FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS	ESN: FAO/WHO/UNU/ EPR/81/26 August 1981
	WORLD HEALTH ORGANIZATION
	THE UNITED NATIONS UNIVERSITY

Provisional Agenda Item 3.1.3

Joint FAO/WHO/UNU Expert Consultation on
Energy and Protein Requirements

Rome, 5 to 17 October 1981

LONG-TERM NITROGEN BALANCE STUDIES AND OTHER CRITERIA
FOR PROTEIN REQUIREMENT ESTIMATIONS

by

V.R. Young and W.M. Rand
Massachusetts Institute of Technology
Cambridge
USA

I. Introduction

Most estimates of adult protein requirements have been based on short-term metabolic nitrogen balance experiments conducted in small groups of young, healthy adults.Furthermore, there is currently a large amount of data being accumulated concerning the short term nitrogen balance responses of different population groups to varying diets.However, questions have been raised about the extent to which such data are relevant for predicting protein intakes required for long-term maintenance of nutritional status.

Motivated in part by these questions, longer-term nitrogen balance experiments have been conducted.These studies provide information of two kinds; (a) they serve to assess the general validity of short term studies and additionally, (b) they give insight into the protein intakes necessary for long-term nutritional health.

This paper first raises some of the problems associated with short-term studies and discusses long-term studies in that context.Next, some insights related to requirement estimations that have emerged from long-term studies are discussed, together with the limitations of these studies.Finally, some additional and alternative criteria for the further examination of protein and amino acid requirements are reviewed.

II. Problems of Short-term Studies

The validity of estimates of population protein requirements that are derived from short-term nitrogen balance studies depends on both the validity of the short-term method for determining the requirements of individuals, and the way in which population requirements are estimated from the usually small restricted samples. that are studied.These two concerns are quite different, and while only minor discussion will be presented of the latter, it will be evident that these two questions are closely linked.

Focusing, however, on determining the requirement in an individual, there are two fundamental questions concerning the results of short-term studies: are they true? and are they relevant?

1. Are the results of the short-term method true, in the sense that they are representative of a nutritionally relevent metabolic response? The question here is, in part, does the short-term method measure the short-term nutritional requirement of an individual, or do the results depend on some aspect of the experimental situation itself? In particular, is the response of an individual to a specific dietary intake determined only by that intake, or is it dependant, at least in part, on the prior dietary intake of the subject and the relationship of that intake to the new, experimental dietary situation? This question leads in turn to the more fundamental question: is short-term protein requirement itself a valid concept? This question must be considered in view of the multiple metabolic systems that allow subjects to adjust successfully to environmental changes, with interactions between the various systems, and each with its own time scale.

2. Are the results of the short-term method relevant to the definition of human protein requirements?

   If it is possible to measure a meaningful short-term protein requirement, that is, the physiological need of an individual for a specific brief time period, then the next question is how much bearing such short-term results have on the requirements for longer-term protein nutritional status.

III.The Significance of Long-term Studies for Interpretation of Data from Short-term Studies

One reason that long-term nitrogen balance studies are important is that they permit investigation of the validity and relevance of the extensive data available from short-term experiments.Indeed, several investigators have analyzed data from long-term studies specifically from this point of view.Thus, Sukhatme and Margen (1) examined data from several individuals studied in the metabolic ward at Berkeley and found significant positive autocorrelations between daily values of nitrogen output, suggesting that short-term results would not be unbiased estimators of that individual's long-term metabolic state.Rand et al. (2), studying less confined individuals at MIT. did not confirm these findings, but did observe long-term trends in many of their subjects.Their results question the concept of a constant long-term requirement for an individual.These findings have been confirmed and extended in a recent survey by Rand (3) which examines data from 42 individuals (including 16 that were involved in the earlier study) all of whom were consuming constant protein intakes in the range 0.73 to 1.8 g/kg/day.The results of these analyses suggest that short-term nitrogen balance within a given subject, whatever meaning it might have in and of itself, has limited meaning within the context of the longer-term Nitrogen balance for that subject.However, these data do show that nitrogen balance, averaged for all the subjects, did not change significantly over the duration of the experiment, suggesting that short-term Nitrogen balance results might be value in relation to a population response and, thus, an estimate of population requirement. A summary of the data on which these conclusions are based is given in the Appendix.

At a recent meeting Margen (4) presented preliminary results of a study of 6 subjects maintained on 0.36 g. protein / kg body weight for 77 days in the metabolic ward at Berkeley.The findings, relevant to short-term studies, were: 1) auto-correlations were present in 5 of their 6 subjects and, 2) time to reach an apparent steady state of nitrogen balance varied from 7 to 28 days.

