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Discussion

Rose's estimates of IAA requirements

The problem which Waterlow tried to examine in his paper is why the MIT estimates are in most cases so much higher than those of Rose. It is logical therefore, to begin by looking at what Rose actually did; a point that was not dealt with in any detail in Waterlow's paper. The group considered that these studies were flawed in many ways. If the results were corrected for the high energy intakes (55 kcal/kg/d) and for skin and other losses, how much difference would it make? In Young's opinion, not much.* Rose's figures, as reproduced by FAO/WHO in their 1973 report, and quoted again by FAO/WHO/UNU in 1985, represented the highest estimate of individual requirement needed to achieve a positive N balance; this Rose called 'the tentative minimum requirement'. He then doubled these figures to give 'a definitely safe intake', which, for many IAAs would bring this safe level closer to the MIT estimates. However, the MIT figures represent average requirements, so that a comparison with Rose's 'highest individual requirement' is not an exact comparison of like with like.**

* On the question of the effect of the high energy intake in Rose's studies: if as evidence suggests, 1 kcal spares' about 1.5 mg N. a reduction of energy intake to 45 kcal/kg d, as in the MIT studies (Marchini et al, 1993) would increase the requirement by about 15 mg N/kg/d, or about 20% of the obligatory losses (54 mg/kg d) after correction for efficiency at 70% This does not go very far towards filling the gap A further problem is that Rose's N balances included faecal nitrogen, whereas the MIT figures take no account of possible faecal losses of I AA. This difference would further enlarge the gap.

** Table 17 of the 1973 report, dealing with the IAA requirements of adults, gives three columns, all in mg/d.

(a) Rose's figures for young men;
(b) a variety of data for women;
(c) data for women recalculated by regression analysis.

A fourth column gives 'combined adult values' as mg/kg/d. It is not clear, without going back to the original papers, what body weights were used to calculate mg kg and how the male and female data were combined. As a result there seem to be some discrepancies, but they are not very important. e.g.

The next question was whether it makes any difference if the studies are done in ascending or descending order of amino acid intake. Millward referred to the work of Atinmo et al (1988), who used both ascending and descending designs and found quite large differences. These differences, however, are not consistent in showing more positive balances with either design. Scrimshaw and Torun quoted studies by UNU (Rand et al, 1984; Torun et al, 1981) and at INCAP (Pineda et al, 1981) done in randomized ascending and descending order; there was no difference. However, Scrimshaw considered that the ascending pattern was preferable for establishing a minimum requirement, since the subjects will be starting from a lower level and therefore will be better adapted. Young, in his carbon balance studies, tried to replicate the design of Rose's work, but could not understand how he did it. At the end of each period he determined the balance and then decided whether to go higher or lower; but how did he do it, since it takes time to analyse urine and faeces?*

*What Rose actually says is: 'to arrive at an amount representing the minimum requirement of the organism for a given amino acid, a negative N balance was induced at some stage of the test, and then the intake of the substance under investigation was raised until a slight but distinct positive balance, as measured by the average for a period of several days was achieved' (Rose's italics) (Rose, 1957).

Allen made the point that these designs, whether of carbon or nitrogen balances, are in any case artificial, since they take no account of the day-to-day and meal-to-meal fluctuations in intake that are found in real life. Her point fits in with Millward's insistence that if the question is whether a particular level is adequate for maintenance, then the measurements have to be made in subjects adapted to that level. However, for Young that is not the question: the whole aim of his work has been to establish a minimum requirement to balance the obligatory loss. In either case, whether the aim is to find a minimum requirement or a requirement at some other level, the question of adaptation and the time needed to achieve it is crucial, but was not discussed in any detail. Scrimshaw just pointed out that in the long term studies sponsored by the United Nations University (UNU) and lasting as much as 6 months, adaptation occurred in a few days.

Protein quality

The discussion began with a consideration of whether there are different patterns of amino acid requirements for growth and for maintenance, as proposed by Waterlow in his paper. There was a general consensus (Reeds, Young, Scrimshaw) that it is illogical to use biological assays in rapidly growing animals such as rats and pigs to make inferences about the amino acid needs of adult humans, and the continuing use of PER in many countries is to be deplored. This conclusion is in line with the recommendations of the 1990 FAO/WHO Expert Consultation.

