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Commentary

1. Introduction and background
2. Methodology
3. Adult protein requirements
4. Energy expenditure
5. Protein-energy interactions
6. Nitrogen balance studies in children
7. Other data
References

1. Introduction and background

The requirements of individuals and populations for protein and energy have been discussed extensively by experts and committees over the past few decades. Although a rigorous definition for either protein or energy needs has not been unanimously agreed upon, various operational definitions have been formulated, and these have varied with time. Early efforts focused on the measurement of habitual protein intakes of healthy populations. Subsequently, metabolic studies provided data on obligatory losses that were used for the definition of requirements and the establishment of recommendations. More recently, N-balance response to graded levels of protein intake over long and short periods has gained acceptance.

During the last decade attention has been directed towards the limitations of these methods. While some of the questions relate to methodology, the most important issues deal with fundamental relevance of the metabolic data to individual and population requirements. The purpose of the Berkeley workshop was to review critically recent data from experimental studies conducted in developing countries in humans. It was inevitable that major issues in the field of protein-energy requirements would be raised. A listing of the more relevant of these as discussed during the meeting has been included in this introduction to indicate a sense of the meeting and to give the framework of the data review.

1.1. Measurement of Nitrogen Balance

General methodological aspects fundamental to metabolic studies have been well addressed in a previous UNU publication (1) that deals with knowledge and research needs of protein-energy requirements under conditions prevailing in developing countries. Further discussion is contained in the 1977 FAD/WHO report on methodology appended here (Appendix A), with special attention to work with children. Of special interest to the participants in the Berkeley workshop were questions of comparability of studies, necessitating some sort of standardization programme, and estimation of skin and miscellaneous losses. Both these topics are addressed in the methodology section.

A major issue that arose continually was how to measure, interpret, and use the variability both within and among subjects. While an aspect of this question is examined in the methodology section. no resolution of the problem was reached. At best it was admitted that potential sources of variability included biological, analytic, genetic, and environmental factors, and that these factors affect both the metabolic responses measured and the measurements themselves.

A further question that was considered important but not discussed in depth was how to estimate nitrogen retention for growing children and how to include this in their age-specific protein needs. It was only agreed that the growth allowance should consider the mean velocity of ponderal gain, the proportion of protein in the new tissue deposited. and the variability in growth observed under real-life conditions.

1.2. Interpretation of Nitrogen Balance

Several concerns were expressed in interpreting the results of nitrogen balance studies.

The Significance of Zero Nitrogen Balance

Day-to-day variation in N balance is inherent to homeostasis, and the shorter the time interval over which data are examined, the less physiological significance or relevance to requirements do deviations have. However, given a reasonable time-span, the level of protein intake sufficient for zero N balance is an appropriate estimate of mean protein requirement, assuming all losses are accounted for in the calculation of N balance.

There is a problem in estimating all N losses. However, we can assume that over long periods of time, N balance, under steady-state conditions, must average zero.

Therefore, any consistent positive deviation from zero with no indication of failure of homeostasis must be assumed to represent unmeasured losses. How much to allow for such losses when determining zero N balance for the purpose of estimating requirement is still an issue that must be resolved.

Variation within the Individual

The protein requirement of an individual is not to be viewed as genetically fixed. The original genetic potential of every individual is continuously modified by environmental circumstances from the time of conception onward. The expression of this genetic potential. the phenotype, is heavily influenced by environmental factors that vary with time, place, and situation. Thus, the protein requirements of an individual may be lower or higher at some time other than the one measured. Some quantitative information is becoming available on such variation and it is thought to be similar in magnitude to the population variation itself. This suggests that metabolic balance studies of individuals over brief periods of time - and even three months is still brief contribute more to population estimates than they do to reliable estimates of an individual's "requirement."

Variation between Individuals

In most studies, one or more individuals behave in an anomolous manner. The method of handling the data from such subjects will influence the mean and have an even greater effect on the standard deviation and confidence intervals. Since the number of subjects in all of these multilevel studies is small, reliable population variance estimates cannot be derived from any one of them. The pooled coefficient of variation for those studies that follow the protocol resonably closely and appear comparable is 17 per cent. It is reasonable to assume that the coefficient of variation of individual studies depends, at least in part, on the degree of confinement and homogeneity of the sample.

Variation between Studies

In arriving at realistic population estimates on the basis of short-term balance studies on small samples, one obviously needs to consider many factors; for example, the degree of departure of the experimental situation from normal free-living conditions and usual diets is very important. Even more troublesome is judgement as to the extent to which recommendations to be applied to countries or regions of the world must take into account not only dietary protein quality, but also effects arising from physical, biological, and social factors in the environment. Examples of these are, respectively, high environmental temperatures, prevalence of infectious disease, and existence of subpopulations with very high physical activity levels or with consistently inadequate or excessive dietary energy intakes.

