.5 9 protein/kg/day and 100 kcal/kg/day.

Phase 2: A similar diet, but one providing a 60:40 protein ratio from corn and beans, is superior to that tested in phase 1 under the same conditions.

Phase 3: Children allowed to choose freely from corn-and-bean dishes satisfy their protein and energy needs and select close to a perfect ratio of corn and bean protein.

The diet in phase 1 was homogenized. In the other two phases, familiar corn and bean preparations were given or offered to the children. They were offered food five times per day; at least three of the meals contained beans.

The four children in this study were between 21 and 32 months of age, with height ages betweeen 16 and 19 months. They remained essentially healthy throughout the study.

Nitrogen balance (three days) was performed periodically. In phase 1, four balances were performed in each child (one every two weeks); in phase 2, five balances were performed, except for one child who had only four balances; in phase 3, three balances were performed except for the same child who had only two.

Results are summarized in figure 2. During phase 1, four balances were either marginal or lower than expected, with intakes ranging between 230 and 240 mg N/kg/day. During phase 2, all balances, except for one unexplained case, were satisfactory, with intakes between 260 and 270 mg/kg/day. During phase 3, nitrogen intake (ad libitum) was higher (between 330 and 370 mg/kg/day), the selected ratio of corn to bean protein was 49:51, and all nitrogen balances were highly positive.

3. The purpose of this study was to define the necessary energy density of corn-bean diets to ensure that such diets fulfil protein and energy requirements when consumed ad libitum by pre-school children.

Four pre-school children, between 21 and 26 months of age (height-ages between 14 and 16 months), were studied. The diets were offered four times a day as follows: First, the children were offered corn and beans in amounts that ensured a protein intake of 1.75 g/kg/day, 50 per cent of the protein coming from each source (corn and beans). Only after the children had consumed this amount of food, given as tortilla and black bean paste, were they offered ad libitum extra amounts of these foods plus banana and lemonade. Three levels of energy density were tested (see table 1); these were achieved by adding vegetable oil to the black bean paste. All children received vitamin supplements.

FIG. 2. Nitrogen Retention of Children Receiving Corn-Bean Diets

TABLE 1. Protein, Fat, and Calorie Contents of the Experimental Diets, Excluding Banana and Lemonade

	Caloric density
	Low	Intermediate	High
Protein (9/100 9 dry wt)	12.9	11.9	11.0
Fat (9/100 9 dry wt)	4	12	21
Intrinsic fat (%)	100	33	19
Added vegetable fat (%)	0	67	81
Fat calories (% of total)	8	22	35
Caloric density (cal/g dry wt)	4.5	4.9	5.4
Caloric density (cal/g protein)	34 9	41.2	49.1

Two children were given the highest-energy-density diet first and followed a descending design; the other two children followed an ascending design. Each energy-density diet was offered for three weeks. The study lasted nine weeks per child. Nitrogen balance was performed during the last three days on each diet.

Table 2 presents actual energy and protein intakes and changes in body weight in g/day. It is evident that with the lowest energy-density diet (4.5 kcal/g of dry diet, 8 per cent fat calories), weight gain was inadequate. Table 3 attempts to provide mean estimates of energy adequacy, although no actual measurements of energy expenditure were performed. Table 4 presents the nitrogen balance data plus other indicators of protein nutrition. All these parameters were satisfactory. Figure 3 expresses graphically the mean weight gain in relation to that expected for the children's height age. It is clear that with the lowest energy-density diet children do not grow adequately.

TABLE 2. Total Calorie and Protein Intakes and Weight and Height Increments with the Three Experimental Diets

	Dietary caloric density (cal/g dry wt)
	4.5	4.9	5.4
Total caloric intake/kg/day	843.3 ± 6.2*	95.7 ± 12.5	102.2 ± 10.4
Total protein intake (g)/kg/day	2.3 ± 0.2	2.3 ± 0.3	2.0 ± 0.2
Change in weight (g/day)	6.5 ± 9.3	14.8 ± 8.7	13.2 ± 9.7
Change in height (mm/day)	0.45 ± 0.39	0.01 ± 0.02	0.37 ± 0.36

TABLE 3. Mean Caloric Expenditures for Children Weighing 10.5 kg

			Cal/kg/day
Basal			59
Activity			24
Growth			6
Total			89
Mean expected weight gain (g/day)			12
Mean expected height gain (mm/day)			0.32
	4%	12%	21%
Dietary caloric density (cal/g dry wt)	4.5	4.9	5.4
Mean caloric intake (%of required)	95	108	115
Mean caloric intake above basal (car/kg/day)	28	40	46
Mean weight gain (%of expected)	54	123	110
Mean height gain (% of expected)*	117	31	142

* The total height gain is 97 per cent of expected for the total period of the study.

TABLE 4. Nitrogen Retention and Biochemical Indices of Protein Nutrition in Children Consuming Three Corn-and-Bean Experimental Diets with Different Caloric Densities

	Dietary caloric density (cal/g dry wt)
	4.5	49	5.4
Nitrogen retained (mg/kg/day)	187 ± 34*	97 ± 32	140 ± 40
Total serum proteins (9/100 ml )	6.25 ± 0.26	6.33 ± 0.31	6.15 ± 0.34
Serum albumin (9/100 ml)	3.75 ± 0.17	3.85 ± 0.26	3.78 ± 0.13
Non-essential /essential amino acid ratio	1.97 ± 0.21	2.14 ± 0.12	2.10 ± 0.20
Creatinine-height index (%)	+5.8 ± 4.4	-4.5 ± 4.4	+0.8 ± 4.0

FIG. 3. Mean Weight Gain Relative to That Expected for the Height-Age

(introduction...)

Objective
Experimental details
Summary of main results
Comments
Conclusions

Benjamin Tord Fernando E. Viteri
Institute of Nutrition of Central America and Panama (INCAP), Guatemala City, Guatemala

Objective

To assess whether a diet considered customary among certain segments of the pre school-age population of Guatemala supplied adequate amounts of protein, provided that:

1. Energy intakes corresponded to the estimated requirements.
2. There were no constraints in the amounts of staple foods available to the children.
3. The children were encouraged to eat as much food as they wanted to.
4. The children were in good health and nutritional status at the beginning of the study.

Experimental details

1. Subjects

1. Eleven boys of mixed Maya and Caucasian descent (Lading).
2. Chronological age: 37 ± 4 months (range: 29 to 46). Height-age: 20 ± 4 months (range: 13 to 26).
3. All had been treated for moderate or severe protein-energy malnutrition. They had recovered fully at least two months before beginning the studies, based on clinical, anthropometric, and biochemical criteria (plasma proteins, non-essential/essential amino acid ratio, haematological indices).
4. Weight: 11.69 ± 0.82 kg (range: 10.47 to 13.00). Height: 83.8 ± 4.3 cm (range: 76.1 to 89.4). Weightfonheight, percentage of expected: 99 ± 5 per cent (range: 92 to 109).
5. The children were happy and active throughout the study, except for short disease episodes. Children (I.D. nos. 404, 405, 408, and 410) had febrile infections in weeks three, one, four, and six, respectively, that lasted from four to seven days. Only child 410 received antibiotics, to treat a periodontal infection. All others received symptomatic medication for upper respiratory infections. During week three, child 406 had an afebrile exanthema of unknown aetiology that disappeared spontaneously, and child 407 had an afebrile upper respiratory infection. Finally, all children except 405 had an afebrile upper respiratory infection characterized by catarrh and coughing during the last one or two weeks of the study.

A few children vomited occasionally and sometimes had slight increments in rectal temperature (<38.5° C) without other signs or symptoms of disease.

2. Study Environment
INCAP's Clinical Centre in Guatemala City; 1,500 metres above sea level. Temperature 18 to 24 C. Relative humidity 40 to 60 per cent. All children spent four to six hours each day outdoors on the grounds and playing area around the Clinical Centre, except on rainy days.

