16a.1    Introduction

Total body protein accounts for about 14‑16% of body mass; approx­imately 11kg of protein in a 70kg reference male and approx­imately 9kg of protein in a 60kg reference female (Zoladz, 2019) which is widely distributed throughout the different tissues of the body. Most body protein is present in the skeletal muscle (approx­imately 43%) (IOM, 2006). Structural tissues (i.e., skin and blood) each contain approx­imately 15% of the total protein while the meta­bolically active visceral tissues (e.g., liver and kidney) contain about 10% of the total protein. The remainder is in the brain, heart, lung, and bone. Approximately 50% of the total protein content of the body is characterized by four proteins, myosin and actin in skeletal muscle, collagen, and hemoglobin. The skeletal muscle protein (termed somatic protein) and the visceral protein pool together comprise the meta­bolically available protein known as the "body cell mass".

The distribution of protein among the organs such as the brain, lung, heart, and bone varies with developmental age, with the neonate having proportionately less muscle and much more brain and visceral tissue than an adult.

16a.1.1 Functions of protein and amino acids

Protein is important in the diet as a source of amino acids, particularly those amino acids which are indis­pens­able and hence must be derived from the diet. There are 20 amino acids required for protein synthesis (Table 16a.1),

Table 16a.1 Indispens­able, dispens­able and conditionally dispens­able amino acids in the human diet (IOM, 2006).

Indispensable	Dispensable	Conditionally dispensable
Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Histidine	Alanine Aspartic acid Asparagine Glutamic acid Serine	Arginine Cysteine Glutamine Glycine Proline Tyrosine

of which nine are indis­pens­able for adults and must be provided through dietary intake (IOM, 2006). The remainder are either dispensable, because they can be formed in the body from carbon and nitrogen precursors, or conditionally indis­pens­able. The term conditionally indis­pens­able recognizes that under certain physiological circumstances (e.g., neonates) the ability to synthesize some of these amino acids endoge­nously relative to the demand becomes limited.

Proteins synthesied from the 20 amino acids are the basic functional units of the cell and carry out multiple functions essential for life. Recently, increasingly complex roles for protein and amino acids have been discovered, including roles in the regulation of body composition, main­tenance of organ / tissues such as bone, gastro­intestinal function, bacterial flora, glucose homeostasis, cell signaling, and satiety. Box 16a.1 provides some examples of selected functions of proteins and amino acids.

In addition to the synthesis of body protein, amino acids are also required for the synthesis of other important nitrogen-containing com­pounds. The latter include creatine, nucleo­tides and nucleic acids, poly­peptide hormones (e.g., insulin, thyroid hormones, growth hormone, and glucagon), and some neuro­trans­mitters and other non-poly­peptide hormones. Functions of some of the individual amino acids are shown in Box 16a.2; for more details, see Wu (2009).

Amino acids consumed in excess of the amounts needed for the formation of nitrogenous com­pounds are not stored but are degraded; the nitrogen is excreted in the urine as urea, and the keto acids, left after removal of the amino groups, are either used directly as sources of energy or are converted to carbo­hydrate or fat. Nitrogen is also lost in feces, sweat, and other body secretions and in sloughed skin, hair, and nails. Therefore, a continuous supply of amino acids must be obtained from the diet to replace these losses, even in adulthood after growth has ceased.

16a.1.2 Absorption and metabolism of protein and amino acids

In the body amino acids exist freely or as part of protein (IOM, 2005). They are made available through dietary protein intake or de novo synthesis. The protein in the diet is denatured by stomach acid and hydrolyzed in the gastro­intestinal tract by a series of proteolytic enzymes. The end products of protein digestion — free amino acids and small peptides — are taken up by mucosal cells of the small intestine by an energy-requiring process involving several carrier systems. Trace amounts of whole proteins are also absorbed through the intestinal mucosa and can enter the blood stream.

Upon entering the portal blood, the free amino acids are taken up by the liver and other organs and metabolized along three possible pathways. These are: (a) incor­pora­tion into tissue proteins, (b) catabolism by degra­dation pathways that involve deamination or trans­amination and, (c) synthesis of new nitrogen-containing com­pounds such as purine bases, creatine, and epinephrine, as well as dispensable amino acids. About one third of the amino acids entering the liver from the portal blood are used for protein synthesis, energy metabolism, or gluconeogenesis. Skeletal muscle is the main site of metabolism of the branched-chain amino acids (leucine, isoleucine, and valine). For further details see Crim and Munro (1994) and the Dietary Reference Intakes (IOM, 2005).

The amount of protein synthesized daily depends on the require­ments for growth, the manu­facture of digestive and other enzymes, and the repair of body tissues. Even in healthy adults, proteins and other nitrogenous com­pounds are being continuously degraded and resyn­the­sized in the body. This process is termed "protein turnover" and varies with age, physiological status, and level of protein intake.

In healthy adults, the rate of protein synthesis is balanced by an equal amount of protein degra­dation, so that the amount of body protein remains approx­imately constant over long periods of time. During growth, however, protein synthesis exceeds protein degra­dation so there is a net deposition of protein leading to an expansion of the body cell mass. When protein intake is inad­equate or diets are limiting in certain indis­pens­able amino acids, then there is a shift in this balance so that rates of synthesis of some body proteins decrease while protein degra­dation continues in an effort to provide an endoge­nous source of those amino acids most in need. Conversely, when amino acids are consumed in excess of the amounts actually needed, the excess are not stored but are degraded, as noted earlier.

16a.1.3 Effects of protein deficiency

There is no long-term storage of proteins in humans (Holt et al., 1962 ), and there­fore loss of body protein results in the loss of essential structural elements as well as impaired function. Initially, during short-term changes in dietary intakes, the main loss of protein occurs from the visceral protein pool (de Blaauw et al., 1996). In chronic deficiency states, however, the largest single contributor to protein loss is the skeletal muscle (Hansen et al., 2000).

Loss of muscle mass (and adipose tissue) characterizes the marasmic form of protein-energy malnu­trition (PEM). This is the form most frequently encountered in low-income countries, where it is known as nutritional marasmus. It generally results from a prolonged reduction of food intake. Marasmus may also occur in more affluent countries in hospital patients with chronic illnesses such as cancer, or from the prolonged use of clear fluid diets and hypo­caloric intra­venous infusions of 5% dextrose (Corish & Kennedy, 2000; Naber et al., 1997).

Kwashiorkor, a second form of PEM, occurs in children from certain regions of low-income countries. In these countries, kwash­iorkor is often precipitated by a series of environ­mental insults in concert with a diet with a low protein content relative to energy (Fuhrman et al., 2004). Obser­vations in Malawi have also implicated the gut microbiome as a causal factor in kwash­iorkor (Smith et al., 2013).

