Further Reading

Fereday A, Gibson NR, Cox M et al. (1998) Variation in amino acid mediated, insulin activated inhibition of proteolysis determines the efficiency of protein utilization. Clinical Science 95: 725-733. Gibson NR, Fereday A, Cox M et al. (1996) Influences of dietary energy and protein on leucine kinetics during feeding in healthy adults. American Journal of Physiology 33: 282-291.

Haussinger D and Lang F (1992) Cell volume and hormone action. Trends in Pharmacological Science 13: 371-373.

Jagoe RT and Goldberg AL (2001) What do we really know about the ubiquitin-proteasome pathway in muscle atrophy? Current Opinion in Clinical Nutrition and Metabolic Care 4: 183-190.

Millward DJ (1995) Insulin and the regulation of amino acid catabolism and protein turnover. In Cynober L (ed.) Amino Acid Metabolism in Health and Disease, pp. 127-136. Boca Raton, FL: CRC Press.

Millward DJ (1995) A protein-stat mechanism for regulation of growth and maintenance of the lean-body mass. Nutrition Research Reviews 8: 93-120.

Millward DJ, Fereday A, Gibson NR et al. (1996) Postprandial protein metabolism. Baillier's Clinical Endocrinology and Metabolism 10: 533-549.

Millward DJ, Fereday A, Gibson NR et al. (2000) Human adult protein and amino acid requirements: [13C-1] leucine balance evaluation of the efficiency of utilization and apparent requirements for wheat protein and lysine compared with milk protein in healthy adults. American Journal of Clinical Nutrition 72: 112-121.

Millward DJ and Rivers JPW (1989) The need for indispensable amino acids: The concept of the anabolic drive. Diabetes Metabolism Reviews 5: 191-212.

Miotto G, Venerando R, Marin O et al. (1994) Inhibition of macroautophagy and proteolysis in the isolated rat hepatocyte by a non-transportable derivative of the multiple antigen peptide Leu8-Lys4-Lys2-Lys-betaAla. Journal of Biological Chemistry 269: 25348-25353.

Pacy PJ, Price GM, Halliday D et al. (1994) Nitrogen homeostasis in man: 2. The diurnal responses of protein synthesis, degradation and amino acid oxidation to diets with increasing protein intakes. Clinical Science 86: 103-118.

Waterlow JC, Garlick PJ, and Millward DJ (1978) Protein Turnover in Mammalian Tissues and in the Whole Body Amsterdam: Elsevier/North-Holland Biomedical Press.

Requirements and Role in Diet

D J Millward, University of Surrey, Guildford, UK

© 2005 Elsevier Ltd. All rights reserved.

Defining minimum amino acid and protein requirements is inherently difficult. Humans are exposed to a wide range of protein intakes, which enable full expression of their genotypical lean body mass throughout the range, and identifying the lower limits of this range has proved intractable. Without unequivocal symptoms of deficiency, the adequacy of an intake can only be assessed in terms of nitrogen or amino acid balance, which is unsatisfactory for several reasons. In particular, adaptation causes major difficulties in designing balance studies and interpreting results. Furthermore, balance methods are inherently imprecise and logistically extremely difficult. It is therefore not surprising that there is much debate about both the nature and the extent of protein requirements.

Terminology

Protein requirements are best discussed in terms of metabolic demand, dietary requirement, and dietary allowances. Metabolic demand concerns amino acids and is determined by the nature and extent of those metabolic pathways (e.g., net protein synthesis) that consume amino acids and that vary with the phenotype and the developmental and physiological state of the individual. The dietary requirement is the amount of protein and/or its constituent amino acids that must be supplied in the diet in order to satisfy the metabolic demand. The requirement will usually be greater than the metabolic demand. Thus, factors associated with digestion and absorption may limit digestibility (i.e., dietary nitrogen lost in the feces) and biological value (i.e., the availability of the absorbed amino acid pattern in relation to cellular needs, which influences urinary nitrogen excretion). Dietary allowances are a range of intakes derived from estimates of individual requirements taking into account variability between individuals. They are designed to meet the dietary requirements of the population. In the United Kingdom, these allowances are described in terms of Dietary Reference Values (DRVs) and in the United States as Dietary Reference Intakes (DRIs).

Metabolic Demands for Amino Acids

Current evidence supports the representation of the metabolic demands as in Figure 1. The metabolic demand for amino acids is to maintain tissue protein at appropriate levels and to provide for all amino acid-derived metabolites and any additional needs during growth, rehabilitation, pregnancy, and lactation. Tissue proteins are diverse, including structural or fibrous insoluble types and soluble globular

Protein intake

Protein intake

Fecal

Urinary

Sweat

Urinary

Protein losses:

N

urea N

N

non-urea N

skin, hair etc.

Figure 1 Schematic representation of the metabolic demands for amino acids.