3. Physical Activity
The children were encouraged to be as physically active as healthy children who live in a good home environment. This was done through daily outdoor walks in the areas around the Clinial Centre and participation in games and other activities that required walking, running, jumping, or climbing. They were never forced to participate in those activities when they did not feel like doing so, nor were they ever pushed to exhaustion. Such activities alternated with periods of rest or sedentary play to avoid boredom or fatigue.

4. Duration of the Study
The children ate the experimental diets for at least 11 weeks, divided into the fol lowing periods:

1. Two weeks: ad libitum intake of indigenous diet to measure each child's individual food intake.
2. One week: ad libitum intake of indigenous diet with a higher density, whenever necessary, to ensure intakes of 87 to 97 kcal/kg/day.
3. Eight weeks: experimental period. Metabolic and other measurements while eating the diet with 87 to 97 kcal/kg/day in ad libitum amounts. The results were analysed as two separate four-week periods, as described below.

TABLE 1. Average Composition and Frequency of Consumption of Diets Customary for Many Children of Pre-school Age in Guatemala

Food	Intake per day (g)	Frequency of intake (days/week)	Weekly intake
			Amount (g)	Protein (g)	Energy (kcal)
Corn tortilla flour	105	7	735	67.6	2,734
Black bean flour	18	7	126	27.8	423
Bread (sweet roll), fresh	37	7	259	19.7	1,000
Vegetables (chayote, squash,or potatoes), raw	44	7	308	5.8	163
Milk products (as fluid milk equivalents)	100	3	300	9.9	195
Fruit (orange, apple, banana),fresh	30	4	120	0.6	60
Egg, fresh	43	2	86	9.9	142
Meat, raw (as beef equivalent)	40	1	40	7.6	97
Sugar	42	7	294	-	1,176
Oil or lard	5	7	35	-	315
Total intake per week				148.9	6,305
Mean intake per day				21.3	901
Mean intake/kg/day (assuming weight of 12 kg)				1.78	75
Bean: corn ratio = 15:85 by weight and 29:71 by protein contents
Animal protein = 18% of total
Energy from fat (including natural fat content of all foods) = 17% of total

TABLE 2. Menus Offered to the Children ^a

Days	Breakfast (7 a.m.)		Lunch (11 a.m.)		Dinner (6.30 p.m.)
	corn and beans b		corn and beans sweet breads		corn and beans sweet bread
	+		+		+
Mon.	egg (1 unit) a		apple		chayote d
Tue.	potatoes		apple		potatoes
Wed.	chayote		beef (40 g) a		apple
Thu.	squash		potatoes		potatoes
Fri.	egg (1 unit) a		squash		squash
Sat.	potatoes		apple		potatoes
Sun.	chayote		chayote		potatoes
		Afternoon snack (3 p.m.): sweet bread (13 g) a + Mon., Wed., Sat.: milk (100 ml) a Tue., Thu., Sun.: lemonade (200 ml) a		Night drink (8 p.m.): lemonade or water

a All foods offered ad libitum, except when noted otherwise.
b Corn-based beverage (atole), soft corn bread (tamal), and mashed black bean puree.
c Sweet bread dough prepared with sugar and lard,
d Chayote = Sechium edule.

5. Diet

1. The diet was based on the proportions of foods eaten by pre-school children from poor socio-economic rural families (see table 1). For practical reasons of food preparation in the metabolic kitchen, fluid milk was used as the only dairy product and the vegetables and fruits were limited to those shown in table 2.
2. The children ate three daily meals and a mid-afternoon snack (see table 2). All meals included the basic staple foods of the region (corn and black beans) plus one or two other foods that varied from meal to meal and from day to day. The snack consisted of a sweet roll and half a cup of milk or lemonade. The daily menus were repeated at seven-day intervals; that is, there were menus for Sundays, Mondays, Tuesdays, and so on. The children ate as much as they wanted and the nurses played the role of mother at meal times, encouraging but not forcing the children to eat all that was served. If a child asked for more of a food served in a meal, it was given to him except for the foods that are scarce and limited in the home environments represented by the study (meat: maximum of 40 9 once weekly; egg: maximum of one twice weekly; milk: maximum of 100 ml three times weekly). No child was forced to eat if he refused to do so. Lemonade or water was offered to the child before retiring at night. By eating ad libitum, each child determined his own intake of proteins and other nutrients for the day. Energy intake, however, was adjusted each week, based on the preceding week's intakes, to approximately 92 ± 5 kcal/kg/day. The adjustments were made by adding more or less oil to the black beans and more or less sugar to the lemonade, or substituting water for the latter before retiring. Regardless of the preceding week's intakes, no more dietary adjustments were made in the last four weeks of the study.
3. Since the purpose of the study was to evaluate protein and energy nutriture, the diet was supplemented with vitamins and minerals, as shown in table 3.

6. Indicators and Measurements
a. Metabolic-balance studies: Most of the children were not toilettrained. To avoid excessive limitations of physical activity, complete 24-hour urine and faecal collections were obtained at 4-day intervals so that every 28 days excrete corresponding to the seven different menus for each day of the week were collected. Urine collection began after the first morning micturition and ended after a micturition 22 to 26 hours later; volumes were adjusted to 24-hour periods. Faeces were collected between carmine red and charcoal faecal markers fed with breakfast on 2 consecutive days. When the two markers were excreted together or when there were other problems, such as losses of excrete, collections were repeated on the same day of the following week. Faeces were homogenized and dried. Their nitrogen and energy concentrations were measured in aliquots of powdered faeces by Kjeldahl analysis and bomb calorimetry, respectively. Urinary nitrogen was also determined by a Kjeldahl procedure. Each food was served in a separate dish or cup at every meal. The amounts eaten by a child were measured by weighing the corresponding containers before and after each meal, accounting for any additional servings or for losses by spillage. Aliquots of each food, as served to the children, were analysed at least four times during the study using the same methods as those for faeces. Tryptophan and benzoic acid were used as standards in each Kjeldahl and bomb calorimeter run, respectively. Total daily intakes of protein (nitrogen x 6.25) and gross energy (by bomb calorimetry) were calculated multiplying by the amounts of each food ingested.

Nitrogen balance ("apparent") was calculated by subtracting urinary and faecal nitrogen from intake. No corrections were made for integumental and other insensible nitrogen losses.

TABLE 3. Vitamin and Mineral Supplements Administered Daily

Vitamin A	2,500	I.U .
Vitamin B1	1	mg
Vitamin B2	0.5	mg
Niacinamide	5	mg
Vitamin B6	0.5	mg
Pantothenic acid	5	mg
Folic acid	30	mog
Vitamin Bl2	2	mcg
Biotin	50	mcg
Vitamin C	25	mg
Vitamin D	500	I.U.
Vitamin E	1.5	mg
Iron (as ferrous sulphate)	60	mg
iodine (as Kl)	100	mcg
Manganese sulphate	0.9	mg
Zinc sulphate	1	mg

Net energy intake was calculated as the gross value intake minus faecal energy (by bomb calorimetry). This value was used to calculate the contribution of dietary protein to total energy intake (P%), assuming that each 9 of protein ingested corresponded to 4 kcal of metabolized energy. The dietary energy retained was calculated by subtracting urinary nitrogen energy (estimated as 5 kcal/g urinary nitrogen) from the net intake. Energy balance was calculated by subtracting the total energy expenditure, as described below, and sweat losses (estimated as 0.1 kcal/kg/day, based on 8 kcal/g sweat nitrogen) from the dietary energy retained.

The daily metabolic-balance data were combined in 28-day periods that included intakes and excrete corresponding to each of the 7 days of the week. These periods were termed I and 11.

Apparent digestibility of nitrogen and apparent absorption of energy were calculated from the combined gross intakes and faecal excrete of a 28-day period.

