Chapter-2 - Ingles
Chapter-2 - Ingles
Chapter-2 - Ingles
2
PROTEIN AND AMINO ACIDS
Yong-Ming Yu1, MD, PhD
Naomi K. Fukagawa2, MD, PhD
1
Massachusetts General Hospital, Harvard University, Boston, MA, United States
2
USDA ARS Beltsville Human Nutrition Research Center, Beltsville, MD, United States
SUMMARY
This chapter briefly introduces basic biochemistry of protein and amino acids (AAs) and their chemical
structures, followed by the discussion on the dynamic process of protein and amino digestion, absorption,
and their turnover in whole body and specific tissues in vivo as the biochemical and physiological basis in
assessing protein requirements. The latter part of the chapter provided an in-depth discussion of the latest
knowledge of the indicators used to assess protein requirements and AA requirements. The chapter
finishes with a discussion of the current recommended intakes for protein and AAs, along with aspects of
their use in nutritional assessment and issues in developing recommendations of protein/AAs for health
maintenance throughout the life cycle.
Keywords: Amino acids; Nitrogen; Protein; Protein requirement; Protein structure; Protein synthesis; Protein
turnover; Recommended Dietary Allowance; Signaling.
Present Knowledge in Nutrition, Volume 1 Copyright © 2020 International Life Sciences Institute (ILSI).
https://doi.org/10.1016/B978-0-323-66162-1.00002-0 15 Published by Elsevier Inc. All rights reserved.
16 2. PROTEIN AND AMINO ACIDS
FIGURE 2.1 Structure of the 19 amino acids and 1 imino acid (proline) used in human protein synthesis. Names of amino acids essential
(indispensable) for humans are underlined.
cannot synthesize them; histidine is essential primarily the process of gluconeogenesis, or converted to other
for children as there is some capacity for it to be synthe- AAs. In healthy subjects, AA oxidation provides about
sized when not required in high amounts such as during 15% of daily energy. In certain clinical conditions, such
growth. The other 11, called nonessential, may be made as severe burn injury or trauma, the oxidation of AAs
by the body from essential AA or glucose. In this chap- is increased leading to increased breakdown of the
ter, the terms essential and nonessential are used. body’s own proteins and a state of protein wasting.
Many factors affect essentiality; for example, some in- Therefore, in many clinical and disease conditions, pa-
fants, especially those born prematurely, or adults under tients require increased protein intake.5
certain health circumstances, cannot make adequate One can consider two pools representing AAs and
amounts of several of them, making them conditionally body protein. The first, a free or labile AA pool, consists
essential.1,2,3 It has been suggested that in the neonate, of free AAs dissolved in body fluids. The proteins in the
only five are truly dietarily nonessential or dispensable tissues and circulation are grouped into a second pool.
(alanine, aspartate, glutamate, serine, and probably These two pools are in constant interaction, due to the
asparagine).4 continual degradation and resynthesis of proteins as
In addition to protein synthesis, AAs are oxidized as proteins turn over at various rates, some of which may
an energy source, converted to carbohydrates through be short and others very long, or not degraded in the
Section A. Macronutrients
I. Introduction 17
body, such as keratin. The free or labile AA pool expands or more AAs bonded together by peptide bonds, and
as AAs are supplied from absorbed AAs derived from an intermediate string between 4 and 10 AAs is an oligo-
dietary proteins and from de novo synthesis in cells peptide. Peptides differ from proteins by virtue of their
(including those of the gut, which are also a source of size. Traditionally, peptide chains that are short enough
nonessential AAs); this labile pool decreases with the to be made synthetically from the constituent AAs are
loss of AAs by oxidation, excretion, or conversion to called peptides, rather than proteins.7
other metabolites.
Dietary protein is the primary source of AAs. Foods
of animal origin, such as meat, poultry, fish, eggs, and C. Functions of Proteins
dairy products, are good sources of essential AAs. Plant Proteins are the most abundant organic molecules in
sources of protein often contain lesser amounts of one or cells and are fundamental to cell structure and function.
more of the essential AAs so a single source of plant pro- All proteins in humans are constructed from the same
tein often cannot provide the full spectrum of essential basic set of 20 AAs linked by peptide bonds. In addition,
AAs to meet requirements, particularly during growth. some contain sulfur AAs and others have ligands with
Soy is an exception as it contains all the essential AAs phosphorus, iron, zinc, and copper.
in amounts adequate for adults. Hence, vegetarians
must consume a variety of plant proteins to balance The roles of proteins are versatile; examples of proteins
the intake of essential AAs. For example, a grain such and their functions are described in Box 2.1. These high-
as wheat is low in lysine while corn has low amounts lighted functions are only a fraction of the many roles
of tryptophan and lysine, but when either is combined that proteins play, but the list conveys some sense of
with legumes such as garbanzo beans (low in methio- the immense variety of proteins and their importance
nine), the diet contains adequate amounts of the limiting in the body.8 More recent research reveals increasingly
AAs, assuring complementarity.6 complex roles for protein and AAs in regulation of
body composition, maintenance of organ/tissues such
Peptides as bone, gastrointestinal (GI) function, bacterial flora,
Peptides are composed of AAs chemically linked by glucose homeostasis, cell signaling, and satiety.9
peptide bonds. The peptide bond always involves a sin-
gle covalent link between the a-carboxy (oxygen-bearing Transport proteins
carbon) of one AA and the a-amino (or imino in the case Transport proteins in plasma bind and carry specific
of proline) nitrogen of a second AA. In the formation of a molecules or ions from one organ to another. Some move
peptide bond from two AAs, a molecule of water is elim- around in the body fluids, carrying nutrients and other
inated. Dipeptides are two AAs linked by a peptide molecules. For example, hemoglobin carries oxygen from
bond, whereas three AAs bonded together by peptide the lungs to the cells throughout the body; lipoproteins
bonds are called tripeptides. Polypeptides consist of 10 transport lipids in the blood; and specific proteins, such
BOX 2.1
Section A. Macronutrients
18 2. PROTEIN AND AMINO ACIDS
as retinol-binding protein, carry vitamins (vitamin A). fluid. Each tRNA carries its AA to the mRNA, which
Other kinds of transport proteins are present in cell mem- dictates the sequence in which the AAs will be attached
branes and are adapted to bind and transport glucose, to form the protein strands. Thus, the mRNA ensures
AAs, and other nutrients across the membrane into cells. the AAs are lined up in the correct sequence. The
sequence of AAs in each protein ultimately determines
Signaling proteins its configuration to support a specific function.
