Protein Quality, Amino Acid Balance
Alfred E. Harper, PhD and Norman N. Yoshimura, PhD
Nutrition Vol 9, No. 5 September/October 1993
Protein Quality, Amino Acid Balance, Utilization, and Evaluation of Diets Containing Amino Acids as Therapeutic Agents
Basic concepts of amino acid nutrition are summarized and quantitative amino acid requirements of different age-groups based on current knowledge are presented. The newer concepts of “conditionally indispensable” amino acids and organ-specific requirements for amino acids are discussed. The concepts of nitrogen balance, limiting amino acid, protein utilization, protein efficiency ratio, and amino acid score for determining protein quality are reviewed, and examples of low- intermediate, and high-quality proteins are provided. Problems in assessing efficiency of nitrogen utilization when single amino acids or amino acids in combination with balanced diets are used as therapeutic agents are discussed in relation to the potential roles of the branched-chain amino acids, arginine, and glutamine in trauma and as immunostimulators. Nutrition 1993;9:460-469.
Key Words: amino acids, protein quality, requirements, immunostimulators
An adequate supply of dietary protein is required for survival, growth and development, reproduction and lactation, and maintaining health throughout life. The amino acids released during the digestion of food proteins are essential for the synthesis of tissue proteins, which comprise ~16% of the human body.
Table I. Nutritional Classification of Amino Acids
Leucine Aspartic acid
Phenylalanine Glutamic acid
Some proteins are structural components of cells and tissues; others function as enzymes (biological catalysts), hormones (chemical messengers), antibodies, carriers for the transport of lipids in the blood, and components of systems for transporting small molecules across cell membranes. The structural proteins of muscles make up the largest proportion of total-body proteins. Also, many small nitrogen containing molecules needed for normal body functions are synthesized from amino acids. Some of the individual amino acids are precursors of the purines and pyrimidines needed for the synthesis of nucleic acids, the hereditary units that carry information from one generation to the next. Other amino acids are precursors of small biologically important molecules such as heme, small hormones such as thyroxine and epinephrine, creatine, neurotransmitters, skin pigments, and nitrogenous constituents of phospholipids.
Besides being building blocks for tissue proteins and precursors of many biologically important molecules, some amino acids also have specific regulatory functions. The rates of protein synthesis and degradation in liver, for example, are influenced by the influx of amino acids after a meal, some of which affect those processes more than others. (1,2) Some amino acids serve as stimuli for the release of hormones from endocrine organs and the gastrointestinal tract. (3) Several of these amino acids, particularly leucine, glutamine, and arginine, have been used as therapeutic agents in the treatment of patients in catabolic states (4 - 6) or with hepatic encephalopathy. (7) Because these amino acids are also components of the proteins or amino acid mixtures that serve as sources of nutrients in diets, enteral-nutrition formulations, or solutions for parenteral nutrition, their nitrogen cannot be distinguished from that of the amino acids that are provided only for nutrition purposes. A question that arises is how should amino acids used in large amounts as therapeutic agents be dealt with in assessing nitrogen utilization by such patients?
3. Nutritional Essentiality of Amino Acids
Food and tissue proteins contain 20 amino acids of nutritional importance (Table I). Nine of these — histidine, isoleucine, leucine, lysine, methionine, phenylalanine, theronine, tryptophan, and valine — cannot be synthesized by the body; they are therefore essential or indispensable nutrients that must be obtained from the diet. The other 11 — alanine, arginine, aspartic acid, asparagine, cystine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine — are also ordinarily obtained from the diet, but the body can synthesize them. They are therefore not essential nutrients; they are nutritionally dispensable or nonessential. They are, nevertheless equally as important as the indispensable amino acids for the nutrition of cells and for normal cell and organ function.
Methionine and phenylalanine are required as specific precursors for the synthesis of the dispensable amino acids cysteine and tyrosine, respectively but the other dispensable (nonessential) amino acids can be synthesized from organic acids that are intermediates in the metabolism of carbohydrates and nitrogen from surpluses of other amino acids or even from ammonium sales.