It was assumed that collection days when a child ate little food or did not defecate much would be balanced by other collection days with higher intakes or greater faecal excretions. During collection days the children who were not toilet-trained remained in a metabolic bed during the hours in which it was expected that they would defecate and while they slept; at other times they moved and played around freely while wearing urine collection bags.

b. Basal oxygen consumption: This was measured with an oxygen diaferometer at 1 B-day intervals, each time on two separate occasions not more than 3 days apart; the lower of the two results was considered as basal. Basal conditions were defined as after a minimum of eight hours of sleep and ten hours of fasting. Measurements were done while the child was sleeping, sometimes after oral administration of chloral hydrate (4 mg/kg). Energy expenditure was calculated by indirect calorimetry, assuming a respiratory quotient of 0.82.

c. Total energy expenditure: Physical activity and energy expenditure were quantified by monitoring the children's heart rate (HR) throughout the day and calculating energy expenditure from individual determinations of heart rate and oxygen consumption (VO2). The HR-VO2 relationship was determined in each child at 14- to 21-day intervals. Heart rate was continuously monitored for at least 10 days within ± 7 days of determining the HR-VO2 relationship. Total daily energy expenditure was calculated from each child's heart rate and his corresponding heart rate-energy-expenditure relationship from 6 a.m. to 8 p.m. (14 hours), and from his basal energy expenditure from 8 p.m. to 6 a.m. of the following day (10 hours).

d. Anthropometry: The children were weighed naked before breakfast each morning. Body length ("height"), right arm circumference, and subcutaneous skin-fold thickness (tricipital, subscapular, and paraumbilical) were measured initially and at 14-day intervals.

e. Urinary creatinine excretion: This was measured in the 24-hour urine collections obtained for nitrogen balance. An alkaline picrate method (Jaffe) was used. The creatinine-height index (CHI ) was computed, and running or weekly averages were calculated, including and excluding data from the days when meat was eaten.

f. Other biochemical and haematological determinations: Venous blood was drawn initially and at 18-day intervals. Packed cell volume (microcentrifuge) and the concentrations of blood haemoglobin (cyanomethaemoglobin), plasma proteins (refractometry), and serum albumin (bromcresol purple) were determined, as well as the ratio of serum non-essential/essential amino acids (Whitehead).

g. Statistical analysis: Changes in weight, anthropometry, and CHI were calculated by regression analysis. Data calculated at 7-,14-, or 18-day intervals were also computed by analysis of variance. Differences between the 28-day periods were examined by the student's paired t test.

Unless otherwise noted, the data in the text and tables are expressed as the mean + standard deviation, and in the figures as the mean + standard error of the mean.

Summary of main results

1. Food Intake
Although there were differences among children, on a group basis food intake did not differ significantly from week to week. Febrile episodes were usually accompanied by anorexia, resulting in diminished food intakes. In most cases these episodes were followed by a transient increase above the food intake preceding the illness.

2. Growth
Table 4 and figure 1 show the anthropometric changes. a. Weight: One child (402) did not gain weight and two (401, 410) gained at a rate slower than the 0.45 to 0.50 g/kg/day expected for healthy children of the same height-age. In contrast, two children (404,405) gained weight at more than twice that rate. b. Height: Five children grew at the expected rate of 0.30 to 0.34 mm/day. The other six children grew more (0.43 to 0.64 mm/day). This resulted in some catch-up growth, as shown in figure 2. c. Weight-for-height: Maximum individual changes were + 3 per cent. There were no changes on a group basis. d. Other anthropometric measurements: A small decrease in tricipital skin-fold thickness resulted in a slight increment of lean arm diameter, since there were no changes in arm circumference.

3. Protein Intake, Digestibility, and Balance
Figure 3 and table 5 give the individual and group data. Protein intake accounted for 8.8 ± 1.1 per cent of the net dietary energy. Mean protein intakes were high (1.75 ± 0.22 g/kg/day), and apparent digestibilities were about 72 ± 5 per cent, greater in period I than 11 by 3 per cent. "True" digestibilities were about 7 per cent higher than apparent digestibilities. The average amount of protein "truly" absorbed was 1.46 ± 0.17 g/kg/day.

TABLE 4. Average Values and Rates of Change in Anthropometric Measurements and CHI of 11 Children during Periods I and II

Average values	Period I	Period II	Mean of I and II	Paired t I vs. II a
Weight (kg)	11.98 ± 0.80*b	12.12 ± 0.89	12.05 ± 0.83	2.367
Height (cm)	84.5 ± 4.3*	85.7± 4.3	85.1±4.2	6.120
Weight-for-height 1%)c	99±4	100±5	99±4	0.810
Arm circumference (cm)	16.1±0.8	16.0±0.9	16.0 ± 0.8	0.088
Lean arm diameter (cm)d	42.9± 1.5*	43.6±1.6	43.2 ± 1.6	3.169
Subcutaneous skin-fold thicknessese	17,6±3.1*	16.5±3.2	17.0 ± 3.1	2.300
CHI (units)f	80±0.13	0.82±0.12	0.82 ± 0.12	1.847
Rates of change
Weight (g/day)	10.6 ± 8.7	3,7 ± 6.4	7.2±8.2	1,987
Weight (g/kg/day)	0.87±0.70	0.29±0,54	0.58±0.68	1.955
Height (mm/day)	0.41 ± 0.16	0.46± 0.23	0.43± 0.12	0.565
Lean arm diameter (mm/day)	0.02± 0.04	0.02± 0,04	0.02± 0.02	0.140
Subcutaneous skin-fold thicknesses (mm/day)	- 0.01 ± 0.06	- 0.04 ± 0.03	- 0.03 ± 0.04	1.342
CHI [units/Period)	0.035 ± 0.103	0,011 ± 0.106	0.023 ± 0.103	0.812

a For 10 degrees of freedom p<0.05 = 2.228 and p<0.01 = 3.169.
b Mean ± standard deviation,
c Weight expected for height: 100 per cent = 50th percentile of Boston standards.
d Corrected for subcutaneous skin-fold thickness.
e Sum of 3 sites: tricipital, subscapular, and paraumbilical.
f Creatinine-height index calculated from urine excreted on days without meat ingestion.
g Weight changes calculated by- individual regression analyses over 28 days. All other changes by individual differences between days 0 and 28 [Period 1) and between days 28 and 56 (period 11).
* Mean values of the two periods differ (see paired t value),

FIG. 1. Cumulative Anthropometric Changes-Periods I and II (11 children)

FIG. 2. Curves of Height Growth

FIG. 3. Nitrogen Balance Study

 

TABLE 5. Metabolic Balance Studies and Energy Expenditures in Periods I and II

	(11 Children) a
	Period I	Period II	Mean of I and IIb	Paired t I vs IIb
Protein
Protein intakec (g/kg/day)	1.85 ± 0.19 d	1.85 ± 0.25	1.85 ± 0.22	0.183
Apparent digestibility (%)	73.6 ± 4.6*	70.6 ± 4.7	72.1 ± 4.8	3.100
"True" digestibility e (%)	80.4 ± 4.6*	77.4 ± 4.7	78.9 ± 4.8
Nitrogen balance f (mg/kg/day) 98.0 ± 20.6	82.3 ± 20.6	90.2 ± 2.16	1.905
p% g (% energy)	8.6 ± 0.9	8.9 ± 1.3	8.8 ± 1.1	1.847
Energy
Gross intakeh (kcal/kg/day)	93.6 ± 4.6	90.9 ± 4.8	92.3 ± 4.8	1.415
Apparent absorption, (%)	91.9 ± 1.6	91.2 ± 1.7	91.6 + 1.6	1.326
Net intakei (kcal/kg/day)	85.9 ± 4.3	83.2 ± 4.8	84.6 ± 4.7	1.383
Total energy expanditure j (kcal/kg/day)	76.6 ± 8.6	73.0 ± 6.3	74.8 ± 7.6	2.188
Energy balancek (kcal/kg/day)	8.2 ± 10.1	9.7 ± 6.2	9,0 ± 8.2	0.589
Basal energy expenditure,
(kcal/kg/hr)	-	-	1.33 ± 0.25	-
(kcal/m² /fur)	-	-	54.2 ± 5.4	-