How should protein quality be measured? The FAO/ WHO/UNO 1985 report concluded that this could appropriately be done by using a chemical score. For Reeds the chemical score, which is an intrinsic property of a protein depending on its amino acid composition, provides a measure that is accurate, precise and objective. Others considered that the chemical score is not appropriate. It is easy to show a difference in protein quality, for example between milk and Incaparina, if the proteins are fed at sub-maintenance level, but at levels above maintenance the difference disappears (Scrimshaw). Studies carried out by UNU (Rand et al 1984; Torun et al, 1984) showed that with diets actually consumed in developing countries, based on rice, wheat or maize, differences in the amount of protein needed resulted from differences in digestibility and not in protein score. The reason that these diets were apparently not limiting in any indispensable amino acid was probably complementation by other components of the diet. Thus the 1985 Report concluded that with real diets quality is not important for adults, once a correction has been made for digestibility. However, this might not apply in situations such as that of refugees, whose diet is limited in composition and may not fulfill their energy requirements.

In balance studies at MIT for the comparative evaluation of wheat protein vs milk protein, the predicted NPU of wheat, based on amino acid composition and the MIT pattern of requirements, was about 70, which agreed with the balance data (Young). This was contested by Millward, who claimed that the amount of wheat protein that achieved balance (0.74 g/kg/d) did not provide an amount of lysine that matched the MlT estimate of lysine requirement. Millward described a study in which subjects were maintained on their usual level of protein intake; there was no difference between wheat and milk, although the lysine content of the wheat diet was below the level of adequacy according to the MIT pattern. This was not an experiment set up to disprove the MIT pattern: it showed that there is enough lysine in the body pool in the early postprandial phase to supplement the wheat lysine and enable it to be used with 90% efficiency.

Millward reiterated the importance of establishing a biological model of how amino acids are utilized to maintain balance. When agreement has been reached on whether amino acid needs are fixed or variable, then it will be possible to design experiments to evaluate the need. In response (Waterlow): is this duality between 'fixed' and 'variable' needs a real question? The amount of an amino acid needed to secure balance is evidently influenced by the preceding or habitual intake, and in this sense requirements are variable; however, most of the discussion, notably that of Young, had been focused on the minimum requirement, which seems to be a reasonably clear-cut concept, even though this minimum may be less than the amounts usually consumed in real life, and less than some so far undefined optimum.

Reeds raised the question of whether, with leucine for example, the requirement for leucine carbon was the same as the quantity of N that the body needs to come from leucine. (The point was not taken up, but the question seems to be answered, at least in part, by the experiments of Walser's group on the relative utilization of leucine and ketoisocaproic acid (Kang & Walser, 1985)).

The MIT pattern

Millward began by stressing the importance of differentiating between values obtained experimentally and those generated theoretically from the pattern of amino acids in body tissues. He himself had focused on lysine because of Young and Pellett's (1990) conclusion that lysine was likely to be limiting in dietaries based largely on cereals. There are no reliable isotopic data for lysine; Zello et al's paper (1993) had been referred to, but the method is inappropriate; to use phenylalanine oxidation rates is very complicated and the results debatable. Only a few of the MIT values are experimentally derived. In reply Young said that there are stable isotope data for leucine, valine, threonine, lysine and methionine, although the early studies can be criticized. The early studies might be criticized. The best experimental data-base is that for leucine, particularly the more recent work of Marchini et al (1993) and el Khoury et al (1994). The measurements of phenylalanine oxidation were entirely consistent with the data for leucine. Balances were consistently negative at the FAO level. The experimentally determined values were compared with those predicted from the composition of body protein (Young & Pellett, 1990). The requirements for those amino acids that had not been determined experimentally were also predicted in the same way. It turned out that these predicted values agreed reasonably well with the estimates for preschool children derived by Torun at INCAP (Pineda et al, 1981) and adopted in the 1985 report. The ratios of predicted requirement: preschool child requirement are: leucine 0.7; isoleucine 1.0; valine 1.0; lysine 0.94; threonine 0.86; aromatic amino acids 0.8; sulphur amioacids 0.96. For this reason the FAO/WHO 1990 report recommended as an interim measure that the preschool pattern should be used for all age-groups.

