ESSENTIAL AMINO ACID REQUIREMENTS: A REVIEW

	FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS	ESN:FAO/WHO/UNU/ EPR/81/30 August 1981
	WORLD HEALTH ORGANIZATION
	THE UNITED NATIONS UNIVERSITY

Item 3.2.3 of the Provisional Agenda

Joint FAO/WHO/UNU Expert Consultation on
Energy and Protein Requirements

Rome, 5 to 17 October 1981

ESSENTIAL AMINO ACID REQUIREMENTS: A REVIEW

Marian E. Swendseid
University of California
Los Angeles

Most of the studies of essential amino acid requirements with semi-synthetic diets were conducted during the 1950s and 1960s using nitrogen balance or nitrogen retention as the criterion of adequacy. Dietary levels of a single amino acid were varied in successive periods of 5 to 10 days duration. These studies have been reviewed in detail by Irwin and Hegsted (1). In addition to studies with varying quantities of single amino acids, investigations with different amounts of complete essential amino acid mixtures or single food proteins of known amino acid composition and digestibility have also provided information and verification of essential amino acid requirements. The results of all of these investigations have been considered and evaluated by both the FAO/WHO Expert Committee on Energy and Protein Requirements (2) and the Committee on Amino Acids of the Food and Nutrition Board (3). In this paper new information published since the deliberations of these committees will be reviewed. Attention will be given to investigations of biochemical indices that might prove useful in assessment of amino acid requirements and factors which may affect amino acid requirements will be considered.

Amino Acid Requirements of Infants and Children

The experimental data on amino acid requirements for infants and 10–12-year-old children have been expanded by Pineda and co-workers (4) to include requirements for the 2-year-old child. Forty-two healthy children received diets consisting of a core of 0.3 g of cow's milk protein/kg/day plus an amino acid mixture in proportions and amounts equal to 0.9 g milk protein/kg/day. Diets provided 100 kcal/kg/day with proper vitamin and mineral supplements. The single essential amino acid under study was partially replaced in the diet by glycine at five different levels. Nitrogen balance (4 day periods) was calculated with an allowance of 8 mg N/kg/day for integumental losses. It was assumed that a retention of 16 mg N/kg/day allowed for normal growth and results were validated with studies conducted in children fed milk or soy protein to assess protein needs. The following requirements were suggested as mg/kg/day: isoleucine, 31; lysine, 64; sulfur amino acids, 27; threonine, 37; and tryptophan, 14. The investigators concluded that for children in this age group the FAO/WHO 1973 estimates may be too high for isoleucine, sulfur amino acids, threonine and valine. Fasting plasma amino acid levels were also measured and it was concluded that reductions in specific amino acids levels occur in plasma as amino acid intakes are decreased to sub-optimal levels. Thus, in young children as in infants, fasting plasma amino acid levels appear useful in estimating amino acid requirements.

Graham, MacLean and co-workers have data in both infants and young children fed soy protein (5) or wheat protein (6), that the amino acid known to be limiting in these proteins is decreased in plasma 3 hrs post-prandial in contrast to other amino acids, which are either increased or stable. Supplementation with methionine for soy and lysine for wheat corrected this condition. Thus the posprandial plasma amino acid response seems to be helpful in quantitating amino acid needs in growing children. Fomon (7) has also shown that in infants fed a soy bean diet when a supplement of methionine was added, weight gain improved and the serum urea nitrogen level decreased. Hence for infants and young children both amino acid and urea concentrations in plasma might be employed to gain information on amino acid requirements.