a. Balance data and digestibilities calculated from intakes and excrete collected seven times at 4-day intervals in each 28-day period.
b. For 10 degrees of freedom p.<0.05 = 2.228 and p<0.01 - 3.169.
c. Protein = nitrogen (Kjeldahl) x 6.25.
d. Mean ± standard deviation.
e. "True" digestibility calculated assuming obligatory faecal nitrogen loss of 20mg/kg/day.
f. Nitrogen balance [apparent) = intake —urinary excretion —faecal excretion. No allowance made for sweat and other insensible losses.
g. P% = proportion of dietary energy derived from proteins = (protein intake, g x 4) + net energy intake x 100.
h. Gross energy intake determined by bomb calorimetry of the foods ingested.
i. Net energy intake = gross intake—faecal energy (bomb calorimetry).
j. Total energy expenditure calculated from heart rate and the corresponding heart rate—energy expenditure relationship during 14 hours of the day 16 a.m. to 8 p.m.) and from basal energy expenditure during 10 hours (8 p.m. to 6 a.m.)
k. Energy balance = net intake —urinary losses 15 kcal/g urinary nitrogen) -- sweat losses 18 kcal/g sweat nitrogen = approximately 0.1 kcal/kg/day)—total energy expenditure.
* Mean values of the two periods differ (see t value).

Apparent nitrogen balance was also high. All children retained at least twice the amount estimated for normal growth and to compensate for insensible losses (about 15 plus 9 mg N/kg/day, respectively).

Figure 3 indicates that only two children (405, 411) had protein intakes below the population estimates given in table 1. Their P%'s were also the lowest in the group, and their apparent digestibilities were near the group mean. Child 405 had the lowest nitrogen balance and child 411 retained 70.5 mg N/kg/day in period 11 when his intake. was only 1.36 9 protein/kg/day. Neither child had clinical signs of protein deficiency, both grew well, and their CHI, haemoglobin concentrations, and other biochemical measurements did not differ from the group. Child 404 had the highest food and protein intakes and the highest P% of the group. He had a high faecal output and his apparent nitrogen digestibility was 64 per cent in both periods of the study. As a result of this, he absorbed 1.46 9 protein/kg/day in the two periods and his nitrogen balance was only 53.1 mg N/kg/day in period 11; in period I it was 92.3 mg N/kg/day.

4. Basal and Total Energy Expenditure
Each child's basal energy expenditure varied little throughout the study. Therefore, each child's mean value was used to compute his total energy expenditure. Table 5 shows the group's basal expenditure. Child 414 was higher than the rest, with 67.2 kcal/m2/hr (2.88 kcal/kg/hr). Basal expenditure varied among the other children from 47.7 to 58.4 kcal/m2/hr (2.11 to 2.65 kcal/kg/hr). These values agree with those of similar children measured by the same method.

Figure 4 shows the range of individual total daily energy expenditures. The average daily energy expenditure did not vary between periods and the medical and nursing staff did not notice changes in the pattern or duration of the children's physical activity, except when they were ill.

5. Energy Intake, Absorption, and Balance
Figure 4 and table 5 give the individual and group data. Gross intakes during the days of excrete collection ranged from 85 to 100 kcal/kg/day. These were to a certain extent independent of total food intake during period 1, since the energy density of each child's diet was adjusted when the preceding week's intake was not between 87 and 97 kcal/kg/day. Apparent absorptions had a low coefficient of variability (1.7 per cent), and, except for child 414 who absorbed 88.6 per cent of the energy ingested, ranged from 91 to 94 per cent. Net energy intakes were, on the average, 8.4 per cent lower than gross intakes.

The estimates of urinary and sweat energy losses varied from 0.8 to 1.1 kcal/kg/day. Total daily energy expenditures and the energy balance results coincided with those from another study with similar children and equivalent dietary energy intakes. The energy balance ranged between-7.6 and 24.6 kcal/kg/day in period 1, and between -4.8 and 21.2 kcal/kg/day in period 11 (see figure 4).

FIG. 4. Energy Expenditure and Balance Studies

TABLE 6. Blood Chemistry and Haematology during Periods I and II

		Days on the study
		0	18	36	54
Packed red cell volume	%	36 ± 2*	35 ± 2**	37 ± 1	37 ± 2
Haemoglobin	g/dl	12.4 ±0.8	12.2 ±07	12.4±0.6	12.3 ±0.6
Plasma proteins	g/dl	7.2 ± 0.4	7.0 ± 0.4	7.1 ±0.3	6.8 ± 0.4 t
Serum albumin	g/dl	3.8 ± 0.6	4.1 ± 0.6	3.8 ±0.4	3.7 ± 0.7
Serum amino acid ratio non-essential/essential		1.68 ± 0.44	1.61 ± 0.40	1.38 ±0.31	1.42 ±0.35

* Mean + standard deviation, n = 11.
** Lower than on days 36 and 54. F (3,40) = 3.072, p < 0.05, L.S.D. = 2.
t Lower than initial (day 0) values, student's paired t = 3.253, p < 0.01

6. Haematological and Biochemical Analyses
Table 6 gives the results of the analyses performed on blood, serum, and plasma. Analyses of variance indicated a small, transient decrease in packed red blood cell volume on day 18 (p < 0.05). The analysis of variance did not show differences among the other haematological and biochemical determinations. However, the paired comparison of initial and final values indicated decrease in total plasma protein concentration (p < 0.01 ) not accompanied by a decrease in albumin concentration.

Comments

1. The results indicate that the diet used in this study satisfied the protein needs of healthy, well nourished, pre-school children. In contrast with the most common dietary practices among people of low socio-economic conditions in developing countries, the preparation and administration of the foods differed in three important ways: (a) the increment in energy density achieved by the addition of oil and sugar to some foods; (b) the availability of staple foods in sufficient amounts to satisfy the children's appetites; and (c) the preparation and administration of the foods in the same way as for adults (e.g., feeding whole beans to small children instead of only a watery bean soup). These three conditions can be met by an increment in local food resources, including fats, combined with an educational programme for the use of such resources.
2. It is not necessary to use large amounts of proteins from animal sources or of commercially processed foods, as these tend to be more expensive and less regularly available and may imply greater changes in food preparation and eating habits than the mere addition of fats and carbohydrates to staples. The diet used in this investigation included small amounts of animal proteins on some days. It remains to be shown if these small additions of high-quality proteins are essential to other diets of exclusively vegetable origin. Our previous investigations with diets based on corn and black beans indicated that such additions or other protein fortification measures are not necessary, provided that the vegetable foods are eaten in such quantities and proportions that a good complementary vegetable protein mixture is absorbed by the children. In any event, the present study demonstrated that it is not necessary for such high-quality protein "complements" to be ingested with every meal or every day.
3. The use of mixed diets with varied menus and free choice of intake departed from the traditional patterns of metabolic studies. However, it resembled everyday life since children do not eat the same foods or the same amounts day after day, and all the nutrients in their diet are not always digested and absorbed simultaneously or in similar time sequences. This experimental design also accounted for some of the possible interactions of various nutrients and other food components, such as the effects that the presence of different amounts and types of dietary fibres may have on nitrogen and fat absorption on different days.
4. Complete collections of daily excrete might yield more accurate metabolic-balance results, but this is feasible only with older children and adults. The 4-day interval surrogate used in this study was adequate, as shown by the replication of the 28-day periods. The results did not vary unless the children became sick for several days with fever and marked anorexia; illness and mild symptoms did not produce any changes in metabolic balances.
5. Growth rates were also similar in the two consecutive periods. Therefore, a 28-day period might be adequate to assess the effects on body weight and metabolic balance of local diets eaten in the usual patterns if the children do not become ill. Otherwise, 28-day replications are necessary. The evaluation of other anthropometric changes, such as length, lean arm diameter, and subcutaneous skin-fold thickness, requires a longer period (42 to 56 days).
6. There is no satisfactory explanation for the apparent high nitrogen retentions. This is known to occur in children and adults with protein intakes well beyond their requirements. The important observation derived from this study was that nitrogen balance remained positive and sufficed to cover the estimated needs for growth and insensible nitrogen losses. This is supported by the stability of the CHI and the small, but consistent, increment in lean arm diameters.
7. The energy expenditure and balance data confirm our previous observations that mean net energy intakes (i.e., dietary intakes measured by bomb calorimetry and corrected for faecal losses) of 82 to 85 kcal/kg/day are adequate for active, well nourished children two to four years old. The energy not available from the protein absorbed, calculated from urinary nitrogen excretion, was about 1 kcal/kg/day. Consequently, the net energy intake is similar to that calculated from food composition and the Atwater energy factors for proteins, fats, and carbohydrates. Since the coefficient of variability of the mean net intake was 5 to 6 per cent, it seems safe to recommend energy intakes of 92 to 95 kcal/kg/day (mean + 12 per cent) for well nourished children of this age group.
8. The question remains open as to whether diets of the types used in this investigation will allow children with mild-to-moderate nutritional deficiencies to catch up. This is highly relevant, since such children make up the majority of the pre-school age population of low socio-economic groups in the developing world. The catch-up in length and high rates of weight gain observed in this investigation suggest that this might be possible. The answer can be explored through a similar study on apparently healthy children with small weight deficits (5 to 15 per cent below that expected for their heights) who do not have clinical signs of malnutrition except for their leanness. A longer period of time (e.g., 84 instead of 56 days) may be necessary to assess the rates of catch-up growth and to account for the impact of disease that might occur.

