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Amino Acid Metabolism: Disposal of Nitrogen ▪ Nitrogen enters the body in a variety of compounds present in
Clinical Biochemistry food, the most important being amino acids contained in
Albert Francis K. Tirado, RMT dietary protein
▪ Nitrogen leaves the body as urea, ammonia, and other
Overview products derived from amino acid metabolism
▪ Unlike fats and carbohydrates, amino acids are not stored by ▪ The role of body proteins in these transformations involves
the body two important concepts: the amino acid pool and protein
▪ Therefore, amino acids must be obtained from the following: turnover
▪ Diet Amino Acid Pool
▪ Synthesized de novo ▪ Free amino acids are present throughout the body, e.g., in
▪ Produced from normal protein degradation cells, blood, and the extracellular fluids
▪ Any amino acids in excess of the biosynthetic needs of the cell ▪ All these amino acids belong to a single entity, called the
are rapidly degraded amino acid pool, which is supplied by three sources:
▪ Amino acid catabolism: ▪ Degradation of body proteins
▪ First phase is the removal of the α-amino groups ▪ Amino acids derived from dietary protein
▪ Usually by transamination and subsequent ▪ Synthesis of nutritionally nonessential amino acids
oxidative deamination
▪ This forms ammonia and the corresponding α-
keto acid ( “carbon skeletons” of amino acids)
▪ A portion of the free ammonia is excreted in
the urine, but most is used in the synthesis of
urea (urea cycle), which is quantitatively the
most important route for disposing nitrogen
from the body
▪ Second phase is the carbon skeletons of α-keto acids
are converted to common intermediates of energy
producing metabolic pathways
▪ Metabolized to CO2 and water
▪ Metabolized to glucose (via gluconeogenesis)
▪ Metabolized to fatty acids and ketone bodies

Overall Nitrogen Metabolism

▪ Amino acid catabolism is part of the larger process of the
metabolism of nitrogen-containing molecules