Unlike marasmus, kwash­iorkor does not result in clinical signs of wasting, but instead with a range of clinical signs, including bilateral pitting, edema, loss of hair pigmen­tation, and skin lesions. Metabolic disturbances are also more severe in kwash­iorkor than marasmus, some of which are poorly under­stood, but include hypo­albumin­emia, hepatic steatosis, and depletion of minerals, vitamins, and antioxidants. Of the latter, a reduced rate of synthesis of red blood cell glutathione, the most abundant antioxidant in the body, has been reported in children with edematous PEM relative to those with non-edematous PEM (Reid et al., 2000). Such a low glutathione status has been associated with a decreased glutathione synthesis secon­dary to a shortage of protein-derived cysteine in children with edematous PEM. Differences in gut micro­biota have also been reported, with more pathogenic species in the kwash­iorkor gut micro­biota. For more details on meta­bolic differences, see Green et al. (2014); Di Giovanni et al. (2016); and Pham et al. (2021).

Kwashiorkor may also occur in adult hospital patients in more affluent countries, where it is termed "adult kwash­iorkor" and tends to arise from an inad­equate intake of dietary protein accom­panied by acute protein losses and meta­bolic reactions induced by the stresses associated with hyper­metabolism (e.g., trauma or sepsis) (Jeejeebhoy, 1981).

16a.1.4 Effects of high protein intakes

There is no formal definition of a high-protein diet, although most of the definitions range between 1.2 to 2.0g/kg/d (Ko et al., 2020). Within this range, a protein intake >1.5g/kg/d can be considered a high-protein diet. According to data from the U.S National Health and Nutrition Examination Surveys (NHANES), the average intake of protein in an American diet is 1.2 to 1.4g/kg/d (Berryman et al., 2018).

High protein intakes have been associated with negative effects on kidney function (Brenner et al., 1982). In a more recent meta-analysis of data from 28 papers from 1975 to 2016, the effects of low (<0.8g/kg/d) and normal protein intakes (0.8 to <1.5 g/kg/d) versus higher protein intakes (>1.5g/kg BW/d) in healthy indi­vid­uals were investigated on kidney health (Devries et al., 2018). The data showed that when changes in glomerular filtration rates were compared, dietary protein had no effect suggesting that high protein intake does not negatively affect kidney health in healthy adult populations. These results are in accordance with statements made by FAO / WHO / UNU (FAO, 2007) and by the US Institute of Medicine in their report on protein requirements (IOM, 2005).

Another concern with high protein diets pertains to bone health. Several systematic reviews and meta-analyses have addressed the benefits and risks of dietary protein intakes for bone health in adults (Darling et al., 2011; Mangano et al., 2014; Shams-White et al., 2018 ). Overall, the evidence suggests no adverse effects of higher protein intake on bone health. Instead, protein has been shown to be positive for bone health. Moreover, mounting evidence demonstrates that older adults require more dietary protein to support good health, recovery from illness, and maintain functionality (Bauer et al., 2013). Higher dietary protein intakes (1.0 to 1.2g/kg/d) are necessary to offset age-related changes in protein metabolism such as high splanchnic sequestration of amino acids and the decline in anabolic responses to ingested protein (Fujita & Volpi, 2006; Volpi et al., 2003). In addition, more protein than current recom­men­dations is required for the elderly to promote an adequate meta­bolic response to inflammatory and catabolic conditions associated with chronic and acute diseases that occur frequently with aging (Bauer et al., 2013).

Despite the lack of evidence suggesting harmful effects of higher protein intakes in healthy populations, under certain conditions/disease states higher protein intakes may be unsafe. The Modification of Diet in Renal Disease (MDRD) study was a large random­ized multi­center, controlled trial evaluating the relationship between dietary protein restriction and renal disease (Levey et al.,1999). Outcomes such as glomerular filtration rate (GFR), were measured in patients with chronic renal disease at baseline and during a 2‑year follow-up period. The results showed that for patients with a lower total protein intake, the time to renal failure was longer. These findings suggest that a lower protein intake post­poned the progression of advanced renal disease (Levey et al.,1999). Addition­ally, a systematic review and meta-analysis by Li Q et al. (2021) investigated the effects of a low protein diet (<0.8g/kg/day) versus a control diet (1.29 g/kg/d) on the progression of diabetic kidney disease (DKD). Patients with DKD who consumed a low protein diet had a signif­icantly reduced decline in GFR and a signif­icant decrease in proteinuria compared to those on the control diet. Therefore, under certain conditions / disease states, particularly renal disease, higher protein intakes are not recommended.

16a.2    Methods to assess protein require­ments

Nitrogen balance is the traditional method used for measuring protein require­ments, and the one adopted for estimating the protein require­ments in healthy adults by several expert groups including the Institute of Medicine (2005), FAO (2007) and EFSA (2015) (see Section 16a.3). For children, pregnant and lactating women, a factorial approach is used whereby the main­tenance needs, based on nitrogen balance, are summed with the addit­ional protein needs associated with the deposition of tissues or secretion of milk. The only other method that has been used to assess protein require­ment is the indicator amino acid oxidation (IAAO) method. All three methods and their limitations are discussed briefly below.

16a.2.1 Nitrogen balance

The nitrogen balance method is the classical approach for determining protein requirements. It is based on several assumptions. These include the assumption that nearly all of the body nitrogen is incorporated into protein so that gain or loss of nitrogen from the body can be regarded as synonymous with gain or loss of protein. Secondly, in healthy adults in energy balance, body nitrogen is constant if the dietary intake of the test protein is adequate, and thirdly if the body nitrogen content decreases or does not increase adequately, then the dietary protein is deficient. The validity of these assumptions is discussed in the FAO /WHO / UNU report (FAO, 2007).

The method for nitrogen balance involves the measure­ment of nitrogen intake (from the diet) minus the sum of nitrogen output in urine, plus nitrogen losses in feces, skin, hair and other miscellaneous losses. However, the majority of nitrogen balance studies only report measure­ments of urinary and fecal losses and not the measure­ments for dermal or miscellaneous nitrogen losses. As a result, adjustments are made to take such losses into account when determining the protein requirements. The intake of nitrogen is quantified by analyzing the nitrogen content of duplicate portions of food and careful collection of all food not consumed, such as spillage and residue on plates. In some cases, nitrogen intake is estimated from the protein intake because most proteins are assumed to contain approximately 16% nitrogen.

To assess protein requirements using nitrogen balance, different levels of a high-quality protein above and below the expected requirement are tested on healthy adults who are in energy balance. For each level of test protein, multiple days (i.e., 6-10 days) are needed for each participant to adapt to reach a new steady state of nitrogen excretion (Danielsen & Jackson, 1992). Therefore, nitrogen balance studies are usually conducted on only a few test protein intakes. After the metabolic adaption period, it is assumed that the requirement is met when the individual comes into "zero nitrogen balance" i.e., when nitrogen intake equals nitrogen excretion. When nitrogen intake is inadequate, more nitrogen is excreted than is consumed, due to net losses of body protein, and the individual is said to be in negative nitrogen balance. Conversely, when the nitrogen intake exceeds nitrogen losses, an individual is in positive balance.

The expression for nitrogen balance is:

N balance = I − (U − Ue) + (F − Fe) + S

where I = intake of nitrogen (protein / 6.25), U = total urinary nitrogen, Ue = endoge­nous urinary nitrogen, F = nitrogen voided in feces (as unabsorbed protein), Fe = endoge­nous fecal nitrogen losses, and S = dermal nitrogen losses.