Conclusions

1. With the diet used in this study, protein intakes were adequate when energy intakes corresponded to the estimates of energy requirements.
2. The protein and energy needs of well-nourished children were satisfied by the diets used in this study if:

(a) dietary energy density was increased, and
(b) sufficient amounts of the staple foods were available.

3. If the diet is supplemented with foods of animal origin or other high-quality proteins, there is no need to provide them every day of the week. Some local vegetable based diets may not require such supplements.
4. It is not necessary to change the composition of the diet during short, acute episodes of disease or during convalescence from them if the foods are offered to the child in sufficient amounts to satisfy his appetite.
5. The energy needs of most well-nourished children two to four years old can be satisfied with dietary intakes of 92 to 95 kcal/kg/day.
6. Measurements made at 4-day intervals over 28 days yield good metabolic-balance data. A 28-day period of observation is adequate to assess the dietary effects on body weight and metabolic balance if the child does not become ill. In that event, other 28-day replications are necessary. The evaluation of other anthropometric changes requires a longer time.
7. It is still necessary to show whether or not the preceding conclusions are correct for pre-schoolers with mild-to-moderate protein-energy deficiencies.

Acknowledgements

The investigations were carried out with the financial assistance of the Danish International Development Agency (DANIDA) and the United Kingdom's Office for Overseas Development. The Food and Agriculture Organization of the United Nations (FAO) administered funds from DANIDA and made the award to INCAP.

(introduction...)

Objectives
Experimental details
Summary of main results
Conclusions and comments

Benjamin Tord Fernando E. Viteri
Institute of Nutrition of Central America and Panama (INCAP), Guatemala City, Guatemala

Objectives

1. Evaluation of the adequacy of a diet based on corn and beans, fed at recommended levels of protein intake with different levels of energy intake.

2. Measurement of total energy expenditure, energy balance, and energy requirements.

Experimental details

1. Subjects

1. Six boys of mixed Maya Indian and Caucasian descent (Lading).
2. Chronological age: 30 8 months (range: 22 to 40 ). Height-age: 17 5 months (range: 15 to 25).
3. All had been treated for severe, oedematous protein-energy malnutrition (kwashiorkor and marasmic kwashiorkor). They had recovered fully at least one month before beginning the studies, based on clinical, anthropometric, and biochemical criteria (plasma proteins, non-essential/essential amino acid ratio, haematological indices, urinary creatinine excretion, and creatinine-height index [CHI]).
4. Weight: 11.93 0.95 kg (range: 10.85 to 13.25). Height: 84.4 3.5 cm (range: 78.2 to 88.6).
5. Weight-for height, percentage of expected: 104 4% (range: 89 to 109).
6. All children were healthy throughout the study, except for occasional minor illnesses, such as upper respiratory infections of viral aetiology, that were treated symptomatically. In a few instances a child had fever for one to three days. If that happened immediately before or at the time scheduled for nitrogen-balance studies, the balance was postponed until seven days after the fever had subsided. Energy expenditure was not measured during days when a child had fever. f. Intestinal parasites: Two children (nos. 387 and 390) had mild infestation with Trichuris trichiura. Two others 1388, 394) had Giardia lamb/ia, and one of them (394) also had mild ascariasis. All were asymptomatic and none was treated before or during the study.

2. Study Environment
INCAP's Clinical Centre in Guatemala City, 1,500 metres above sea level. Temperature 18 to 24 C. Relative humidity 40 to 60 per cent, except during rainy days. All children spent two to four hours each day in outdoor playing facilities, except on rainy days.

3. Physical Activity
The children were encouraged to be about as active as those living in a free environment, rather than leading the sedentary life usually observed in children who live in an institution. This was accomplished by encouraging them to participate throughout the day in various games that required walking, running, and climbing ramps and stairs. They were never forced to participate, and the games never exhausted them.

4. Duration of the Study
Five children were studied for 120 days, 40 days with each of three levels of dietary energy. The sixth child was studied for 160 days with four levels of dietary energy, as described below.

5. Diet
a. The diets were based exclusively on vegetables, with 95 per cent more of the protein derived from black beans (Phaseolus vulgaris) and corn. The diets were designed to provide 1.75 9 protein/kg/day. The proportion of black beans to corn protein was 42:58. Three different levels of energy intake were used: initially about 100 kcal/kg/day and after 40 day intervals about 92 and 83 kcal/kg/day.

The 100 kcal level was that used in previous experiments when the requirement for corn-and-bean protein was established. This energy intake was excessive for several children who became obese during those experiments. Only one child (KG) ate a higher energy-dense diet after he failed to gain the expected weight with the initial intake of 100 kcal/kg/day and lost weight with 92 kcal/kg/day. His energy intake was then raised to 120 kcal/kg/day, and at 40-day intervals it was lowered to 110 and 100 kcal/kg/day. It should be pointed out that while this child was recovering from protein-energy malnutrition before participating in the present study he required a diet with 150 kcal/kg/day for a longer period than was needed by the other children for recuperation. The changes in dietary energy were made by adding more or less vegetable oil to the black bean preparations, as shown in table 1.

b. As the components of the diet were those used locally in Central American rural and low-income urban homes, no multivitamins and mineral supplements were added, except for supplementary iron and vitamin A provided with sugar fortified with NaFeEDTA (13 mg of iron per 100 9 of sugar), and retinol palmitate (15 micrograms of vitamin A in 1 9 of sugar).

c. Table 2 shows the proportions of energy provided by fat, protein, and carbohydrate. The diet provided 0.54 9 crude fibre/kg body weight/day.

d. The foods were prepared and cooked in ways similar to those followed in Guatemalan homes, except that with the higher levels of energy intakes more oil was used than is customary in low-income homes. Table 3 shows the foods served in every meal. The same menus and amounts of food were served each day throughout the study, adjusted for each child's average weekly body weight.

6. Indicators and Measurements
a. Nitrogen and energy balance: Every 20 days, urine and faeces were collected for 96 hours and analysed to determine their nitrogen and energy contents. The same was done with an aliquot of the diets. Nitrogen was determined by an automated method using alkaline phenol-hypochlorite-nitroprussiate (Berthellot reaction) after a Kjeldahl digestion; food and faecal aliquots were homogenized and solubilized with H₂O₂-H₂SO₄ before digestion. "True" nitrogen balance was calculated by subtracting urinary, faecal, and insensible losses from the amount of nitrogen ingested. It was assumed that the insensible losses were 5 mg/kg/day.

