REFERENCE PROTEIN PATTERNS

P.R. Payne
Department of Human Nutrition
London School of Hygiene and Tropical Medicine

1. The concept of a fixed pattern of amino acid, which may be used as a yardstick with to compare the nutritive value of foods and diets, is subject to the same limitations and qualifications as is the concept of 'protein quality'. The suggestion is that the nutritive value can be expressed as a single figure which is to be regarded as applicable under all circumstances. In fact, the relative proportions in which the essential amino acids are needed almost certainly depend upon the species of animal, its physiological state, and upon numerous interelationships and interactions, not only between the amino acids themselves, Lewis & Boorman (1), but probably with other dietary components as well. Further difficulties arise in connection with the method of calculation of protein scores on the basis of a chosen reference pattern. For example the choice between Chemical Score according to Block & Mitchell, or the essential Amino Index of Oser or A/E and A/T ratios as suggested by the previous committee. The relatively poor accuracy of amino acid determinations in foods, and the problem of biological availability of the amino acids, present further complications. The sum total of these limitations and doubts is formidable. However the advantages of a method of dietary assessment in terms of amino acids are also great, even if the precision attained is limited. In fact in most circumstances such a method is probably the only practical one at this time, the ideal being direct biological assay using human subjects of each and every diet and food mixture under consideration.

2. Essential Amino Acids of practical importance

There is good experimental evidence in the form of N balance or growth response e.g. Rose et al (2) McLoughlan et al (3), Bigwood (4) that the amino acids found to be limiting in human foods and diets are in order of their relative frequency of occurrence methionine/cystine, lysine and possibly tryptophan. Although Lysine is the limiting factor in the cereal staples, in most practical circumstances, this deficiency is made up by the addition of the legumes and small amounts of animal proteins that customarily go to make up diets. Thus Miller and Donoso (5) found that of 25 diets based upon food consumption data from all parts of the world, all showed an improvement of nutritive value on addition of methionine.

The practical implications of this are that any method of establishing a reference pattern must be judged in relation to its success in predicting these three requirement levels, and secondly, any reference pattern must be suspect if it suggests contrary to biological evidence that other amino acids are frequently limiting in normal diets.

3. Methods of expression of reference patterns

3.1 The recommendations of the 1965 FAO/WHO committee

The last committee on Protein Requirements suggested that attention should be paid not only to the relative proportions of the various essential amino acids, the so called A/E ratios, but also to the proportion of the whole group of essential amino acids in relation to the total N, i.e. the E/T ratio. The committee did not specify in what way these two ratios should be used to assess the quality of a protein, but merely suggested that protein scores could be calculated by a comparison of A/E ratios with the corresponding ratio in a reference pattern, and further, that some proteins might be found to be limited by their E/T ratio.

3.2 The Practical value of Protein scores using A/E ratios

It has been found in practice that protein scores based only upon A/E ratios are much worse correlated with biological assessments of protein quality, than are those based upon conventional A/T ratios. Thus Cresta et al (6) found for 274 materials a correlation of only 0.39 between scores based upon A/E ratios using egg as a reference pattern, and biological measurements. For a smaller number of materials comprising of the common staples legumes and animal protein sources, Payne (7) showed that using egg as a reference the correlation ceofficient for A/T scores versus net protein utilization was 0.93 (n = 18) while for A/E scores on the same materials it was only 0.51.

3.3 The significance of E/T ratios

It is reasonable to suppose that there must exist some level for the ratio in a protein above which some of the essential amino acids are simply used by an animal to satisfy its requirements for non essential N. For such a protein, dilution with any source of non-essential N should cause no decrease in biological value. Bender (8) showed that addition of up to 15% of non-essential amino N to whole egg produced a mixture which still had an NPU of 100% when fed to rats. Snyderman et al (9) showed that addition of urea and glycine to the diet of infants receiving 1.1g/kg of milk protein; brought about an increase in N retention. However Daniel et al (10) found an increase in urinary N, and a decrease in biological value of milk protein fed to older children (10 - 11 years) when 25% of the milk N was replaced with a mixture of nonessential amino acids. Scrimshaw et al (11) & (12) found no increase in the urinary N excretion of young men when up to 25% of either egg or milk proteins was replaced with non essential N. Clarke et al (13) on the other hand have shown that in human adults given an amino acid mixture similar to the FAO reference pattern, N balance is simply related to the total essential amino acid intake and does not depend upon the E/T ratio, even when this was varied from 0.84 to 3.15. To some extent the discrepancies between the findings of these groups of workers may be partly due to the fact that those who have failed to find a beneficial effect of addition of non specific N (10) and (13) have used low intakes of N which were clearly demonstrated to be less than sufficient to meet the subjects N requirements.