In most of the nitrogen balance studies on which the protein requirements have been based, only the nitrogen contents of the diet (I), urine(U) and feces (F) have been measured directly. For the endogenous urine and fecal nitrogen losses and dermal nitrogen losses, the estimated allowances are based on data from a limited number of studies, as noted earlier.

Nitrogen balance studies are very challenging, with difficulties in quantifying all routes of both intake and loss of nitrogen. In practice, intake is often over-estimated due to unconsumed food (crumbs, plate residue etc) and output under-estimated due to inaccurate collection techniques. As a consequence, nitrogen balance results tend to be biased toward a positive balance which leads to the under-estimation of nitrogen/protein requirements (Elango et al., 2010). For more details on the nitrogen balance method and its limitations to estimate protein requirements for adults, see the FAO / WHO / UNU report (FAO, 2007).

Nitrogen balance is also used to guide protein intake in critically ill patients in whom excessive protein catabolism (e.g., from trauma, sepsis, infection, and burns) can result in high negative nitrogen balance values. Results of a recent systematic review and meta-analysis analysis suggest that improved nitrogen balance is associated with a better prognosis in critically ill patients (Zhu et al., 2022).

Other factors that may precipitate a negative nitrogen balance include excessive loss of nitrogen arising from fistulas or excessive diarrhea, inadequate intakes of protein or energy, and an imbalance in the dispensable : indispensable amino acid ratio.

In the past, total urinary nitrogen levels were rarely measured in routine hospital laboratories, so urinary urea nitrogen was determined and used to replace the estimation of total urinary nitrogen in some circumstances. To account for the nonurea nitrogen components of the urine not measured (e.g., ammonia, uric acid, and creatinine), a correction factor of 2g of nitrogen per day was commonly applied. This factor was derived from studies examining the relationship between measured total urinary nitrogen and measured urinary urea nitrogen in a variety of nutritional and clinical conditions (Blackburn et al., 1977; MacKenzie et al., 1985). An additional correction factor of 2g was also added for the dermal and fecal losses of nitrogen that were also not measured ( MacKenzie et al., 1985). Nitrogen intake was determined accurately by analyzing the nitrogen content of the diets or parenteral/enteral formulas using the micro-Kjeldahl technique. Alternatively, specific conversion factors were used to calculate the nitrogen content of parenteral solutions containing free amino acids, and for mixed diets, 16% of the protein intake was assumed to be protein.

However, many factors can affect the validity of adjusted urinary urea nitrogen as a measure of nitrogen balance in clinical patients (Konstantinides et al., 1991), and the use of the estimated nitrogen balance method has declined.

16a.2.2 Factorial approach

For infants >6mos, children, pregnant, and lactating women, current protein require­ments are based on the factorial approach (FAO, 2007; (IOM, 2005; EFSA, 2015) as noted earlier. In this approach the additional nitrogen (as protein) needed for the deposition of newly formed tissues or secretion of breast milk is added to the requirement for maintenance for an adult. As the main­tenance need is based on nitrogen balance, the factorial approach is subject to the limitations described above in Section 16a.2.1. Moreover, whether the calculated additional amounts of nitrogen required for depositing newly formed tissues and secretions for children, pregnant and lactating women, are correct is unclear, especially in view of the debate surrounding the efficiencies of protein utilization (Millward, 2012). For more discussion, see Sections 16a.3.2 and 16a.3.3.

16a.2.3 Indicator amino acid oxidation

In light of the limitations of nitrogen balance, the FAO, 2007 report notes nitrogen balance has ..serious technical drawbacks that may result in require­ment values that are too low. Moreover, the U.S Institute pf Medicine (IOM, 2005) has stated, ...due to the shortcomings of the nitrogen balance method noted earlier, it is recom­mended that the use of nitrogen balance should no longer be regarded as the 'gold standard' for the assess­ment of the adequacy of protein intake and that alternative means should be sought. However, the only other method that has been used to assess protein require­ments to date is the indicator amino acid oxidation (IAAO) method, described below.

The IAAO method was developed to determine amino acid and protein require­ments in baby pigs and later used to determine amino acid and protein require­ments in humans (Ball & Bayley, 1986; Kim et al., 1983; Humayun et al., 2007). It is a minimally invasive technique employing a stable isotope-labeled indis­pensable amino acid to determine amino acid or protein require­ments (Elango & Ball, 2016). The premise of the method is that there is no long-term storage of amino acids in the body so excess amino acids must be partitioned between incor­pora­tion into protein and oxidation (Elango, et al, 2012; Holt et al., 1962) . This principle was validated in an animal study by Ball & Bayley (1986).

When protein or a single indis­pensable amino acid is provided below its require­ment in the diet, all others (including the indicator amino acid), are in relative excess and will be oxidized as shown in Figure 16a.1 (Ball & Bayley, 1986). As the intake of protein increases (from deficient to excess) there is a decline in indicator oxidation, reflecting whole-body protein synthesis. Oxidation of the indicator amino acid is highest when the dietary intake is below the protein pr amino acid require­ment. Once the require­ment is met for the limiting amino acid / protein, there is no further change in oxidation of the indicator amino acid, and the point of inflection, where oxidation stops declining and plateaus, is denoted as the breakpoint (Figure 16a.1). The breakpoint is determined using two-phase linear regression and indicates the requirement (equivalent to the Estimated Average Requirement or EAR) of the limiting (test) amino acid or protein (Elango et al., 2012). The upper 95% confidence interval provides a surrogate estimate of the FAO safe indi­vid­ual intake (equiv­alent to the IOM Recommended Dietary Allowance (RDA).

For the method, an oral dose of the stable isotope is given, breath and urine samples are collected, and only a single day is required to adapt to each of the test intakes, unlike the nitrogen balance method. For more details, see Bross et al. (1998).

Several limitations of the IAAO method in determining protein require­ments have been highlighted by Courtney-Martin et al. (2016). They include (a) whether the indicator amino acid (i.e., phenyl­alanine) becomes limiting and impacts the esti­mated protein require­ment; (b) the protein require­ment is determined in the fed state and does not capture the fasted state; and (c) the diet is provided as continuous hourly formulas comprised of free crystal­line amino acids and not as whole protein foods in a 3‑4 meal format comparable to that eaten by most people. For responses to these concerns, see Humayun et al., (2007), Rafii, et al., (2015), and Mao et al. (2020). For discussion on the use of the IAAO method to redefine protein require­ments, see Section 16a.3.4.

16a.3    Nutrient Reference Values for protein

Nutrient reference values (NRVs) for protein have been set by several expert groups, although inconsistencies exist on the terminology and approaches applied and the recom­men­dations set. In most cases, two NRVs for protein have been set for life-stage groups from aged 6mos to >70y. The Average Requirement (AR) (or equiv­alent) is defined as the median protein require­ment for healthy indi­vid­uals of a particular sex and life-stage group. Hence, the AR (or equiv­alent) refers to the intake level that meets the protein require­ment of half of the healthy indi­vid­uals in that sex and life-stage group. The AR is used to evaluate the adequacy of protein intakes of population groups. In contrast, the second NRV, termed a Safe Individual Intake by FAO (2007), and the Recommended Dietary Allowance by IOM (IOM, 2005) is defined as the 97.5th percentile of the distribution of indi­vid­ual require­ments, nominally the average + 1.96SD. Hence any indi­vid­ual with a safe indi­vid­ual intake (or equivalent) will have a very low risk (i.e., <2.5%) of deficiency (intake less than require­ment) (FAO, 2007).