The MIT and Berkeley groups thus differ with respect to the results of their long-term studies.These differences presumably result, at least in part, from differences in how the studies were conducted and how the results were analyzed. For example, the participants in the Berkeley experiments were confined under controlled conditions throughout the duration of the study, while the MIT subjects were required only to eat their meals in the metabolic kitchen and continued their everyday college activities.Thus, the MIT subjects were exposed to environmental stresses that do not apply in the Berkeley study.In addition, in the time series analysis of the data, where autocorrelation was sought, the MIT group removed the long-term trend in nitrogen output prior to analysis since long-term trend can produce spurious significant autocorrelations.

IV.Long-term Nitrogen Balance as an Indication that Requirements are Being Met

Long-term experiments involving constant nutrient intake are perhaps most important because they provide data on the relationship between nitrogen balance and long-term nutritional status.In general, although there is some disagreement on the level of intake necessary to maintain protein nutritive balance, in adult subjects, there is a growing concensus that nitrogen balance alone is not a sufficient sole measure of overall protein nutritional status.The few published studies support this view.

In a 12-week nitrogen balance study of protein requirements by Yoshimura (5), two young men received a diet providing 0.77 g protein/kg from cereal and pulses, and two others a diet providing 0.57 g protein/kg from meat and eggs.To determine the long-term physiological effects of adaptation to low protein intakes, measurements of body weight, basal metabolism, total body water, extracellular water, total hemoglobin, total serum protein content, albumin:globulin ratio, protein-bound iodine, serum ADH and daily urinary excretion of 17-ketosteroids and 17-hydroxycoricosteriods were included.Decreases were observed in BMR, hemoglobin, and urinary 17-ketosteroids and 17-hydroxycorticosteriods, while serum ADH increased even though nitrogen balance was well maintained and had a tendency to be positive.Because of the persistent adverse physiological changes observed, Yoshimura concluded that nitrogen balance alone could not serve as an adequate criterion of dietary adequacy.

Bricker et al. (6) estimated the protein requirements (70% cereal diet of ten college women age 19 to 30 years from three 10 to 15-day nitrogen balance periods at intakes ranging from 0.2 to 3.2 g nitogen/kg per day.When these subjects were given their estimated requirements for 70 days, they remained positive nitrogen balance.However, their estimated requirement included an allowance of 0.77 g nitrogen/m² for skin and miscellaneous losses.This was the equivalent of an average allowance of 1.29 g nitrogen/day, or about four times higher than suggested by the FAO/WHO committee.If the 1973 FAO/WHO estimated protein allowances were adjusted according to this higher allowance, they would be about 25% higher.Even this might be any underestimate, however, because Bricker's subjects experienced an average weight increase of 1 kg over the 70 days of the study, suggesting an excess energy intake.Moreover, a preceding low-protein period of approximately 35 days would result in a higher efficiency of dietary nitrogen utilization during the test period, above that expected by non-depleted individuals receiving an adequate but not excessive energy intake.

Garza et al. (7) conducted a study in six Caucasian students, age 19 to 23 years, who were given a formula diet providing 0.59 g egg protein/kg body weight/ day and energy intakes approximately 10% above their usual requirements.Four subjects continued on this diet for 81 to 89 days, but for two the protein intake was increased after 50 and 59 days because of an excessive rise in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels.The elevated serum AST and ALT activities fell to normal rapidly when these subjects received a diet providing 1.5 g protein/kg/day from skim milk powder.Body weight increased in five of the subjects.Four showed cumulative negative nitrogen balances, and all showed a net loss of total body ⁴⁰K, which was significant in three.These findings were interpreted as indicating that the 1973 FAO/WHO “safe level” of egg protein intake of 0.57 g/kg was not sufficient for long-term protein nutritional maintenance of most healthy young men.Furthermore the greater nitrogen losses calculated form ⁴⁰K and creatine measurements than from the nitrogen balance data suggested that integumental nitrogen losses may be higher than the 5 mg level included in the 1973 FAO/WHO allowance.