Energy in diets and faeces was measured by bomb calorimetry using benzoic acid standards. Energy balance was calculated by subtracting from the dietary energy intake (measured by bomb calorimetry): the faecal energy (measured by bomb calorimetry); urinary energy losses (estimated as 5 kcal/g urinary nitrogen); sweat (estimated as 0.1 kcal/kg/day, based on 8 kcal/g sweat nitrogen; and the total energy expenditure, as described below.

b. Absorption: Twenty mg N/kg/day were used as the obligatory faecal losses to calculate "true" nitrogen digestibility.

TABLE 1. Ingredients and Amounts of Foods Eaten

	Amount (g)	Protein (g)	Fat (g)	Energy (keel)	Amount eaten per kg/day
Black bean paste (pur					16.6 g/kg
Black bean flour	18.1	4.2	0.4	61
Salt	0.7	-	-	-
Vegetable oil	11.8	-	11.8	106
	(6.3)*	(6.3)	(57)
	(0.0)		(-)
Water enough to reach	100.0
Total	100.0	4.2	12.2	167
			(6.7)	(118)
			(0.4)	(64)
Corn tamale					15.1 g/kg
Lime-treated corn flour	35.4	3.1	1.5	131
Salt	1.7	-	-	-
Water	62.9
Total	100.0	3.1	1.5	131
Corn gruel (atole)					50 g/kg
Lime-treated corn flour	11.4	1.0	0.5	42
Sugar	6.5	-	-	26
Water	82.1	-	-	-
Total	100.0	1.0	0.5	68
Lemonade	—(10 g sugar/100 g = 40 kcal/100 g)				25 g/kg
Banana	—(1 g protein and 106 kcal/100 g)				5 g/kg
Cooked carrots	—(0.2 g protein end 31 kcal/100 g)**			approx.	5 g/kg
Cooked squash	—10.5 g protein and 20 kcal/100 g)**			approx.	2 g/kg
Boiled potatoes	—(1.1 g protein and 42 kcal/100 g)**			approx.	1.3 kg/kg
Cooked spinach	—(2 g protein and 17 kcal/100 g)**			approx.	1.7 g/kg

* 11.8 per cent oil added to provide a total of about 100 kcal/kg/day. Figures in parentheses correspond to diets that provided about 92 or 83 kcal/kg/day.
** Only two of these vegetables were served each day

TABLE 2. Nutrient Contents of the Diet as Energy Sources*

	Theoretical dietary energy levels (kcal/kg/day)
% Energy from:	120	110*	100	92	83
Total fats	27.0 (23)+	24.8 (21)	22.5 (18)	15.5 (10)	5.9 (0)
Proteins	5.8	6.3	7.0	7.6	8.4
Carbohydrates	67.2	68.8	70.5	76.9	85.7

* Based on Atwater factors and proximal analysis of beans, corn, tamale, corn gruel, and lemonade, and on food composition tables for banana and other vegetables.
** Diets with 120 and 100 kcal/kg/day were consumed only by child E,G. (no, 387).
+ In parentheses: percentage of energy derived from vegetable oil added to black beans.

c Total energy expenditure: Physical activity and energy expenditure were quantified by monitoring the children's heart rate throughout the day and calculating energy expenditure from individual determinations of heart rate and oxygen consumption. The heart-rateoxygen-consumption relationship was determined in each child at 20-day intervals. Total daily energy expenditure was calculated from each child's heart rate and his corresponding heart-rate-energy expenditure relationship from 5 a.m. to 8 p.m. ( 15 hours), and from his basal energy expenditure from 8 p.m. to 5 a.m. of the following day (9 hours).

Basal energy expenditure was measured by indirect calorimetry with an oxygen diaferometer at two day intervals, each time on two separate occasions not more than three days apart; the lower of the two results was considered as basal. Basal conditions were defined as after a minimum of eight hours of sleep and ten hours of fast. Measurements were done while the child was sleeping, sometimes after oral administration of chloral hydrate (4 mg/kg). Energy expenditure was calculated by indirect calorimetry, assuming a respiratory quotient of 0.82.

d. Growth and body composition: The children were weighed naked before breakfast every morning. Anthropometric measurements were taken at 14-day intervals. Basal oxygen consumption was measured on 2 consecutive days at approximately 20-day intervals. Urinary creatinine excretion was determined at 20-day intervals when the nitrogen-balance determinations were carried out.

e. Other determinations: Initially and at two-day intervals, packed blood cell volume and total plasma proteins were determined.

f. The experimental design is summarized in table 4.

TABLE 3. Menu Used in the Study

Breakfast:	corn gruel (corn flour + sugar + cinnamon + calcium) * tamale (corn flour + salt + calcium) beans (black bean flour + salt + vegetable oil)
Mid-morning snack:	banana beans tamale lemonade (lemon juice & sugar)
Lunch:	corn gruel tamale beans cooked carrots or spinach
Mid-afternoon snack:	banana beans tamale lemonade
Dinner:	corn gruel tamale beans mashed potatoes or squash

* Ingredients are shown in parentheses.

TABLE 4. Summary of Experimental Design and Schedule

Experimental days
	0	4		8	12		16	20	24		28		32	36	40
		44		48	52		56	60	64		68		72	76	80
		84		88	92		96	100	104		108		112	116	120
Procedures	A, B	BEE, HR				A		NE, B		A	BEE, HR				A,B,NE

Diet: Protein: 1.75 g/kg/day throughout the study. Energy: initially 100/kcal/kg/day and decreased to 92 and 84 kcal/kg/day on days 41 and 81, respectively (only exception: child E,G., see text).
A: Anthropometric measurements: height; perimeters of arm and leg; tricipital, subscapular, and abdominal subcutaneous skin-fold thicknesses. Weight was measured every day.
B: Packed red blood cell volume, plasma proteins, and urinary creatinine.
NE: Nitrogen and energy balance.
HR: Heart-rate-energy-expenditure relationship. Heart rate continuously monitored 5 days before and 5 days afterwards.
BEE: Basal energy expenditure.

TABLE 5. Mean Daily Intakes of Dietary Protein and Energy During the 4-Day Balance Periods

Patient	Protein (g/kg)	Energy (kcal/kg)*
		120**	110	100	92	83
379	1.73 0.03 (6) ***			99 (2)	92 (2)	83 (2)
387	1.75 0.06 (8)	118 (2)	106 (2)	100 (3)	97 (1)	-
388	1.69 0.04 (6)			99 (2)	89 (2)	78 (2)
390	1.75 0.01 (6)			95 (2)	84 (2)	76 (2)
394	1.79 0.01 (6)			102 (2)	94 (2)	86 (2)
395	1.63 0.04 (6)			100 (2)	92 (2)	84 (2)
Average	1.73 0.06 (38)	118 (2)	106 (2)	99 3 (13)	91 4 (11)	81 4 (10)
Mean net energy intakes ****		106	96	90	82	71

* Gross intakes, i.e., bomb calorimetry values not corrected for faecal energy excretion.
** Theoretical levels of energy intakes.
*** Mean standard deviation (number of balance period).
**** Net intakes = gros sintekes - faecal losses (bomb calorimetry).

Summary of main results

1. Dietary Intakes
Table 5 gives the average intakes of dietary energy and protein for each child and for the group.

2. Energy and Protein Absorption
Table 6 gives the apparent energy absorption of each child for all levels of energy intake. The overall average absorption was 88 ± 6 per cent. With gross intakes of 100, 92, and 83 kcal/kg, apparent absorptions were, respectively, 91 ± 6 per cent, 80 ± 7 per cent, and 85 ± 4 per cent. Although the mean values decreased with decreasing levels of intake, they were not different from each other.