Signaling proteins serve as signaling molecules that
Stage 4: Termination and release: As the AAs are lined
modulate multiple cellular processes, including protein
up in the right sequence and the ribosome moves
synthesis and a cell’s response to chemicals in the envi-
along the mRNA, an enzyme connects one AA after
ronment. Signal transduction plays an important role in
another in a peptide bond to rapidly form the
controlling cell function and homeostasis through a set
growing protein strand of 40 to over a 100 AAs. The
of chemical reactions that occur when a molecule, such
tRNA is freed from its corresponding AA to pick
as a hormone, attaches to a receptor on the cell mem-
another of its specific AA. When all the AAs have
brane. This is followed by a cascade of biochemical reac-
been attached based on the mRNA coding, the
tions inside the cell that eventually reach the target
completed protein is released.
molecule or reaction. The process thus triggers a series
of intracellular events and invokes a cellular response. Stage 5: Folding and processing: To achieve its
It is a method by which molecules inside the cell can biologically active form, the polypeptide that was
be altered by molecules outside. Each step of these formed undergoes folding into its proper three-
events involves biochemical reactions of proteins. An dimensional conformation. Before or after folding, the
example of this sequence is the multiple set of proteins new polypeptide may undergo processing by
that mediate the insulin transduction pathway. Insulin enzymatic action to remove initiating AAs; to
binds to a cell receptor called “insulin receptor sub- introduce phosphate, methyl, carboxyl, or other
strate” on cell surfaces; the receptor triggers a series of groups into certain AA residues on the polypeptide; or
biochemical reactions through a chain of signal proteins, to attach oligosaccharides or other prosthetic groups.
which eventually modulates the functions of insulin in
Stages 2e5, occurring in the cytosol, together are
the target cells, such as increasing or decreasing glucose
called translation. Transcription and translation are
transport into adipose and muscle cells.10
summed up by the central dogma of molecular biology:
DNA / RNA / Protein, with the formed protein pep-
tide chains undergoing posttranslational modifications.
II. NUTRIENT METABOLISM The chemical properties of AAs in a protein molecule
and their spatial configurations in a protein determine
A. Protein Synthesis its biological activity.
The information necessary for its production resides
A protein’s structure and shape determine its func- in the cellular nucleus as DNA, which makes up a large
tionality. Fundamentally, the process of protein synthe- component of genes. If a genetic error alters the mRNA
sis consists of the following stages; a detailed review code and thus the AA sequence of a protein, or if a
of synthesis, breakdown, and turnover is available11: mistake is made in copying the sequence, an altered pro-
Stage 1: Transcription: To inform a cell of the sequence tein will result, sometimes with dramatic consequences.
of AAs for a needed protein, a stretch of
deoxyribonucleic acid (DNA) in the cell nucleus Resulting protein structure
serves as a template for making a strand of Proteins are large polypeptide molecules with
ribonucleic acid (RNA), called messenger RNA molecular weights of 6500 to 205,000 Da. The three-
(mRNA), that carries a code which lists the order of dimensional configuration of a protein molecule deter-
the AAs that will be needed to make a given protein. mines the function of the protein. Two classes of strong
Each mRNA strand copies exactly the instructions for bonds (peptide and disulfide) and three classes of weak
making a specific protein that the cell needs. bonds (hydrogen, hydrophobic, and electrostatic or salt)
stabilize most protein molecular configurations. A single
Stage 2: Initiation of the polypeptide chain: The
protein molecule may contain one or more protein struc-
mRNA leaves the nucleus through the nuclear
ture types: primary, secondary, tertiary, and quaternary
membrane bearing the code for the polypeptide to be
structure.
made and attaches itself to the protein-making
Primary structure refers to the “linear” sequence of
machinery of the cell, the ribosomes.
AAs in a polypeptide chain and the location of disulfide
Stage 3: Elongation: Another form of RNA, transfer bonds, if present. Secondary structure refers to “local” or-
RNA (tRNA), collects specific free AAs from the cell dered structure via hydrogen bonding, mainly within the
Section A. Macronutrients
II. Nutrient Metabolism 19
peptide backbone. Tertiary structure refers to the “global” of organs and tissues. Proteolysis is typically catalyzed
folding of a single polypeptide chain. Hydrogen bonding by cellular enzymes called proteases and may also occur
involving groups from both the peptide backbone and by intramolecular digestion. Low pH or high tempera-
the nonpolar side chains is important in stabilizing ter- tures can also cause nonenzymatic proteolysis.
tiary structure. Quaternary structure refers to the stable Intracellular proteolysis removes damaged and ab-
association of multiple polypeptide chains resulting in normal proteins and prevents their accumulation, thus
an active unit united by forces other than covalent bonds serving to regulate cellular processes by removing en-
(not peptide or disulfide bonds). The forces that stabilize zymes and regulatory proteins that are no longer
these aggregates are hydrogen bonds and electrostatic (or needed. Intracellular proteins are degraded into free
salt) bonds formed between AA residues on the surfaces AAs through two mechanisms: lysosomal proteolysis,
of the polypeptide chains. which degrades cytoplasmic components via autophagy
The protein structure is formed through the processes into the intracellular membrane-enclosed lysosome,
of translation and posttranslational modification. The and the ubiquitin-proteosome system. The autophagy-
configuration of a protein determines the physical prop- lysosomal pathway is normally a nonselective process,
erty and the function of the protein. For example, the but it may become selective upon starvation whereby
configuration of an enzyme protein determines the cata- proteins containing peptide sequences biochemically
lytic sites of action and the ability of the individual related to Lys-Phe-Glu-Arg-Gln (KFERQ) are selectively
enzyme to function. Another example of such posttrans- broken down.12 Ubiquitin is a basic 76-AA peptide that
lational modification is of the residues of proline and joins to lysine residues selectively on the proteins
lysine in the protein procollagen, which are hydroxylat- marked for degradation through the action of a multiple
ed to hydroxyproline and hydroxylysine residues, group of enzymes of the proteasome to degrade proteins
permitting cross-linking of proteins to increase strength into free AAs. The free AAs can then be oxidized, con-
and stability of the resulting collagen. verted to other metabolic intermediates, or reused for
protein synthesis in the cycle of protein turnover.13,14
B. Protein Breakdown
Protein breakdown or proteolysis is the biochemical
C. Digestion
process by which proteins are “disintegrated” into Dietary protein is digested in the GI tract to free AAs
smaller polypeptides or AAs. Protein degradation oc- and peptides by the action of gastric acid and enzymatic
curs during the digestion and absorption of ingested hydrolysis. Multiple AA transport systems exist for sub-
and endogenous GI proteins as well as within the cells sequent absorption of the freed AAs and peptides.15
Section A. Macronutrients
20 2. PROTEIN AND AMINO ACIDS
Section A. Macronutrients
II. Nutrient Metabolism 21
TABLE 2.2 Some proteolytic enzymes responsible for digesting protein.