Some amino acids that are ordinarily nutritionally dispensable may not be synthesized in large enough amounts to meet the body’s needs if the metabolic pathways for their synthesis are immature or impaired. This appears to be the case with cystine and tyrosine in premature infants and possibly with taurine (8) there is also evidence that, after severe trauma, glutamine may not be synthesized in adequate amounts (6) Thus, under some conditions, certain dispensable amino acids may become conditionally indispensable. (8) The requirement for protein is thus a dual requirement — for nine amino acids that the body cannot synthesize and some that may not be synthesized in adequate amounts and for nitrogen needed for the various nitrogenous compounds that are synthesized continuously. (9)
The carbon skeleton of amino acids can be oxidized by the body, so surpluses of protein and individual amino acids can also serve as sources of energy. Organs and tissues differ greatly in their ability to use amino acids as energy sources. The liver has the capacity to oxidize most amino acids and, if they are in surplus, will oxidize them in preference to other energy-yielding molecules. (10) Most of the indispensable amino acids are not oxidized in other tissues, but the branched-chain amino acids — leucine, isoleucine, and valine — like many of the dispensable amino acids, can be oxidized by most tissues and organs. (11) Glutamine and glutamic acid are preferential energy sources for the intestine and lymphocytes. (12)
4. Human Requirements for Protein and Amino Acids
Body proteins are in a dynamic state. They undergo continuous breakdown and resynthesis. A large proportion of the amino acids released during the breakdown of tissue proteins are reutilized for the synthesis of new proteins. Only in pathological conditions are proteins and amino acids excreted from the body unaltered in significant amounts. Amino acids that are not reutilized and those that are consumed in excess of the amounts needed for tissue synthesis are degraded completely, and their nitrogen, after being incorporated into urea, is excreted quantitatively in the urine.
5. Nitrogen Balance
The nitrogen in foods is almost entirely in proteins, and proteins contain, on the average, 16% of nitrogen; hence, the amount of food nitrogen consumed multiplied by 6.25 gives a measure of the amount of protein consumed. Measurement of the amount of nitrogen excreted from the body provides an estimate of the amount of body protein that has been completely degraded and cannot e reutilized and of the amount of dietary protein consumed in excess of that needed for tissue protein synthesis. the difference between the amount of nitrogen consumed and the amount lost from the body provides a measure of nitrogen balance.
As protein intake declines, the efficiency with which amino acids are reutilized increases, but small amounts of amino acids are are continuously degraded, and their nitrogen is lost from the body even when no protein is being consumed. These losses are termed obligatory nitrogen losses. If protein intake declines below the amount of these obligatory losses, the amount of nitrogen excreted by the body will exceed the amount consumed, and the body will be in a state of negative nitrogen balance. As protein intake is increased, nitrogen balance will become less negative until, at some point, a steady state will be reached such that nitrogen intake will equal nitrogen loss; i.,e., nitrogen balance will be zero. An estimate of adult requirements for protein or amino acids can therefore be obtained from measurement of the minimum amount of protein or amino acid nitrogen that must be consumed to just balance body nitrogen losses. (9 13)
To determine protein requirements, human subjects are initially fed a diet containing insufficient protein to maintain nitrogen balance and then, in sequence, a series of diets in which the protein content is increased incrementally every few days. Throughout this time, the amount of nitrogen in the food consumed and the amounts excreted in the urine and feces each day are measured. As protein intake is increased, a point is reached at which nitrogen intake just balances nitrogen loss. Some nitrogen is also lost in sweat, in sloughed skin and hair, and by other minor routes. These losses are not ordinarily measured, so the amounts estimated in the few studies in which they have been determined must be added to the urinary and fecal losses to obtain the true value for nitrogen losses. (9 13) Nitrogen intake required to maintain zero balance after this adjustment multiplied by 6.25 is then taken as the estimate of the adult protein requirement.
If protein consumed exceeds the amount required to maintain zero nitrogen balance, the extra amino acids will be degraded, their carbon skeletons will be oxidized for energy, and the nitrogen released will be converted to urea and excreted in the urine. Thus, when nitrogen intake exceeds the amount needed for zero nitrogen balance, nitrogen excretion increases, but nitrogen balance is maintained at zero with a greater inflow and outflow of nitrogen and with a greater proportion of the dietary protein being used as a source of energy.
6. Protein Requirements
The amount of protein needed to meet the requirements for indispensable amino acids differs with the source of protein in the diet. Protein needs are minimal when the dietary protein is of the highest quality, i.e., it is highly digestible and provides indispensable amino acids in the proportions in which they are required for the synthesis of body constituents. Requirements for protein are therefore established from the results of nitrogen balance studies in which human subjects have been fed the highest-quality proteins. The results of such studies have been reviewed regularly (9 13); the most recent comprehensive reassessment of protein requirements was done by an international committee in 1985. (14) This committee concluded that the average adult requirement for high-quality protein was 0.6 g kg -1 body wt day -1.
Requirements of individuals for nutrients differ depending on their genetic makeup. It is not possible to distinguish between individuals having low or high requirements without elaborate metabolic studies, but the range of protein requirements expected in a large population can be estimated from statistical analysis of the individual values obtained in experimental studies. From such information, the coefficient of variation of adult protein requirements is estimated to be 12.5%; a value 25% above the average is considered high enough to cover the needs of most individuals in the population. (14) The “safe intake” of high-quality proteins such as those of milk, eggs, or meat, was set on the basis of these considerations at 0.75 g kg -1 day -1. In the United States the Recommended Dietary Allowance (RDA) for protein for adults, based on similar considerations, was set in 1974 at 0.8 g kg -1 day -1. (15) It has recently been reduced to conform with the international safe intake of 0.75 g kg -1 day -1, (16) but the protein allowances for adults in the RDA table are still based on the value of 0.8 g kg -1 day -1.