Currently, due to lack of data, no Tolerable Upper Intake Level (UL) for protein has been set by FAO (2007), IOM (2005), or EFSA (2015). The UL represents the highest level of daily nutrient intake that is likely to pose no risk of adverse effects for almost all people.

In the following sections, the NRVs for protein set by FAO (2007) for global use are outlined. Discrepancies in the NRVs for protein set by FAO (2007), the IOM (2005), and ESFA (2015) are also highlighted.

16a.3.1 Protein requirements for adults

A meta-analysis of nitrogen balance studies (Section 16a.2.1) has been used to estimate protein require­ments for adults by all three expert groups. Protein require­ments were defined as the minimum amount of nitrogen required to achieve zero balance (FAO, 2007; EFSA, 2015; IOM, 2005). When based on per kg body weight basis, adult values for the AR and safe indi­vid­ual intake for protein are the same for all three expert groups. These are 0.66 g/kg BW/d for the AR (or equiv­alent) and 0.83 g/kg BW/d for the safe level of intake (or equiv­alent) based on a protein digestibility corrected amino acid score of 1.0. Note that currently these values remain the same for the elderly and for those consuming only plant-based products due to insufficient available data for these subgroups.

16a.3.2 Protein NRVs for infants, children, and adolescents

All three agencies also applied the factorial model to derive protein NRVs based on protein needs for main­tenance plus growth for infants from 6mos onwards and for children and adolescents up to 18y. FAO applied the adult main­tenance value (0.66g/kg/d), plus growth costs from total body potassium (TBK) studies of protein gain adjusted with an efficiency value of 58%.

Table 16a.2 NRVs for protein (g/kg/d) for weaned infants and children (0.5‑10y) and for children and adolescents (11‑18y) by sex. From FAO (2007).

Ages (years)	Average Requirement (median)	Safe individual intake (+1.96 SD)
0.5	0.12	1.31
1	0.95	1.14
2	0.79	0.97
3	0.73	0.90
4	0.69	0.86
5	0.69	0.85
6	0.72	0.89
7	0.74	0.91
8	0.75	0.92
9	0.75	0.92
10	0.75	0.91
11	M 0.75    F 0.73	M 0.91    F 0.90
12	M 0.74    F 0.72	M 0.90    F 1.89
13	M 0.73    F 0.71	M 0.90    F 1.88
14	M 0.72    F 0.70	M 0.89    F 0.87
15	M 0.72    F 0.69	M 0.88    F 0.85
16	M 0.71    F 0.68	M 0.87    F 0.84
17	M 0.70    F 0.67	M 0.86    F 0.83
18	M 0.69    F 0.66	M 0.85    F 0.82

The two protein NRVs expressed as g/kg/day are presented in Table 16a.2. EFSA (2015) adopted these protein NRVs, but IOM (2005) did not. The IOM adopted different values for main­tenance and for the efficacy of protein utilization for growth; see IOM (2005) for more details.

For infants aged 0‑6 mos, different methods were used by FAO (2007) and IOM (2005) to set the protein NRVs; none were set by EFSA (2015). For infants <6mos FAO applied the factorial approach, applying a main­tenance value derived from nitrogen balance studies in infants and children plus growth costs from TBK measure­ments (Butte et al., 2000) and an efficiency of 0.66 for main­tenance and growth (FAO, 2007) (2007). See Table 16a.3 for two NRVs for protein set by FAO (2007).

Table 16a.3 NRVs for protein (g/kg/d) for infants <6mos set by FAO (2007).

Age (months)	Average Requirement (median)	Safe individual intake (+1.96 SD)
1	1.41	1.77
2	1.23	1.50
3	1.13	1.36
4	1.07	1.24
6	0.98	1.14

In contrast, the IOM (2005) did not consider there was enough scientific data to establish protein NRVs for infants aged 0‑6mos. Instead, IOM established an Adequate Intake (AI) based on the average amount of human milk consumed (0.78L/d) and an average protein content of human milk (11.7g/L) during the first 6mos of lactation. The resulting AI for protein is 9.1g/d or 1.52g/kg for a 6kg infant.

16a.3.3 Protein NRVs for pregnant and lactating women

A factorial method was also used by all three expert groups to estimate the protein require­ments of pregnant and lactating women. For pregnant women, the require­ment was derived from adult protein main­tenance needs (0.66g/kg/d) esti­mated from mid-trimester weight gain plus mean protein deposition esti­mated from TBK accretion in normal healthy pregnant women gaining 13.8kg by the end of the third trimester (Emerson et al., 1975; FAO, 2007; King, 1975; Pipe et al., 1970). However, estimates for efficiency of dietary protein utilization varied across the three expert groups. FAO adopted an estimate of 42% (Emerson et al., 1975; IOM (2005) of 43%, whereas EFSA (2015) applied 47%, the value derived for adults. FAO (2007) include gestational stage-specific recom­men­dations for additional protein intakes to support a 13.8kg weight gain during pregnancy (Table 16a.4), an approach also adopted by EFSA (2015). In contrast, IOM (2005) provide only one value equivalent to the FAO Average Requirement and Safe Individual Intake for pregnancy. For more details see FAO (2007), IOM (2005), and EFSA (2015).

Table 16a.4: Recommended additional protein require­ments during pregnancy and lactation set by FAO / WHO / UNU (FAO, 2007) *For pregnancy: Safe indi­vid­ual intake calculated as the average require­ment plus allowance for estimation of variation of 12% **For lactation: Safe indi­vid­ual intake calculated as mean + 1.96 SD with 1 SD calculated from a coefficient of variation of 12% For lactation: Average require­ment represents efficiency of milk protein synthesis applied to true protein.

	Pregnancy additional protein requirement (g/d)
	Average requirement (median)	Safe individual intake (+1.96 SD)
1st Trimester	0.5	0.7
2nd Trimester	7.7	9.6
3rd Trimester	24.9	31.2
	Lactation additional protein requirement (g/d)
1 mo	16.2	20.2
2 mo	15.6	19.5
3 mo	14.8	18.5
4 mo	14.3	17.0
5 mo	14.4	18.1
6 mo	15.5	19.4
> 6 mo	10	12.5

For lactating women (Table 16a.4) the mean equiv­alent milk protein output was esti­mated from: (a) mean rates of milk production from well-nourished women exclusively breastfeeding (first 6mos postpartum), and partially breastfeeding (second 6mos), and (b) mean concentration of protein and non-protein nitrogen in human milk (Dewey et al., 1996). The efficiency of protein utilization associated with milk protein production was esti­mated as 47% based on data from non-lactating adults. This approach was consistent across the three expert groups. However, in contrast to FAO (2007) and EFSA (2015), IOM only specified a single value intended to cover the increased protein needs (i.e., 1.1g/kg/d) throughout the entire lactation period. For more specific details, see IOM (2005) and EFSA (2015).