the duodenum deactivates the pepsin and provides an result of the cross talk between protein metabolism
optimal pH for the activity of the pancreatic enzymes. and host immune response.22
By releasing proteolytic enzymes initially in their inac-
tive form, the enzyme-forming cells are protected from
self-digestion.19,11 D. Absorption
Enzymatic hydrolysis The final end products of protein digestion (e.g., free
AAs, dipeptides, and tripeptides) are now ready for ab-
Enterokinase (also known as enteropeptidase) is an sorption within the small intestine. To a lesser extent, the
enzyme secreted from the brush border of the small intes- absorption of intact proteins may occur through leaks at
tine, also in response to secretin and CCK. Enterokinase cell junctions, which accounts for the immune response
serves to activate trypsinogen to trypsin, which in turn to foreign proteins associated with food allergies. 11,23
converts many of the other proenzymes into their respec- However, the vast majority of AAs are absorbed in the
tive enzymatic forms for ongoing protein hydrolysis.11 At proximal small intestine, leaving less than 1% of dietary
this point, endogenous protein sources from sloughing protein to be excreted in the feces.20,11 Through both
mucosal cells or other secretory proteins are also recycled sodium-dependent and sodium-independent active
through the digestive process to form the general pool of transport systems, AAs pass from the lumen of the small
AAs. intestine into the enterocyte. The various AA carrier sys-
The presence of protein within the gut signals further tems have an affinity for specific AAs.24 The rate of AA
enzyme secretion. When the majority of the protein has absorption may vary depending upon the chemical
been digested, the presence of unbound trypsin acts as a properties of the AA. Essential AAs are absorbed faster
feedback mechanism to turn off further secretion of than nonessential AAs: methionine and the BCAAs
trypsinogen by the pancreas.19 At this point, the by- leucine, isoleucine, and valine are the most rapidly
products of pancreatic protease digestion, free AAs absorbed.25 Competition can occur between AAs for
and peptides of two to six AA components, now enter transport by a carrier system.
the final phases of digestion. The brush border of the The active transport of di- and tripeptides across the
small intestine produces a variety of peptidases, which brush border membrane of the enterocyte also involves
allow peptide digestion to continue through the distal a competition for transporters but utilizes different car-
small intestine and ileum.20,21,11 rier systems than those of the free AAs. In fact, peptide
absorption occurs more rapidly than an equivalent
Role of gut microbes mixture of free AAs.26,27 It is estimated that 67% of die-
In recent years, the importance of gut microbes in the tary protein is absorbed as di- and tripeptides with the
digestion, absorption, metabolism, and transformation remaining 33% absorbed as free AAs.11,28,29 Once inside
process of dietary protein in the GI tract has been recog- the enterocyte, these peptides are finally converted to
nized. Like all dietary components, AAs can be metabo- free AAs by peptide hydrolases. The final phase of AA
lized by gut microbes into numerous microbial absorption occurs across the basolateral membrane of
metabolites that may affect gut microflora. It has been the enterocyte by diffusion or active transport into the
proposed that the ratio between protein and carbohy- portal circulation (Fig. 2.2).
drate or even a low-protein diet may be advantageous
compared to a diet high in dietary protein, which may
lead to an increase of pathogenic microorganisms with
E. Protein and Amino Acid Metabolism
associated higher risk of metabolic diseases due to cross Body protein is in a constant dynamic state; the body
talk between dietary protein and gut microbiota compo- breaks down protein into free AAs which enter the free
sition and function. It has been proposed that this is a AA pool. There are constant fluxes among different AA
Section A. Macronutrients
22 2. PROTEIN AND AMINO ACIDS
pools, especially the flux between muscle and visceral tissue may provide qualitative information on the
organs. The free AAs are reutilized to synthesize proteins possible changes occurring in either protein synthesis
or serve as intermediates of energy metabolism, via gluco- or breakdown in specific tissues (such as muscle),
neogenesis and oxidation. Therefore, AAs are constantly instead of the dynamic fluxes of intermediate
trafficking between protein-bound and protein-free pools. metabolites. The methods developed to quantify the
The turnover rates of individual proteins vary dynamic aspects of protein metabolism are listed in
depending on the function of the protein and its loca- Table 2.3.
tion. For example, peptides and hormones have rela-
tively high rates of synthesis and degradation, in Nitrogen balance; Nitrogen balance is the difference be-
contrast to structural or plasma proteins. In a normal tween the amount of nitrogen taken in and the amount
subject during a 24-h period, the nitrogen in the dietary excreted or lost and has been the traditional approach
protein consumed matches nitrogen output, indicating to evaluate protein nutritional balance.
that the rates of protein synthesis and breakdown reach Mathematically,
a balance without net protein gain or loss. Almost all ni-
trogen is lost in urine, with small amounts excreted in Nitrogen balance ¼ Nitrogen intake Nitrogen output
feces and much smaller amounts as sweat and miscella-
neous losses. Urinary nitrogen losses in healthy adults where nitrogen intake is the total nitrogen administered
are about 11e15 g when consuming 70e100 g of protein. either by an enteral or parenteral route, and nitrogen
These losses are primarily urea, with small amounts of output is the sum of urinary, fecal, dermal, and other
ammonia, creatinine, uric acid, and other nitrogen- body fluid losses of nitrogen.
containing end products of protein metabolism. In a clinical setting (see the classical work of Wil-
However, the balance between protein synthesis and more37), since the majority of nitrogen output is urine,
breakdown is altered by disease and certain physiolog- and about 80% of urinary nitrogen content is from
ical states, as well as changes in nutrient intake.30 urea, approximate nitrogen output is estimated as
The body of a 70-kg man is about 16% or 11 kg of pro- Nitrogen output (g/day) ¼
tein. About 43% is in skeletal muscle, 15% in structural Urinary urea nitrogen (mg/100 mL) urinary volume
tissues and 15% in blood. 31 About 50% of total body (L/day)/100 / 20% of urinary urea losses þ 2 g37.
protein is made up of four proteins, actin and myosin
in skeletal muscle, collagen, and hemoglobin, with However, the concentration of urinary urea nitrogen is
collagen comprising 25% of the total protein. In malnu- affected by stress and can be increased with increased uri-
trition, this proportion can rise to 50% because of the nary excretion of nonurea nitrogen coming from nonpro-
substantial loss of noncollagen proteins, whereas tein compounds such as creatinine or nucleic acids.
collagen itself is retained.32 Visceral tissues (e.g., kidney Stool, dermal, and other losses are usually estimated to
and liver), while containing together only about 7 kg or be about 2 g nitrogen/day, unless there is diarrhea, os-
10% of the total protein, are very active in metabolism. tomy/fistula losses, or skin exudate such as with large
Other organs such as the brain, lung, heart, and bone area burns.38 There are also other losses not accounted
contribute the remainder. Newborn infants have less in for clinically, such as menstrual losses, seminal fluid,
muscle and much more in brain and visceral tissue and nasal secretions. In clinical settings, a 24-h urine
than adults proportionally.33 Protein turnover in infants collection is required for urea nitrogen measurements.
on a body weight basis is greater than in young adults, However, others have used shorter collection times and
which is greater than in elderly.33 Visceral tissues such extrapolated those results to a 24-h collection. Adjust-
as liver and intestine together are thought to be respon- ments need to be made in certain patients.39,40
sible for up to 50% of whole body protein turnover34,35 Many other factors affect the accuracy of nitrogen
while even though skeletal muscle contains the largest balance measurement. In addition to renal dysfunction,
component of body protein, it is estimated to contribute errors in estimating intake or incomplete collections of
only w25% to daily turnover.34,36 urine, stool, fistula, or ostomy losses may affect balance
results. It is worth mentioning that the measurement of
whole body nitrogen balance is relatively simple and
Methods to measure protein turnover noninvasive, is a rough estimate of the difference be-
Protein turnover rates in both healthy individuals tween body protein synthesis and breakdown, and is
and critically ill patients may be measured by several a poor indicator for characterizing inter-organ AA
different methods. Each of them has some limitations, flux and changes in protein turnover.41 More sensitive
but these in vivo measurements have provided most of tools exist for determining substrate balance across an
the quantitative information of protein metabolism in organ and AA circulation such as isotopically labeled
living humans. Measurements of gene expression in tracers.