Table II. Estimated Safe Intakes or Recommended Dietary Allowances for Protein
Age g/ kg Body Wt /Day
1-3 mo 2.00
6 mo 1.50
1 yr 1.20
6 yr 1.00
Protein requirements are highest during the period of rapid growth after birth. Protein requirements of infants have been estimated from measurements of the amounts of protein consumed by infants growing satisfactorily on breast milk or formulas of comparable quality. Requirements of older children are estimated by a factorial method. The maintenance requirement for protein has been estimated in short-term nitrogen balance studies on children consuming high-quality proteins; then, with knowledge of growth rates and the protein content of the tissue being deposited, the average amount of protein needed for growth can be calculated. From the sum of the maintenance and growth needs, and estimate of the average amount of protein required at various ages is obtained. These values are increased, in the same way as for adults, to allow for individual variability in requirements and permit estimation of safe intakes or RDA. (14) For infants, safe intakes or protein from breast milk or from formulas of comparable quality are estimated to be -2 g kg -1 day -1 shortly after birth and decline to 1.5 g Kg -1 day -1 by 6 mo of age, 1.2 g kg -1 day -1 by 1 yr, 1 g kg -1 day -1 by 6 yr, and gradually thereafter to the adult RDA value of 0.75 g kg -1 day -1 (Table II).
7. Amino Acid Requirements
Adult requirements for indispensable amino acids have been estimated with the nitrogen balance procedure in the same way as for establishing protein requirements, except that the protein in the basic diet is replaced by a mixture of amino acids so the quantity of each amino acid can be adjusted separately. Diets containing adequate quantities of all by one indispensable amino acid and increasing increments of the missing one are then fed in sequence to experimental subjects, and the intake at which zero nitrogen balance is achieved, as in estimating the protein requirement, is taken as the requirement for the indispensable amino acid. This process was used to determine the adult requirements for eight of the indispensable amino acids. (17 18)
The indispensable amino acid requirements of infants and young children have been estimated by observing changes in weight after the amount of one amino acid in an otherwise fully adequate amino acid diet is reduced incrementally. If intake of one amino acid falls below the requirements, weight gain will decline. To ensure that growth will not be impaired by this procedure, the amount of the amino acid that is limiting growth is increased in the diet as soon as the first evidence of reduced weight gain is detected. Indispensable amino acid requirements of infants have also been estimated from amino acid intakes of infants growing satisfactorily, calculated from knowledge of the amounts of breast milk or formula they have consumed and the amino acid composition of these foods. Requirements determined via this procedure, except that for tryptophan, were lower than those obtained with amino acid diets. (9 14)
The most recent estimates of amino acid requirements of infants, children, and adults are shown in Table III. (14) Requirement values for infants reported by a National Research Council committee (9) using essentially the same information ranged from 20% lower to 20% higher than the Food and Agriculture Organization (FAO)/World Health Organization (WHO) values. The values for each age grouping were, nonetheless, in the same range, and differences between the age-groups were similar.
Table III. FAO/WHO/UNO Amino Acid Requirements (mg/kg Body Wt/day)
(4-6 Mo) Children
(2 Yr) Boys
(10-12 Yr) Adults
Histidine 28 ? ? ?
Isoleucine 70 31 30 10
Leucine 161 73 45 14
Lysine 103 64 60 12
Methionine + Cysteine 58 27 27 13
Phenylalanine + Tyrosine 125 69 27 14
Theronine 87 37 35 7
Tryptophan 17 12.5 4 3.5
Valine 93 38 33 10
Total amino acids 742 352 261 84
Proteine 1650 1200 1000 750
E/T 45 29 26 11.2
Amino acid requirements decline much more rapidly with increasing age than protein requirements. Snyderman, (19) in a carefully controlled study of phenylketonuric infants, observed that, during the first 2 yr of life, the phenylalanine requirement fell by 75%, whereas the protein requirement declined by only ~50% over this period. For most amino acids, the difference between the amino acid and protein requirements becomes greater as maturity is approached. Thus, the proportion of protein or total amino acids required as indispensable amino acids is much lower for the adult than for the infant (Table III). In other words, a protein that may not meet the indispensable amino acid requirements of the child when it is consumed in an amount that meets the total nitrogen requirement may provide amounts of amino acids in excess of the requirements for adults consuming enough protein to meet the nitrogen requirement. (9 14)
Requirements of adults for several indispensable amino acids have been reinvestigated with an isotopic procedure. (20 21) Adult human subjects were fed diets containing a mixture of amino acids instead of protein, with one carbon-labeled indispensable amino acid being included at a time in graded amounts in a series of diets. Expired air was collected for several hours after feeding each diet, and the amount of labeled amino acid oxidized was estimated from the amount of isotopic carbon in the expired carbon dioxide. From the values obtained for the amounts of several amino acids oxidized, the investigators concluded that adult requirements for indispensable amino acids had been underestimated by 50-75% by the nitrogen balance procedure. Other investigators questioned this conclusion, largely on the basis of theoretical considerations, but did acknowledge that amino acid requirements of adults are probably underestimated by the nitrogen balance procedure. (15 22)