Questions have been raised concerning the NRVs for protein. Millward (2012) argues that the values for the efficiencies of protein utilization applied in the factorial models, especially during pregnancy, are too low. Pencharz (2013) suggests that the protein NRVs recommended by FAO, especially for children, are also too low based on data using the IAAO method. Details of the IAAO method have been discussed earlier (Section 16.2.3); details of its application for protein requirements are outlined below.

16a.3.4 Protein NRVs based on the IAAO method

The IAAO method (Section 16a.2.3) was first applied to the study of protein require­ments in humans by determining the protein require­ments of the same young adult males who participated in the original nitrogen balance study (Humayun et al., 2007). The IAAO response was measured when feeding graded intakes of protein ranging from 0.1 to 1.8g/kg/d. NRVs corresponding to the FAO AR and safe indi­vid­ual intake of 0.93 and 1.2g/kg/d respectively, were determined. Subsequently, the IAAO method has been used to determine protein require­ments in other life-stage and physiological groups (Table 16a.5) (Rafii et al., 2015; Elango et al., 2011; Stephens et al., 2015). All the IAAO studies were conducted under measured energy needs and the derived protein require­ments range from 10‑18% of the energy from protein.

Table 16a.5: Summary of protein requirement estimates determined by IAAO method. Note EAR (Estimated Average Requirement) equiv­alent to Average Requirement; RDA (Recommended Daily Allowance) equiv­alent to Safe Individual Intake set by FAO.

Life-stage group	EAR	RDA
Children (6-10y)	1.3	1.55
Young Adult Males	0.93	1.2
Pregnant Females: Early Gestation	1.2	1.66
Pregnant Females: Late Gestation	1.52	1.77
Older Adult Males (>60y)	0.94	1.24
Older Adult Females (>60y)	0.96	1.20

Note these IAAO protein require­ments are consistently higher (by approx­imately 30‑40%) across the lifespan compared with the estimates determined by the traditional nitrogen balance studies. For debate on the use of IAAO to determine protein require­ments, see Hoffer 2012; Millward & Jackson, 2012; Pencharz, 2013; Elango & Ball, 2016.

16a.4    Methods for assessing amino acid require­ments

Several methods have been developed to improve the nitrogen balance method to determine the require­ment for an indi­vid­ual indis­pens­able amino acid. Of these, amino oxidation methods are described briefly below. These methods are now used to determine the amino acid requirements in adults (>19y) by FAO / WHO / UNU (2007).

16a.4.1 Direct Amino Acid Oxidation (DAAO)

This method was the first to demonstrate that amino acid require­ments derived using the nitrogen balance method were underesti­mated by a factor of 2 to 3 (Zello et al., 1995). However, because the DAAO method can only be used to estimate the require­ments of those amino acids whose carboxyl carbon is released into the bicar­bonate pool and measurable in breath (Pencharz & Ball, 2003), other methods with broader application have been developed.

16a.4.2 Indicator amino acid oxidation (IAAO)

This method can also be used to determine amino acids require­ments (Section 16a.4.3), when instead of feeding graded intakes of protein, graded intake levels of the test amino acid are provided. The protein source is a crystalline amino acid mixture patterned after egg protein pattern and provided at 1.0g/kg/day in adults. Additionally, the indicator amino acid is a different indis­pens­able amino acid than the amino acid being tested, often ¹³C‑labeled L‑phenylalanine or ¹³C‑labeled L‑leucine. When determining amino acid require­ments, the IAAO study day (day 3) diet is formulated to be isocaloric and isonitrogenous. The nitrogen content of the diets is adjusted according to the level of amino acid intake level being studied. For all dietary indis­pens­able amino acid require­ments except the branched chain amino acids, L‑alanine is used to keep the diets isonitrogenous. When assessing branched chain amino acid require­ments, L‑serine is used to balance nitrogen content.

As discussed in Section 16a.2.3, opponents argue that the IAAO fed-state amino acid require­ments do not capture 24h fasted and fed cycles. Additionally, concern about whether prior adaptation to the test amino acid intake is required, led to the modification termed the 24h indicator amino acid balance (IAAB) method and described below.

16a.4.3 24h indicator amino acid balance (IAAB)

The IAAB method is an adaption of the IAAO method led by concerns that the IAAO fed-state amino acid require­ments do not capture 24h fasted and fed cycles, and the need for prior adaptation to the test amino acid intake. The method includes a 6d adaptation period to the test amino acid intake, followed by measure­ment of an indicator amino acid oxidation over a 24h period (12h fasted and 12h fed) (Elango et al., 2012). The indicator amino acid used is usually ¹³C‑labeled L‑leucine. Balance is calculated as the difference in leucine oxidation and leucine intake. The ¹³C‑labeled L‑leucine isotope is administered intra­venously over 24h and during this time blood and breath samples are collected to measure isotopic enrichment in the plasma. Thus, the IAAB protocol is more invasive than the IAAO protocol, so a fewer number of amino acid levels are assessed in each participant: between 2 to 4 intakes.

As reviewed by Elango et al., (2012), no systematic difference in amino acid require­ment estimates have been found using the 24h IAAB or 8h fed state IAAO protocol. Also, for a given amino acid require­ment determined by the 24h IAAB method, the require­ments in the fed and fasted state are the same (Kurpad, Raj, et al., 2002; Kurpad, Regan, et al., 2002). Prior adaptation to the test amino acid intake does not alter the IAAO-derived require­ment for threonine (Szwiega et al., 2023).

16a.4.4 Protein NRVs for indispensable amino acids

Unlike the require­ments for protein, amino acid require­ments in adults (>19y), including older adults, have been set by FAO / WHO / UNU using data from studies conducted using both IAAO (Section 16a.4,2) and IAAB (Section 16a.4.3) methods; see FAO (2007) for more details. All IAAO and IAAB studies were conducted under measured energy needs and the derived protein require­ments range from 10‑18% of the energy from protein.

It is noteworthy that EFSA (2015) did not derive requirements for the indispensable amino acids . They argued that amino acids are not provided as individual nutrients but in the form of protein. They were also of the view that more data are needed to obtain precise values for the individual indispensable amino acids. The US Institute of Medicine has published recommendations for amino acids as part of the Dietary Reference Intakes (DRI) (IOM, 2005)

Table 16a.6: Average indis­pens­able amino acid requirements (mg/kg per day) set by FAO / WHO / UNU (FAO, 2007). Males and females combined.
HIS: histidine; ILE: isoleucine; LEU: leucine; LYS: lysine; SAA: sulfur amino acids; THR: threonine; TRP: tryptophan; VAL: valine.

