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6. Evaluation and choice of assay procedures

References

7. Research suggestions

Table 13 provides a comparison of various assay procedures for protein quality evaluation. The choice of procedure is to some extent determined by the various criteria desired. Thus, some assays are more suitable for screening because they use a small sample, are relatively inexpensive, have little complexity, and often provide information on the limiting amino acid and on complementary potential. Scoring and digestibility data can require moderately complex analytical techniques, although in some circumstances published data may be used for calculation of mixed dietaries, as discussed in chapter 3. The limitation of these procedures, however, is that they have only moderate capacity for discriminating among proteins of differing quality. Table 13 also presents the characteristics of human and rat assays that can be performed at single or multiple levels The complexity of these techniques, the size of the sample required, and the expense, preclude their use for screening purposes. Furthermore, by themselves (i.e., without performing the assays several times in the presence of added amino acids}, they do not identify limiting amino acids or provide information on their complementary nature. The values obtained are, however, inclusive of digestibility and availability and also provide the ability to recognize the presence of toxins. Their main strength lies in their ability to discriminate among proteins in the determination of quality, especially the procedures involving multiple levels of protein.

TABLE 13. Some Criteria for the Selection of an Appropriate Protein Assay Procedure

Table 14 describes in more detail individual rat assays of protein quality. Five of the tests, as have been described in chapter 4 and summarized in table 8, are performed at only one protein level with or without a non-protein control group, whereas the remaining three are multi-level tests. It will be noted that NPU can be performed in two ways, either by carcass analysis or by balance technique. If digestibility data are required, faecal analysis will be necessary in addition to the carcass analysis in the former procedure for NPU. The eight tests largely fall into the two categories of single-level and multi-level when their ability to rank proteins and to discriminate among proteins is examined. Most single-level tests are poor or moderately good in these capacities, while the multi-level tests may be more easily reproduced in different laboratories. Furthermore, by definition, the single-level assays cannot provide any indication of linearity of response to dose, whereas the multi-level assays are constructed to give this information. The complexity of the testing procedures tends to be greater for multi-level techniques than for the single-level procedures except for NPU (balance), which requires urinary and faecal collection procedures demanding considerable precision.

TABLE 14. Some Criteria for the Selection of an Appropriate Rat Assay Procedure

For proteins extremely low in Iysine, all bioassay procedures have limitations. This is because, for at least the period of time used in these assay procedures, rats have the ability to conserve Iysine, and thus their response is not directly proportional to Iysine level. The RPV procedure, which does not include consideration of the zero-protein level, is better in this respect than the others but is still only moderately accurate. The length of test period required varies from 9 to 21 days, except for PER, which, in its official version, requires 28 days. The cost of these procedures is generally less for single-level assays than for multi-level ones, except that the longer period of food consumption at the 9 to 10 per cent level, and the larger number of animals demanded by some standardized PER procedures, per group, increase costs. It will be noted that all three of the multilevel procedures tend to be the most expensive because of the larger number of diets and animals used.

In summary, PER is the poorest of current animal tests of protein quality, and if a single-level assay is chosen, procedures other than PER are preferable on the grounds that, by using a zero-protein control, they provide a response that may be more proportional to the quality of the protein. NPU by balance can be a reliable and precise procedure, especially if corrected by use of a standard protein. It does, however, require many analyses of urine and faecal samples and demands high-quality analytical facilities. If a growth method is to be used, RNPR would be the recommended procedure because it incorporates both a zero-protein control group and a standard protein for comparison. These both increase its reproducibility and ability to discriminate.

Among multi-level tests of protein quality, RNV may be more sensitive than the other two because its statistical design improves its ability to discriminate among proteins. While RPV is not problem-free for proteins low in Iysine, RNV has more limitations than RPV in this respect because it includes non-protein data. In general, the greater complexity and cost of multi-level assays may not be justified unless analyses are being performed on sources of protein of low quality, especially those

TABLE 15. Some Criteria for the Selection of an Appropriate Human Assay Procedure

* This has been termed RPV in some human studies since the approach is identical. ** At suboptimal intake of test protein. Note: There are insufficient data to judge the other criteria listed in table 14 (rat assays) since the methods used have not been standardized. limited in Iysine, or unless one is concerned with detecting small differences in quality. In these circumstances, RNV and NGI may be less reliable than RPV because of statistical deflection in the slope of the line caused by the inclusion of the non-protein control group.

For many purposes, the final test of quality involves evaluation of the protein sources using human subjects as the consumers. Table 15 compares the merits of four commonly used procedures. All procedures provide a direct measure of digestibility because faecal analyses are required. On a limited intake of the test protein, nitrogen balance by itself is generally unrewarding; in particular, it has a low capacity for ranking proteins in order of qua)ity and poor applicability to proteins that are very low in Iysine. The other three procedures have a good record for both of these criteria, but they are more difficult to perform, take a longer time, and are more costly. It is not currently possible to express a definitive opinion on the relative values of these three procedures because of the limited amount of published work on which to make a judgement.

