Protein and Amino Acid Requirements

Protein and Amino Acid Requirements

Crystal L. Levesque, PhD and Ronald O. Ball, PhD

The dietary requirement for protein represents the need for the amino acids that constitute it and, in fact, dietary protein can be replaced by mixtures of amino acids. It might seem then that a measure of the amount of each amino acid needed for metabolic processes (i.e., maintenance and growth) would define the need for protein. However, because 11 out of 20 of the amino acids can be synthesized de novo, not all amino acids need to be provided in the amounts that are actually used for anabolic processes. Some amino acids can be synthesized from common intermediates of metabolism if amino acids that can donate an amino group are available. Thus the diet does not need to provide all amino acids in the exact amounts used by cells. Rather, the total amount of protein or total amino acids provided by the diet is an important consideration along with the requirements for specific amounts of particular amino acids.

Classification of Dispensable and Indispensable Amino Acids

The 20 amino acids required for protein synthesis have been divided into two categories: (1) indispensable (or essential), and (2) dispensable (or nonessential). The term dispensable may be preferred over the term nonessential because all the amino acids found in protein are metabolically essential, even though some are dispensable in the diet.

Indispensable Amino Acids

Borman and colleagues (1946) defined an indispensable amino acid as “one which cannot be synthesized by the animal organism, out of materials ordinarily available to the cells, at a speed commensurate with the demands for normal growth” (p. 593). Humans do not possess the pathways for the synthesis of nine amino acids from compounds ordinarily available to cells: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine (Box 15-1). Thus these nine amino acids are always indispensable.

Semidispensable, Conditionally Indispensable, and Dispensable Amino Acids

The other eleven amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, and tyrosine) can be synthesized by cells from materials ordinarily available to the cells. However, the extent of synthesis of some of these amino acids depends upon the dietary supply of particular precursor amino acids or the biosynthetic capacity of the organism. In addition, the amount of an amino acid needed may vary under specific circumstances such as injury or parenteral feeding. These factors have led to subclassifications of the dispensable amino acids and the use of the terms semiessential, conditionally indispensable, and truly dispensable (Reeds, 2000; Institute of Medicine [IOM], 2005).

Semidispensable Amino Acids

Cysteine, or its disulfide cystine, and tyrosine are considered semiessential because their synthesis depends upon an adequate dietary supply of an indispensable amino acid precursor. Cysteine can be synthesized in the body from serine and the sulfur group of methionine. Tyrosine can be formed in the body by the hydroxylation of phenylalanine. Both of these syntheses are irreversible; therefore a lack of tyrosine in the diet can be compensated by an excess of phenylalanine, but the reverse is not true: excess tyrosine cannot compensate for a deficiency of phenylalanine. Likewise, methionine cannot be synthesized from cyst(e)ine. Each of these semiessential amino acids must be considered along with its indispensable amino acid precursor when evaluating indispensable amino acid intake for adequacy; both the sum and the proportion of methionine and cyst(e)ine or of phenylalanine and tyrosine must be taken into account. The requirements for methionine and cysteine and for phenylalanine and tyrosine are referred to as total sulfur amino acids and total aromatic amino acids, respectively.

Conditionally Indispensable Amino Acids

Arginine, proline, glutamine, and glycine are considered to be conditionally indispensable from a dietary viewpoint because the rate at which they are provided by endogenous synthesis may fall below the rate at which they are used. In a state of rapid growth, such as in the neonate, the essentiality of these amino acids is much more dependent on their rates of de novo synthesis. For example, in neonatal piglets, arginine is synthesized primarily in the small intestine from proline and citrulline, but the gut synthesis of arginine is insufficient to meet the whole body arginine demand (Urschel et al., 2007). Conditions that affect gut metabolism and function (i.e., intestinal disease, gut atrophy, or parenteral nutrition) can also affect the requirement for arginine as well as proline. Dramatic decreases in both arginine and proline synthesis occurred when piglets were fed parenterally, demonstrating the conditional essentiality of arginine and proline during total parenteral nutrition when amino acids bypass the gut, limiting synthesis of proline and citrulline (Bertolo et al., 2003).

During critical illness, glutamine is used as a fuel for the immune system and gastrointestinal tract with increased uptake of glutamine by immune cells, the intestinal mucosa, and the kidney. Although large amounts of glutamine are released from the muscle, the increased demand for glutamine during critical illness may not be met sufficiently by an increase in synthesis, such that glutamine often becomes conditionally indispensable (Wischmeyer, 2008). Further contributing to a deficiency in glutamine during critical illness can be a decline in total body muscle tissue with progressing disease state. Glutamine-supplemented total parenteral nutrition reduced infection rate and incidence of pneumonia in critically ill patients in a large double-blind, randomized clinical trial in France (Déchelotte et al., 2006).