Age/life-stage group	HIS	ILE	LEU	LYS	SAA	THR	TRP	VAL
1 month	36	95	165	119	57	76	29	95
2 month	26	69	121	87	42	55	21	69
3 month	23	60	105	75	36	48	19	60
4 month	21	54	95	68	33	44	17	54
6 month	20	52	90	65	31	41	16	52
1-2 yr	15	27	54	45	22	23	6.4	36
3-10 yr	12	23	44	35	18	18	4.8	29
10-14 yr	12	22	44	35	17	18	4.8	29
14-18 yr	11	21	42	33	16	17	4.5	28
> 18 yr	10	20	39	30	15	15	4	26

Table 16a.6 presents the values (mg/kg/day) for the average amino acid require­ments for adults set by FAO / WHO / UNU (FAO, 2007). Variability of require­ments for indi­vid­ual amino acids is unknown so values for the safe indi­vid­ual intake levels are approximated, by applying the same inter-indi­vid­ual coefficient of variation of the require­ments for the amino acids as that for total protein (i.e., 12%) (FAO, 2007). Conse­quently, the safe indi­vid­ual intake levels for the indis­pens­able amino acids are 24% higher than the corresponding average require­ment levels shown in Table 16a.6. Note the latter are 2 to 3 times higher than earlier 1985 FAO / WHO / UNU recom­men­dations based on nitrogen balance data (Millward, 2012). See FAO (2007) for a comparison table.

Unlike FAO / WHO / UNU (2007), the US IOM (2005) used data from studies that measured the minimum intake of each indispensable amino acid needed to maintain nitrogen balance in healthy adults.

For infants <6mos, values for the indis­pens­able amino acids require­ments are based on the average intake of indis­pens­able amino acids from breast milk from a healthy well-nourished mother (FAO, 2007). FAO recognizes that intakes derived in this way may be generous in relation to actual demands.

The US IOM (2005) bases the indispensable amino acid requirements for infants <6 mos on their average intake from breast milk from healthy, well-nourished mothers.

For older infants, children and adolescents, current FAO / WHO / UNU recom­men­dations for indis­pens­able amino acids were set using the factorial approach, and as such based on the main­tenance and growth components of the protein require­ment. The pattern of main­tenance amino acid require­ments was assumed to be the same as for adults on a mg/kg body weight basis, whereas for growth the pattern is based on the amino acid composition of the whole body; for more details, see FAO (2007).

Table 16a.7: Amino Acid requirements (mg/kg/BW) for pregnancy set by IOM (2005)
HIS: histidine; ILE: isoleucine; LEU: leucine; LYS: lysine; SAA: sulfur amino acids; THR: threonine; TRP: tryptophan; VAL: valine.

Life-stage	HIS	ILE	LEU	LYS	SAA	THR	TRP	VAL
Pregnancy	15	20	45	41	20	21	5	25

There is very little change in both the require­ments and pattern of indis­pens­able amino acids after aged 2y until adulthood, as shown in Table 16a.6.

No require­ments for indis­pens­able amino acids during pregnancy have been set by FAO / WHO / UNU (FAO, 2007). For more details on these requirements, see Millward (2012a). and (2012b). IOM (2005) has determined EARs for pregnancy (equate with FAO / WHO "average" require­ment) (Table 16a.7). The EAR is derived from factorial estimates based on adult amino acid requirements × 1.33 to take into account increased protein demands of pregnancy and the RDA is the factorial estimate based on 24% variability from the EAR. See Elango and Ball 2016 for a discussion of the IOM indispensable amino acid requirements for pregnancy.

16a.5    Food sources, dietary intakes, and quality of dietary protein

In many affluent countries, foods of animal origin (e.g., meat, fish, milk and dairy products) are the major source of protein in the diets of adults, with the proportion from the meat and dairy group varying with age and region. Most animal-source foods are considered to have high quality protein because their protein has both an optimal indis­pens­able amino acid composition and a high digestibility. Some plant-based foods such as legumes, nuts and seeds, and soy products are also considered high-quality sources of plant proteins because they provide a higher proportion of indis­pens­able amino acids than other plant foods. However, certain anti-nutritional factors present in plant foods or formed during processing may reduce the protein digestibility.

In the US NHANES 2015‑2018 survey, approx­imately two-thirds of total protein intake of adults was from animal sources, and one third from plant sources. Of the latter, the primary protein source was grains, mainly as refined grains, whereas the intake of protein from high quality plant protein sources was low (Hoy et al., 2022). Similar patterns have been reported among Canadians (Auclair & Burgos, 2021) and French adult populations (Salome et al., 2020). In general, the proportion of adults in these Western countries with protein intakes considered inad­equate is low, although in the United States, 12% of adults over 70y had inad­equate intakes which is of concern (USDA, 2019; Berryman et al., 2018).

A recent systematic review compared the intake and adequacy of nutrients in adults (mostly in Europe or North America) consuming plant-based diets compared to meat-eaters (Neufingerl & Eilander, 2021). Protein intake (as percent energy) was lower in those following plant-based diets compared to meat-eaters, although on average within recommended levels, a trend consistent with that in the US NHANES 2015‑2018 study (Hoy et al., 2022). Nevertheless, nutrient inadequacies existed across all the dietary patterns reviewed, including vegan, vegetarian, and meat-based diets. In general, however, replacing protein from animal sources with plant foods does provide a better intake of those dietary components associated with reducing chronic disease risk (i.e., lower intake of cholesterol and saturated fat and higher intake of dietary fiber) (Hoy et al., 2022).

Both quantity and quality impact the adequacy of dietary protein intakes. Protein quality of diets is mainly dependent on its composition of indis­pens­able amino acids, and their digestibility and bioavailability. In Western countries, the amino acid pattern of mixed diets meets the require­ment levels for most age groups. Even vegetarian mixed diets are likely to meet the require­ment levels if they contain complementary mixtures of plant proteins (IOM, 2005).

A recent study comparing the habitual indispensable amino acid intake of participants of the US NHANES, 2001-2018 reported that the intakes of the US population exceeded the recommended minimum population requirements. Less than 1% of those aged at least 19y did not meet the EAR for each indispensable amino acid Berryman et al., 2023).

In low-income countries, unlike affluent countries, plant-based foods often supply more than 50% of the total protein. Moreover, there is often excessive reliance on a single low protein plant-based staple which is a poor source of certain indis­pens­able amino acids. For example, cassava and maize are the main sources of dietary protein in many parts of Africa, and poor sources of leucine and methionine, and tryptophan and lysine, respectively. Hence, some concerns have been raised, especially among young children in low‑ and middle-income countries, that poor protein quality may be a factor limiting their growth (Ghosh et al., 2012).

The protein content of staple foods in low-income countries are often expressed as the proportion of the energy in the food provided by protein: i.e., the FAO / WHO / UNU protein : energy ratio (FAO, 2007). Using this approach, foods such as cassava, plantains, sweet potatoes and taros are classified as examples of poor sources of protein (in relation to their energy). Indeed, cassava has the lowest protein : energy ratio of any staple crop, common cassava cultivars having only 1% protein. Potatoes, unrefined maize, rice and sorghum are classified as "adequate" sources of protein, and groundnuts, beans and peas, cow's milk (skimmed), soybean, and dried fish as "good".