Figure 1 Schematic representation of the metabolic demands for amino acids.

species, with characteristic properties and functions that are determined by their amino acid sequence. All proteins are in a dynamic state of constant turnover (i.e., breakdown to constituent amino acids and resynthesis), although for the structural proteins this is slow or minimal. Nonprotein products include nucleic acids and a diverse range of smaller molecules, such as creatine, taurine, glutathione, hormones (e.g., catecholamines and thyroxine), neurotransmitters (serotonin and dopamine), and nitric oxide, a key regulator of blood flow and other physiological processes.

The metabolic demand is supplied from the free amino acid pool, the size of which, for most amino acids, is regulated within narrow limits. Regulation involves supply from three sources: dietary proteins after digestion and absorption from the upper gastrointestinal tract (GIT), tissue protein after proteo-lysis during protein turnover, and de novo formation, which may include amino acids and ammonia, deriving from urea salvage after hydrolysis and bacterial metabolism in the large bowel. Removal of free amino acids occurs by reactions in which they act as substrates, and these reactions are shown as three pathways, one of which is the metabolic demand. This pathway involves a number of irreversible pathways, including net protein synthesis and other irreversible metabolic transformations of individual amino acids. The second and quantitatively largest pathway is the removal for protein synthesis during protein turnover. At nitrogen equilibrium, because turnover involves the reversible removal of amino acids, with replacement through proteolysis, it does not exert a net metabolic demand (other than for those amino acids irreversibly modified during or subsequent to protein synthesis). The third pathway is the irreversible removal of amino acids by oxidation and nitrogen excretion provoked, for example, by the transient increases in some or all free amino acids after a protein meal. This would represent an inefficient utilization.

The metabolic demand for amino acids appears to involve obligatory and adaptive components. The obligatory component for subjects at equilibrium (i.e., maintenance) comprises conversion of some individual amino acids into important metabolites that are further transformed into nitrogenous end products, mainly urea and other compounds in urine, feces, or sweat, as well as net synthesis of proteins lost from the body as skin, hair, and any other secretions. These diverse biological demands for amino acids for maintenance represent an essential but probably quite small intrinsic metabolic demand for protein. The magnitude of this maintenance component is assumed empirically to be equal to the obligatory nitrogen loss (ONL)—that is, the sum of all nitrogen losses from the body observed in subjects fed a protein-free but otherwise nutritionally adequate diet after 7-14 days, by which time nitrogen losses have declined to a stable and reproducible low level with the subjects losing body protein at a constant daily rate. In normal adults, the obligatory urinary, fecal, and subcutaneous and other losses are approximately 29, 13, and 5 mgN/kg, respectively (i.e., 47mg/kg/day), in total equivalent to 0.29gprotein/kg/day tissue protein mobilized to meet such demands. The ONL is a function of body weight and, as far as is known, varies little with age. After adaptation to a protein-free diet, net tissue proteolysis is assumed to provide for the nonprotein components of the obligatory demand at a rate determined by the metabolic consumption of the rate-limiting amino acid (the amino acid with the highest ratio of molar proportion in the metabolic demand to molar proportion in protein). Because the obligatory metabolic demand is for a mixture of amino acids with a profile that is unlikely to match that of tissue protein, the actual nitrogen content of the metabolic demand is likely to be less than that indicated by the ONL (i.e., less than an equivalent of 0.29gprotein/kg/day). This is because all amino acids mobilised to provide for the metabolic demand must be oxidized and will contribution to the nitrogen excretion, whereas only some of them will serve useful functions. The evidence for this is the lowering of the ONL in response to feeding selective amino acids, such as threonine, tryptophan, and methionine. In addition to these metabolic demands for maintenance, any net protein synthesis associated with growth, pregnancy, and lactation also constitutes an obligatory metabolic demand.

The adaptive component of the metabolic demand represents amino acid oxidation at a rate varying with the habitual protein intake that occurs as a result of the increasing activities of the pathways of oxidation of amino acids that regulate free amino acid pool sizes. Although this aspect of amino acid metabolism is least understood, it is likely that it is a consequence of the fact that humans grow slowly or maintain constant weight on diets that contain protein considerably in excess of minimum needs. Thus, in order to be able to rapidly dispose of excess protein and maintain the very low tissue concentrations of those amino acids, such as the branched-chain, aromatic, and sulfur amino acids, that may be toxic at higher concentrations, pathways of oxida-tive amino acid catabolism adapt (increase their Vmax), enabling them to operate at the appropriate rate set by habitual protein intakes. Importantly, the adapted rate of amino acid oxidation, characteristic of habitual intake, changes only slowly in response to either a change in dietary protein intake level or feeding and fasting. This has two main consequences. First, when intake falls below habitual intake mobilization of tissue protein occurs with a negative nitrogen balance for as long as it takes to adapt to the lower level of intake. This was previously identified as the labile protein reserves. It can be assumed that for intakes greater than the minimum requirement, full adaptation to the new level will include not only a change in the adaptive metabolic demand to match intake but also repletion of most tissue nitrogen lost during the adaptive transition. Second, because the adaptive rate of amino acid oxidation continues to some extent into the postabsorptive state, there are varying postab-sorptive losses of tissue protein and nitrogen excretion with varying habitual intake—that is, a diurnal cycle of postabsorptive losses and postprandial gains with an amplitude that increases with the increasing habitual level of protein intake as shown in Figure 2. As such, the adaptive metabolic demand includes a component of net protein synthesis that repletes postabsorptive losses. The magnitude of this varies in a complex way with eating pattern and with the amount and quality (amino acid score) of the habitual protein intake.