The rate of endogenous glycine production during recovery (catch-up growth) from severe childhood malnutrition may be less than the rate of glycine utilization (Badaloo et al., 1999). There are suggestions that glycine may similarly behave as an indispensable amino acid in infants (especially premature infants), who have a limited capacity for glycine synthesis (Jackson et al., 1981).

In preterm infants, cysteine, tyrosine, glutamine, arginine, proline, and glycine are typically considered conditionally indispensable because of their low endogenous synthesis. Courtney-Martin and colleagues (2010) demonstrated that the total sulfur amino acid requirement in preterm infants could be met by methionine alone. This suggests that cysteine may not be a conditionally indispensable amino acid in preterm infants. High plasma phenylalanine and excretion of abnormal metabolites of phenylalanine were observed when phenylalanine was added to commercial total parenteral nutrition solutions fed to neonatal piglets (Wykes et al., 1994), suggesting a low conversion of phenylalanine to tyrosine in neonatal piglets. Interestingly, the mean tyrosine requirement in parenterally fed neonates was three to four times higher than that supplied by commercial total parenteral nutrition solutions (Roberts et al., 2001). Some of these conditionally essential amino acids may need to be present in higher concentrations in formulations used for enteral or parenteral feeding, particularly of preterm infants.

Requirement for Protein (Amino Acids)

The requirement for protein must be stated in such a way that the total need for protein (amino acids) and the need for individual indispensable or semidispensable amino acids are both considered. Usually, this is done by stating a total requirement for protein with a consideration of protein quality that relates to the level of indispensable amino acids in the protein sources. In general, the requirements for amino acids or protein are the sum of the physiological requirements for net tissue accretion during growth and pregnancy or for milk secretion during lactation and the requirements for tissue maintenance. Maintenance needs represent the largest fraction of the protein/amino acid requirement and nearly all of the requirement for adults. During growth and in pregnancy, when protein synthesis is greater than protein degradation, the use of amino acids for protein accretion is a major component of the requirements. Likewise, in lactation, the needs of the mammary gland for milk protein synthesis become a large component of requirements.

Protein (Amino Acid) Requirement for Maintenance

Maintenance is the state in which there is no net change in body protein mass. Although protein degradation returns amino acids to the free amino acid pool for use in protein synthesis, some irreversible loss of amino acids occurs such that there is a dietary requirement for amino acids at maintenance. Even when there is no change in body protein mass, amino acids or proteins are lost due to irreversible modification of amino acids, loss of proteins through the epithelia, loss of amino acids in the urine, use of amino acids for synthesis of nonprotein substances, and oxidation of amino acids as fuels (see Chapters 5, 9, 13, and 14). Except for the first months of life, maintenance requirements account for the major proportion of the dietary protein needs of children, adolescents, and adults. For example, the proportion of the total requirement due to maintenance is approximately 80% at 1 to 3 years and more than 98% by age 18 years.

The maintenance requirement can be defined as the dietary intake needed to replace the obligatory losses of amino acids. Maintenance requirements can be estimated using the factorial method to add up obligatory losses of protein or nitrogen and then to adjust this for an estimate of the efficiency of utilization of dietary protein to replace these obligatory losses. Alternatively, maintenance requirements can be determined by the nitrogen balance technique.

Obligatory Losses of Proteins and Amino Acids

Estimation of the magnitude of obligatory amino acid and nitrogen losses is usually done by feeding protein-free diets to adults and measuring the loss of nitrogen from the body in the urine and feces, as well as cutaneous and miscellaneous losses. The association between obligatory nitrogen losses of individuals adapted to protein-free diets (i.e., ≤ 5 mg N·kg-1·day-1, or ≤ 2.2 g protein·kg-1·day-1) and the magnitude of the negative nitrogen balance experienced by these subjects is illustrated in Table 15-1. It should be noted, however, that obligatory nitrogen losses are expected to be somewhat higher in individuals consuming protein in their diets; this is especially true for losses due to amino acid catabolism.