These published studies raise a number of intriguing problems concerning the nature and meaning of body nitrogen balance measurements.For example, the urinary nitrogen changes observed by Garza et al. (7) indicate a more complex response of body nitrogen metabolism to a continued low-protein intake than had been predicted.Thus, Forbes (8) suggested that in subjects going from a high to low protein intake, nitrogen losses should contine to decrease for 50 to 150 days, depending on the change in intake and the fractional turnover rate of body protein that is assumed.This response did not occur in any of the subjects in Garza's experiment and only one showed any significant fall in urinary nitrogen with time. Thus based on this study a slow drift of body nitrogen balance toward equilibrium was not observed.Actually there is a lack of published data to adequately explore the relationships between nitrogen intake and nitrogen output in healthy subjects receiving various levels of nitrogen intake and under differing environmental conditions. However a recent unpublished report (4) presented by Margen does suggest that when subjects received 0.36 g protein/kg/day nitrogen balance did appear to show a drift toward equilibrium as the 77 day study period progressed.Also no adverse biochemical changes were detected at this low protein intake.

V.Limitations of Long-term Studies

Long-term nitrogen balance studies have been suggested as the next step beyond short-term studies as the way to improve estimations of requirement or safe levels of intake for nutritional “health”.They are of course a step in that direction but they raise additional and specific problems as follows:

1. How long is long? Few studies have been carried out for longer than a few months, and while many metabolic processes respond within this time scale, others may not be readily revealed during a two-three month time frame (eg. calcium homeostasis in relation to protein intake).

2. In short-term experiments many variables (environmental and otherwise) are either ignored or merely measured.This is because they either do not vary significantly over the short-term, or they are assumed to have only long-term significance, and thus not affect short-term nitrogen balance. However, when long-term studies are conducted these factors must be carefully examined with respect to relevance and importance.This point is not only of theoretical significance but of practical importance in reference to experimental design; for example, long-term experiments must be much more carefully designed since they need to be more complex in order to deal with all these additional variables.

3. Assuming that the above problems of long-term experiments can be handled, the question remains as to how relevant such results are to the ultimate goal of estimating the requirements for maintenance of protein nutritional status. For example an important problem is that carefully conducted long-term experiments do not mimic the usual dietary situation in where the body responds to variable intakes over short and longer-term periods of time (ie. the real world is not a constant intake of protein and energy.) It is necessary to know whether the body responds, in terms of maintenance of nitrogen balance and other parameters of protein nutritive, in the same way to constant protein and energy intake as it would to intakes that are the same average level but where protein and energy vary according to habitual patterns of daily food intake.

4. The studies by Yoshimura (5) and by Garza et al. (7) indicate the inadequacy of using nitrogen balance as the sole criterion for estimating minimum physiological protein requirements.Experiments that do not attempt to measure changes in other physiological parameters and ignore factors that may lead to overestimation of retention, such as the possible compensatory decreases in urinary nitrogen excretion because of changes in sweat nitrogen losses, cannot be used to justify fully the adequacy of levels of protein intake that are substantially lower than those characteristic of well-nourished populations. Similarly, for the various reasons stated, conclusions based exclusively on short-term nitrogen balance studies must be made with considerable caution.

   There is a clear need for the conduct of additional studies in which the possible functional costs of tolerance to protein intakes lower than those habitually consumed by healthy populations are explored.

VI.Other Criteria

In view of the problems associated with use of nitrogen balance measurements as a sole basis for estimating protein need, other criteria or measures of adequacy might be considered.At the outset we recognize that the health significance of many, if not all, of the biochemical criteria that could be proposed (including nitrogen balance) for this purpose cannot be stated clearly. Nevertheless, a considerable advance in the further definition of human protein and amino acid requirements might be achieved if the use of multiple indicators was developed and explored more extensively.To accomplish this a more complete understanding of the biochemical and physiological changes in protein and amino acid metabolism following dietary alterations in human subjects will be necessary.

In spite of the incomplete state of our knowledge at present, some attempts have and are being made to explore the possible utility of other measures.Thus, in the case of the adult human, plasma free amino acid responses to charges in amino acid intake have been explored (eg. 9).However, this approach has not led yet to a general or more satisfactory method than nitrogen balance determinations because the pattern of changes in plasma amino acid levels varies according to the specific amino acid under test.With the further study or plasma amino acid changes under differing metabolic and nutritional conditions it seems likely that determination of plasma amino acid levels might lead to improved assessment of the amino acid states of human subjects.

Recent studies involving use of stable isotope tracer techniques, carried out in adult human subjects (eg. 10), together with findings in growing and adult rats (11–13) based on use of radio-labeled amino acids, indicate that the biochemical mechanisms responsible for maintaining whole body protein and amino acid homeostasis may be closely integrated with the individual's requirement for these nutrients. Thus, it seems likely that the dynamic responses of the whole body metabolism of specific amino acids and of protein, might differ for those intakes of protein and/ or specific amino acids that are generous on the one hand or inadequate on the other.