Largely in response to criticisms of the nitrogen balance procedure, adult protein requirements have been extensively reinvestigated and reassessed, leading to the conclusion that they had earlier been underestimated by ~20%. (14) It should not be surprising, therefore, if adult amino acid requirements have also been underestimated. The question is, by how much? although the results of oxidation studies indicate that, with the exception of the methionine requirement, amino acid requirements of adult humans have been underestimated by a factor of 2-3, (20 21) values for amino acid requirements of young rats (23) and piglets (24) estimated by the oxidation procedure and growth assays have been in good agreement. Also, young adults have been maintained in nitrogen balance and good health for 50 days while consuming cereal grain diets that provided lysine at ~50% above the current requirement. (25) These observations raise several questions. Is the nitrogen balance procedure so much less reliable for estimating adult requirements for amino acids than for protein? If it is, why? why are methionine requirements of adults estimated by the nitrogen balance and oxidation procedures similar but values for most other amino acid requirements so widely divergent? Is the oxidation procedure for estimating amino acid requirements for maintenance subject to sources of error that are not obvious? Unfortunately, both the nitrogen balance and oxidation procedures for estimating amino acid requirements for maintenance are indirect methods, and there is no direct method of validating them in human subjects. Thus, the question of amino acid requirements for adults remains unresolved.
From a practical viewpoint, even if amino acid requirements for adults have been underestimated by 50-75%, the proportion of total amino acids needed as indispensable amino acids would increase only from ~10 to 20 or 30%. Therefore, any protein that meets the indispensable amino acid needs of young children would be more than adequate for adults, provided that the amount consumed was sufficient to meet the nitrogen requirement.
8. The Concept Of Amino Acid Balance
Indispensable amino acids are required in specific proportions, as seen from examination of the amino acid requirement values listed in Table III. Proteins that provide amino acids in the proportions in which they are required have well-balanced amino acid patterns. Provided such proteins are readily digested, their amino acids will be used highly efficiently for the synthesis of tissue proteins. If a protein contains a disproportionately low amount of one or more amino acids, e.e., has a poorly balanced or unbalanced amino acid pattern, it will be used inefficiently for tissue protein synthesis. The greater the deviation in the amino acid pattern of the dietary protein from the pattern of amino acid requirements, the less efficiently it will be used. This occurs because, if one amino acid is provided in less than the amount required, its concentration in tissues will fall, and it will become limiting for protein synthesis. Other amino acids can then be used for tissue protein synthesis only in amounts equivalent to the proportion of the requirement of the limiting amino acid that has been met, e.g., if a diet provides only 50% of the amount of the limiting amino acid required, then only amounts of the other amino acids equivalent to 50% of the amount of the requirements can be used. Quantities in excess of this will be degraded. If a protein with a well-balanced pattern of amino acids is consumed in an amount in excess of that needed to meet amino acid and nitrogen requirements, it will also be used inefficiently.
Proteins of most cereal grains have unbalanced amino acid patterns; they are disproportionately low in lysine. Such proteins are sometimes referred to as incomplete proteins; this is a misnomer. Although they are used inefficiently, they do contain all amino acids. In fact, requirements for indispensable amino acids are readily met with such proteins if the amount consumed is sufficiently in excess of the amount of high-quality protein needed. Nitrogen balance can be maintained in individuals consuming cereal grains as the sole source of protein, but efficiency of utilization of such proteins will be low (9) because, when the requirement for lysine is met, most other amino acids will be consumed in amounts well in excess of the amounts needed, and the excesses will be oxidized for energy. (10)
Although requirements for all indispensable amino acids, including the limiting one, can be met from proteins with unbalanced amino acid patterns if the total amount of protein consumed is sufficiently high, requirements for some amino acids will not be met if the protein content of the diet is low. When such conditions were produced experimentally by including a mixture of amino acids devoid of one indispensable amino acid in a low-protein diet fed to experimental animals, not only was the mixture of amino acids and protein used inefficiently, but food intake and growth rate were depressed. These effects have been attributed to amino acid imbalances. (26) They have been observed especially with young, growing animals fed low-protein diets in which there is a severe disproportion of indispensable amino acids, with one or more of them resent in the diet in an amount well below the requirement. Under these conditions, the concentration of the limiting amino acid in the blood and brain falls sharply in association with the depressed food intake. Lesions in the prepyiform cortex of the brain prevent this response, (27) suggesting that amino acid concentrations in this area of the brain are monitored and that depletion of the brain pool of the limiting amino acid serves as a signal for depression of food intake, thereby limiting intake of the imbalanced diet and preventing more severe distortion of blood or brain amino acid patterns. If the diet provides amounts of all indispensable amino acids that meet or exceed amino acid requirements, or if the requirements are met by tube feeding (force feeding), even young animals will tolerate severely unbalanced dietary amino acid patterns. (26)
9. Evaluation of Protein Quality
Values for the nutritional quality of proteins are relative measures of the efficiency with which proteins are used to meet requirements for amino acids and nitrogen. The value for a protein depends on its amino acid composition and digestibility. If a protein contains a disproportionately low amount of one or more amino acids or is not completely digested, the amount needed to meet protein requirements will be greater than for a protein that has a well-balanced pattern of amino acids and is highly digestible. (28) Examples of high-, intermediate-, and low-quality proteins are listed in Table IV (based on total essential/total proteins x 100 values and utilization (29).