Because dietary intake can vary enormously, the body must have mechanisms to adjust the rate of amino acid disposal according to the supply, and the major mechanism is the modulation of amino acid oxidation. This adaptive component changes only slowly with a sustained change in intake, as illustrated by the pattern of urinary nitrogen excretion by subjects switched from a diet containing adequate protein to one providing only a minimal level of protein; an example is shown in Figure 15-1. Thus obligatory losses will be affected by prior intake as well as by current intake unless a period of adaptation to the new intake is allowed before measurement of losses.

The major loss of amino acids is due to oxidation, or catabolism. Even when dietary intake is less than the requirement, a residual rate of amino acid catabolism occurs, and although small in comparison with the rate in normal diets, amino acid catabolism remains the major route of obligatory loss. When amino acids are catabolized, the nitrogen is excreted in the urine, mainly as urea and to a lesser extent as ammonium. Amino acid catabolism therefore is commonly assessed by measuring the amount of nitrogen excreted in the urine (as urea and ammonium). Urinary nitrogen losses also include amino acids that are irreversibly modified after incorporation into proteins (i.e., methylhistidine, hydroxylysine) as well as very small amounts of amino acids that escape renal reabsorption.

Obligatory fecal nitrogen loss represents nitrogen released from the body into the gastrointestinal tract but not reabsorbed; the sources of obligatory nitrogen lost via the gut are proteins secreted into the gastrointestinal tract in the salivary, gastric, pancreatic, hepatic (bile) and intestinal secretions and proteins of cells shed from the gastrointestinal epithelium. Most of the endogenous proteins released into the gastrointestinal tract are digested and reabsorbed, but about 20% of the endogenous proteins (particularly proteins such as mucin that are inherently resistant to proteolysis) escape digestion and pass into the large intestine where they are used by the gastrointestinal microflora. Accurate measurement of obligatory losses of amino acids from the gastrointestinal tract has raised considerable debate, and although the protein-free diet method is most commonly used, no one method is without limitations (Stein et al., 2007).

Dermal losses of protein (skin, hair, nails), sweat, sputum, nasal fluids, menstrual fluids, seminal fluids, and breath ammonia represent integumental and miscellaneous losses that collectively account for the loss of approximately 0.03 g protein·kg-1·day-1.

Synthesis of essential substances such as hormones, neurotransmitters, pyrimidines, and purines makes up the final route of obligatory amino acid loss. The contribution of the synthesis of nonprotein compounds to the daily requirement for protein and amino acids is typically small but still significant. As these nonprotein compounds are degraded in the normal process of turnover, their metabolites or end products are excreted in the urine or feces or lost in bodily secretions and thus are included with the urinary, fecal, and integumental and miscellaneous losses of N.

As summarized in Table 15-1, the sum of obligatory nitrogen losses in adults amounts to 48 mg N·kg-1·day-1. Using the general conversion factor of 6.25 g protein per g of nitrogen, the obligatory protein loss is 0.3 g protein·kg-1·day-1. The amount of protein required to maintain N equilibrium is much greater than the sum of the obligatory losses, however, because the efficiency with which absorbed amino acids are used for protein synthesis is not 100%. The efficiency of dietary protein utilization is about 50% (47% for adults and 58% for children), as determined from the slope of N balance response curves. This means that 0.5 g N is retained per 1 g N consumed. Thus (0.3/0.5) g protein·kg-1·day-1, or 0.6 g protein·kg-1·day-1, would be required to replace losses measured under the protein-free condition, and even more might be required in practice because obligatory losses are higher with normal diets than with protein-free diets. (With regard to this issue, note that the average protein requirement determined by nitrogen balance studies is about 0.66 g protein·kg-1·day-1.)

Protein (Amino Acid) Requirements for Nitrogen Equilibrium or Maintenance

The nitrogen balance method, in which the difference between nitrogen intake and nitrogen losses is determined, is the basis of current estimates of maintenance requirements of adults. Use of the nitrogen balance method is not a perfect measure of protein requirements because the body can maintain nitrogen equilibrium over a wide range of intakes. In cases of low intake of protein, N balance may be achieved only at the expense of the loss of significant lean body mass. Therefore the protein or amino acid “requirement” is usually defined as the minimum intake consistent with nitrogen equilibrium (no change in body protein mass).