Section A. Macronutrients
II. Nutrient Metabolism 23
TABLE 2.3 Methods in place to measure protein metabolism. Tracer methodology to measure protein turnover; To further
trace how ingested protein is utilized and influences
Methods Characteristics
protein catabolism, nonradioactive, stable isotope tracer
Nitrogen balance • Measure of nitrogen input (diet) AAs to measure whole body protein turnover were first
minus nitrogen output (urineþ used in the 1950s but were fraught with methodological
estimates of fecal and other difficulties. It was not until 1969 that a simpler tracer
endogenous losses)
• Can be used in clinical setting
method was developed by Picou and Taylor-Roberts
• Decreased accuracy in certain using 15N-glycine.42 It was based on the assumption
clinical conditions (renal that administered 15N-glycine tracer is evenly
dysfunction, high output fistula, distributed throughout the whole body nitrogen pool.
and ostomy) The estimate was based on the following series of
• Provides information on the net
difference between protein
equations:
synthesis and breakdown; 15
N-glycine administration rate=
however, the specific changes in
either protein synthesis or 15
N-nitrogen excretion in urine rate
protein breakdown are unknown (2.1)
¼ whole body nitrogen turnover rate=
Tracer methodology using • The constant IV infusion of an
stable isotope-labeled amino amino acid(s) or bolus injection
total urinary nitrogen excretion
acids method is used in human studies
Therefore, from the rate of 15N-glycine entry via
• The subject is required to be at a
metabolically steady state administration into the body nitrogen pool, and the rate
15
• Ideal for measuring acute N-nitrogen excretion in urine, and estimation of total
changes in subjects urinary nitrogen excretion, the whole body nitrogen
• Important research tool that turnover rate, Q, can be calculated at a steady condition:
allows direct in vivo
measurements of protein N influx ¼ N outflux ¼ Q (2.2)
synthesis and breakdown rates
in the whole body
Tracer plus imaging technology • Using radioactive positron Q ¼ N influx
(27a) emission tomography (PET)
with tracer amino acid, such as ¼ N released from protein breakdown
11
C-methionine, which does not þ dietary N intake (2.3)
have degradation pathway in
muscle, can image the kinetics of
this tracer under PET camera to Q ¼ N outflux
quantify the rate of protein
synthesis in muscle tissue ¼ N incorporated into proteinðprotein synthesisÞ
• Drawback to its use is the short- þ urinary N excretion ð2:4Þ
lived radioactivity of the gamma
tracer
• Does not measure protein
breakdown
Therefore, from the measurements of total isotope-
labeled nitrogen intake, 15N-output in the urine, the total
Arteriovenous amino acid • Invasive methods for subjects as dietary nitrogen intake rate, and the urinary nitrogen
differences across organs a tracer amino acid must be
infused. Samples of blood are
excretion rate, the rates of whole body protein break-
obtained from both the arterial down (Eq. 2.3), and whole body protein synthesis
and venous catheters (Eq. 2.4) can be determined.
• These measurements are made The tracer methods thus can provide quantitative in-
across muscle beds. Samples of formation on the rates of both protein synthesis and
blood are obtained from both the
arterial and venous catheter
breakdown compared to the nitrogen balance method.
The isotope tracer method provides the fate of the die-
Urinary creatinine excreted • Indirect measurement of muscle tary protein within the body, in terms of its influence
over a 24 h period mass
• Has high variation
on protein anabolism and catabolism of the host.
3-Methyl histidine excretion • Can be used as a measure of Measurements of protein turnover in specific tissue organs
protein degradation as 3-methyl
histidine is not oxidized
in vivo; Understanding how nutrition supports individ-
• Compromised accuracy with ual organ protein metabolism is important in physiology
organ dysfunction and pathophysiology. There are a number of methods
used to measure protein synthesis in specific organs.
Section A. Macronutrients
24 2. PROTEIN AND AMINO ACIDS
1. One approach is to directly measure the rate of stable splanchnic beddcirculation in the GI tract, liver, spleen,
isotope-labeled AAs incorporated into specific and pancreas.50 In the presence of a low-protein diet, GI
proteins, into muscle or gut mucosa, or into multiple tract growth is preserved over that of other peripheral
circulating proteins. The measurement is conducted tissues or muscle.51,52 A large proportion of the AAs uti-
by a constant infusion of stable isotope-labeled amino lized by the GI tract, especially the small intestine, are
tracer until a steady state of the tracer in the protein is used for the synthesis of secretory proteins. Within the
reached; the increment of tracer enrichment in the intestinal cells, AAs may also be used for synthesis of
protein-bound AA pool is used to calculate the nucleic acids, the antioxidant glutathione, Apo proteins
protein synthesis rate.43 as components of lipoproteins, or other nitrogen-
2. Another approach is the measurement of containing compounds.51
arteriovenous (A-V) difference in concentration of As an important energy source to the GI tract, dietary
AAs in arterial blood inflow to a tissue or organ and glutamate and aspartate account for a significant pro-
the venous blood outflow of the same organ tissues; portion of the energy used by the small intestine.51
from the difference between the nitrogen inflow and Glutamine released by the lung and skeletal muscle
outflow, the protein metabolism in the organ can be into the circulation is a primary source of energy for
assessed. A positive nitrogen balance indicates net the enterocyte and may have trophic effects on the
nitrogen accretion in the organ, while negative mucosal cells of the GI tract. Glutamine, as a precursor
balance indicates net loss of protein in that organ.44 to nucleotide synthesis, is also used by the immune sys-
3. More complicated models combine the A-V tem, which is abundant within the GI tract as gut-
difference of concentration of stable isotope labeled- associated lymphoid tissue.53
AA tracers.45,46 These models involve complicated
mathematical calculations to quantify net protein Liver
balance and rates of protein synthesis and breakdown Liver is a key organ for protein and AA metabolism
in specific tissues such as muscle, under different due to its high capacity for uptake and metabolism of
nutritional conditions, and disease conditions.47 AAs. Because of its anatomical location in the GI tract,
4. Positron emission tomography (PET) imaging is a the liver helps to regulate the flow of AAs and other
possible new approach to measure protein nitrogenous compounds into the systemic circulation
metabolism in muscle. Because methionine is not from ingested food or from endogenous protein degra-
degraded in muscle tissue, injection of the short-lived dation in tissues.47 Approximately 57% of the AAs
(T1/2 ¼ 20 min) 11C-labeled methionine followed by extracted by the liver are either oxidized or used to syn-
imaging the metabolic behavior of the methionine thesize plasma proteins in the liver.11 Important roles
tracer in muscle provides protein synthesis rates in that the liver plays in metabolism are discussed below.