Table 6 also gives the "true" and apparent digestibilities of nitrogen (i.e., with or without correction for endogenous faecal nitrogen). The average apparent digestibility was 59 ± 6 per cent for all levels of energy intake. With net energy intakes of 90, 82, and 71 kcal/kg/day, apparent nitrogen digestibility was 56 ± 7 per cent, 59 ± 6 percent, and 62 ± 9 per cent, respectively.

TABLE 6. Absorption of Energy and of Nitrogen as a Percentage of the Amounts Ingested (Mean of all Levels of Dietary Energy)

	Energy absorption,	Nitrogen digestibility
Patient	apparent	apparent	"true"* *
379	94 ± 3*	64 ± 4	71 ± 4
387	88 ± 4	59 ± 11	66 ± 11
388	84 ± 9	60 ± 3	67 ± 3
390	85 ± 4	61 ± 9	68 ± 9
394	88 ± 7	47 ± 4	54 ± 4
395	87 ± 5	65 ± 7	73 ± 7
Average	88 ± 6	59 ± 9	66 ± 9

* Mean + standard deviation.
** Assuming obligatory faecal losses of 20 mg N/kg/day.

These values were not different from each other. "True" digestibilities were higher by about 7 per cent of nitrogen intake. One child (394) consistently had low nitrogen digestibilities. If his results were not included in the analysis, the average apparent digestibility for the other five children at all levels of energy intake would be 62 ± 4 per cent, and with the three levels of net dietary energy they would be 58 ± 6, 61 ± 3, and 66 ± 8 per cent. These values do not differ from each other.

There was no association between the apparent absorptions of energy and protein (r= 0.173).

3. Nitrogen Balance
The values of the two four-day balance periods at each dietary energy level were averaged for each child. Table 7 gives the nitrogen balance data of five children at each level of energy intake. There were no differences among the three dietary energy intakes. Child 387 retained 54.1, 95.7, and 43.6 mg N/kg/day when he ingested 106, 96, and 90 kcal/kg/day, respectively. The mean and standard deviations of the seven balances performed on him were 61.5 ± 31.8 mg N/kg/day.

TABLE 7. Nitrogen Balance (Nitrogen Intake-Urinary Nitrogen- Faecal Nitrogen-5 mg/kg/day) Expressed as mg N/kg/day

Child	Net dietary energy (kcal/kg/day)			Mean and S D of 6 balances*
	90	82	71
379	76.6	51.4	59.9	62.6 ± 18.7
388	59.9	88.3	60.8	69.6 ± 20.6
390	51.6	53.1	86.7	63.8 ± 18.1
394	52.5	50.9	49.9	51.1 ± 14.6
395	78.1	69.4	66.0	72.2 ± 9.2
Average	63.7 ± 11.5	58.0 ± 6.8	64.9 ± 15.2
(n= 10)*

* Two balance periods with each level of energy intake.

4. Energy Expenditure
Table 8 gives the energy expended by five children at each of the three levels of dietary energy. The children spent more energy in physical activity with the highest level of intake (p < 0.05); there was no difference between the two lower levels of intake. The nurses and the investigators, however, did not notice any changes in the children's behaviour or in the pattern and intensity of their activities throughout the study. The sixth child (387) also expended more energy when he had the highest intake. His daily expenditure with net dietary intakes of 90,106, 96, and 90 kcal/kg were 80.4, 90.4, 73.4, and 78.9 kcal/kg, respectively.

5. Energy Balance
Table 8 also gives the energy balance of the children. There were no differences in energy balance with the different levels of dietary energy intake. The corresponding figures for child 387 were ±9, 14, ±19, and ±5 kcal/kg/day with net dietary energy intakes of 90, 106, 96, and 90 kcal/kg/day, respectively.

6. Anthropometry and Growth
The only consistent anthropometric change was a decrease in bodyweight gain with the net dietary energy intake of 71 kcal/kg/day. Weight gains calculated in five children by regression analysis during the 40 days at each level of energy intake were (mean ± S.D.) 10.7 ± 4.8, 11.4 ± 3.4, and 0.6 ± 4.4 g/day for 90, 82, and 71 kcal/kg/ day, respectively. Paired comparisons confirmed that the last value differed from the preceding two (p < 0.05 and <0.01), respectively.

TABLE 8. Total Daily Energy Expenditure and Energy Balance (kcal/kg/day)*

Net dietary energy (kcal/kg/day)
Patient	90**			82			71
	exp.	bal.	exp.		bal.	exp.		bal.
379	104.0	- 11	89.8		- 3	81.6		- 7
388	84.8	7	76.0		4	63.3		5
390	77.8	0	77.8		- 4	72.0		- 10
394	93.5	- 6	65.6		22	65.6		3
395	85.2	- 3	69.7		13	74.9		- 3
mean	89.1***	- 3	75.8		6	71.5		- 2
±
S.D.	9.0	6	8.3		10	6.5		6

* Energy expenditure was calculated from heart rate and the corresponding heart-range-energyexpenditure relationship between 5 a.m. and 8 p.m., and from basal energy expenditure during the remaining 9 hours. Energy balance was calculated from energy intake {bomb calorimetry! —faecal energy (bomb calorimetry)—urine losses 15 kcal/g urinary nitrogen)—sweat losses 18 kcal/g sweat nitrogen = approx. 0.1 kcal/kg/day) — energy expenditure (as described above).
** Dietary-faecal energy, measured by bomb calorimetry.
*** Differs from the two other levels of intake, p < 0.025.

The sixth child (E.G., no. 387) lost 2 to 3 g/day with intakes of 90 and 82 kcal/kg/day. With 106 and 96 kcal/kg/day he gained 25 and 13 g/day, respectively, and only 3 g/day when he again received 90 kcal/kg/day.

The level of dietary energy did not affect growth in terms of height. Three children showed a continuous tendency to catch up, regardless of the amounts of energy intake.

7. Other Biochemical Measurements
Fluctuations within normal ranges were observed, without relation to the amounts of dietary energy intake.

Conclusions and comments

1. An all-vegetable diet can fulfil the protein requirements of pre school children when black beans and corn provide about 0.7 and 0.95 9 protein/kg/day, respectively. This is true when the children are physically active and net dietary energy intakes range between 71 and 90 kcal/kg/day.

2. Within those ranges the level of energy intake did not affect nitrogen balance. The amounts of nitrogen retained by the children were greater than those that have been estimated to allow adequate growth, suggesting that the diets used in this investigation provided more protein than required. Therefore, it may be that the amounts of protein provided by the diet surpassed requirements to a point where the protein-sparing effect of energy was obscured, and it is conceivable that the amounts of dietary energy used might have influenced nitrogen balance if the protein intake had been closer to the requirement level.

3. Nitrogen retentions were high even though the "true" protein digestibilities were low, corresponding to an average of 1.14 9 protein absorbed/kg/day (1.73 g/kg with a digestibility of 66 per cent). Nitrogen digestibility was lower than we have observed in other studies using similar diets (67 to 83 per cent), and it seemed to be related to large faecal volumes that averaged 197 g/day in this study. The apparent absorption of energy (88 per cent) differed less from our other investigations (89 to 92 per cent).

4. Diets of the type used in this study failed to fulfil pre-school children's energy requirements unless their energy density was increased. At least 10 per cent additional energy had to be added to the diets as vegetable oil to ensure adequate weight gains. This provided an average of 82 net kcal/kg/day.

5. There were no changes in weight gain when the net dietary energy decreased from 90 to 82 kcal/kg/day. However, this change was accompanied by a decrease in total energy expenditure, although no changes in physical activity patterns were noticed, and energy "balance" (defined as intake minus faecal, urinary, and sweat losses, and minus total energy expenditure) remained constant. Basal energy expenditure and the energy lost through excrete also remained constant. The children seemed to adjust to the initial decrease in intake by a decrease in physical activity or by a greater efficiency in performance (i.e., spending less energy to do the same things) and continued to gain weight at an adequate rate. These findings could also indicate that, within certain limits, the children avoid becoming obese by increasing their energy expenditure when there is an excessive dietary energy intake. When the net dietary intake was further decreased to 71 kcal/kg/day, energy expenditure did not diminish any more and the children lost or reduced their weight gains. Therefore, we conclude that a net dietary intake of 71 kcal/kg/day was insufficient and the children could not compensate for this low intake by a decrease in physical activity under the experimental conditions that prevailed.