A number of criticisms were raised. The FAO/WHO 1990 report was criticized because it was the outcome of a small, not widely representative meeting, and the discussion of the amino acid reference protein was relatively brief. However, Torun pointed out that it was an official FAO/WHO consultation. Even if the discussion and review were not satisfactory, it is now the report accepted by the Agencies, and represents the established pattern until there is another formal UN meeting. A second criticism was that the INCAP studies on preschool children have never been published in peer reviewed journals (Pineda et al, 1981). A question can be raised about those studies because there were substantial positive N balances, greater than would be expected in normally growing children of the same age; they were children who had recovered from malnutrition, but they may still have been depositing lean tissue at a greater than normal rate. Torun replied that the studies were done with diets containing a certain amount of milk protein and a mixture of amino acids in the pattern of milk protein, except for the amino acid being tested. They were done in both ascending and descending order. The criteria used were: maintenance of a positive plateau in N balance, a plateau in urinary urea and changes in free circulating amino acids. The diets were fed at a level of 1.2 g protein/kg/d, with glycine to make up the amount of non-essential N. Other studies were done with various sources of protein (milk, soy) and the results were in good agreement. Young had back-calculated the amounts of lysine that would be needed to achieve a lower and more reasonable level of N balance: this amount fitted exactly with his prediction. The INCAP data are entirely consistent with classical fortification studies that have been done at MIT.

There followed a brief discussion of individual amino acids. Questioned about the confidence that he would put on his isotopic estimate of the lysine requirement, Young conceded that his data were limited; however, new 24 h studies, not yet published, clearly show that the FAO level is far too low. The lysine content of the MIT pattern is, in fact, less than that of body protein. Reeds expressed concern about the sulphur amino acids (SAA). The N balance literature is consistent in suggesting a high SAA requirement for maintenance. It was noted that the Rose value for SAA was quite close to that proposed by Young et al even though his values for the other amino acids were much lower.

Policy implications

Ferro-Luzzi was apprehensive about changing from the 1985 recommendations to higher values. The key question here is whether the conclusion of the 1985 report, that almost all real diets are adequate in IAAs, would still hold if the MIT pattern is adopted. Scrimshaw referred again to the UNU studies showing that natural diets, eaten in usual amounts, fulfilled IAA requirements, as judged by the criterion of N balance. However, the 'key' question is not answered unless the IAA content of these diets is compared with the MIT pattern. Ferro-Luzzi was concerned that the conclusions of these studies, based on a small number of subjects, may not hold at the national level, because of the poor quality of FAO data on national diets. Young pointed out that in some countries, particularly in Africa, where cereals provide more than two-thirds of the protein intake, if there is a risk of IAA deficiency, the MIT pattern suggests that it would be of lysine. However, a small amount of milk powder or fish would make up for any deficiency. In the study of Nicol and Phillips in Nigeria (Nicol & Phillips, 1976), about 5 g of dried fish used as a condiment apparently made it possible to achieve N balance on protein intakes that otherwise seemed remarkably low. Ferro-Luzzi was still worried about the implications of adopting the MIT pattern, even though Young & Pellet (1990) refer to risk rather than actual deficiency of lysine. In China she had seen people sprinkle lysine on top of rice, like Parmesan cheese.