skeletal muscles of limbs. It has been validated in
animal studies48 and applied to human subjects.49 Degradation of amino acids; Most of the AAs are degraded
in the liver. The biochemical processes include deamina-
Other approaches to measure protein breakdown; Protein tion and transamination of AAs, followed by conversion
turnover in skeletal muscle accounts for a substantial of the nonnitrogenous part of those molecules to glucose
portion of whole-body protein turnover.34 Muscle or lipids. Several of the enzymes used in these pathways
wasting has been a metabolic feature of multiple (for example, alanine and aspartate aminotransferases)
disease conditions, especially in critical illness.39 Since are commonly assayed in serum to assess liver damage.54
N-s-methylhistidine (3-methylhistidine) is a
component of actin and myosin, it is not reutilized for Nonessential amino acid synthesis; Liver is the primary or-
protein synthesis following the breakdown of muscle gan for synthesis of most of the nonessential AAs from
protein but is quantitatively excreted in the urine. Its essential AAs, such as tyrosine from phenylalanine
presence can thus indicate the degree of muscle and cysteine from methionine, or de novo synthesis of
breakdown occurring at the time of measurement. AAs from carbohydrates such as alanine from pyruvate
and aspartate from oxaloacetate. Carrier systems also
exist for AA transport into the hepatocyte as the only
F. Protein Metabolism in Specific Organs site for oxidation of five of the essential AAs, but not
the BCAAs.20,11
Gastrointestinal tract
The GI tract is a metabolically active organ and is the Protein synthesis; AAs for use in the liver are transported
primary vehicle by which dietary nutrients enter the across the basolateral membrane of the enterocyte into
body. It has been estimated that a minimum of 25% of the portal vein circulation (Fig. 2.2). Most of the plasma
protein consumed is used in its first pass within the proteins, including apo proteins and acute-phase
Section A. Macronutrients
II. Nutrient Metabolism 25
proteins, are synthesized and released from liver to the as compared to 80% for phenylalanine.56 Therefore, a
general circulation. Albumin, the most abundant large portion of ingested BCAAs enter the systemic cir-
plasma protein, is synthesized almost exclusively by the culation for metabolism, largely by the muscle.57
liver. Some plasma proteins synthesized in the liver Skeletal muscle uptake of AAs following a protein-
play important roles, including maintenance of oncotic containing meal leads to net protein synthesis within
pressure in the blood (albumin), transport functions the muscle.20 The fed state supplies AAs from dietary
(albumin, prealbumin, transferrin, lipoproteins, some protein as a source of energy and helps replenish AA
globulins), clotting functions (fibrinogen, prothrombin), stores. Muscle mass is maintained in a relative steady
and enzymatic reactions (alanine aminotransferase, state with a fraction of the muscle broken down and
aspartate amino-transferase). resynthesized daily (protein turnover).11 Most AAs are
released from the muscle in proportion to their concentra-
Urea synthesis; The liver removes ammonia from the tions within the muscle.58,59 The release of BCAA is more
body through the urea cycle (KrebseHenseleit) conservative than that of other AAs. Alanine and gluta-
pathway. Ammonia, a metabolite of nitrogen com- mine are released from muscle at higher concentrations
pounds, has high toxicity. It can accumulate from exog- than their concentrations within muscle, playing a key
enous protein intake or from protein breakdown during role in muscle BCAA metabolism by participating in
catabolic states such as trauma or sepsis.54 If not rapidly removal of the excess nitrogen produced during BCAA
and efficiently removed from the circulation, it impairs degradation, which mostly occurs in muscle.58,60
the central nervous system function. The normal blood The glucoseealanine cycle is another biochemical
concentration of the ammonium ion (NHþ 4)
process for skeletal muscle to eliminate nitrogen waste
is <50 mmol/L; a doubling to 100 mmol/L can lead to a while replenishing its energy supply. Glycolysis, the
disturbance in consciousness, and at 200 mmol/L, process of glucose oxidation within the muscle, pro-
coma and convulsions may occur. The major biochem- duces alanine via the action of alanine transaminase or
ical pathway to eliminate ammonia is via the urea cycle alanine aminotransferase. Alanine is then transported
exclusively found in the liver. Amino groups donated by through the bloodstream to the liver where its carbon
ammonia and L-aspartate are converted to urea in this atoms are used in gluconeogenesis to produce glucose,
cycle, while L-ornithine, citrulline, L-argininosuccinate, which is then delivered back to the muscle. The amino
and L-arginine act as intermediates. Urea (NH2eCOe group from alanine is converted in the liver to urea
NH2) is subsequently released into the circulation and and eventually excreted by the kidneys.61
excreted in the urine by the kidneys. Within muscle, leucine is the only AA completely
oxidized for energy. During a fast, leucine levels rise
both in the bloodstream and within the muscle. Leucine
Carbon metabolism; For many AAs, ketoacid analogues
oxidation by skeletal muscle in turn increases, produc-
are formed when the amino group(s) is removed, and
ing acetyl coenzyme A that acts as an energy source
some are thus ready to enter oxidative metabolic path-
for the muscle while simultaneously sparing pyruvate
ways. For example, both a-ketoglutarate (from gluta-
oxidation in the tricarboxylic acid cycle. Pyruvate is
mate) and pyruvate (from alanine) are intermediates of
then reduced to lactate in the absence of oxygen (anaer-
the tricarboxylic acid pathway in glucose oxidation.
obic respiration) by the action of lactate dehydrogenase
The carbon skeletons of others participate in degrada-
and released by the muscle. The carbon skeleton of py-
tion pathways that result in intermediates that are then
ruvate is thus transferred to the liver in the form of
metabolized in these oxidative pathways. Thus, protein
lactate as the precursor for gluconeogenesis. Leucine
contributes to the energy supply of the body, with about
oxidation by skeletal muscle may have some effect on
15%e16% of dietary energy consumed as protein in the
sparing these essential gluconeogenic precursors.62
United States.55 This contribution to energy needs in-
creases during periods of caloric restriction or when car- Kidney
bohydrate stores (such as from glycogen) are depleted.
The kidney plays an important role in the interorgan
exchange and synthesis of a variety of AAs.51 The kid-
Skeletal muscle
ney preferentially extracts specific AAs, such as glycine,
After passing through the splanchnic organs (mainly alanine, glutamine, glutamate, phenylalanine, and
the liver), comparatively little of the BCAAs are aspartate. The kidney is also a major site for arginine,
oxidized versus other AAs that are metabolized within histidine, and serine production.20 Additionally, the kid-
liver. Using stable isotope-labeled tracers, it was esti- ney plays an important role in the conversions of
mated that 2% of ingested leucine was oxidized phenylalanine to tyrosine and glycine to serine.62 Aside
when it passed the splanchnic region, mainly the liver, from the liver, the kidney is the only organ with the
Section A. Macronutrients
26 2. PROTEIN AND AMINO ACIDS
Section A. Macronutrients
III. Dietary Requirements 27
is reflective of energy nutritional status, while the arm included when appropriate. Since the major losses of ni-
muscle circumference (or diameter) is reflective of pro- trogen under most conditions are via urine and feces, the
tein nutritional status (unless a myopathy or neuropathy protein requirement can be estimated by extrapolating
is present).76 In addition, urinary creatinine excretion the obligatory losses to a point where it is assumed
has been used as a reflection of muscle mass,74,77,75 but that protein needs (as nitrogen) equal the obligatory pro-
it is not very sensitive. Serum proteins such as albumin, tein lost as nitrogen (termed nitrogen equilibrium) (see
prealbumin, transferrin, and retinol binding protein are Table 2.3 for the various methods).