If this is so then is might be possible to use whole body amino acid kinetic data to expand knowledge of protein and amino acid requirements.

Measurements of whole body amino acid kinetics, with the aid of stable isotope tracers and simultaneous determination of plasma amino acid and nitrogen balance responses are being undertaken in our laboratories to provide a more comprehensive picture of the status of whole body amino acid metabolism, in adults, for intake above and below those considered necessary for maintenance of protein nutriture.Our preliminary data suggest that this approach might be valuable for improving upon present estimations of amino acid requirements.For example summary of this multiple metabolic parameter approach for assessment of the lysine requirement is illustrated in Table 1.Interpretation of these combined data leads to the conclusion that current recommendations for the lysine requirement in adults may be too low.The number of test amino acids examined by this approach are limited to date and based only on a small series of experiments, each involving few subjects and studied under highly controlled conditions that are quite different from normal free-living circumstances.Of course, this criticism applies to the data base used by the 1971 FAO/WHO Committee to arrive at estimates of amino acid requirements.Thus, we cannot yet be precise as to the minimum physiologic requirements for amino acids in healthy young men as approached in this way.However these developments are mentioned in order to suggest a possible approach for improvement in knowledge concerning human protein and amino acid requirements.

REFERENCES

1.Sukhatme, P.V. and Margen S. 1978.Models of protein deficiency.Am. J. Clin. Nutr. 31:1237–1256.

2.Rand, W.M., Scrimshaw, N.S., and Young V.R., 1979.An analysis of temporal patterns in urinary nitrogen excretion of young adults receiving constant diets at two nitrogen intakes for 8 to 11 weeks.Am. J. Clin. Nutr. 32:1408–1414.

3.Rand, W.M., 1981.Patterns in urinary nitrogen experiments in long-term studies with constan protein intake.(Presented at UNU/FAO/WHO Berkeley workshop on protein-energy requirements, Aug. 1981.)

4.Durkin, N., Ogar, D.A., Tilve, G., and Margen, S. 1981.Human protein requirements: autocorrelation and adaptation to a low-protein diet containing .356 gm protein/kg or 57 mg nitrogen/kg body weight. (Presented at the UNU/FAO/WHO Berkeley workshop on protein-energy requirements, Aug. 1981.)

5.Yoshimura, H. 1972.Physiological effect of protein defeciency with special reference to evaluation of protein nutrition and protein requirement. World Rev. Nutr. Dietet. 14:100–133.

6.Brecker, M.L., Slively, R.F., Smith, J.M., Mitchell, H.H. and Hamilton, T.S., 1949.The protein requirements of college women in high cereal diets with observations on the adequacy of short balance period.J. Nutr. 37:163–183.

7.Garza, C., Scrimshaw, N.S., and Young, V.R., 1976.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.J. of Nutr. 107:335–352.

8.Forbes, G.B., 1973.Another source of error in the metabolic balance method. Nutr. Rev. 31:297–300.

9.Young, V.R.. and Scrimshaw, N.S., 1978.Nutritional evaluation of proteins and protein requirements.In: Protein Resources and Technology: Status and Research Needs. (eds. N. Milner, N.S. Scrimshaw and D.I.C. Wang) Chpt. 10, p. 136–173, AVI Publishing Co., Inc., Westport, Connecticut.

10.Motil, K.J., Matthews, D.E., Bier, D.M., Burke, J.F., Munro, H.N., and Young, V.R., 1981.Whole body leucine and lysine metabolism: response to dietary protein intake in young men.Am. J. Physiol. 240:E712–E721.

11.Kang-Lee, T.A., and Harper, A.E.. 1977.Effect of histidine intake and hepatic histidase activity on the metabolism of histidine in vivo.J. Nutr. 107:1427–1443.

12.Kang-Lee, T.A., and Harper, A.E., 1978.Threonine metabolism in vivo: effect of threonine intake and prior induction of threonine dehydratase in rats.J. Nutr. 108:163–175.

13.Simon, O., Adam. K., and Bergner, H., 1978, Stoffwechselorientierte Lysin-bedarfsbestimmung bei ausgewachsensen Ratten anhand der Katabolisierung-strate von ¹⁴C- und ¹⁵N-markiertem Lysin.Arch. Tierernahrung 28:609–617.

¹ According to Munro (17).
² Estimated upper range of individual requirements.
³ Unpublished results.