Evidently the quantitative nature if any of the relevance of the E/T ratio to defining the nutritive value of proteins has not been clearly established and at the present time the use of separate A/E and E/T ratios cannot be justified. It should be noted in fact that the more conventional A/T ratios can in any case be regarded as a combination of the two, and hence these should clearly be retained as the basis for expression of reference patterns, and for the calculation of protein scores.

4. Methods of determining reference patterns

4.1 Direct assessment of amino acid requirements

Table I shows some of the more recent values obtained for the amino acid requirement levels in growing rats and chicks, and also for purposes of comparison, values for children and for adult man.

TABLE I

Amino acid Requirements of Different Species - expressed as
mg amino acid per gr.N

1. Rama Rao, Chalam Metta, V. and Connor Johnson, B. J. Nutrition 69 387 (1959).

2. Rogers, Q.R. and Harper A.E. Fed. Proc. 23 186 (1964).

3. McLaughlan, J.M. and Illman, W.I. J. Nutrition 93 21 (1967).

4. Dean W.F. and Scott H.M. Poultry Science 44 803 (1965).

5. United States National Research Council.

6. Evaluation of Protein Nutrition NRC publication No. 711 Nat. Acad. Sci. U.S.A. Washington D.C.

The range of values is considerable, even within the same species, and in fact although interspecies variations in requirements have been the subject of much speculation, it is not possible to conclude from these figures anything about the existence or nature of such variability. Evidently the technical difficulties involved in the measurements are such that we cannot choose any one of these as a reference pattern. Nevertheless we can draw some tentative conclusions about the FAO provisional pattern, particularly with regard to the more important amino acids. In most cases, the level of lysine indicated is greater in absolute terms than that of methionine and cystine combined. This is also true of the patterns based on the composition of egg or milk (Table II). In practice this means that the level of lysine in the FAO pattern is too low, so that protein scores of lysine limited foods are overestimated by the FAO pattern (Table III). The level of tryptophan in the FAO pattern is higher than that required by the rat or chick, and a reduction to 70 mg/gN has been suggested by experiments on human adults (14).

4.2 The suitability as reference patterns of the composition of proteins of high biological value

Table II which is taken from Cresta et al (6) shows the composition of egg according to three different authorities; and of cow and human milks. A major objection to all these as reference patterns, is that they are likely to contain superfluous amounts of some or all of the essential amino acids since a high biological value only provides information about the single amino acid which happens to be limiting, and in the case of egg for which non essential N is limiting, all of the individual amino acid; total N levels are likely to be too high. For this reason, scores based upon the lysine and sulphur amino acid levels in egg tend to underestimate nutritive values (Table III).

TABLE II

Amino Acid Composition of Proteins of high Biological Value

1 Mitchell, HH., Block, E.J. Some Relationships between the Amino Acid Contents of Proteins and their Nutritive Value for the Rat. J. Biol. Chem. 163 (1946), 599 - 620.

2 Report of Joint FAO/WHO Expert Group (1963), Table 6B.

3 Amino acids content of foods and biological data on proteins. Average data by column chromatography.

4 Report of Joint FAO/WHO Expert Group (1963), Table 6B.

5 Report of Joint FAO/WHO Expert Group (1963), Table 6B.

6 Report of the FAO Committee on Protein Requirements (1955).

Similary, the levels of isoleucine, valine and tryptophan are almost certainly excessive in both egg and milk, so that scores based upon these patterns frequently indicate them to be the limiting factors in human diets and single foods Autret et al (15), Cresta et al (6). In fact with the single exception of haemoglobin, no protein or food mixture has ever been demonstrated by biological tests to be primarily deficient in isoleucine, so that such results can only be regarded as artefacts due to the reference pattern, and can have no biological significance.

4.3 Reference levels based upon mathematical analysis of the nutritive values and amino acid composition of foods

The recent publication by FAO of amino acid composition and biological values of a large number of foods and food mixtures (16) has made it possible to search for empirical relations between nutritive value and composition, the criterion being simply the greatest accuracy of prediction of nutritive values. Cresta et al (6) carried out a multiple regression analysis relating all of the essential amino acids to the nutritive values of a large number of diets and foods. This approach gives prediction equations for PER and NPU which are complex (they contain terms for each amino acid) and do not seem to be justified by the degree of accuracy of prediction they provide. Further, a multiple regression analysis of this kind assumes that there are linear interactions between the effects of the different amino acids upon nutritive values. However all of the available evidence still supports the original concept of Block end Mitchell of the dependence of nutritive value upon a single amino acid, which operates up to definite threshold value above which the relationship becomes dependent upon another amino acid, i.e. to a very non-linear interdependence.