Amino Acid Requirements of Adults

Evidence is accumulating that histidine is an essential amino acid for adults. It was found that when uremic patients receiving mixtures of the Rose eight essential amino acids were given a histidine supplement, hemoglobin increased (8) and nitrogen retention improved (9). Later Kopple et al. (10) studied 3 normal (ages 48, 38 and 24 yrs) and 3 uremic men (ages 55, 54, 43 yrs) who ingested a histidine-deficient amino acid diet of approximately 6.5 g N/day. These subjects except for one individual went into negative nitrogen balance after periods of 20 to 30 days. Strong positive nitrogen balance was immediately restored on the addition of 1200 mg of dietary histidine in all but the one subject mentioned previously who was in negative balance after 5 days of receiving the histidine-deficient diet. In 24 hrs plasma histidine levels fell to 52% of control values with the 40 g protein diet. By the end of the histidine-depletion period, plasma histidine levels were 17% of control values. Muscle free histidine concentrations also decreased. Serum albumin and hematocrits fell and serum iron rose. Subjects felt unwell and in five subjects a erythematous skin lesion appeared. All clinical symptoms and skin lesions disappeared with the histidine supplement. Anderson et al. (11) have also investigated six college-age men consuming for 1 week 6.3 g N amino acid diets with or without histidine or histidine plus arginine. Mean nitrogen balances (last 4 days of period) were positive only when histidine was present in the diet. Cho et al. (12) observed reduced plasma histidine levels when the young men referred to above ingested low-histidine diets. In further studies of the amounts of histidine that may be required to maintain nitrogen balance over prolonged time periods, 9 normal and 4 uremic men were fed amino acid diets containing 6.5 g N and 4, 8, or 12 mg/kg of histidine for 27 ± 5 days per period (13). The average age of subjects was 44 years. Fasting plasma histidine levels rose with increasing histidine intake and 1 hr postprandial levels were higher only in subjects ingesting 12 mg histidine/kg. Likewise the uptake of radio-iron into red cells was lower than the normal range for all subjects ingesting 4 mg histidine/kg, lower in 3 of 6 subjects receiving 8 mg/kg and within the range for 5 subjects fed 12 mg/kg. From these data, it appears that the dietary histidine requirement in both normal and uremic men is more than 8 mg/kg/day and may be as high as 12 mg/kg/day. For a 70 kg man the range is from 560 to 840 mg of histidine per day.

These observations indicate that histidine is unique among essential amino acids in that short-term nitrogen equilibrium is maintained with extremely low histidine-containing diets. There are several hypotheses to explain this phenomenon: 1) histidine comprises eight percent of the hemoglobin molecule and the breakdown of hemoglobin contributes more histidine in proportion to other essential amino acids; 2) the hydrolysis of the dipeptide carnosine from muscle releases histidine. There is good evidence that in rats and chicks the muscle carnosine content is decreased with histidine-free diet and replenished with histidine supplementation (14, 15) and 3) there is some biosynthesis of histidine (16). A combination of these hypotheses may provide the explanation for the unique response of body tissues to the dietary removal of histidine. No reports of histidine requirements of women have been found. However, it is possible that in women fed histidine-deficient diets negative nitrogen balance might develop more rapidly than in men because of a decreased muscle mass and thus perhaps more limited carnosine stores.

Young and co-workers have presented additional data on valine, lysine (17), tryptophan (18, 19) and threonine requirements (20) using nitrogen balance and the plasma amino acid response curve. In human subjects, for some amino acids at least, as dietary intake is increased, plasma levels remain stable for a time, then rise abruptly to be followed by another plateau. Young has attempted to quantitate the amino acid intake at the point where plasma levels begin to increase, the “breakpoint” and compare this intake level with the amount needed to maintain nitrogen equilibrium. These investigators have also used postprandial amino acid levels as indicators of dietary requirements. They have found that in general the lower “breakpoint” of the plasma amino acid response curve corresponds to the minimum levels of amino acid intake that is necessary to maintain nitrogen balance. It is possible therefore in the case of essential amino acids with plasma levels responsive to dietary intake that criteria based on plasma amino acid concentrations can be developed to assess requirements.