Considering only the major fluxes of amino acids, the following equations apply to an individual in nitrogen (N) equilibrium or zero N balance:

Nitrogen balance data have been used by both the IOM (2005) and the World Health Organization (WHO, 2007) as well as many other groups for estimating protein requirements. For nonpregnant, nonlactating adults in nitrogen equilibrium, the nitrogen intake required for balance represents the requirement for maintenance. The average requirement of adults for protein was based on a meta-analysis of published nitrogen balance studies, as illustrated in Figure 15-2 (Rand et al., 2003). For each study, the lowest continuing intake of dietary protein that was sufficient to achieve body nitrogen equilibrium was considered as the individual requirement. The median nitrogen requirement derived from the meta-analysis was 105 mg·kg-1·day-1, and this value, along with the conversion factor of 6.25 g protein per g of nitrogen, was used to set the average requirement for protein at 0.66 g·kg-1·day-1 for men and women (≥19 years of age). Maintenance requirements of children aged 6 months to 18 years are similar to those of adults on a per unit body weight basis.


Does Muscular Work Require the Breakdown of Muscle Protein for Fuel?

Justus Liebig, in his influential Animal Chemistry published in 1840, stated that protein broke down during the release of energy, with the nitrogenous fraction being converted to urea and excreted by the kidney, so that the total amount of work performed (both internally and externally) was proportional to the nitrogen excreted. These points were tested by two Swiss physiologists, Adolf Fick and Johannes Wislicenus, in 1866 (Carpenter, 1994). Using themselves as subjects, Fick and Wislicenus spent a day walking up a steep mountain path carrying urine collection equipment. From noon the day before the climb and until 6 hours after the climb, Fick and Wislicenus ate only cakes from starch paste fried in fat. Both men excreted approximately 5.6 g of N during a 14-hour period including the 8-hour climb plus the 6-hour period after the climb. Relying on the concurrent studies of Sir Edward Frankland on the heat of oxidation of organic materials and of James Prescott Joule in establishing the mechanical equivalent of heat, they calculated that the urinary N was equivalent to the breakdown of approximately 35 g of protein, and that 35 g of protein would be equivalent to approximately 154 kcal, or a work equivalent of 65 x 103 kg-m. The net external work required to ascend the 1,956-m path was calculated to be approximately 139 × 103 kg-m. Even without considering the energy required for basal or internal work or a correction for incomplete efficiency of the muscle in converting chemical energy to work, the quantity of protein metabolized was clearly insufficient to have provided the energy needed for their climb. Therefore Fick and Wislicenus (1866) concluded that the burning of protein could not be the only source of muscular power, and although their conclusion was not immediately accepted, we now know this to be true.

Protein (Amino Acid) Requirements for Growth, Pregnancy, and Lactation

The additional protein above the maintenance requirement that is needed for growth and pregnancy is usually estimated based on tissue protein accretion corrected for the efficiency of dietary protein utilization. Similarly, protein requirements for lactation are based on milk protein secretion corrected for efficiency of dietary protein utilization.

The protein needs for growth are an important component of the protein requirement of infants, children, and adolescents. A newborn infant deposits about 0.55 g protein·kg-1·day-1; this rate of protein deposition gradually decreases to about 0.11 g·kg-1·day-1 by age 18 months, and then to less than 0.06 g·kg-1·day-1 by age 3 years (WHO, 2007). Using an efficiency of dietary protein utilization of 58%, the protein requirements for growth would be 0.95, 0.19, and 0.10 g·kg-1·day-1 at ages 1 month, 18 months, and 3 years, respectively. By adding the growth requirement to an assumed maintenance requirement of 0.66 g protein·kg-1·day-1, the total protein requirement would be 1.61, 0.83, and 0.76 g·kg-1·day-1 at age 1 month, 18 months, and 3 years, respectively. Thus during the first 2 months of life, an infant’s requirement for growth exceeds the infant’s requirement for maintenance with almost 60% of the newborn’s protein requirement being for growth. The proportion of the total protein requirement needed for growth decreases to about 22% at 18 months and then to 12% or less by 3 years, remaining in the range of 0% to 12% throughout the remainder of childhood and adolescence.

Protein is deposited in fetal and maternal tissues during pregnancy. The Food and Agriculture Organization (FAO) of the United Nations, the WHO, and the United Nations University (UNU) arms of the United Nations system used estimates of 0, 1.9, and 7.4 g/day for amounts of protein deposited during the first, second, and third trimesters of pregnancy, based on a mean gestational weight gain of 13.8 kg (WHO, 2007). The efficiency of dietary protein intake for protein accretion during pregnancy was conservatively set at 0.42, yielding additional protein requirements of 4.5 and 17.7 g/day for the second and third trimesters, respectively. It should be noted that maintenance requirements also increase during pregnancy on a g/day basis due to the increase in total body mass. The IOM (2005) used a similar approach in estimating needs during pregnancy but averaged total protein accretion during gestation over the second and third trimesters (5.4 g/day). They used this average, along with an efficiency of 0.43, to set the need for additional protein at 12.6 g/day for protein accretion during pregnancy. Based on a reference weight of 57 kg, this corresponds to an increase of 0.22 g·kg-1·day-1 or a total protein requirement of 0.66 + 0.22 = 0.88 g·kg-1·day-1 during the later two trimesters.