Finally, it is appropriate to summarize the application of all the preceding tests of protein quality under the five major circumstances into which the reasons for assessment of protein quality can be divided.

In general, the systematic study of protein in any of these circumstances involves initial) chemical testing followed by biological assays. For the purposes of evaluating plant protein made available in the course of breeding new varieties of grains or legumes, the first step is usually chemical analysis for protein content and amino acid profile. Sometimes amino acid analysis at this stage may be for a single critical amino acid, or for a restricted number, such as tryptophan, sulphur amino acids, and Iysine. If these two criteria are promising, then the subsequent assays, including bioassays, are described in detail in several excellent publications (1-3) that are designed for the express purpose of following a defined sequence of analyses in circumstances where the initial sample is often very small. For exploring new sources of protein, the initial approach is to examine protein concentration and amino acid pattern from which an amino acid score can be derived followed by an in vitro test for digestibility. If these criteria suggest a potentially useful new protein source, then a bioassay, both to test for availability of amino acids and as a further test of quality, should be undertaken. If amino acid pattern and score indicate a significant limiting amino acid, then the bioassay should be repeated with the limiting amino acid added to the diet as a confirmation of the prediction. Finally, tests should be performed together with another protein source to examine the complementary potential of the test protein.

These biological tests may reveal a less favourable picture than indicated by amino acid scoring alone. In this case, the product should be examined for non-available amino acids, as for example by use of tests for available Iysine, or should be evaluated for possible toxic materials present in the foodstuff. The biological testing should include a measure of digestibility, because this can be a significant cause of discrepancy between chemical and biological evaluations of quality. This could be done either by use of a technique such as NPU (balance), or by additional determination of faecal nitrogen.

For the third use, namely the monitoring of variables introduced by food processing, it is common to begin with availability of Iysine and of methionine, as these are usually the amino acids most sensitive to processing. If it is desirable to test the product further, the sequence outlined above for testing new sources of protein will serve as a guideline. Many guidelines and statements have been published by PAG that list in detail the testing sequence required for new protein products (see appendices A and B2).

For routine regulatory purposes, the examination of the sample should begin with chemical analysis for nitrogen, amino acids, and toxins, including microbial toxins. The regulatory requirements may also indicate bioassays of protein quality that are commonly. specified in detail by the regulatory agency but that may not necessarily represent the choice of the investigator. For example, the customary use in North America of the PER assay for regulatory purposes has been highly criticized by many investigators, and represents a failure to adopt more rigorous procedures.

Finally, tests for protein quality to assess human protein requirements for such proteins should be conducted. These need to be performed with the greatest care. In addition to a knowledge of the amino acid composition of the protein and relevant animal data, usually originating from multi-level tests, the examination of nitrogen balance in response to a single level or several levels of protein is usually the criterion of protein quality in the assessment of requirements. The merits of different techniques for measuring nitrogen balance are discussed earlier and are shown in tables 13 - 15.

These comments are only intended as guidelines for users not familiar with the detailed literature. It is clear that many techniques are available and that local circumstances and availability of facilities, as well as familiarity with the tests, will determine the final choice. The wide range of costs of different tests, and the limitations of human assays, may make it impossible under some circumstances to deploy the complete battery of appropriate tests outlined above. If the investigator is indeed restricted by such circumstances, it is nevertheless hoped that recognition will be given to fact that perhaps only a partial picture has been obtained.

In conclusion, it must be emphasized that even when a protein is assayed using the best available procedures, ancillary studies, both bioligical and chemical, are necessary to assess fully the potential value of a protein in a real-life situation. Health, nutritional status, age, and physiological status of the individual consuming the protein, together with the complete dietary composition, including other proteins and the total energy value, all combine and interact to affect the final value of the protein to the consumer.

References

1.Nutritional Evaluation of Cereal Mutants, proceedings of an Advisory Group meeting,Vienna, July 1976, ST1/Pub. 444 (International Atomic Energy Agency, Vienna, Austria,1 977).

2.J.H. Hulse, K.O. Rachie, and L.W. Billingsley, eds., Nutritional Standards and Methods of Evaluation for Food Legume Breeders, IDRC-TS7e (International Development Research Centre, Ottawa, Canada, 1977).

3 "Protein Methods for Cereal Breeders as Related to Human Nutritional Requirements," PAG Guideline No. 16,PAG Bulletin, V (2): 22 - 4811975).