1.3. Protein Requirement

The fundamental underlying question of how to estimate protein requirements of course arose continually during the week of meetings at Berkeley. It was felt that any recommendations for requirement need to be based not only on metabolic and

TABLE 1. Laboratory Comparisons

Entries in parentheses represent the immediately previous exercise recalculated with one laboratory deleted

biochemical indices of protein nutrition, but also on functional and health consequences. Further, there was an evident need for better understanding of the implications of adaptations to changes and variability in dietary intake and environment. While doubts were raised regarding the long-term validity of methods that use 10 to 15 days to assess nitrogen balance response to an intake level, it was agreed that this method does contribute to a first approximation of requirements. Subject to the errors and variabilities discussed, and assuming that they are minimized, short-term, multiple-level nitrogen balance studies give a mean zero balance intercept that is a useful estimate of mean requirements for the population studied, given the dietary protein fed under the conditions of the study.

TABLE 2. Results of Berkeley Standardization Exercise

a. One laboratory (No 5) was removed from all calculations - its reported values were consistently more than 5 SDs from the mean One lab (No 9) was removed from all ammonium sulphate calculations its reported values were consistently more than five SDs from the mean
b. One lab did not report values for this sample
c. One lab was removed for high values

2. Methodology

2.1. Standardization of Laboratory Determinations

To ensure that the studies conducted at the various laboratories around the world involved in UNUsponsored research could be compared with one another, a standardization procedure was set up under the direction of Christine Bilmazes, the technician in charge of the MIT Human Nutrition Laboratories. Over the past three years she has visited most of the participating laboratories to consult with laboratory personnel and advise on the procedures in use and to be used. Additionally, during this same time, four sets of samples, each including urine, faeces, and protein sources, were sent to all the laboratories for analysis The results of these analyses are summarized in table 1 and discussed as part of the general discussion of research results. More detailed summaries were prepared for the participating laboratories.

Because of the importance of this aspect of the studies, and its fundamental nature in assessing individual laboratory results, a fifth set of samples was prepared during the workshop under the direction of Dr. Sheldon Margen, to be carried home by the participants, analysed, and reported back immediately. The results are shown in table 2.

TABLE 3. Skin and Miscellaneous N Losses in Adults

a. Chapter 1 below.
b. Ref. 3.
c. Ref. 4.

2.2. Skin and Miscellaneous Losses

Most investigators participating in UNU-sponsored studies have used 3 mg N/kg/day for skin and 2 mg/kg for miscellaneous N losses, as suggested by the 1973 FAO/WHO report (2), in calculating true N balance. These estimates were derived mainly from studies conducted under moderate ambient temperatures and correspond to obligatory losses. The applicability of this figure for N-balance studies conducted under conditions of high environmental temperatures and at protein intake in the submaintenance range was questioned, since increased sweating and N intake affect skin losses. Inoue et al. (paper 1 below) provided data concerning this problem, while Bourges et al. (paper 6) also reported sweat N concentrations in subjects receiving different protein intakes and undergoing exercise. Tables 3 and 4 summarize their findings in comparison with results previously published by Huang and Lin (3) and Calloway et al. (4).

Calloway reported recent studies (5) indicating that up to 100 mg N. nitrate-nitrogeniday are formed in the body, and lost daily in urine and faeces. This loss is not measured as N by the Kjeldahl method.

These data suggest that 5 mg N/kg/day may not be an appropriate figure to use for miscellaneous losses when estimating "true" N balance. In particular, the estimate probably should be adjusted upward when ambient temperature is above the thermoneutral range, since there is evidence that higher environmental temperatures are correlated with increased N losses in sweat, and further that these losses are compensated for by lower urinary losses of N. This results in an underestimate of the dietary N required for zero balance unless the allowance for integumental loss is appropriately increased. In theory, the same effect can result from sweat losses associated with heavy physical activity. Yet, for individuals accustomed to such work in a tropical climate, these losses appear to be less than would be predicted from experimental studies with non-adapted, non-acclimatized subjects.

TABLE 4. Skin Loss in Exercise-Induced Sweating

2.3. Standard UNU Short-Term Protocol

Many of the investigators (papers 8-12) followed the standard short-term protocol as it was formulated at the Costa Rica meeting in 1977 (1). This procedure for estimating protein requirement for a particular protein source and population is based on feeding each of several individuals several different, constant levels of protein for successive periods of 10 to 15 days each. These data are used to estimate individual responses and individual mean requirements that, in turn, are used to estimate the needs of the population. Details of this design and notation are as follows.