The additional amino acid requirements for lactation derive primarily from the quantity and composition of the protein and amino acids secreted in the milk. The IOM (2005) based the increase in protein requirement for lactating women on an average output of 10 g protein per day in the milk (g N × 6.25) and the adult efficiency of protein utilization of 0.47, yielding an increased protein requirement of 21 g protein per day, or 0.39 g·kg-1·day-1. Adding the lactation requirement to the maintenance requirement, the total protein requirement for lactating women is 0.39 + 0.66 = 1.05 g·kg-1·day-1. The FAO/WHO/UNU (WHO, 2007) used a similar approach but made separate recommendations for the first 6 months and second 6 months of breast-feeding. Based on estimates of milk protein content and milk volume, along with correction for an efficiency of 0.47, WHO set the average increase in requirement for protein at 15 g/day for the first 6 months and at 10 g/day for the second 6 months, assuming partial breast-feeding during the second 6 months (WHO, 2007).

Consideration of Protein Quality

The degree to which a food protein, if consumed in an amount that meets the requirement for total protein, is able to meet the requirements for all of the indispensable amino acids is called its protein quality. Protein quality of a particular food source is influenced by the amino acid composition of the food source (i.e., the composition of amino acids making up the protein) and the digestibility of the protein contained in the food source. The digestibility determination for a protein source includes assessment of the availability of amino acids for absorption and thus accounts for modifications of amino acids that prevent their absorption. Availability refers to whether or not the amino acid can be used in the body for protein synthesis (i.e., whether amino acids have been irreversibly modified in a way that allows absorption but not entrance into common pathways of amino acid metabolism). Different individual proteins (e.g., casein, gelatin) or different food proteins (e.g., milk proteins, soy proteins), which are actually mixtures of proteins, are not identical in terms of their amino acid composition and hence are not identical in their ability to replace losses or support net protein accretion.

The protein requirements established by the IOM and the FAO/WHO/UNU are based on nitrogen balance studies in which subjects were fed high-quality proteins or protein mixtures. In setting the Dietary Reference Intakes (DRIs) for the populations of the United States and Canada, the IOM (2005) did not include an adjustment for protein quality, because it was assumed that these North American populations consume varied diets with both high-quality and complementary proteins, such that protein quality is not of concern for most of the population. However, recommended intakes would need to be higher for populations or individuals consuming diets in which the overall protein quality is not high. The FAO/WHO/UNU (WHO, 2007) made their recommendations with provisions for additional adjustments for protein quality when appropriate. The topic of protein quality is considered in more detail later in this chapter.

DRIs for Protein

Requirements for protein are the amounts of dietary protein that will meet the needs of the organism for maintenance and for protein accretion during growth and pregnancy or milk production during lactation. Safe requirement levels or recommended intakes are conventionally set as the average requirement plus 2 standard deviations (SD) based on between-individual variance so that the recommended intake should cover the needs of 97.5% of the population. For protein requirements, the coefficient of variation is typically about 12.5%, so the actual recommended intakes are about 25% (2 × 12.5) higher than the average requirement.

The IOM (2005) set DRIs for protein intake of various age and sex groups. In the DRIs Across the Life Cycle box, the Estimated Average Requirement (EAR) and Recommended

Dietary Allowance (RDA) are shown as g protein·kg-1·day-1, and RDAs are also given as g/day for reference size males and females (e.g., 70-kg man and 57-kg woman). Thus the RDA for protein is 0.8 g·kg-1·day-1 for adults, or 46 g/day for the 57-kg reference woman and 56 g/day for the 70-kg reference man.

There is considerable debate whether the requirement for protein is higher in elderly adults. The IOM (2005) and the FAO/WHO/UNU (WHO, 2007) concluded that the lack of demonstrated improvement in biochemical indicators of protein sufficiency or measured nitrogen balance with higher protein intakes suggests that the requirement for protein does not increase with age. However, it was noted that the protein to energy ratio for diets of the elderly needs to be higher than for younger individuals because of the decreased energy expenditure of the elderly, despite no change in their protein requirement per se.