▪ Conversely, the amino acid pool is depleted by three routes:
▪ Synthesis of body protein
▪ Amino acids consumed as precursors of essential
nitrogen-containing small molecules
▪ Conversion of amino acids to glucose, glycogen, fatty
acids, ketone bodies, or CO2 + H2O
Protein Turnover
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▪ Although the amino acid pool is small (comprised of about 90-
100 g of amino acids) in comparison with the amount of
protein in the body (about 12 kg in a 70-kg man), it is
conceptually at the center of whole-body nitrogen metabolism
▪ In healthy, well-fed individuals, the input to the amino acid
pool is balanced by the output, i.e., the amount of amino acids
contained in the pool is constant
▪ The amino acid pool is said to be in a steady state, and the
individual is said to be in nitrogen balance
▪ Most proteins in the body are constantly being synthesized
and then degraded, permitting the removal of abnormal or
unneeded proteins
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▪ For many proteins, regulation of synthesis determines the proteins (used in receptor-mediated
concentration of protein in the cell, with protein degradation endocytosis)
assuming a minor role
▪ For other proteins, the rate of synthesis is relatively constant, Digestion of Dietary Proteins
and cellular levels of the protein are controlled by selective ▪ Most of the nitrogen in the diet is consumed in the form of
degradation protein, typically amounting to 70 to 100 g/day
▪ Rate of turnover ▪ Proteins are generally too large to be absorbed by the
▪ In healthy adults, the total amount of body protein intestine – an exception to this rule is that newborns can take
remains constant, because the rate of protein up maternal antibodies in breast milk
synthesis is just sufficient to replace the protein that is ▪ Therefore, proteins must be hydrolyzed to yield di- and
degraded tripeptides as well as individual amino acids, which can be
▪ This process, called protein turnover, leads to the absorbed
hydrolysis and re-synthesis of 300 to 400 g of body ▪ Proteolytic enzymes responsible for degrading proteins are
protein each day produced by three different organs:
▪ The rate of protein turnover varies widely for ▪ Stomach
individual proteins ▪ Pancreas
▪ Short-lived proteins (e.g., many regulatory proteins ▪ Small intestine
and misfolded proteins) are rapidly degraded, having
half-lives measured in minutes or hours
▪ Long-lived proteins, with half-lives of days to weeks,
constitute the majority of proteins in the cell
▪ Structural proteins, such as collagen, are metabolically
stable, and have half-lives measured in months or
years
▪ Protein degradation
▪ Two major enzyme systems responsible for degrading
damaged or unneeded proteins:
▪ ATP-dependent ubiquitin-proteasome system
of the cytosol – degrades mainly endogenous
proteins
▪ ATP-independent degradative enzyme system
of the lysosomes – degrade primarily
extracellular proteins (taken into the cell by Digestion of Proteins by Gastric Secretion
endocytosis), and cell surface membrane
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▪ Digestion of proteins begins in the stomach, which secretes ▪ Release and activation of the pancreatic zymogens is mediated
gastric juice – a unique solution containing HCl and the by the secretion of cholecystokinin and secretin, polypeptide
proenzyme, pepsinogen hormones of the digestive tract
▪ Hydrochloric acid ▪ Enteropeptidase (formerly called enterokinase)
▪ Stomach acid is too dilute (pH 2–3) to hydrolyze ▪ Enzyme synthesized by and present on the luminal
proteins surface of intestinal mucosal cells of the brush border
▪ HCl, secreted by the parietal cells, functions instead to membrane
kill some bacteria and to denature proteins, thus ▪ Enteropeptidase (formerly called enterokinase)
making them more susceptible to subsequent ▪ Converts the pancreatic zymogen trypsinogen to
hydrolysis by proteases trypsin by removal of a hexapeptide from the N-
▪ Pepsin terminus of trypsinogen
▪ Acid-stable endopeptidase is secreted by the chief cells ▪ Trypsin subsequently converts other trypsinogen
of the stomach as an inactive zymogen (or molecules to trypsin by cleaving a limited number of
proenzyme), pepsinogen specific peptide bonds in the zymogen
▪ In general, zymogens contain extra amino acids in their ▪ Enteropeptidase thus unleashes a cascade of
sequences that prevent them from being catalytically proteolytic activity, because trypsin is the common
active – removal of these amino acids permits proper activator of all the pancreatic zymogens
folding required for an active enzyme
▪ Pepsinogen is activated to pepsin, either by HCl, or
autocatalytically by other pepsin molecules that have
already been activated
▪ Pepsin releases peptides and a few free amino acids
from dietary proteins
Digestion of Proteins by Pancreatic Enzymes
▪ On entering the small intestine, large polypeptides produced
in the stomach by the action of pepsin are further cleaved to
oligopeptides and amino acids by a group of pancreatic
proteases
▪ Each of these enzymes has a different specificity for the amino
acid R-groups adjacent to the susceptible peptide bond
▪ For example, trypsin cleaves only when the carbonyl group of
the peptide bond is contributed by arginine or lysine
▪ These enzymes, like pepsin described above, are synthesized
and secreted as inactive zymogens
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▪ In individuals with a deficiency in pancreatic secretion (e.g.,
due to chronic pancreatitis, cystic fibrosis, or surgical removal
of the pancreas), the digestion and absorption of fat and
protein are incomplete
▪ Results in the abnormal appearance of lipids and undigested
protein in the feces
▪ Celiac disease (celiac sprue) is a disease of malabsorption
resulting from immune-mediated damage to the small
intestine in response to ingestion of gluten (or gliadin
produced from gluten), a protein found in wheat, barley and
rye
Digestion of Oligopeptides by Enzymes of the Small Intestine
▪ Luminal surface of the intestine contains aminopeptidase
▪ Aminopeptidase is an exopeptidase that repeatedly cleaves
Amino Acid Transport into Cells
the N-terminal residue from oligopeptides to produce even
▪ The concentration of free amino acids in the extracellular
smaller peptides and free amino acids
fluids is significantly lower than that within the cells of the
Absorption of Amino Acids and Small Peptides
body
▪ Free amino acids are taken into the enterocytes by a Na+-
▪ This concentration gradient is maintained because active
linked secondary transport system of the apical membrane
transport systems are required for movement of amino acids
▪ Di- and tri- peptides, however, are taken up by a H+-linked
from the extracellular space into cells
transport system
▪ At least seven different transport systems are known that
▪ The peptides are hydrolyzed in the cytosol to amino
have overlapping specificities for different amino acids
acids that are released into the portal system by
▪ The small intestine and the proximal tubule of the kidney
facilitated diffusion
have common transport systems for amino acid uptake
▪ Therefore, only free amino acids are found in the
▪ Therefore, a defect in any one of these systems results in an
portal vein after a meal containing protein
inability to absorb particular amino acids into the gut and into
▪ These amino acids are either metabolized by the liver or
the kidney tubules
released into the general circulation
▪ For example, one system is responsible for the uptake of
▪ Of note, branched-chain amino acids are important examples
cystine and the dibasic amino acids, ornithine, arginine, and
of amino acids that are not metabolized by the liver, but
lysine (represented as “COAL”)
instead are sent from the liver primarily to muscle via the
▪ In the inherited disorder cystinuria, this carrier system is
blood
defective, and all four amino acids appear in the urine
▪ Cystinuria
▪ Occurs at a frequency of 1 in 7,000 individuals
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▪ One of the most common inherited diseases
▪ Most common genetic error of amino acid transport
▪ Expresses itself clinically by the precipitation of cystine
to form kidney stones (calculi), which can block the
urinary tract
▪ Oral hydration is an important part of treatment for
this disorder
▪ Cystinuria is distinct from cystinosis
▪ Cystinosis is a rare defect in the transport of cystine
out of lysosomes that results in the formation of
cystine crystals within the lysosome, and tissue
damage
▪ Of note, defects in the transport of tryptophan (and
other neutral amino acids) can result in Hartnup
disorder and pellagra-like dermatologic and neurologic
symptoms