often used clinically in evaluating protein status in hos- While this method was used in the 1960’s through
pitalized patients but often of limited use as values may 1980’s, the weakness in the method is that feeding the
be affected by fluid retention, drugs, etc. They may be of amount of protein estimated as being lost did not result
greater use as a screen for the general population. in nitrogen equilibrium, in part due to the need for addi-
In determining adequacy of requirements, defined as tional nitrogen required to metabolize dietary protein
the lowest daily intake for a nutrient that meets the need (considered the efficiency of utilizing dietary protein)
of apparently healthy individuals, approaches have and because of the curvilinear relationship between pro-
been taken to estimate requirements from short-term tein intake and nitrogen retention.83,84 An additional
metabolic studies. Unlike most required nutrients, the issue is how to estimate the additional nitrogen (as pro-
body has many mechanisms to adapt to the varying tein) needed to meet the needs for growth for infants and
levels of intake of dietary protein. This adaptation oc- children and during pregnancy and lactationdamounts
curs due to the relatively large body pools that can be where efficiency of utilization is known to be less than
accessed when specific AAs or nitrogen is needed. 100%.
When no protein is consumed, protein breakdown con-
tinues at a comparatively higher than protein synthesis, Nitrogen balance method
with a decrease in nitrogen excretion. Nitrogen excretion The balance method is typically used for nutrients
decreases to a relatively constant level provided energy that are not metabolized or whose excretion products
is adequate; and this lowest level of nitrogen loss when can be obtained. A given intake is consumed, and after
not consuming protein has been termed basal or endog- equilibrium is reached at this level of intake, all routes
enous losses. These endogenous losses have been corre- of excretion are measured or estimated. When intake is
lated with basal metabolic rate and body weight.78,79 equal to excretion, or “0” balance, it is assumed that
In older individuals, these losses are the same as for the individual’s need for that nutrient is met at that level
younger individuals based on body weight but higher of intake. It does not provide an understanding of the
based on lean body mass.80 Energy must be adequate mechanisms in place that result in changes in excretion.
to prevent the degradation of body protein to meet the Since the major component of dietary protein is nitro-
needs of glucose and carbon skeletons of nonessential gen, and for most proteins approximately 16% is nitro-
AAs.81 Unless the correct balance of essential AAs is pre- gen, nitrogen intake can be measured, as can excretion
sent in the diet in the correct amounts (refer below to in carefully controlled studies in apparently healthy
protein quality), utilization of dietary protein will be people. Nitrogen balance occurs when nitrogen intake
diminished. equals the amount of nitrogen excreted in urine, feces,
skin, and miscellaneous losses.
This method of estimating human protein require-
B. Approaches to Assess Protein Adequacy/ ments generated sufficient data for use in estimating
Status total protein (nitrogen) requirements.85 One assump-
tion in using this method is that when adult needs
Factorial method are met or exceeded, after a period of 6e10 days
This method has been employed to determine protein consuming a specific intake level, nitrogen intake
requirements in the past 82 and is based on estimating equals nitrogen excretion (and no growth occurs such
endogenous nitrogen losses (also called obligatory nitro- as might with muscle building). When intakes are inad-
gen losses) as described above when a person is fed a equate, negative nitrogen balance results, meaning that
diet over a period of time, usually >6 days, that is essen- more nitrogen is excreted than is consumed, due to net
tially free of protein, but with adequate energy intake to losses of body protein. In order to ensure that the
maintain body weight. Estimates (usually by measure- amount of protein is being evaluated and not the
ment) of the amounts lost in feces, hair, seminal fluid, need for a specific essential AA, high-quality proteins
menstrual losses, sweat, and skin losses are added to are utilized as test proteins to prevent negative nitro-
the amount of nitrogen excreted in urine. Additional gen balance due to a limiting essential AA86,87
amounts for growth or losses due to lactation are (Table 2.3).
Section A. Macronutrients
28 2. PROTEIN AND AMINO ACIDS
As mentioned, multiple days of consuming a constant Plasma amino acid response method
level of dietary protein are needed for adaptation to This method depends on the use of a test essential AA
reach a new steady state of nitrogen excretion.88,89 that is followed as the intake of the AA is increased from
Such studies also require accurate nitrogen balance mea- an inadequate amount to levels above adequacy; at levels
surements: if not all, the dietary protein is consumed below requirement, the circulating concentration of the
thus overestimating actual intake and not all the losses essential AA is not only low but changes little in response
are obtained or accurately estimated thus underestimat- to increases in intake of the AA; as the intake amount is
ing nitrogen losses, false positive nitrogen balances may near the requirement level, the level in plasma becomes
be obtained.90 more sensitive and starts to increase and continues to in-
crease at higher levels. The point at which the relation-
Statistical analysis of nitrogen balance data ship between intake and plasma concentration starts to
While studies in healthy adults have shown that pro- increase (becomes a new linear relationship) is consid-
tein requirements can be met through the use of nitrogen ered to be an estimate of the requirement. This method
balance methodology, the relationship between intake has been extended to measure the changes in plasma con-
and requirements near the point of nitrogen equilibrium centration of the essential AA from the postabsorptive to
does not appear to be linear. In assessing the data avail- the fed state postconsumption,98 expecting to rise only
able at the time, the Institute of Medicine (IOM) report when the dietary supply of the AA is greater than the in-
on protein requirements released in 200285 evaluated dividual’s requirement.
three methods to compare data from multiple research
studies that evaluated protein requirements in men, Amino acid oxidation methods
women, elderly, and from nonanimal protein-based di- A number of methods have been developed to
ets.85 Using only data from nitrogen balance studies in improve on nitrogen balance to determine essential
which levels of nitrogen (protein) intake that were found AA requirements. With the direct amino acid oxidation
to be inadequate as well as adequate, three methods of method, the essential test AA is labeled with 13C, and
interpolation were evaluated: a smooth nonlinear production of the label as 13CO2 is then taken as the mea-
model,91,83 a two-phase linear model 92,93 and a linear sure of irreversible oxidative loss of the AA. For almost
model.89,94 The linear interpolation model was thus all of the essential AAs, the inability of the human to
used to estimate both the individual requirements (the synthesize the carbon skeleton is responsible for its es-
intakes predicted to result in zero balance) and thus sentiality. When the requirement is reached, then the
the distribution of protein requirements.85 low constant oxidation rate starts to increase proportion-
Some have argued that this may underestimate actual ately. The most salient problem arises from the reliance
protein needs, as it depends on equilibration to a steady on the determination of a breakpoint in the oxidation
state at the lowest level of protein intake at which nitro- of the test AA. One limitation of this method is that it
gen balance may be achieved, and given that little is can only be used for BCAA, phenylalanine, and lysine
known about corresponding changes in body pools, given their oxidation steps. A subsequent modification
lean body mass may be adversely affected at such min- of this method was to conduct studies over 24 h (24 h
imal levels.95,96 In particular, reference has been made to AA balance method) to include a period of fasting and
the possible need for additional levels of dietary protein feeding.