7. Research suggestions

The concept of dietary protein quality seems at first sight to be self-evident. However, upon closer examination, a precise definition becomes more difficult. The capacity of a protein source to meet the amino acid and nitrogen requirements of the organism depends not only upon the amino acid composition and digestibility of the protein source or mixture, but also upon the composition and adequacy of the diet as a whole, and on the physiological, nutritional, and health status of the consumer. Among the dietary factors that might be included are level and source of carbohydrate and lipid intake, mineral and vitamin content, water intake, and size and frequency of meal ingestion. Nutritional status, age, and health factors interact in a complex way to modify the utilization of dietary protein. Studies of the effects of these various factors and their interactions are necessary in order to define adequately the variability and nutritional significance of dietary protein quality. However, the following suggestions for research are readily apparent.

Standard Proteins in Biological Assays

The biological assessment of protein quality depends upon an appropriate comparison with a suitable reference protein. In the past, casein has been used as the standard or "reference protein," and standards of protein quality in foods have been related most frequently to the nutritive value of casein. However, there are a number of reasons why casein does not provide the ideal reference standard. First, due to its limiting concentration of sulphur amino acids, it is not as efficiently utilized in meeting the nutritional needs of the rat, in comparison with proteins such as lactalbumin. Thus, it would appear more desirable to utiiize a protein of higher quality for assay purposes. Another difficulty is that even the high-nitrogen ANRC casein may not be as well standardized a product of constant nutritional quality as is commonly assumed. Therefore, it is recommended that research be focused on the development and standardization of a suitable reference protein. A defined mixture of L-amino acids should be explored as a possible reference protein of standard nutritive value. While lactalbumin has been used in slope-assay procedures, its availability and lack of standardization do not make this protein an ideal standard protein at the present time. ANRC casein supplemented with 1 per cent DL-methionine has proved to be of high nutritional value in the rat and is recommended for further evaluation as a possible standard protein for comparative purposes.

Choice of Animal Species in Bioassay

Bioassay procedures utilizing the laboratory rat require amounts of test protein that may be in excess of the amount readily available. Thus, it would be worthwhile to explore the use of other species for protein quality estimations. Another problem is the relevance of findings in the rat, or any other species, to quantitative aspects of protein quality in human subjects. Although some evidence indicates, as discussed earlier, that there is a close and quantitative relationship between studies in growing rats and nitrogen-balance indices in children who have recovered from malnutrition, the data are limited and it is not known whether results in children who have always been healthy would give similar close agreement. Furthermore, there are insufficient data in the literature to provide a critical comparison of protein quality in humans of different ages, although it is assumed that protein quality is of lesser significance in the adult. In order to develop appropriate bioassay procedures in rats that have relevance to human nutrition, these problems must be explored.

Assay Procedures

It might also be questioned whether any one of the common bioassay procedures is adequate or significantly better than other assays for all protein sources irrespective of the limiting amino acid. A particularly difficult problem is the appropriate choice of assay procedure with rats in the case of low-quality protein sources, especial)y those limited in Iysine. Further critical examination of this problem is warranted.

Plasma Amino Acids

The earlier studies relating plasma amino acid levels and protein quality were carried out without full appreciation of all the various factors affecting plasma amino acid levels. With current information it may be possible to develop a more reliable and rapid protein quality assay with growing rats, using changes in plasma amino acid patterns. This approach may also be of potential value for the clinical evaluation of protein quality. Thus, it would be desirable to explore critically those experimental conditions under which the measurement of plasma amino acid levels would provide useful predictive data on protein nutritional quality in human feeding. This may be accomplished initially through examination of the plasma amino acid levels at the termination of standard rat bioassay procedures. An index of protein quality and identification of the limiting amino acid might be obtained through this approach.

Rapid Procedures

With the greater requirement for protein quality data for use in food labelling and other regulatory purposes, there is a tremendous need by both the food industry and the regulatory agencies for improved, rapid but reliable tests of protein quality. Animal bioassays are time-consuming and expensive, and they are not ideal for meeting the needs of the food industry for nutrition labelling of food products. The ideal test would be reproducible, rapid, inexpensive, and applicable to a wide variety of food products. Perhaps such a single test is unattainable. Because protein quality is not dependent on a single factor but rather is the result of an interaction of a complex set of variables and interrelated factors, more than one test may be needed. Such rapid tests should perhaps be concerned with the separate major components of quality - i.e., amino acid composition (score), processing damage, digestibility, and amino acid retention and utilization. Criteria might be devised for each of these categories rather than for a single quality measurement.

Finally, there is still a need to undertake collaborative studies of protein quality assay procedures, ranging from chemical and in vitro studies to rat and human bioassays. These collaborative investigations should involve laboratories both in developed and developing countries. It is hoped that the United Nations University will take the initiative for this research effort.

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