Although amino acid oxidation and urea synthesis is assumed to be irreversible, this is not entirely true because of urea salvage. Thus, the rate of urea synthesis is usually in excess of the rate of urea excretion because some urea enters the large bowel and is hydrolysed by bacteria. Most of this nitrogen is utilized by bacteria, and since little is lost as fecal nitrogen, it is eventually returned to the systemic pool as ammonia and amino acids after bacterial death and proteolysis, including indispensable amino acids. Although the extent and nature of this salvaged urea nitrogen are poorly understood, it may provide nutritionally important amounts of amino acids.

The main practical implications of the previously discussed model are that true minimum metabolic demands and consequent protein requirements will occur when the adaptive metabolic demand has fallen to the lowest possible level, and it is not known with any certainty how long such adaptation would take. However, studies that have examined balance responses to changes in protein intakes suggest it is likely to be longer than the periods employed in short-term balance studies. This implies that short-term balances from which our estimates of the minimal protein requirement (MPR) derive may overestimate the value and also that some of the variability in protein requirements between studies may reflect variable completeness of adaptation to the test diets. Another implication of the adaptive metabolic demands model is that intakes and requirements are correlated, which has implications for the definition of risk of deficiency and safe intakes.

Protein Requirements Plant versus Animal Sources

The nutritional requirement for protein will be the minimum intake that satisfies metabolic demands and that maintains appropriate body composition and growth rates, after taking into account any inefficiency of digestibility and metabolic

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Regulated level .

Body protein content

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Body protein content

Protein intake cycle with increasing habitual protein intakes.

Î Variation in splanchnic protein

Protein intake

Figure 2 Balance regulation throughout the diurnal cycle with increasing habitual protein intakes.

consumption. With continuous and extensive amino acid interconversion, the pattern of dietary amino acids need not match that of the composition of tissue proteins or the maintenance metabolic demand exactly because some amino acids (aspartic acid, asparagine, glutamic acid, alanine, and serine) are dispensable and can be replaced by sufficient total amino acid nitrogen supplied from other amino acids or sources of nonessential nitrogen. However, there will be a minimum dietary requirement for those amino acid that are not intercon-verted, classified as indispensable amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine), and for those that are formed only slowly from other amino acids and become indispensable under specific physiological or pathological conditions (conditionally indispensable; e.g., cysteine, tyrosine, taurine, glycine, arginine, glutamine, and proline). Traditionally, dietary proteins have been classified by their nutritional value (quality) measured in terms of their ability to provide for tissue growth in rapidly growing rats. In this case, marked differences are observed between most animal protein and plant protein sources, with relative nutritional value reflecting mainly relative amounts of specific indispensable amino acids. The similarity between overall tissue protein amino acid composition and that of most animal dietary protein sources and the contrast with plant protein sources resulted in clear distinctions between their quality, although when combined it is clear that plant proteins can provide the appropriate balance of essential amino acids.

However, in human nutrition with growth occurring very slowly after the first few months of life, the nutritional demand for indispensable amino acids for tissue growth is much less and may be minimal. Little metabolic demand for amino acids is generated by protein turnover because of amino acid recycling. Some net protein synthesis is associated with skin and hair growth and with gastric secretions (e.g., threonine-rich mucus glycoproteins) that pass into the colon to be utilized for bacterial metabolism. The metabolic demand for maintenance of normal function and composition is a poorly understood pattern of amino acids utilized in the various metabolic pathways other than protein synthesis, but this pattern is almost certainly different from that required for growth (i.e., mainly the amino acid pattern of tissue protein) and may contain a much lower overall amount of indispensable amino acids. As such, a distinction between plant and animal dietary protein sources is much more difficult to demonstrate and is probably less relevant in human nutrition. Currently, there is considerable controversy regarding the magnitude of the requirements for indispensable amino acids in the human diet and there are different views about the relative importance of dietary protein quality in human nutrition. Some national bodies have stressed that in most mixed, nutritionally balanced diets, sufficient indispensable amino acids will be provided regardless of the relative amounts of plant or animal protein sources (e.g., UK Department of Health). However, others (e.g., US FNB/ IOM) have argued that the requirements for indispensable amino acids in the human diet are higher than previously believed and that protein quality is important.

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