Table IV. Examples of High-, Intermediate-, and Low-Quality Proteins
10. Methods for Evaluating Protein Quality
Animal assays are usually used to estimate protein quality. The amount of weight gained by laboratory rats per unit of protein consumed provides a measure of the protein efficiency ratio. The proportion of dietary nitrogen retained in the body measured directly by carcass analysis or indirectly by the nitrogen balance procedure provides a measure of net protein utilization. These methods take into account both the digestibility of the protein and effects of deficits of indispensable amino acids. They provide information about differences in the quality of different food proteins but, because the assays must be done with diets low in protein, the values obtained are relative; they usually exceed the efficiency or utilization observed for proteins being consumed at the requirement level in human diets. (28)
Efficiency of nitrogen utilization or protein quality can be determined from nitrogen balance measurements made on human subjects consuming their usual diets or diets containing only specified protein sources. Considerable information about the biological value (the percentage of absorbed nitrogen retained) and the net protein utilization (the percentage of ingested nitrogen retained) of various proteins and mixtures of proteins has been obtained from such measurements made on human subjects, usually in experiments designed to investigate protein requirements. (9 30 31) The nitrogen balance procedure is expensive, time consuming, and impractical for routine determinations of protein quality. Estimating the effectiveness of proteins in meeting human amino acid and nitrogen requirements is greatly simplified, however, by use of an amino acid scoring procedure, (13) a modified chemical score method, (32) and an estimate of digestibility.
11. Amino Acid Score
The amino acid scoring procedure provides a method of predicting how efficiently a food protein will be used in meeting human amino acid needs from knowledge of its amino acid composition. The assumptions underlying the procedure are those of the amino acid balance concept: 1) tissue protein synthesis will be limited unless all amino acids are present together in appropriate amounts at sites of tissue protein synthesis, and 2) the proportion of amino acids from the dietary protein that will be used for tissue synthesis will be limited by the amount of the indispensable amino acid present in the dietary protein in least amount in relation to the amount required, i.e., by the proportion of the requirements for the limiting amino acid that is met from the dietary protein.
The initial step in the amino acid scoring procedure is to identify the limiting amino acid. To do this requires a reference amino acid scoring pattern in which amino acid requirements are expressed as milligrams needed per gram of protein, or as percentages needed in a theoretically ideal dietary protein, to meet the requirements of all of the indispensable amino acids when the amount consumed meets the nitrogen requirement. Amino acid scores are then calculated by expressing the amount of each amino acid in the dietary protein as a percentage of the amount in the scoring pattern. Values > 100% are considered to be 100; then, from among those >100, the limiting amino acid is identified as the one having the lowest score. To illustrate, if the lysine content of a whole wheat flour is 2.6% and the value for lysine in the scoring pattern based on the amino acid needs of the young child is 5.1%, the amino acid score for lysine in wheat proteins is 2.6/5.1 x 100 = 51. The scores for all other amino acids are higher, so lysine is the limiting amino acid, and the amino acid score for wheat proteins is 51. The score for whole egg proteins is 100; therefore, to meet the requirement for lysine, a young child would have to consume twice as much protein from whole wheat as from whole egg. (9, 14, 28-32)
A limitation of the amino acid scoring procedure is that it does not take into account protein digestibility. (9 13 28) It can be used directly to compare the nutritional quality of food or dietary proteins that are highly digestible, e.g., most animal products and refined foods that have not been heated excessively. Many foods of plant origin, however, are not completely digested, so a correction must be made for this in assessing the nutritional quality of their proteins. Digestibility of food proteins by humans can be determined from measurements of only nitrogen intake and fecal nitrogen excretion corrected for the amount of nitrogen in the feces when the diet contains no protein. The procedure has been used extensively and remains the standard method for obtaining information about digestibility. (14) Recently, a group of consultants convened by FAO/WHO to reassess current knowledge of evaluation of protein quality concluded that digestibility measurements made with rats as the experimental subjects are as satisfactory as those obtained from measurements on human subjects. (33) If, in the example cited above, the proteins of wheat product are only 90% digestible, protein quality based on the amino acid score must be adjusted for this in estimating the quantity of protein needed to meet requirements.