Nitrogen Removal from Amino Acids

▪ The presence of the α-amino group keeps amino acids safely
locked away from oxidative breakdown
▪ Removing the α-amino group is essential for producing energy
from any amino acid, and is an obligatory step in the
catabolism of all amino acids
▪ Once removed, this nitrogen can be incorporated into other
compounds or excreted, with the carbon skeletons being
metabolized
▪ This section describes transamination and oxidative
deamination – reactions that ultimately provide ammonia and
aspartate, the two sources of urea nitrogen
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▪ All amino acids, with the exception of lysine and threonine,
participate in transamination at some point in their catabolism
▪ Lysine and threonine lose their α-amino groups by
deamination

Transamination
▪ In transamination, there is funnelling of amino groups to
glutamate
▪ First step in the catabolism of most amino acids is the transfer
of their α-amino group to α-ketoglutarate
▪ Products are an α-keto acid (derived from the original amino
acid) and glutamate Substrate Specificity of Aminotransferases
▪ α-ketoglutarate plays a pivotal role in amino acid metabolism ▪ Each aminotransferase is specific for one or, at most,
by accepting the amino groups from a few amino group donors
most amino acids, thus becoming glutamate ▪ Aminotransferases are named after the specific amino group
▪ Glutamate produced by transamination can be oxidatively donor, because the acceptor of the amino group is almost
deaminated or used as an amino group donor in the synthesis always α-ketoglutarate
of nonessential amino acids ▪ Two most important aminotransferase reactions are catalyzed
▪ Transfer of amino groups from one carbon skeleton by alanine aminotransferase (ALT) and aspartate
to another is catalyzed by a family of enzymes called aminotransferase (AST)
aminotransferases (formerly called transaminases) ▪ Alanine aminotransferase (ALT)
▪ Aminotransferases are found in the cytosol and mitochondria ▪ Present in many tissues
of cells throughout the body –
especially the liver, kidney, intestine, and muscle
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▪ Catalyzes the transfer of the amino group of
alanine to α-ketoglutarate, resulting in the formation
of pyruvate and glutamate
▪ Reaction is readily reversible
▪ However, during amino acid catabolism, this enzyme
(like most aminotransferases) functions
in the direction of glutamate synthesis
▪ Thus, glutamate, in effect, acts as a “collector” of
nitrogen from alanine
▪ Aspartate aminotransferase (AST)
▪ An exception to the rule that aminotransferases funnel
amino groups to form glutamate
▪ During amino acid catabolism, AST transfers amino
groups from glutamate to oxaloacetate, forming
aspartate, which is used as a source of nitrogen in the
urea cycle
▪ Reaction is also reversible