16a.5.1 Factors affecting the quality of dietary protein

The quality of dietary protein is largely evaluated based on its composition of indis­pens­able amino acids, and their digestibility and bioavailability. The latter affect how well the amino acids within the protein are digested, absorbed, and utilized for protein synthesis. All three factors are discussed below. For a detailed review of protein quality, see Boye et al. (2012).

Composition of amino acids affects protein quality. High quality or animal proteins provide all nine indis­pens­able amino acids in the correct proportions for the synthesis of body protein and other non-protein substances. Poorer quality proteins such as plant proteins are low in one or more indis­pens­able amino acid as noted earlier (IOM, 2005). The indis­pens­able amino acid that is present in the smallest quantity relative to the require­ments and thus limits the body's ability to fully utilize other amino acids in a given food or diet, is termed the "first limiting amino acid".

There is no official standardized method for amino acid analysis. The methods involve three steps: hydrolysis to break down protein into their constituent amino acids, followed by derivatization to improve their detectability, and finally chromatography to separate the derivatized amino acids. This final step is performed using techniques such as High-Performance Liquid Chromatography (HPCL) or Gas Chromatography (GC).

Digestibility refers to the net absorption of an amino acid following digestion (FAO, 2013). Assessment of digestibility is challenging due to the presence of endoge­nous nitrogen com­pounds (digestive enzymes, mucoproteins etc.) which are mixed with the ingested food in the intestinal tract. Digestibility can be measured as fecal or ileal digestibility. Both methods have limitations.

Fecal digestibility measures the amino acid absorbed across the entire gastro­intestinal tract (mouth to anus) (FAO, 2007). A limitation of this method is that a portion of the dietary protein that enters the large intestine is degraded by microbial activity. This leads to disappearances and/or appearances of amino acids from the digestive tract which have no nutritional contribution to the availability of the amino acids for protein metabolism. Hence, this results in an over‑ or under-estimate of digestibility depending on the food consumed.

Ileal digestibility quantifies the absorption of amino acids across the small intestine (mouth to terminal ileum). It has been adopted by FAO as more representative of the bioavailability of dietary amino acids, overcoming some of the limitations of fecal digestibility (FAO, 2013). When measuring ileal digestibility, endoge­nous amino acid components must be accounted for to yield the true ileal digestibility value. This step requires insertion of a naso-intestinal tube in humans and is highly invasive. However, in vitro digestibility methods are being explored, such as INFOGEST, that mimic oral, gastric, and intestinal phases of digestion (Brodkorb et al., 2019). Some hold promise for the measure­ment of ileal amino acid digestibility (Xipsiti, 2024).

It is posible that dual stable-isotope tracer method can be used to determine true ileal indis­pens­able amino acid digestibility in humans but requires validation. The method requires development of an isotopically intrinsically labeled food, which is then fed to subjects in small meals, after which the appearance of the labeled amino acids in the plasma is measured (Mansilla et al., 2020).

Bioavailability refers to the quantity of amino acids that are digested and absorbed in a form that is utilizable for synthesizing body proteins. Heat and processing affects the bioavailability of some amino acids (e.g, lysine, lysine, methionine, tryptophan and threonine) which can lead to overestimates of ileal digestibility (Batterham, 1992; Van Barneveld et al., 1994). Certain anti-nutritional factors present in plant foods or formed during processing can also interfere with bioavailability and digestibility. Examples of those naturally occurring include: trypsin inhibitors and haemagglutinins found in legumes; tannins in legumes and cereals; and phytates in cereals, legumes, and oilseeds. Antinutritional factors formed during processing include: Maillard reaction products (MRP) from heating or alkaline treatment, oxidized forms of sulfur amino acids, D‑amino acids and lysino-alanine; for more details, see Gilani (2012).

16a.5.2 Methods to evaluate protein quality

Evaluating protein quality involves assessing the capacity of sources of protein in both foods and diets to meet the protein and indis­pens­able amino-nitrogen require­ments (FAO, 2013). Two methods for evaluating protein quality are described, the first originally recommended by FAO / WHO in 1993, is termed the Protein digestibility-corrected amino acid score (PDCAAS) in which digestibility is adjusted using nitrogen fecal digestibility factors. The second, recommended by FAO / WHO in 2013 is called the Digestible Indispensable Amino Acid Score (DIAAS) and includes measure­ment of ileal digestibility.

Table 16a.8: FAO / WHO / UNU amino acid requirement pattern based on amino acid requirements of preschool-age children (2-5yrs).
^a Methionine & Cysteine; ^b Phenylalanine & Tyrosine. From WHO / FAO / UNU (2007).

Amino acid	Requirement (mg/g crude protein)
Isoleucine	28
Leucine	66
Lysine	58
Total sulfur amino acidsa	25
Total aromatic amino acidsb	63
Threonine	34
Trytophan	11
Valine	35
Total	320

Protein digestibility-corrected amino acid score (PDCAAS) is a method used to evaluate the quality of a protein based on both its amino acid composition and its digestibility. The method was recommended by FAO / WHO in 1993 and WHO / FAO / UNU in (2007). It is based on comparison of the first limiting amino acid in the test protein (i.e., the amino acid present in the lowest concentration in a food) with the concentration of that amino acid in a reference scoring pattern, typically the amino acid require­ments of preschool-age children (i.e., 1‑2y) (considered the most demanding in terms of protein quality of indis­pens­able amino acids) (Table 16a.8). For school children and adolescents, the amino acid scoring pattern for 3‑10y range is recommended (WHO / FAO / UNU, 2007). This comparison generates a limiting amino acid score for the test protein which is then multiplied by digestibility. For the PDCAAS method, nitrogen fecal digestibility (i.e., digestibility over the whole intestine) is applied. The calculation is shown below:

\[\small \mbox {PDCAAS =} \frac{\mbox {mg of limiting amino acid in 1g test protein}} {\mbox {mg of the same amino acid in 1g of the reference protein}}\] \[\small \mbox { × true fecal digestibility}\]

Note initially, the highest PDCAAS score any protein could achieve was 1.0. This indicated that the protein provided all the indis­pens­able amino acids in the amounts required by the body. Any scores exceeding 1.00 were truncated to a maximum of 1.0, reflecting the view that amino acids supplied above the require­ments do not have additional physiological value and will be catabolized. Truncation was also said to simplify the comparison of protein quality among different foods.

Truncation, however, has been criticized and is no longer practiced in the modified PDCAAS described below. The choice of an indis­pens­able amino acid pattern for preschool children has also been of concern as it can underestimate the protein quality of a food consumed by adults who require protein for main­tenance and not growth (Mansilla et al., 2020). For more details of the calculation of PDCASS scores, see Marinangeli & House (2017). For PDCAAS for selected foods, see Boye et al. (2012).

Digestible Indispensable Amino Acid Score (DIAAS) relies on measures of true ileal digestibility of indi­vid­ual amino acids and lysine bioavailability because they better reflect the true quantity of amino acids digested and absorbed. It also avoids truncation of the score obtained. The most limiting digestible indispensable amino acid content (DIAA) defines the DIAAS value of a protein. For DIAAS, FAO (2013) updated the amino acid reference scoring patterns and recommended three age-related reference patterns: 0‑6mos (infant based on breast milk pattern); 0.5‑3y (based on pattern for 0.5y infant); and >3y (based on pattern for 3‑10y old child) for the remainder of the population; see Table 16a.9.