For infants, the IOM (2005) did not determine an EAR/RDA, but an Adequate Intake (AI) was set based on the known adequacy of breast milk for infants. The AI for infants up to 6 months of age, set by the IOM, is based on the average volume of milk intake (0.78 L/day) and the average protein content of human milk during the first 6 months of lactation (11.7 g/L) and is 9 g/day. The AI can be stated as 1.5 g·kg-1·day-1, based on a reference weight of 6 kg for 2- to 6-month-old infants. A similar estimate based on milk plus complementary food intake is the basis of the AI for infants during the second 6 months of life. The FAO/WHO/UNU (WHO, 2007) has calculated safe protein intakes for infants based on estimates of their maintenance

requirements (0.58 g protein·kg-1·day-1 from nitrogen balance studies) and growth requirements (protein deposition adjusted for an efficiency of 0.66), with the average requirement increased by 2 SD, to yield safe levels ranging from 1.8 g protein·kg-1·day-1 (for the 1-month-old) to 1.1 g protein·kg-1·day-1 (for the 6-month-old infant).

Requirements for Individual Amino Acids

Protein is made up of all the indispensable and dispensable amino acids; therefore the expression of protein requirements must also include a consideration of the supply of indispensable amino acids that cannot be synthesized in the body. Over the course of the last century, several approaches have been developed and applied to the determination of amino acid requirements, resulting in refinements in our ability to precisely estimate these requirements for humans. Essentially two approaches have been used to assess amino acid requirements: empirical analysis of dose response and factorial method.

Amino Acid Requirements Based on Empirical Analysis of Dose Responses

The empirical analysis of dose responses measures a physiological response (i.e., growth rate, nitrogen balance, or amino acid oxidation) at graded intakes of protein or amino acid. The nitrogen balance technique has been used to determine protein requirements for maintenance, and the criterion of nitrogen balance also provided the basis of the first quantitative estimates of the amino acid needs of human adults. However, direct amino acid oxidation or indicator amino acid oxidation has been used as the response criterion in more recent studies. Regardless of the response criterion, a range of amino acid levels greater than and less than the requirement (six or more levels) should be tested in each individual subject. The basal diet must be adequate in all other amino acids and energy, as well as in all other essential nutrients, such that the amount of the amino acid being tested is the only factor that limits the extent of protein synthesis.

Depending on the approach used to determine an indispensable amino acid requirement, three different patterns of response are possible; these are illustrated in Figure 15-3. One pattern is that seen when either nitrogen balance in the adult or growth in a growing animal or child is plotted against amino acid intake. Nitrogen balance or growth will increase as the intake of the limiting indispensable amino acid (i.e., test amino acid) increases up until the requirement amount is reached, at which point nitrogen balance or growth will plateau. A second pattern is seen for the indicator amino acid oxidation (IAAO) method where oxidation of the indicator amino acid is high when amino acids are not being used maximally for protein synthesis but decreases to a plateau once the requirement of the limiting amino acid for protein synthesis has been met. For nitrogen balance, growth, and IAAO, once the requirement is reached the measured response will plateau despite the addition of higher levels of protein or limiting amino acid. A third pattern occurs when oxidation of a test amino acid is measured directly, called the direct amino acid oxidation (DAAO) method. In this case, oxidation of the test amino acid remains low with little change until the requirement is approached or met, after which its oxidation increases progressively as intake is further increased. A similar pattern may be observed by following the plasma level of the test amino acid; the level of the test amino acid will begin to increase once intake exceeds the requirement. When lines are drawn for the slope of the two phases (change and plateau) of the response curve, the crossover point or breakpoint of the two lines is used to determine the requirement level as illustrated in Figure 15-3.

Estimates of Indispensable Amino Acid Requirements by Nitrogen Balance Studies

In nitrogen balance experiments, the rate of body nitrogen retention is estimated as the difference between the dietary nitrogen intake and the sum of the losses in urine and feces and by other routes (integumental and miscellaneous). Nitrogen balance is based on the premise that protein or lean body tissue is the major nitrogen-containing component in the body; thus nitrogen loss or gain equates to protein loss or gain. It is assumed that body nitrogen increases in the growing child and remains constant in the adult (WHO, 2007). As described previously, it is generally presumed that the protein requirement of an adult is achieved when the individual is in zero nitrogen balance (i.e., nitrogen equilibrium). Infants, children, pregnant women, and individuals recovering from disease states in which lean body mass was lost should be in positive nitrogen balance, reflecting net deposition of protein.