The final question was whether the MIT pattern should be endorsed, at least as an interim recommendation for practical purposes. Torun considered that if the recommendation, e.g. for lysine, should prove to be too high, it will be erring on the side of safety. Thus, there would be no danger from a public health point of view. Millward dissented; he agreed that a scoring pattern is needed to supplement balance data that are inevitably limited. However, in his view the MIT pattern is not sufficiently secure, based as it is on predictions from body composition for which the justification is disputed, on balance data on pre-school children, which are open to some criticism, and on limited isotope data. In his view, if we are not able to come up with an agreed scoring pattern, we should say so. There is no intellectual justification for accepting the MIT pattern, just because it is the best we have. As for it being an interim recommendation, any decision made here will have important implications. Once even a grudging commitment is made it is very difficult to change it. In the present state of knowledge he was not in favour of adopting the recommendation.

Scrimshaw summed up the debate as follows: 'Those dealing with developing countries do feel the need for a scoring pattern. Not having one would cause a serious problem for the agencies and users. Secondly, every expert group has been faced with the problem of having to make the best recommendations that they can with inadequate data, and have always come up with a long list of research recommendations. We are no different in this respect. Thirdly, there is clear evidence that when new data become available, groups do change their recommendations and views. There will be another meeting in the next 3 to 4 years, and one of the issues that will be discussed is precisely this issue of amino acid requirements; hopefully in the meantime your criticisms of this work will have helped to generate new data that will put the new group into a better position to take a decision.'

References

Atinmo T. Mbofung CMF, Egun G & Osotumehin BO (1988): Nitrogen balance study in young Nigerian male adults using four levels of protein intake. Br. J. Nutr. 60, 451-458.

Calloway DH & Spector H (1954): Nitrogen balance as related to calorie and protein intake in active young men. Am. J. Clin. Nutr. 2, 405-412.

El Khoury AK, Fukagawa NK, Sanchez M, Tsay RM, Gleason RE, Chapman TE & Young VR (1994): The 24-h pattern and rate of leucine oxidation, with particular reference to tracer estimates of leucine requirements in healthy adults. Am. J. Clin. Nutr. 59, 1012-1020.

FAO/WHO (1973): Energy and protein requirements. FAO Nutrition Meetings Report Series no. 52. Rome: FAO.

FAO/WHO/UNU (1985): Energy and protein requirements. Technical Report Series no. 724. Geneva: WHO.

FAO/WHO (1990): Protein quality evaluation. Rome: FAO.

Kang CW & Walser M (1985): The nutritional efficiency of a-ketoisocaproate relative to leucine, assessed isotopically. Am. J. Physiol. 249, E355-E359.

Marchini JS, Cortiella J. Hiramatsu T. Chapman TE & Young VR (1993): Requirements for indispensable amino acids in adult humans: long-term amino acid kinetic study with support for the adequacy of the Massachusetts Institute of Technology requirements pattern. Am. J. Clin. Nutr. 58, 670-683.

Nicol BM & :Phillips PG (1976): The utilization of dietary protein by Nigerian men. Br. J. Nutr. 36, 337-351.

Pineda O. Torun B. Viteri FE & Arroyave G (1981): Protein quality in relation to estimates of essential amino acid requirements. In Protein quality in humans: assessment and in vitro estimation, eds CE Bodwell, JS Adkins & DT Hopkins, pp 29-42. Westport, CT: AVI.

Rand WM, Uauy R & Scrimshaw NS, eds (1984): Protein-energy requirement studies in developing countries: results of international research. Food Nutr. Bull. Suppl. 10. Toyko: UNU.

Rose WC (1957): The amino acid requirements of adult man. Nutr. Abstr. Rev. 27, 631-647.

Torun B. Young VR & Rand WM, eds (1981): Protein-energy requirements of developing countries: evaluation of new data. Food Nutr. Bull. Suppl. 5. Tokyo: UNU.

Young VR, Bier DM & Pellett PL (1989): A theoretical basis for increasing current estimates of the amino acid requirements in adult man, with experimental support. Am. J. Clin. Nutr. 50, 80-92.

Young VR & Pellett PL (1990): Current concepts concerning indispensable amino acid needs in adults and their implications for international nutrition planning. Food Nutr. Bull. 12, 289-300.

Zello GA, Pencharz PB, Ball RO (1993): Dietary lysine requirement of young adult males determined by oxidation of L-1-¹³C-phenylalanine. Am. J. Physiol. 264,E677-E685.

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