Acknowledgements

This research was made possible through the financial assistance of the World Health Organization, as part of its support of a series of investigations related to protein and energy requirements of children, and through the United Nations University World Hunger Programme.

(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 more precise knowledge of the energy requirements of young babies, which is essential for the planning of rational infant-feeding strategies.

Experimental details

1. Subjects
Twenty infants were studied (13 male and 7 female), as part of a longitudinal investi gation on maternal nutrition and lactation. All were breast-fed up to the sixth month, and 16 were still being breast-fed by the end of the seventh month. Fifteen of the mothers were recruited through the Cambridge Maternity Hospital, and five via the National Childbirth Trust.

2. Anthropometry
The babies' weights, lengths (using a Harpenden Infant Measuring Table), and triceps skin-fold thicknesses (using Holtain callipers) were measured at monthly intervals.

3. Dietary Intakes
From four weeks of age, breast-milk intakes were measured in the home on four consecutive days each month by test-weighing, using Salter Baby weigher Model 40 Scales, after the mothers had received careful instruction in this technique. All other food and drink, including medicaments such as gripe water and "vitamin syrups," were also quantitatively recorded by the mothers for the same four days. First, supplementary foods introduced were usually proprietary cereal preparations, "baby dinners," or foods from the rest of the family's supply; infant formulas based on cow's milk were never given. Energy intakes were calculated using food composition tables and from information provided by manufacturers. The energy content of breast milk was taken to be 69 kcal/100 9, the average value found in a recent national survey of British mothers'milk.

FIG. 1. Total Energy Intake of 20 Babies Compared with the Recommendations of WHO/FAD and the United Kingdom's DHSS (Values are mean + S.E.M.)

Summary of main results

1. Figure 1 shows that after two months total energy intakes were substantially below FAD/WHO recommendations for the average child and there was little difference whether the children were wholly or partially breast-fed. After two to three months there was almost no further rise in intake, although between then and the seventh month the children increased in weight by a further 43 per cent.

2. Body weight ranged within about 3 kg at any given age, as shown in table 1. The total energy intake of individual babies depended mainly on how heavy they were, but it became more consistent when adjusted for body weight.

3 Figure 2 shows the infants' anthropometric data compared with the Tanner-Whitehouse standards for British babies. The biggest difference between the measurements made and the standard was in triceps skin-fold thickness, which suggests that one consequence of the lower energy intakes might be a reduction in body-fat stores.

Conclusions and comments

1. Total energy intakes of both fully breast-fed and mixed-fed infants were considerably below the internationally recommended levels. They were, however, identical with energy intakes calculated from recent Swedish studies on breast-fed children and with those from the much earlier and classical investigations of Wallgren (Acta Paediat Scand., 32: 778, [1944-1945] ).

TABLE 1. Energy Intakes Adjusted for Body Weight in Breast-fed Babies (values are means + S.D.)

Mean age (months)	No.	Weight (kg)	Energy intake/kg body wt. (kcals)
1.26	20	4.42 ± 0.63	115 ± 18
2.31	20	5.42 ± 0.73	101 ± 13
3.41	20	6.17 ±0.83	90 ± 12
4.49	20	6.80 ± 0.86	86 ± 14
5.62	20	7.35 ± 0.87	85 ± 13
6.66	16	7.75 ± 0.81	84 ± 12

FIG. 2. Weight, Triceps Skin-fold, and Length of 20 Breast-fed Babies Compared with Tanner Whitehouse Standards. For weight and length, the standards represented are the 75th, 50th, and 25th centiles; for triceps skin-folds, the 50th, 25th, and 10th.

2. Initial growth was good, but after about three months the lower average intakes began to be associated with a general deviation away from the Tanner-Whitehouse growth standards. However, these standards are higher than other standards.

3. From the present data, it would seem reasonable to conclude that the present FAD/WHO recommended energy intakes for young infants are too high. Up to three months of age, the recent UK Department of Health and Social Security (DHSS) values probably represent a more realistic estimate of the true needs of the average child. Whether even the DHSS recommendations are excessive from then onward depends on the interpretation placed on the anthropometric findings and the standards used.

4. A very careful and long-term evaluation would be required to decide whether deviations in growth of the magnitude found really did reflect processes disadvantageous to the child. The safer conclusion is the DHSS recommendation that dietary energy intake should remain at 100 kcal/kg after three months of age and throughout the rest of infancy.

Acknowledgements

We thank the mothers for their co-operation in this study, Mrs. J. Evans for the anthropometric measurements, Miss J.J. Whichelow for the recruitment of the mothers, and Dr. N.R.C. Roberton and the staff at the Cambridge Maternity Hospital. The study was financially supported by the Department of Health and Social Security.

(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.

List of participants

Dr. Hector Bourges R.
Division of Nutrition
National Institute of Nutrition
Tlalpan, Mexico D.F., Mexico

Dr. Ricardo Bressani
Institute of Nutrition of Central America and Panama (INCAP)
Guatemala City, Guatemala

Dr. Chen Hsue-cun
Institute of Health
Chinese Academy of Medical Sciences
Beijing, China

Dr. Edouard M. DeMaeyer
World Health Organization
Geneva, Switzerland

Dr. J.E. Dutra de Oliveira
Faculdade de Medicina de Ribeiaro Preto
Universidade de Sao Paulo
Estado de Sao Paulo, Brazil

Dr. Luis F. Fajardo
Universidad del Valle
Cali, Colombia

Dr. Lars Garby
Department of Physiology
Odense University
Odense, Denmark

Dr. Po Chao Huang
Department of Biochemistry
College of Medicine
Taiwan University
Taipei, Taiwan

Dr. Goro Inoue
School of Medicine
Tokushima University
Tokushima, Japan

Dr. Carmen Intengan
Food and Nutrition Research Center
National Science Development 80ard
Manila, Philippines

Dr. Jin Soon Ju
Department of Nutrition and Biochemistry
Korea University Medical College
Seoul, Republic of Korea

Dr. Sheldon Margen
Department of Nutritional Sciences
College of Agriculture
University of California
Berkeley, California, USA

Dr Julien Pssbr> Nutrition Division
Food and Agriculture Organization
Rome, Italy

Dr. William M. Rand
Department of Nutrition and Food Science
Massachusetts Institute of Technology
Cambridge, Massachusetts, USA

Dr. MarE. Rio
Departamento de Bromatology Nutricixperimental
Universidad de Buenos Aires
Buenos Aires, Argentina

Dr. Nevin Scrimshaw
Senior Adviser, UN University;
Institute Professor, Massachusetts Institute of Technology
Cambridge, Massachusetts, USA

Dr. Kraisid Tontisirin
Faculty of Medicine
Ramathibody Hospital
Bangkok, Thailand

Dr. Benjamin Torbr> Institute of Nutrition of Central America and Panama (INCAP)
Guatemala City, Guatemala

Dr. Ricardo Uauy
Instituto de Nutricion y Tecnologia de los
Alimentos Universidad de Chile
Santiago, Chile

Dr. Fernando E. Viteri
Pan American Health Organization (PAHO)
Washington, D.C., USA

Dr. John C. Waterlow
London School of Hygiene and Tropical Medicine
University of London
London, England

Dr. Roger G. Whitehead
Dunn Nutritional Laboratory
Cambridge, England

Dr. Vernon R. Young
Department of Nutrition and Food Science
Massachusetts Institute of Technology
Cambridge, Massachusetts, USA

* Chairman