Nitrogen balance studies were used to obtain the first estimates of the amino acid requirements of human adults. Rose and colleagues, in a series of nitrogen balance studies in the 1940s, determined the amino acid requirements of young men (see Rose, 1957). First, men were given diets devoid of a single amino acid so as to establish which amino acids were dispensable and which were indispensable. Eight amino acids were determined to be indispensable. In these studies, removal of histidine from the diet did not lead to a negative nitrogen balance; this was interpreted to mean that histidine was dispensable, and this conclusion was only reversed much later by longer-term experiments. Rose and colleagues then conducted a series of quantitative studies in which they gave each subject a succession of diets with different concentrations of the amino acid under investigation. From the changes in nitrogen balance that ensued, they attempted to identify the intake of each amino acid that was required for nitrogen equilibrium to be achieved. Similar experiments were conducted on young women, but using more subjects and with several improvements in experimental procedures (Leverton et al., 1959). The results of these early experiments in determining the amino acid requirements of men and women formed the basis of the international estimates of adult amino acid requirements (WHO, 1985) that were widely used before 2000.

One of the greatest challenges with the nitrogen balance method is accurate measurement of nitrogen losses. Due to difficulties in measuring integumental and miscellaneous losses, they are not always included in the calculation of nitrogen balance. Not accounting for integumental and miscellaneous losses results in underestimation of nitrogen loss. Urinary and fecal nitrogen can be measured relatively accurately; however, incomplete measurement of food spillage and food residues results in overestimation of nitrogen intake. The tendency to overestimate intake and to underestimate losses both contribute to the tendency of nitrogen balance studies to underestimate protein or amino acid requirements (WHO, 2007). This means that a subject apparently receiving sufficient amino acid (or protein) to maintain nitrogen equilibrium may actually be in negative nitrogen balance and need more of the amino acid (or protein) to maintain true nitrogen equilibrium.

A further problem with the nitrogen balance approach, especially in short-term studies, may arise through the implicit assumption that a subject in nitrogen equilibrium must also be in amino acid equilibrium. This is not necessarily so. Histidine, for example, is stored in the form of the peptide carnosine (β-alanylhistidine). Carnosine, in experimental animals at least, is depleted during dietary histidine deficiency. This allows obligatory losses of histidine to be met for a time without loss of body protein, despite an inadequate histidine intake. There may also be a potential to adapt to an indispensable amino acid deficiency through modification of the amino acid composition of the body protein pool as a whole. Of course, it is not possible to modify the amino acid composition of any of the body’s proteins, but it is possible to vary the relative amounts of the various proteins. Because proteins have different amino acid compositions, there is the potential to adapt, at least temporarily, to a deficiency of one amino acid by depleting the body of proteins rich in that amino acid. This also happens in histidine deficiency, in which hemoglobin, which is very rich in histidine, can be gradually depleted. The mechanism of this adaptation is not known. However, this ability of the body to use endogenous sources of histidine explains why histidine was not identified as an indispensable amino acid in the early balance studies (Rose, 1957; Leverton, 1959).

Estimates of Indispensable Amino Acid Requirements by Amino Acid Oxidation Studies

Both direct and indirect amino acid oxidation are based on the principle that there is no storage of amino acids; therefore amino acids not used for protein synthesis are oxidized. In both direct and indirect methods, the oxidation of an amino acid that is labeled with 13C in the α-carboxyl group is assessed. The 13C is released to the body bicarbonate pool in an early step of the amino acid’s committed degradation pathway, and its oxidation is measured by the appearance of labeled 13CO2 in the breath.

In DAAO, it is the amino acid of interest (i.e., the test amino acid) that is labeled. Thus, application of the DAAO method is limited to amino acids whose α-carboxyl group is irreversibly released early in the degradation pathway. In the DAAO method, the test amino acid is labeled and its content in the diet is varied. As long as the level of the test amino acid remains below the requirement for protein synthesis, it will be efficiently incorporated into protein with little excess for oxidation. Once the requirement is exceeded, the excess test amino acid will be oxidized, giving rise to a rapid increase in the amount of labeled 13CO2 in the breath.

In the IAAO method, a separate indispensable amino acid is labeled (i.e., an indicator amino acid) and its oxidation is measured. The indicator amino acid is another indispensable amino acid present in adequate amounts while intake of the test amino acid is altered. When intake of the test amino acid is low and protein synthesis is limited by a lack of the test amino acid, the excess indicator amino acid (i.e., that not used for protein synthesis) will be oxidized. As the intake of the test amino acid is increased, incorporation of the indicator amino acid, as well as all other non-test amino acids, into protein will increase, such that oxidation of the indicator amino acid to 13CO2 will decrease. The IAAO method is not dependent upon release of the carboxyl carbon of the test amino acid and thus can be used to assess the requirement of any indispensable amino acid. Phenylalanine has been found to be the most responsive and accurate of the indispensable amino acids for use as the indicator amino acid in determining amino acid requirements (Levesque et al., 2010).