The question of the appropriate amino acid scoring pattern to use for evaluating the quality of dietary proteins has been the subject of much debate. If the amino acid scoring pattern based on the amino acid requirements of the youngest age-groups is used to evaluate the quality of proteins, nutritional quality of proteins with unbalanced amino acid patterns will be underestimated for adults because the amino acid requirements of adults are so much lower than those of young children. The international committee that recently reassessed protein requirements proposed that, to avoid this problem, separate patterns based on the amino acid requirements shown in Table III should be used. (14) After assessing the amino acid scores of many diets via these scoring patterns for infants, preschool children, and adults, the committee concluded that there was no need to make corrections for differences in protein quality for older children (age > 12yr) or adults consuming mixed diets. Thus, digestibility becomes the major factor in determining how much protein from mixed diets is needed to meet the protein requirements of these groups.
This subject has also been reconsidered by the recent FAO/WHO group of consultants on evaluation of protein quality. (33) The consultation concluded that, in view of questions about the validity of amino acid requirement values for school age children and adults, the amino acid scoring patterns for these groups should no longer be recommended. Instead, it proposed that the FAO/WHO amino acid scoring pattern for children of preschool age be used to evaluate dietary protein quality for all age-groups except infants. The scoring patterns (milligrams of amino acid per gram of protein) recommended for infants and preschool children (Table V) are identical to those proposed previously. (14) The pattern for infants is based on the amino acid composition of human milk, but in the 1985 FAO/WHO/UNO report, it is acknowledged that “infants consuming cow’s milk proteins at the same level as breast milk show satisfactory growth and nitrogen balance.” (14) Use of a single amino acid scoring pattern for all ages except infants is logical; however, the pattern proposed, although it has been accepted as satisfactory by two international expert groups, (14 35) is based on a single set of requirement values from a conference report (30) that was not subjected to peer review. The appropriateness of this scoring pattern will undoubtedly be examined further. For the populations of industrialized nations generally, whose average protein intake exceeds the requirement by ~>50%, there is little likelihood of healthy adults or older children not meeting their amino acid and protein nitrogen needs, even from diets with unbalanced amino acid patterns, unless food intake is low.
Table V. Suggested Amino Acid Scoring Patterns
Infant Preschool Child
Histidine 26 (19)
Isoleucine 46 28
Leucine 93 66
Lycine 66 58
Methionine + Cystine 42 25
Phenylalanine + Tyrosine 72 63
Theronine 43 34
Tryptophan 17 11
Valine 55 35
Total 460 339
From FAO/WHO/UNO (14) and Food and Agriculture Organization. (33) Histidine value in parenthesis obtained from interpolation from smooth curve of requirement vs. ag. See Ref. 33
12. Protein Intake, Protein Quality, and Utilizable Protein
The various methods for determining protein quality are, in essence, methods for determining the proportion of the protein in a food or diet that is “utilizable” or, more specifically, the proportion of the indispensable amino acids in the protein that can be used for tissue protein synthesis. (34) The remainder, in contrast is the proportion likely to be used only as a source of energy. The amino acid score, with an adjustment for digestibility, provides a direct measure of the proportion of utilizable protein. Although measures of protein quality or utilizable protein, e.g., amino acid scores, are nutritional characteristics of food proteins, they are not absolute characteristics; protein quality varies with the amount of protein consumed.
The concept of protein quality applies only under conditions in which the amount of protein consumed is equal to or less than the amount needed to meet the requirement for the limiting amino acid. When protein intake exceeds this amount, efficiency of protein utilization, or protein quality, will decline regardless of the balance of the amino acid pattern. This will occur even with the highest-quality proteins because, after the requirement for the limiting amino acid has been exceeded, all indispensable amino acids will be present in tissues in excess of the amounts needed to saturate protein-synthesizing system, and because amino acids cannot be stored, the extra amounts of all of them will be degraded and used only a sources of energy. (28)
This relationship between protein intake and protein quality has implications for interpretation of measurements not only of the nutritional quality of individual proteins or mixtures of protein but also of diets containing proteins with unbalanced patterns of amino acids to improve the quality of the protein and reduce the quantity needed to meet amino acid requirements. Effects of amino acid supplements other than the limiting one have been examined in numerous studies of young, growing animals fed low-protein diets. The quality of the total dietary protein is lowered by such additions because, without an increase in the amount of the limiting amino acid, the other ammo acids added can be used only as a source of energy. In some of these studies, amino acid imbalances occurred, food intake and growth of the animals were depressed by the additions, and these responses were associated with a sharp decline in the concentration of the limiting amino acid in blood and tissue free pools. (26)
These effects were overcome by increasing the amount of protein in the diet or providing a supplement of the limiting amino acid, both of which improve the quality of the total dietary protein. They were also overcome, without any improvement in the amino acid balance or quality of the dietary protein, if the animals were exposed to a cold environment, which greatly increased their energy expenditure. (35) Under these conditions, they consumed much more of the low-protein diet with the unbalanced amino acid pattern and therefore obtained a much larger amount of usable protein and were able to meet their requirement for the limiting amino acid. Adult animals, which have much lower requirements for indispensable amino acids, and younger animals receiving enough utilizable protein to meet their requirements for all of the indispensable amino acids can consume diets in which the amino acid pattern is sufficiently unbalanced to result in low overall efficiency of protein utilization without evidence of ill effects.