The sample consists of a number of individuals representative of a specific population (indexed by j).

For each individual data was recorded for four or more periods at different intakes (indexed by i), where each period is 10 days or more at constant intake I_i.

Measured and calculated are, for each individual,
= average urinary nitrogen over the last five days of period i
S_i = standard deviation of urinary nitrogen over the last five days of period i
= average faecal nitrogen over entire study
M = estimated miscellaneous nitrogen losses
O_i = + + M = nitrogen output at level I_i
NB_i = I_i - O_i = nitrogen balance for level I_i
O = a + b . I estimated response curve; a, b estimated by weighted regression of O_i on I_i
(weighted by 1/S², if S is known, if S is unknown, use unit weights).

This produces a slope and intercept (a_j, b_j), for each individual, and from this can be calculated
R_j = a_j/(1 - b_j) = estimated mean requirement for jth individual.

Population estimates are then calculated as
= average requirement over all individuals
SD = standard deviation of requirements.

During the week of presentations and discussion at the workshop, the ability of the individual to reach a plateau in 10 days was questioned. This critical point is a key assumption of short-term studies. Although examination of individuals on low protein intakes showed that 10 days were sufficient (6), it was felt that experiments were needed on individuals at higher levels of intake. Thus, subsequent to the workshop, Ozalp et al. (paper 7) followed 15 young adults for 15 days and compared results based on days 6 to 10 to those results based on days 1.1 to 15. Paired comparison of their balance showed no significant difference between the two periods, supporting the assumption that 10 days are sufficient for the short-term study.

For experiments that follow, in general, the short-term protocol, there are still many factors that can influence the results Several of the more important ones are listed below.

Factors Generally Under Investigators' Control

A-1 Order of presentation of protein intake levels.
A-2 Dietary quality at different intake levels.
A-3 Variability and bias in laboratory determinations.
A-4 Short-term stress - infectious or psychological.
A-5 Over- or underestimation of maintenance energy intake.
A-6 Standardization of experimental conditions.
A-7 Representativeness of subjects.
A-8 Availability of vitamins and minerals.

Factors Generally Not Under Investigators' Control

B-1 Variability of skin and miscellaneous nitrogen losses.
B-2 Activity level of subjects.
B-3 Stress due to conditions of the study.

Since the effects of these factors can be very important, a point-by-point discussion of them is presented.

Factors Generally Under Control of Investigator

A-1. If successive levels are studied in a sequence from high to low, the intercept values may be higher, and in addition the results are likely to be more variable than if the sequence is from low to high. The best way of avoiding the biases resulting from one or the other pattern is to randomize the levels with break periods in between.

A-2. If diet composition changes so that its quality differs at different levels of intake for a single experiment, then the very meaning of the response curve and the zero balance intercept is unclear, since essentially one is examining a different dietary protein at each intake level. At best, if the variation in quality is slight, and if the dietary quality is poorer at the lower levels, the resulting N balances at these levels will, on the average, be proportionately lower than at the higher levels, and the opposite is true if the quality is higher at the lower levels In the former case, the intercept will tend to be falsely low, and in the latter case, falsely high.

A-3. It is necessary that each laboratory maintain an ongoing quality control programme, and that checks be made routinely with reference laboratories. High variability of determinations within a given laboratory reduces the usefulness of its results. Consistent differences between laboratories can be corrected for, but only if the investigators are aware of them.

A-4. Urinary N loss is increased with stress, whether of psychological or infectious origin. The result is an increase in protein requirements during the subsequent recovery period when N retention increases in a compensatory manner Moreover, stress of infectious origin not only increases urinary nitrogen excretion, but also anorexia reduces food intake and there are changes in foods offered or selected It may also reduce absorption if the gastrointestinal tract is involved. In studies done under usual living and working conditions any effect of stress that is compatible with continuation of normal activities can be considered an approximate estimate of the influence of usual environmental factors on the protein requirements of that population. However, the influence of clinical disease requiring hospitalization or confinement to bed should be excluded from population estimates and dealt with as a therapeutic problem.

A-5. Underestimation of dietary energy requirements in balance studies can result in values that are abnormally high unless a compensatory reduction in physical activity can occur. Conversely, excessive caloric intakes can result in high retentions and lead to an underestimation of the protein requirements of individuals in energy balance. Although such caloric deficiencies and excesses will eventually be reflected in the corresponding weight changes, these may not be obvious over the short duration of a balance study. If they do not occur with energy intakes clearly higher or lower than habitual intakes or approximately estimated normal requirements, adaptation must have taken place.