Adult Requirements for Indispensable Amino Acids and Indispensable Amino Acid Requirement Patterns

There has been considerable debate over the most appropriate requirement estimates for indispensable amino acids in adults. However, extensive reviews (Young, 1986; Millward, 1988; Fuller and Garlick, 1994) of the original nitrogen balance studies by Rose (1957) and Irwin and Hegsted (1971) all conclude that previous estimates (WHO, 1985) based on nitrogen balance were too low. The greatest concern with these early studies was the lack of measurement of miscellaneous nitrogen losses, resulting in low estimates of the indispensable amino acid requirements for achieving nitrogen balance. The current FAO/WHO/UNU (WHO, 2007) and IOM (2005) estimates of indispensable amino acid requirements (mg amino acid·kg-1·day-1) are based primarily on amino acid oxidation studies. As shown in Table 15-2, the current estimates of requirements for 6 out of 10 of the indispensable amino acids are close to double the previous 1985 estimates.

The estimates of amino acid requirements can be used to generate safe intakes for individual indispensable amino acids and for generation of the pattern or amounts of indispensable amino acids needed in a protein source for it to meet both the dietary protein and the indispensable amino acid requirements. In order for the required amount of dietary protein to meet all amino acid needs, it must contain the indispensable amino acid in amounts equal to or greater than the required pattern. The requirement patterns for indispensable amino acids are also shown in Table 15-2. Even based on the highest requirement pattern, only 27% of the total amino acids in the dietary protein need to be indispensable amino acids. If the pattern of the dietary protein mixture of a population has an inadequate amount of one or more indispensable amino acids, adjustments should be made to increase the total protein requirements of that population to allow intake of a sufficient amount of the limiting indispensable amino acids. Thus for lower-quality protein mixtures, higher total protein intakes will be needed. Another approach, of course, would be to improve the quality of the protein mixture by using a mixture of complementary proteins or adding a small amount of high-quality protein to the mixture.

Estimates of Indispensable Amino Acid Requirements of Children and of Pregnant and Lactating Women by the Factorial Method

As with setting protein requirements, the requirements for indispensable amino acids in children, pregnant women, and lactating women were based on the factorial approach and include a component for both maintenance and growth or milk secretion. The factorial method is the approach in which the relevant components of the requirement (maintenance, growth, accretion of maternal tissues and growth of the fetus during pregnancy, and milk secretion during lactation) are estimated separately and then added to set the requirement. Although this approach ostensibly provides a logical system for determination of requirements, the direct estimation of the component requirements is difficult. This is particularly true during pregnancy, where direct estimates of protein and amino acid requirements for fetal growth are largely unknown. For example in school-aged children, the factorial estimate for total sulfur amino acids (15 mg·kg-1·day-1) and lysine (30 mg·kg-1·day-1) were similar to the mean requirement determined by IAAO (13 and 35 mg·kg-1·day-1, respectively) (Turner et al., 2006; Elango et al., 2007). However, the factorial estimate for total branched-chain amino acids (85 mg·kg-1·day-1) in school-aged children was significantly lower than the mean requirement based on IAAO (147 mg·kg-1) (Mager et al., 2003).

To estimate the indispensable amino acid requirements of children, the IOM (2005) used the factorial method. The maintenance requirements for children were based on the EARs for indispensable amino acids in adults. To this was added the requirements for accretion of protein, which were based on the amino acid composition of body proteins and an estimated efficiency of utilization of amino acids derived from the diet (0.58). Safe intakes for infants were based on adequate intakes from breast milk during the first 6 months and from breast milk and complementary foods during the second 6 months. FAO/WHO/UNU (WHO, 2007) used a similar approach to estimate indispensable amino acid requirements of children and infants. However, to estimate the amino acid requirements of infants from 6 months to 1 years of age, the FAO/WHO/UNU applied the factorial method instead of simply relying on the amino acid composition and intake volume of human milk. Table 15-3 shows the indispensable amino acid intake of breast-fed infants and the indispensable amino acid requirements of children from 6 months to 18 years of age.

Feb 26, 2017 | Posted by in PHARMACY | Comments Off on Protein and Amino Acid Requirements
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