These observations illustrate that, although the quality of the protein in a diet is critical when protein intake is low, especially for young, growing subjects, the total amount of utilizable protein is critical for meeting indispensable amino acid requirements and ensuring nutritional adequacy. The proportion of well-balanced protein needed by human adults to meet the indispensable amino acid requirements is assumed to be ~>15% of the total protein requirement. This is the basis for the conclusion of the FAO/WHO/UNO committee that only digestibility, not protein quality, need be considered in estimating protein needs of adults. (14) This does not apply to young children, whose indispensable amino acid requirements are several times those of adults.(9 13)
13. Evaluation Of Diets Containing Amino Acids as Therapeutic or Pharmacological Agents
Besides fulfilling specific nutritional or physiological roles, e.g., serving as components of body structures or metabolic systems, some nutrients may also have therapeutic or pharmacological actions. The amounts required for pharmacological effects are usually much greater than those required for nutritional function, and such effects are generally observed in individuals with some degree of metabolic or physiological impairment. This can be illustrated with tryptophan as an example. To maintain protein synthesis, the synthesis of molecules derived from tryptophan, and for regulation of the release of gastrointestinal hormones, the human adult requires ~250 mg tryptophan/day (14); however, in adults who have mild insomnia, tryptophan administered in a dose of > 1 g/day (~> 4 times the daily requirement) will induce sleep. (36) This effect is assumed to result from the increase in the concentration of the neurotransmitter serotonin (5-hydroxytrptamine) in the brain, which has been observed in animals administered a load of tryptophan. Use of tryptophan as a pharmacological agent has been associated with toxic effects, but these appear to be attributable to contaminants in some preparations. (37)
When amino acids are used in this way as therapeutic agents, a question arises as to whether the nutritional and therapeutic functions of an amino acid should be assessed independently or whether the consequences of administering a large amount of an amino acid as a therapeutic agent should be taken into consideration in assessing nitrogen utilization and the nutritional quality of the protein component of the diet as is done with amino acid supplements. When tryptophan is administered as a pharmacological agent separately from the diet at a level of only 1 g/day, the nutritional and pharmacological effects can readily be considered independently. When other amino acids are utilized in much higher doses as therapeutic agents, the situation is more complex. The quantities used may range from 10 to 30 g of each of several amino acids per day; also, they are usually administered as part of the total diet in enteral or parental products rather than separately from the diet.
A comprehensive review of the use of amino acids and their derivatives as therapeutic agents is beyond the scope of this article. A survey of the literature for the past 10 yr, however, reveals scattered reports of amino acids being tested as therapeutic agents, but such effects have been investigated in detail in controlled studies for only a few amino acids. A few examples from among those illustrate the nature of the problems encountered in evaluating protein or nitrogen utilization by subjects consuming diets in which large quantities of amino acids have been included for specific therapeutic purposes.
Parenteral solutions enriched with branched-chain amino acids leucine, isoleucine, and valine, have been used to improve nitrogen retention in septic and uncomplicated postoperative patients. (4 38) Formulations enriched with branched-chain amino acids along with lowered amounts of the aromatic amino acids have been used to improve plasma amino acid patterns in hepatic encephalopathy patients. (7) Leucine is the effective component in improving nitrogen retention in patients in catabolic states, but to maintain appropriate balance among the three branched-chain amino acids, they are included together in most parenteral and enteral preparations. The branched-chain amino acids may comprise ~<50% of the total amino acids in such preparations.
Arginine stimulates the release of several hormones including growth hormone and insulin, in human subjects. Arginine has been shown to reduce nitrogen loss in surgical patients with moderate trauma and improve lymphocyte function in healthy human volunteers. (5) It has been proposed that arginine is a conditionally indispensable amino acid in individuals who have been subjected to trauma. The quantities of arginine with which these therapeutic effects have been demonstrated are in the pharmacological range and may be as high as 30 g/day.