The investigations of Young and co-workers have included elderly subjects and have provided new data on specific essential amino acid requirements for subjects in this age group. When subjects were ingesting 0.5 g protein/kg body wt, the mean minimum requirement of elderly people for tryptophan was 2 mg/kg body weight/day and for threonine it was 8 mg/kg. These values are similar to those estimated for young subjects.

Plasma response curves have also been used in an attempt to evaluate histidine requirements (21). Five normal and two uremic men received amino acid diets in which histidine intakes were varied from 60 to 2800 mg/ day at 8-day intervals. Postabsorption plasma and urinary histidine levels were correlated with histidine intake but the plasma response curve did not demonstrate a consistent breakpoint which could be used to indicate histidine requirement. When 6 normal subjects were fed 2 mg/kg/day or less, nitrogen balance (not corrected, for integumental losses) was -0.18 ± 0.5 mg/kg/day, suggesting that histidine requirements are greater than this amount.

Summary of Current Evidence on Amino Acid Requirements:

The conclusions of the reviewers of amino acid requirement studies in 1971 (1) appear to be quite relevant even today. Data are still lacking in children of some age groups and in women during pregnancy and lactation. The number of adult subjects who have been studied in terms of needs for specific essential amino acids is small and there is an apparent marked variability among individuals. Also, differences are great between study designs. The NRC/FN Committee on Amino Acids (22) has indicated that with present criteria of estimation, it seems unlikely that there is a difference in essential amino acid requirements expressed per kilogram of body weight between men and women. This conclusion would appear to be still valid.

It would appear that although there are some conflicting data, the preponderance of studies suggests that essential amino acid requirements of the elderly do not differ from younger age groups, at least when the total nitrogen intake is low.

Factors to be Considered in Assessing Essential Amino Acid Requirements:

It is becoming increasingly apparent that a knowledge of essential amino acid requirements is of great practical value for evaluating food supplies and instituting supplementation when it is needed. It would appear that a considerable expansion of the amino acid requirement studies is a necessary prerequisite for obtaining a valid data base and factors to be considered in designing future investigations should be carefully evaluated.

Energy intake: Recent studies of protein requirements have clarified the quantitative relationships between total nitrogen and energy intake (23, 24). Calloway (24) has found that 68 ± 15 mg/kg of egg N maintained nitrogen balance and weight in young men with a dietary intake of 43 ± 4.4 kcal/kg but with an intake of 39.6 ± 4.4 kcal, 89 ± 18 mg N per kg was required. A slight adjustment in energy intake with marginal N intake can substantially perturb N balance. It has been often stated that amino acid requirements are based on dietary energy contents that in many instances were higher than the amount required to maintain weight and were above the usual intake. In the ideal study of amino acid needs, weight and body composition should be maintained.

Length of study period: Amino acid requirements were formulated on the basis of short-term nitrogen balance studies. However protein allowances sufficient to promote short-term nitrogen balance have now been found to be inadequate to maintain long-term nitrogen balance and body potassium content (25). Although there is evidence that the limiting nitrogen component in these studies is non-protein nitrogen (26) these results bring into focus the possibility that the minimum or “safe” amount of one or more of the essential amino acids also may not be sufficient for prolonged periods. It is now known that dietary histidine inadequacy affects nitrogen balance only after a period of 15–20 days or longer (vide infra)(10).

Total nitrogen intake: There is now good evidence, including the study referred to above (26) that the limiting nitrogen component in high quality proteins is not the essential but rather the nonessential amino acids (27, 28). When nonessential nitrogen is added to high quality protein, nitrogen balance is promoted. There are also other circumstances related to nonessential nitrogen additions where nitrogen balance could be enhanced. When energy intake is limited, additional nonessential nitrogen through provision of extra calories could increase nitrogen retention. It is also possible that extra nonessential nitrogen may increase requirements for essential amino acids. In older individuals there is limited evidence that the requirement for an essential amino acid mixture increases as the total nitrogen intake is increased from 7 g to 10 g/day and the requirement lessened as total nitrogen intake is reduced from 7 g to 3.5 g/day (29). It is also possible that the oxidation rate of certain amino acids may be affected by protein intake. Limited evidence with injection of labeled tyrosine indicates less expired ¹⁴CO₂ and therefore decreased oxidation in uremic men fed 20 as compared to 40 or 60 g protein diets (30). Enhanced oxidation of amino acids could also be a factor in increasing amino acid requirements with higher protein intakes.