in the elderly,97 where decreased muscle mass is Subsequently, the indicator amino acid oxidation
frequently observed. method was developed and is based on measuring AA
oxidation; it uses the catabolism of the carbon skeleton
of an indicator (nonlimiting) AA as a carbon analogue
C. Assessment of Amino Acid Adequacy of nitrogen balance. When a single essential AA is pro-
vided below its requirement, it acts to limit the ability
Indicators for assessing essential amino acid needs
to retain other nonlimiting AAs in body protein, which
The primary method to determine the requirement then are oxidized at higher rates until the essential AA
for an individual essential AA focuses on measuring is at a level at which protein synthesis and breakdown
the relationship between the intake of the AA in an is constant.85 For further discussion of these methods,
otherwise adequate diet and a marker of nutritional ad- see the DRI report.85
equacy. Markers can be the effect of the AA on nitrogen
balance or the concentration of the AA resulting from its
metabolism and utilization. Research studies over the
last three decades using nitrogen balance-derived values
D. Reference Intake Levels
for essential AAs in adults are lower than values derived Reference intakes for dietary protein established in
by the other methods.85 2002 as part of the Dietary Reference Intake (DRI)
Section A. Macronutrients
III. Dietary Requirements 29
TABLE 2.4 Dietary Reference Intakes for dietary protein for Canada and the United States.44
AI, Adequate Intake; EAR, Estimated Average Requirement; RDA, Recommended Dietary Allowance.
process of the IOM for the United States and Canada are requirements were evaluated for elderly, for women,
listed in Table 2.485 The Recommended Dietary Allow- and for studies using only plant proteins, no differences
ance (RDA) is defined as the average daily level of intake were identified. Thus the EAR remains the same for these
sufficient to meet the nutrient requirements of nearly all subgroups.
(>97%) healthy individuals in a specific population Given that protein requirements for children need to
group, while the Estimated Average Requirement reflect needs for maintenance and for growth, a factorial
(EAR) is the amount that is sufficient to meet the approach was used to estimate the average requirement
nutrient requirements of half of a population group (EAR) for children.85
and is used to evaluate adequacy of nutrient intakes.85
Recommended dietary allowance
Estimated average requirements The RDA for the United States and Canada is calcu-
As part of the DRI process for the United States and lated at 2 standard deviations above the EAR. Since
Canada, a metaanalysis of available nitrogen balance the nitrogen balance data for adults were distributed
studies94 was used to the estimated the EAR for nitrogen in a log normal fashion, the CV was 12% based on the
(and thus for dietary protein) in healthy adults85 (see statistics employed, and the RDAs were rounded after
Table 2.4). The criterion of adequacy used for the protein multiplying the EAR by 1.24.85 This CV of 12% was
EAR is based on the lowest continuing intake of dietary also used to increase the EARs to obtain the RDAs for
protein that is sufficient to achieve body nitrogen equi- other age groups (see Table 2.4).
librium (zero balance).
Although the data indicated that women had a lower Adequate intake
nitrogen requirement than men per kg body weight, As with other nutrients, the DRI category established
perhaps because women on average have a larger fat for young infants is an Adequate Intake (AI), based on
mass (15 vs. 28%, respectively) when controlled for lean the average amount in human milk consumed in the
body mass, no gender differences in protein requirements age group (0.78 L/d for 0e6 mo).85 The average protein
were found. The reference body weights of 70 and 57 kg content of human milk for this age group was estimated
for men and women, respectively, were used to establish to be 11.7 g/L. The resulting AI is 9.1 g/d or 1.52 g/kg
the daily EAR (see Table 2.4). The metaanalysis included for a 6 kg infant (Table 2.4).
19 published nitrogen balance studies from many coun- While an AI was estimated to be 14.4 g/d or 1.6 g/
tries for 235 individuals given at least three levels of nitro- kg/d based on the reference weight of 9 kg for older in-
gen intake for periods of 10e14 days of nitrogen intake or fants 7e12 mo, concern that other protein sources might
that only provided group data for multiple levels of not be equivalent to that in human milk resulted in an
intake (n ¼ 174 individuals).94 While differences in EAR being established for older infants 7e12 mo based
Section A. Macronutrients
30 2. PROTEIN AND AMINO ACIDS
on the factorial method.85 The EAR for older infants incomplete proteins, even if they have similar amounts
7e12 mo is thus 1.0 g/kg with the RDA set at 11 g/ of total protein per gram of the food item (meaning
d (Table 2.4). that they have higher amounts of nonessential AAs).
For example, a serving of 3 oz of meat contains about
25 g of protein, whereas 8 oz of cooked oatmeal contains
E. Tolerable Upper Intake Level 6 g of protein and is limited in lysine based on compar-
ison with protein scoring patterns (see Table 2.5 and dis-
As part of the DRI process, the scientific literature was
cussion below).
evaluated regarding effects of consuming very high pro-
tein diets to determine if there was adequate informa-
Estimates of protein intake in the United States
tion upon which to establish a Tolerable Upper Intake
Level (UL). Populations worldwide have consumed The 2013e16 cohort of the What We Eat in America
significantly higher protein intakes above the typical component of the NHANES survey found that 5% of
15%e16% of energy as seen in the United States; e.g., pro- men and 8% of adult women in the United States had
tein accounts for just under 50% of energy in the diets of inadequate diets when compared to the EAR of
Eskimos when eating only meat. There are a few nitrogen 0.66 g/kg body weight.55 Of interest in this analysis,
balance studies at high protein levels in the literature in 12% of those over 70 years of age had inadequate in-
which subjects were fed 200e300 g/d; they have all takes of dietary protein,55 meaning that the estimates
shown positive nitrogen balances99,100,101 but no detri- of their dietary intake were below 0.66 g protein/kg
mental effects on protein homeostasis were noted. Of body weight.
concern is whether there is a maximum rate of urea syn- It has been argued that the protein requirements of
thesis to counter the effects of high ammonium ion con- elderly are greater than that established as the RDA in
centrations. Although there are some reports of effects 2002 by the IOM, in part due to improvements in lean
of high protein supplements, there was insufficient evi- body mass in elderly provided supplemental essential
dence upon which to base a UL.85 AAs.102,103 Subsequent studies have pointed to an
increased need for total protein in elderly due to changes
in protein turnover irrespective of the essential AA con-
F. Dietary Sources tent.104 Given that national surveys show that a compar-
atively large proportion of older adults in the United
Almost all protein in the diet is in the form of AAs States (w12%) have intakes below the EAR, the minimal
linked by peptide bonds; free AAs are minimal except amount of protein need in the group, protein intake in
from tissues with large AA pools (such as liver or this age group is of concern.