Glutamine is also considered by some investigators to be conditionally indispensable in critically ill patients. (6) Its concentration declines sharply in muscle of human subjects or animals in a catabolic state. (39) It is a preferential energy source for the rapidly proliferating cells of intestinal mucosa and is used extensively for energy by lymphocytes when they are stimulated to proliferate. In view of its therapeutic potential in the treatment of human subjects in a stressed state, it has been tested for safety in healthy subjects in doses in the range considered appropriate for therapeutic effects, (~> 40g/day i.v. for 5 days). No untoward physiological effects were observed with these doses.(40)
The quantities of these amino acids that may be administered individually as therapeutic agents can greatly exceed the amounts ingested daily by healthy individuals consuming their usual diets. in fact, administration of several of these amino acids together for therapeutic purposes would result in ingestion of a total quantity of amino acids as therapeutic agents in the range of 50-100 g/day, equal to the usual daily intake of protein. Obviously, it would be inappropriate to substitute a therapeutic mixture of these amino acids for part of the dietary protein because this would reduce the quantities of indispensable amino acids in the diet, lower the proportion of balanced (utilizable) protein, and result in modification of the diet so it resembled those used experimentally in investigations of amino acid imbalances. Assuming that the amount of balanced protein in the diet is not reduced when amino acids functioning as therapeutic agents are included, the diet would provide indispensable amino acids in amounts that would meet the requirements, but as much as half of the total nitrogen intake would be from an incomplete mixture of amino acids that could not contribute to synthesis of tissue proteins. A question that must be considered, then, is what is the appropriate way to evaluate nitrogen utilization by an individual consuming a high proportion of total dietary nitrogen from an incomplete mixture of amino acids, which has been included only to fulfill a pharmacological or therapeutic function? if the therapeutic agent were unrelated to the dietary protein, this problem would not arise.
If the incomplete amino acid mixture is considered to be strictly therapeutic, not nutritional, it would seem appropriate to evaluate protein nutriture only in relation to the amount of well-balanced or utilizable protein in the diet and ignore the nitrogenous contribution of the therapeutic components. On the other hand, if nitrogen utilization by the patient is to be evaluated via measurements of nitrogen retention or nitrogen balance, nitrogen contributed by the therapeutic agents cannot be distinguished from that contributed for nutritional purposes by the dietary protein. With equal quantities of high quality protein and an incomplete amino acid mixture, the value for the quality of the dietary nitrogen source would be low, and its efficiency of utilization would probably be ~<50%. If the therapeutic component were disregarded, efficiency of protein utilization based only on utilization of the high-quality protein component of the diet would be high and might approach 80%, close to the maximum observed for high-quality proteins consumed at the requirement level.
The situation can be viewed in two ways. it might be considered analogous to a situation in which subjects are receiving a diet in which the protein component consists of a mixture of the highest-quality proteins consumed at a level that will ensure achievement of zero nitrogen balance. Viewed in this way, the therapeutic component would not be taken into account because it cannot contribute to nitrogen retention; the amount of nitrogen it provided would be subtracted from both the amounts of nitrogen consumed and excreted. Nitrogen utilization would then be estimated solely on the basis of the quantity of high-quality protein consumed. A second approach might be to view the situation as analogous to one in which subjects are receiving a diet containing an unbalanced protein that can be used only inefficiently for protein synthesis and achievement of nitrogen balance. The value of the quality of the mixture of nitrogenous constituents making up the dietary "protein" would thus be only ~50%, but the amount of nitrogen provided would be about double that needed if the protein were of high quality.
Viewed either way, the amount of utilizable protein consumed would be the same. needs for indispensable amino acids should be met equally well, as should needs for total nitrogen. The state of protein nutriture should also be the same; the extra amino acid would serve as a source of energy to substitute for carbohydrate that might have been displaced form the diet. Whichever approach is seed in assessing protein nutriture of subjects or patients consuming such diets or products, it is important that comparisons be made on the basis of utilizable protein and not total nitrogen or total protein.
Problems should arise only if extra amino acids are provided in amounts exceeding their tolerance in individuals. High intake of the amino acids used in various therapeutic mixtures have been tested individually; they should be tested together. The very high nitrogen intake associated with such therapeutic regimens may result in falsely high nitrogen retention or excretion in short-term studies. It may require consumption of the unbalanced mixture for a considerable period until a new steady state is established with the higher nitrogen intake (41) before full nutritional and pharmacological consequences can be established.
These observations on problems in evaluating the efficiency of utilization of diets that serve as vehicles for providing amino acids as therapeutic agents represent only one part of a much larger canvas. Several nutrients, their precursors or derivatives, are being considered as therapeutic, prophylactic, and pharmacological agents. Use of large doses of nutrients in these ways has implications not only for evaluating the efficiency of nutrient utilization but also for classification of nutrients, recommended intakes of nutrients, and assessment of nutrition status. As such uses increase, to avoid confusion in the field of nutrition, it will be important to establish criteria for distinguishing clearly among physiological, pharmacological, and medicinal effects of nutrients and to investigate interactions between physiological and therapeutic effects of nutrients.
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