A-6. There is no doubt that careful screening of volunteers to eliminate infections and other disease problems, together with maintaining temperature control and relieving subjects of normal work and other responsibilities, will give more uniform results than studies with free-living individuals. For some purposes, studies under closely controlled conditions are preferable or even necessary, but for estimation of population requirements, studies on free-living subjects are necessary

A-7. Similarly, exclusion of individuals who differ markedly from the norm will tend to produce more homogeneous results than would be obtained if such individuals were included. This is basically a question of the purpose of the experiment-whether it is to obtain a good estimate of the "normal" population requirement or to gauge the range of variability that exists within the population.

A-8. Some vitamin and mineral deficiencies can impair N retention and growth and can result in incorrect estimation of protein requirements.

Factors Generally Not Under the Control of Investigator

B-1. The appropriateness of using 5 mg/N/kg in adults for integumental and miscellaneous losses is discussed in the previous section.

B-2. There is some evidence that active individuals need less protein per kg than do those not active. Studies with a high proportion of very active subjects may give lower values than appropriate for the general population. The best indicator of the inherent activity level of an individual may be the number of calories necessary for energy balance.

B-3. For some individuals and in some populations, the necessary conditions of a balance study may in themselves constitute a stress that results in an apparent increase in protein requirements. This may be unavoidable, but the possibility should be recognized.

2.4. Long-Term Studies

Two papers were presented that studied individuals maintained for 2 to 3 months on constant intakes; these papers examined the results statistically to describe the nature of intra-individual variability. Rand (paper 2) examined 42 individuals in five separate experiments with intakes from 0.73 to 1.89 protein/kg body weight. He found that the daily urinary nitrogen of 25 (60 per cent) of the individuals could be satisfactorily described in terms of a constant mean and random error (with a standard deviation of 0.74 9 nitrogen) The other 17 (40 per cent) individuals had significant long-term trends (linear, quadratic, or cubic). Examination of the data for autocorrelation, an indication of data on successive days being more alike than data an arbitrary time apart, found only four individuals (10 per cent) who showed significant autocorrelation of values. These autocorrelations were calculated after the long-term trends were removed statistically, since such trends would appear as a false autocorrelation. Obviously, if an individual's values were steadily increasing, successive data would be similar. These findings are consistent with earlier work by Rand et al. (6), but are at variance with a report by Sukhatme and Margen (8), who described the presence of autocorrelation as the rule rather than the exception in their five subjects. Margen presented data (Durkin et al., paper 3) from an additional six subjects maintained on a 0 42 9 protein intake in a metabolic ward Of these individuals, five (83 per cent) showed an autocorrelation; however, all took several weeks to reach an apparent steady state. The differences between the two studies were attributed to: (a) the lower variability of the subjects under the very confined conditions in Margen's laboratory, and (b) Rand's removal of any long-term trend before calculating autocorrelation,

2.5. Very Short-Term Balance Studies

Several investigators have explored the use of procedures shorter than the 50 to 53 days of the standard UNU protocol for estimating the amount of protein in a diet required to achieve zero nitrogen balance. This work is based on the assumption that, although a steady state may not be achieved at each intake level, the balance value measured at a level is proportional to a steady state at that level.

Scrimshaw et al. (paper 4) presented the results of two studies. The first used one-day balances at each of three intake levels and showed that the variability involved is so large as to make this design useless The second experiment used five different levels with two-day balance periods at each level Four individuals followed both the ascending and the descending design The data showed that most responses could be adequately fitted with a straight line, with the variability of the ascending design much less than that of the descending design. However, the intercepts calculated from data from the ascending order were consistently below those calculated from the descending design.

Bressani et al. (paper 5) presented data showing that his ascending design with two-day balance periods at three to five levels gives good agreement in terms of zero balance intercept with standard protocol data for the same proteins preceded initially by three days of a protein-free diet.

TABLE 5 Comparison of Critical Experimental Conditions for Short-Term Multiple Level N Balance Studies Following the UNU "Standard Protocol"

TABLE 5 - continued

While these results are very promising, it was felt that the zero balance intercept values of six to eight subjects studied for two-day periods at four levels using the procedure of Bressani, equally divided between ascending and descending patterns. need to be explicitly compared with the standard procedure. Such agreement must be adequately demonstrated before this methodology can be accepted as a substitute for the standard procedure.

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