Mechanism of Action of Aminotransferases

▪ All aminotransferases require the coenzyme pyridoxal
phosphate (a derivative of vitamin B6), which is covalently
linked to the ε-amino group of a specific lysine residue at the
active site of the enzyme
▪ Aminotransferases act by transferring the amino group of an
amino acid to the pyridoxal part of the coenzyme to generate
pyridoxamine phosphate
▪ Pyridoxamine phosphate then reacts with an α-keto acid to
form an amino acid, at the same time regenerating the original
aldehyde form of the coenzyme
▪ Mechanism of action of enzyme: covalent catalysis
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▪ For example, physical trauma or a disease process can cause
cell lysis, resulting in release of intracellular enzymes into the
blood
▪ AST and ALT are of particular diagnostic value when they are
found in the plasma
▪ Liver Disease
▪ Plasma AST and ALT are elevated in nearly all liver
diseases, but are particularly high during extensive cell
necrosis, such as severe viral hepatitis, toxic injury, and
prolonged circulatory collapse
▪ ALT is more specific than AST for liver disease, but AST
is more sensitive because the liver contains larger
amounts of AST
▪ Serial enzyme measurements are often useful in
determining the course of liver damage
▪ Figure on the right shows the early release of ALT into
the serum, following ingestion of a liver toxin
▪ Of note, elevated serum bilirubin results from
Equilibrium of Transamination Reactions
hepatocellular damage that decreases the hepatic
▪ For most transamination reactions, the equilibrium constant is
conjugation and excretion of bilirubin
near one
▪ This allows the reaction to function in both:
▪ Amino acid degradation through removal of α-amino
groups – for example, after consumption of a protein-
rich meal;
▪ And, biosynthesis through addition of amino groups to
the carbon skeletons of α-keto acids – for example,
when the supply of amino acids from the diet is not
adequate to meet the synthetic needs of cells
Diagnostic Value of Plasma Aminotransferases
▪ Aminotransferases are normally intracellular enzymes, with
low levels found in plasma representing release of cellular
contents during normal cell turnover
▪ Presence of elevated plasma levels of aminotransferases
indicates damage to cells rich in these enzymes
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▪ Glutamate is the only amino acid that undergoes
rapid oxidative deamination – a reaction catalyzed
by glutamate dehydrogenase
▪ Therefore, the sequential action of transamination (resulting
in the collection of amino groups from
most amino acids onto α-ketoglutarate to produce glutamate)
and the oxidative deamination of glutamate (regenerating α-
ketoglutarate) provide a pathway whereby the amino groups
of most amino acids can be released as ammonia

▪ Non-hepatic Disease
▪ Aminotransferases may be elevated in non-hepatic
disease, such as myocardial infarction and muscle
disorders
▪ However, these disorders can usually be distinguished
clinically from liver disease

Oxidative Deamination
▪ In contrast to transamination reactions that transfer amino
groups, oxidative deamination by glutamate dehydrogenase
results in the liberation of the amino group as free ammonia
(NH3)
▪ Reactions occur primarily in the liver and kidney
▪ Provide α-keto acids that can enter the central pathway of
energy metabolism, and ammonia, which
is a source of nitrogen in urea synthesis
Glutamate dehydrogenase (GDH)
▪ Amino groups of most amino acids are ultimately funnelled to ▪ Coenzymes
glutamate by means of transamination with α-ketoglutarate
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+
▪ Glutamate dehydrogenase can use either NAD or
NADP+ as a coenzyme
▪ NAD+ is used primarily in oxidative deamination (loss
of NH3 coupled with oxidation of the carbon skeleton)
▪ NADPH is used in reductive amination (gain of NH3
coupled with reduction of the carbon skeleton
▪ Direction of reactions
▪ Depends on the relative concentrations of glutamate,
α-ketoglutarate, and NH3, and the
ratio of oxidized to reduced coenzymes
▪ For example, after ingestion of a meal containing
protein, glutamate levels in the liver are elevated, and
the reaction proceeds in the direction of amino acid
degradation and the formation of ammonia
▪ The reaction can also be used to synthesize amino
acids from the corresponding α-keto acids
▪ Allosteric regulators
▪ Guanosine triphosphate (GTP) is an allosteric inhibitor
of GDH
▪ Adenosine diphosphate (ADP) is an activator
▪ Thus, when energy levels are low in the cell,
amino acid degradation by GDH is high, facilitating
energy production from the carbon skeletons derived
from amino acids