Table 16a.9:Amino-acid scoring patterns (mg/g protein requirement) for children for protein quality.
HIS: histidine; ILE: isoleucine; LEU: leucine; LYS: lysine; SAA: sulfur amino acids; AAA: Aromatic amino acids;THR: threonine; TRP: tryptophan; VAL: valine.
From FAO / WHO / UNU (2013).

Age (y)	HIS	ILE	LEU	LYS	SAA	AAA	THR	TRP	VAL
0.5	20	32	66	57	27	52	31	8.5	43
1–2	18	31	63	52	25	46	27	7.4	42
3–10	16	31	61	48	23	41	25	6.6	40

To calculate DIAAS of single protein source, data on the complete indispensable amino acid (IAA) composition of the protein, the crude protein content (CP), and the IAA standardized ileal digestibility (SID) are required. For a given IAA_y, DIAA_y ratio is calculated as follows: \[\small \mbox {DIAAy ratio =} \frac{\mbox {IAAy × SIDy}} {\mbox {Reference pattern score IAAy}}\] where IAAy is expressed as mg/g CP. The lowest DIAA ratio leads to the DIAAS value of the protein. \[\small \mbox {DIASS = 100 × lowest DIAA ratio among IAAs}\] Based on the 0.5 to 3-year-old reference pattern (see Table 16a.9), Herreman et al. (2020) classified pork meat, casein, egg, and potato proteins as excellent quliaty proteins with an average DIAAS above 100, whey and soy proteins as high-quality protein with an average DIAAS ≥75, whereas gelatin, rapeseed, lupin, canola, corn, hemp, fava bean, oat, pea, and rice proteins were in the no quality claim category (DIAAS <75).

Values for DIAAS can also be calculated for protein mixtures. To accomplish this, average values of each single protein for IAAy and SIDy in the mixture must be obtained. See Herreman et al. (2020) for details of the calculation. The maximum DIAAS calculated among all possible ratios represents the optimal protein mixture. Care must be taken to ensure, where possible, that the values for IAAy and SIDy selected have taken into account any processing and cooking conditions. A protein source or protein mixture with a DIAAS of 100 or above indicates that none of its indispensable amino acids is limiting and this source of protein has the potential to meet physiological requirements.

This method was first introduced by FAO (2013), but at that time there was a lack of human digestibility data available that utilized DIAAS. However, in a later joint FAO‑IAEA meeting in 2022, valid in vitro models of ileal amino acid digestibility were presented and a database of ileal digestibility of proteins and indi­vid­ual amino acids in foods created. Collection of data on the effects of processing and storage on protein quality was also emphasized. Access to such a database in the future will facilitate calculation of the protein quality of indi­vid­ual foods and mixtures of foods. (Tome et al., 2024).

16a.5.3 Importance of protein quality

The call by the United Nations to promote sustainable diets and health (FAO, 2013), has sparked renewed concern about the impact of protein quality on the adequacy of dietary protein intakes. Until recently, most studies have assessed dietary protein adequacy by comparing crude total protein intake (g/day) with estimates of protein require­ments, with no adjustments for the potential effect of protein quality using the methods outlined in Section 16a.4.2. These studies have suggested there is a low risk of protein inadequacy in developed countries based on national survey data (USDA, 2019; EFSA, 2015). Recently, protein adequacy has been re-examined after correcting for protein quality (using DIAAS, Section 16a.4.2) and based on usual protein intakes from adults in U.S NHANES (2001‑2018) dataset (Moughan et al. 2024). When protein quality was assumed to be 100% (i.e., DIASS of 1.0 (100% utilizable protein), 11% of adults 19‑50 y had utilizable protein intakes below the EAR. This proportion increased markedly as the assumed DIASS declined (e.g., 25% for DIASS=0.8), and was even higher in the elderly (i.e.,72% for DIASS=0.8). These prevalence estimates are higher than any NHANES estimates based on unadjusted crude total protein intake (USDA, 2019); and prompt concern with the global trans­ition to plant-based proteins (Willett et al., 2019).

In many low-income countries, plant-based foods can supply up to 50% of the total protein intake, especially in resource-poor households. Conse­quently, whether the intakes of protein, after adjustment for quality and digestibility, are adequate, especially for optimal growth in children, warrants investigation. Ghosh et al. (2012) examined the effect of adjusting total dietary protein for quality and digestibility based on PDCAAS (Section 16a.5.2) on risk of protein inadequacy in a meta-analyses of 2005 food balance sheet data from 186 developing countries and regions. They showed a higher risk of protein inadequacy in the food supply in these regions after accounting for protein quality. Moreover, adjusted protein supply (i.e., utilizable protein) was negatively associated with the prevalence of stunting.

However, food balance sheet data provide information on protein supply, and not intake at the indi­vid­ual level. A few studies in young children in low-income countries have examined the adequacy of their protein intakes at the individual level and accounted for protein quality. In the earlier studies, protein intakes were adjusted to account for digestibility and amino acid score in some studies (Beaton et al., 1992; Yeudall et al., 2005). At the time, the adjusted protein intakes were not deemed inad­equate based on the adjustment methods employed, and unlikely to be a primary factor limiting the growth of these preschoolers in Egypt, Kenya, Mexico, and Malawi, despite cereals being their primary protein source.

More recently, Arsenault and Brown (2017) applied the PDCAAS method to whole diets to estimate the amount of utilizable protein in the complementary diets of children aged 6‑36 mos from seven low-income countries. Most children were found to consume utilizable protein intakes greater than their esti­mated require­ments, apart from younger breastfeeding children (6‑10mos) whose energy intakes from complementary foods were low. However, in settings where cassava is the major dietary staple (e.g., Nigeria and Kenya), there are concerns that protein intake (unadjusted for protein quality) may be inad­equate in children aged 2‑5y (Stephenson et al., 2014). In a study in Nigeria, cassava was reported to have a very low protein quality, with leucine being the first limiting amino acid, resulting in DIAAS being inadequate (<75) (de Vries-ten Have et al., 2020). Accumulating evidence indicates that infection, intestinal dysfunction, and catch‑up growth are likely to increase protein require­ments in malnourished children, so their intakes of utilizable protein may not be adequate to meet their needs.

Clearly, when evaluating the adequacy of protein intakes in both developed and low-income countries, issues of protein digestibility and protein quality must be considered. Such data could inform future revisions of the protein require­ments and support the development of food-based dietary guidelines, especially in low-income countries.

Information on protein quality has several other important applications. For instance, food security and nutrition could be improved by cultivating locally available high-protein quality crops (Xipsiti, 2024) and enhancing the protein quality of traditional plant-based diets by supplementing with appropriate limiting indis­pens­able amino acids or combining different foods that complement one another. Finally, there is a growing need for data on the protein quality of climate-resilient crops to address sustainability issues (Xipsiti, 2024).

Acknowledgments

The author is grateful to Michael Jory for the HTML design and his tireless work in directing the trans­ition to this HTML version.