A simpler and necessarily much more limited analysis of the same data is presented in Table III. These figures were selected on the following basis. (1) Only those foods were included for which there is biological evidence of the identity of the limiting amino acid. In practice this means that they have been shown to give a response of either PER of NPU on addition of either lysine or methionine. (2) In view of the degree of variability involved both in the measurement of NPU and of amino acid content, and of the problem of sample variability, the foods have been restricted to those for which at least two independent NPU values were listed, and for which the amino acid levels were based on at least 5 samples measured by column chromatography. In spite of these restrictions, the list contains most of the main sources of protein in human diets.

The table shows that in both the lysine and SAA limited groups there is a good correlation between either NPU or PER and the level of the limiting amino acid. This is again reflected in the high correlation of both measures of nutritive value and A/T scores based upon either egg or the FAO pattern. Those based upon the A/E ratios using egg are however much more poorly correlated with NPU or PER. The mean deviations (score - NPU) are high, and negative for A/T (egg) scores in both groups of foods, showing that this reference pattern tends to underestimate nutritive values in particular for the SAA limited foods. The deviations for the A/T (FAO) scores are small for the SAA group, but very large and positive for lysine limited materials, lending further support to the suggestion that the lysine level in this pattern is too low. The final column of scores is calculated using A/T ratios with a modified FAO pattern having a value of 360 mg lysine/gN chosen so as to give zero mean deviation for the prediction of NPU for the lysine limited materials.

This modification to the FAO pattern not only much reduces the average deviation of scores from measured NPU values, but also as the table shows gives an improved correlation between scores and either NPU or PER.

TABLE III

Amino acid composition, protein scores and net protein
utilizations of a number of common foods

Numbers in parenthesis are numbers of independent NPU or PER determinations or of samples analysed by column chromatography.

Recommendations

1. The use of A/E ratios for calculation of chemical score should be discontinued.

2. The use of high biological value proteins such as egg or milk as the basis for reference patterns should be discontinued. Firstly because published figures for their composition change as more analytical data accumulate so that a reference pattern based on egg for instance has to be arbitrarily specified as 'egg FAO 1963' or 'egg FAO 1968'. Secondly reference patterns of this kind will always contain excessive levels of some amino acids, giving rise to spurious identification of less important amino acids e.g. isoleucine as first limiting factors in many diets, and consequently reducing the accuracy of prediction of nutritive values.

3. The FAO reference pattern still represents a useful basis for development. Recent work with human adults Morse et al (17) has shown that foods adjusted to this pattern can sustain N balance at levels of intake close to the minimum requirement. However in order to meet the needs of young growing animals, the level of lysine should be raised from 270 to 360 mg/gN. The level of tryptophan should be reduced from 90 to 70 mg/gN.

The resulting modified FAO pattern gives protein scores which are in better agreement with nutritive values than either the original or the amino acid composition of whole egg.

REFERENCES

1. Lewis D and Boorman KN. In Proteins as Human Food (Ed. R.A. Lawrie) Butterworth London (1969)

2. Rose W.C., Haines W.J. and Warner DT. Journal Biol. Chem. 206 421 (1956).

3. McLaughlan J.M., Rogers C.G., Chapman D.G. and Campbell, J.A. Canadian J. Biochem. 37 1293 (1959).

4. Bigwood E.J. Nutrio et Dieta 4 17.

5. Miller D.S. and Donoso G. J. Sci. Fd. Agric. 14 345.

6. Cresta M, Périssé J. and Autret M. Nutrition Newsletter 7 No. 2 1 (1969).

7. Payne P.R. Voeding 30 No.4 182 (1969).

8. Bender A.E. In Meeting Protein Needs of Infants and Children. Nat. Acad. Sci. Pubn. No.843 (1961).

9. Snyderman S.E., Holt LE, Dancis J. Roitman E. Boyer A and Balis M.E. J. Nutrition 78 57 (1962).

10. Daniel V.A., Doraiswamy T.R., Swaminathan M and Rajalakshmi D. Brit. J. Nutr. 24 741 (1970).

11. Scrimshaw, NS. Young VR, Schwarts R, Pich ML and Das J.B. J. Nutrition 89 9 (1966).

12. Scrimshaw, NS, Young VR, Huang PC, Thanangkul O and Cholakos BV. J. Nutrition 98 9 (1969).

13. Clarke H.E. Fugate K and Allen P.F. Amer. J. Clin. Nutr. 20 233 (1967).

14. Committee on Protein Malnutrition Nat. Acad. Sci. Publn. No. 1100 (1963).

15. Autret M., Périssé J., Sizaret F. and Cresta M. Nutrition Newsletter 6 No.4 1 (1968).

16. Amino-Acid Content of Foods and biological data on proteins FAO Rome 1970.

17. Morse E.H., Tucker, R.E. Braucher P.F. Dawson V.T. Keyser D.E. Merrow S.B. Clarke R.P. and Brown P.T. Amer. J. Clin. Nutr. 22 851, 1970.