Labile body protein: There is firm information that the protein content of some organs such as liver, pancreas and gut respond rapidly to changes in protein intake (reviewed by Munro, 31). The amount of nitrogen lost in the urine during the first few days of ingesting a protein-free diet is considered to represent the labile protein reserve which accumulates when dietary protein is high and recedes when protein intake is lowered. Munro estimates the capacity of the human body to accumulate labile protein as 300–400 g. Maintaining this labile protein must represent a metabolic cost of extra protein and extra energy. When subjects are given protein-free diets prior to test treatments with amino acid mixtures or when subjects are in negative nitrogen balance for prolonged periods of time, the labile protein of the body is depleted and as a consequence it can be presumed that the amino acid requirement would be decreased. In the study by Young on tryptophan requirements (19), when the dietary tryptophan intake was 4 mg/kg/day, 6 subjects who had received protein-free diets for 2 days were in positive nitrogen balance and 8 subjects who had not been given the protein-free diet were in negative nitrogen balance. In future studies of amino acid requirements more attention should be given to the state of body labile protein stores. In addition to the amount of nonessential nitrogen supplied to maintain these stores it is possible the kind of non-essential nitrogen has some influence (32). The limited evidence that older individuals might have increased essential amino acid requirements (30, 33) was obtained only in studies with subjects receiving diets of 7 or 15 g of nitrogen. Since each diet trial period was interspersed with an isonitrogenous period of ordinary food, labile body protein stores would appear to have been well-maintained.

Methods of assessment: The limitations of the nitrogen balance method have been reviewed many times and the recent results of long-term studies have made it clear that this method cannot be the sole criterion for estimating amino acid requirements in adult subjects. Thus other methods must be introduced and validated to accurately assess essential amino acid needs. There is quite good evidence that in infants and children plasma amino acid levels are responsive to dietary intake and can indicate amino acid deficiencies. Graham and co-workers (5, 6) are actively exploring the usefulness of both fasting and postprandial amino acid levels in relation to estimating limiting amino acid needs when various vegetable proteins are fed. In adult subjects, Young and co-workers are investigating the plasma acid response curve as described above (17, 18, 19, 20) and also postprandial amino acid levels as indicators of amino acid needs. These would appear to be methods of promise for evaluating amino acid requirements.

In the development of new approaches for evaluating essential amino acid requirements, perhaps there can be methods appropriate for only a single amino acid which can be based on a specific metabolic need for this amino acid.

The usefulness of alterations in some of the blood proteins with short half lives such as transferrin and retinol binding protein should be explored. The sophisticated studies of Young and Bier and co-workers using infusions of stable isotope-labeled amino acids to measure turnover may provide the ultimate approach to assessing amino acid requirements with different dietary conditions (34, 35).

Requirement for histidine: It has been established that short-term nitrogen balance studies are not useful in assessing daily histidine needs (10, 11). The daily requirement estimates of 8 to 12 mg/kg are based on results obtained from labeled iron uptake into red cells and the data have not been published in detail. The true histidine requirement would appear to consist of maintaining not only body protein but also carnosine stores. The response curve of plasma histidine to dietary histidine did not demonstrate a consistent breakpoint that could be useful in indicating the histidine requirement (21). However both postabsorptive plasma histidine and urinary histidine levels are correlated with dietary histidine intake (21) and it would appear that these parameters might ultimately prove useful in evaluating histidine requirements.

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