Transport of NH3 to the Liver
▪ Two mechanisms in humans for the transport of NH 3 from
peripheral tissues to the liver:
▪ Glutamine synthetase
▪ Found in most tissues
▪ Glutamine synthetase combines NH3 with
glutamate to form glutamine (a nontoxic
transport form of ammonia)
▪ Glutamine is then transported in the blood to
the liver where it is cleaved by glutaminase to
produce glutamate and free NH3
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▪ Transamination of pyruvate to alanine
▪ Used primarily by muscle
▪ Alanine is transported to the liver, where it is
converted to pyruvate, again by transamination
▪ In the liver, gluconeogenesis can use pyruvate
to synthesize glucose, which can enter the
blood and be used by muscle (a pathway called
the glucose-alanine cycle or Cahill cycle)
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Urea Cycle
▪ Urea is the major disposal form of amino groups derived from
amino acids
▪ Accounts for about 90% of the nitrogen-containing
components of urine
▪ Two nitrogen atoms in urea:
▪ One nitrogen of urea is supplied by free ammonia
▪ Other nitrogen is supplied by aspartate
▪ Glutamate is the immediate precursor of both ammonia
(through oxidative deamination by GDH)
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and aspartate nitrogen (through transamination of
oxaloacetate by AST)
▪ Carbon of urea is from CO2 and the oxygen is from H2O
▪ Urea is produced by the liver via the Urea Cycle or Krebs-
Henseleit cycle
▪ It is then transported in the blood to the kidneys
for excretion in the urine
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Overall Stoichiometry of the Urea Cycle

▪ Four high-energy phosphate bonds are consumed in the

synthesis of each molecule of urea
▪ Therefore, the synthesis of urea is irreversible, with a large,
negative ΔG
▪ One nitrogen of the urea molecule is supplied by free NH 3, and
the other nitrogen by aspartate
▪ Glutamate is the immediate precursor of both ammonia
(through oxidative deamination by glutamate dehydrogenase)
and aspartate nitrogen (through transamination of
oxaloacetate by AST)
▪ In effect, both nitrogen atoms of urea arise from glutamate,
which, in turn, gathers nitrogen from other amino acids
Regulation of the Urea Cycle
▪ N-acetylglutamate is an essential activator for carbamoyl
phosphate synthetase I – the rate-limiting step in the urea
cycle
▪ N-acetylglutamate is synthesized from acetyl-CoA and
glutamate by N-acetylglutamate synthase, in a reaction for
which arginine is an activator
▪ Therefore, the intrahepatic concentration of N-
acetylglutamate increases after ingestion of a
protein-rich meal
▪ This provides the substrate (glutamate) and the regulator of
N-acetylglutamate synthesis
▪ This, in turn, leads to an increased rate of urea synthesis
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Fate of Urea
▪ Urea diffuses from the liver, and is transported in the blood to
the kidneys, where it is filtered and excreted in the urine
▪ A portion of the urea diffuses from the blood into the
intestine, and is cleaved to CO2 and NH3 by bacterial urease
▪ This NH3 is partly lost in the feces, and is partly reabsorbed
into the blood
▪ In patients with kidney failure, plasma urea levels are
elevated, promoting a greater transfer of urea from blood into
the gut
▪ The intestinal action of urease on this urea becomes a
clinically important source of ammonia, contributing to the
hyperammonemia often seen in these patients
▪ Oral administration of neomycin reduces the number of
intestinal bacteria responsible for this NH3 production

Metabolism of Ammonia