plasma). Other sources of nitrogen include nucleic acids
and small nitrogen-containing molecules. Dietary pro-
teins are usually assumed to be about 16% nitrogen by TABLE 2.5 Indispensable amino acid scoring patterns, mg/g
weight (thus a factor of 6.25 is used when converting ni- protein.
trogen to equivalent protein), but this percentage varies
FAO/WHO/ Recommended FAO/WHO/
with the protein’s AA composition. For example,
UNU 1985 FNB/IOM UNU 2007
approximately 20%e27% of the nitrogen in human Amino acid patterna patternb patternc
milk is from nonprotein nitrogenous substances such
as urea, resulting in a lower protein content than would Histidine 19 15 18
be expected when using a factor based on 16%. When Isoleucine 28 25 31
the weights of analyzed AAs in a food (taking into ac- Leucine 66 55 63
count water of hydrolysis) are summed, the protein/ni-
trogen ratio is 5.38 for egg, 5.62 for whole milk, 4.86 for Lysine 58 51 52
cooked ham, 5.70 for whole-meal wheat bread, and 6.07 Methionine þ cysteine 25 25 25
for soymilk.85 Thus, the factor used is important when Phenylalanine þ tyrosine 63 47 46
converting the amount of nitrogen present in a specific
foodstuff to total protein present. Threonine 34 27 27
All nine essential AAs are present in foods from an- Tryptophan 11 7 7
imal sources such as milk, eggs, fish, and cheese; thus Valine 35 32 41
animal proteins are considered complete proteins.
Foods that are derived from plants, such as grains, a
Based on requirements for 2e5 years olds for protein (0.99 g/kg/d).97
b
nuts, seeds, and vegetables, are usually low in one or Based on estimated average requirements for 1e3 years olds for protein (0.66 g/kg/d)
and indispensable amino acids.44
more of the AAs essential for humans, and thus c
Based on average requirements for 1e2 years olds for protein (0.86 g/kg/d) and
when consumed individually, they are considered indispensable amino acids.96
Section A. Macronutrients
IV. Issues Related to Protein and Amino Acids in Human Health 31
Determining protein quality protein quality. The comparative ease of measuring ileal
Protein quality focuses on the relative composition of digestibility in the DIAAS method has advanced it as a
AAs present in a protein source compared to a protein possible successor to PDCAAS.110
pattern than is considered to represent the highest quality As part of the DRI process, a scoring pattern to eval-
protein. Methods used in some countries for labeling pro- uate proteins was also proposed, based on the EARs for
tein content of a food item, or methods proposed for use essential AAs established for 1e3 year old children85
for labeling, include the protein efficiency ratio (PER), (see Table 2.5).
required in Canada; the protein digestibility corrected
Methods to analyze for protein content in food
amino acid score (PDCAAS), originally recommended
by FAO/WHO in 1993 and in the United States by the Historically, the protein content of foods was obtained
Food and Drug Administration; and the digestible essen- by determining total nitrogen in a food sample as
tial amino acid score (DIAAS), most recently recommen- measured by the Kjeldahl method.111 Nitrogen content is
ded by FAO/WHO,107 which includes measurement of then multiplied by a factor 6.25 to arrive at the protein
ileal digestibility.107 While PER requires an animal content, based on two assumptions: (1) carbohydrates
growth study comparing utilization of a test protein and fats do not contain nitrogen and (2) almost all
with casein protein, other methods evaluate a food’s pro- nitrogen in the diet is present as AAs in proteins.
tein quality by comparing its essential AA composition to As mentioned earlier, the average nitrogen content of pro-
the ratio of AAs estimated to be needed by humans. The teins was approximately 16%, hence the factor of 6.25.112
practice has been to compare the amount of each essential
AA in a food to a reference (scoring) pattern based on the
estimated essential AA requirements of a 2- to 5-year-old IV. ISSUES RELATED TO PROTEIN
child 106,108 (see Table 2.5). AND AMINO ACIDS IN HUMAN
For example, when a protein source has essential AAs HEALTH
in which some are less than the scoring pattern per gram
of protein, the AA with the lowest percent of the scoring A. Adaptation to Fasting and Starvation
pattern’s amount is said to the limiting AA. For
Fasting
example, when compared to the PDCAAS score, if an
essential AA such as lysine has the lowest percentage, Individuals are in a fasting state as blood glucose
e.g., 40, then only 40% of that protein can be used for levels return to a baseline level before the next meal,
protein synthesis unless another protein is consumed with a decrease in insulin levels and rise in glucagon
that is high in lysine. The highest PDCAAS score any levels.113 Tissues such as the brain and red blood cells
protein can achieve is 100. This would mean that 100% require a constant supply of glucose.
of that protein can be used for protein synthesisdthat
Starvation
it contains all essential AAs in appropriate ratios per
gram of protein. Given that typical diets contain many In prolonged starvation, gluconeogenesis is the major
sources of dietary protein, foods can function as “com- source of circulating glucose. The major substrates for
plementary proteins” if consumed simultaneously and gluconeogenesis are AAs, which are derived primarily
are higher in the AA that is limiting in the other pro- from the breakdown of skeletal muscle protein. Gluco-
tein.109 Comparison with a scoring pattern indicates neogenesis from AA substrates occurs in both the liver
the overall quality of a protein because it represents and the kidney.
the relative adequacy of its most limiting AA as needed
by the most vulnerable individuals in the population,
B. Transition to Ketone Bodies as the Primary
young children, whose needs for essential AAs for
growth are great.
Fuel Source
An important benefit of using the PDCAAS score is Gluconeogenesis from AA substrates occurs in both
that this approach uses human AA requirements, the liver and the kidney. The biochemical process begins
whereas animal bioassays are used by the PER method, within 4e6 h of the last meal.114,61 After approximately
which was used in the past in the United States.54 A step 2 days of starvation, the brain gradually switches its
used in both methods is to multiply the lowest AA ratio fuel source from glucose to ketone bodies; however,
by its true protein digestibility. While this ensures that blood cells and the adrenal medulla continue to rely on
the appropriate levels of essential AAs are available glucose as the primary energy substrate. This adaptation
from the protein source, it does require determining di- to starvation involves the conversion in the liver of free
gestibility in an animal model. This aspect has spurred fatty acids to ketone bodies. As the metabolically active
research to obtain an improved method to evaluate muscle mass is diminished with starvation, metabolic
Section A. Macronutrients
32 2. PROTEIN AND AMINO ACIDS
processes occurring within these tissues are also reduced, with a ketone-based fuel system minimizes gluconeogen-
accounting for a reduction in resting energy expenditure. esis and slows further protein breakdown.115,116
Within approximately 1 week, adaptation to starvation
RESEARCH GAPS
• How alternative protein sources can meet the needs of the growing population.
• Considerations about sustainability of present and future production of protein rich foods.
• Development of new biomarkers for adequacy that may rely on new approaches to data using nonlinear
modeling.
• Additional focus on whether there are problems with consuming too much protein.
• Effective methods to teach consumers about complementarity of proteins as many move toward plant-based
diets.
• Increased focus on how gut microbiota affect protein/AA metabolism in nutrition and neurological function.
Section A. Macronutrients
V. References 33
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