Amino Acids

M. Zaharna Clin. Chem. 2009

 Introduction
• Amino acids play central roles both as building
blocks of proteins and as intermediates in
metabolism.
• The 20 amino acids that are found within
proteins convey a vast array of chemical
versatility.
• The precise amino acid content, and the
sequence of those amino acids, of a specific
protein, is determined by the sequence of the
bases in the gene that encodes that protein.
• The chemical properties of the amino acids of
proteins determine the biological activity of the
protein.
M. Zaharna Clin. Chem. 2009
 Introduction
• In addition, proteins contain within their
amino acid sequences the necessary
information to
• determine how that protein will fold into a three
dimensional structure,
• and the stability of the resulting structure.
• It is important to keep in mind that one of the
more important reasons to understand amino
acid structure and properties is to be able to
understand protein structure and properties.
• The vastly complex characteristics of even a
small, relatively simple, protein are a
composite of the properties of the amino
acids which comprise the protein.

M. Zaharna Clin. Chem. 2009

 Amino Acid Structure
• An α-amino acid consists of:
– a central carbon atom, called the α carbon,
– linked to an amino group,
– a carboxylic acid group,
– a hydrogen atom,
– and a distinctive R group.

• The R group is often referred to as the side

chain.
• The two mirror-image forms are called the L
isomer and the d isomer.
M. Zaharna Clin. Chem. 2009
 L & D isomers

M. Zaharna Clin. Chem. 2009

 • Twenty kinds of side chains varying in
size, shape, charge, hydrogen-bonding
capacity, hydrophobic character, and
chemical reactivity are commonly found.
• The remarkable range of functions
mediated by proteins results from the
diversity and versatility of these 20
building blocks.

M. Zaharna Clin. Chem. 2009

M. Zaharna Clin. Chem. 2009
 Essential Amino Acids
• Humans can produce 11 of the 20 amino acids.
• The others must be supplied in the food.
• Failure to obtain enough of even 1 of the 9
essential amino acids, those that we cannot
make, results in degradation of the body's
proteins—muscle and so forth—to obtain the
one amino acid that is needed.
• Unlike fat and starch, the human body does not
store excess amino acids for later use—the
amino acids must be in the food every day.

M. Zaharna Clin. Chem. 2009

 Metabolism
• Proteolytic enzymes such as pepsin and trypsin
digest dietary proteins into their constituent
amino acids, the breakdown of body proteins is
a source of amino acids.
• The amino acid pool is used for the synthesis of
body proteins e.g. plasma, intracellular and
structural proteins.
• Amino acids are also used for the synthesis of
nonprotein nitrogen "NPN" – containing
compounds such as purines, pyrimidines,
creatine, histamine, thyroxine and others

M. Zaharna Clin. Chem. 2009

• All tissues have some capability for synthesis of
the non-essential amino acids, and conversion
of non-amino acid carbon skeletons into amino
acids.
• However, the liver is the major site of nitrogen
metabolism in the body.
• In times of dietary surplus, the potentially toxic
nitrogen of amino acids is eliminated via
transaminations, deamination, and urea
formation;
• The carbon skeletons are generally conserved
as carbohydrate, via gluconeogenesis, or as
fatty acid via fatty acid synthesis pathways.
M. Zaharna Clin. Chem. 2009
 • In this respect amino acids fall into three
categories: glucogenic, ketogenic, or
glucogenic and ketogenic.
• Glucogenic amino acids are those that give
rise to a net production of pyruvate or TCA
cycle intermediates, such as α-ketoglutarate
or oxaloacetate, (Alanine can be deaminated
to pyruvate, arginine to  – ketoglutarate )
• Lysine and leucine are the only amino acids
that are solely ketogenic, giving rise only to
acetylCoA or acetoacetylCoA.
M. Zaharna Clin. Chem. 2009
• A small group of amino acids such as
phenylalanine, and tyrosine give rise to
both glucose and fatty acid precursors and
are thus characterized as being
glucogenic and ketogenic.

M. Zaharna Clin. Chem. 2009

M. Zaharna Clin. Chem. 2009
 Aminoacidopathies
• They are rare inherited disorders of amino
acid metabolism.
• Hereditary disorders of amino acid processing
can be the result of :
– defects either in the breakdown of amino acids
(activity of a specific enzyme )
– or in the body's ability to get the amino acids into
cells (membrane transport system ).
• More than 100 diseases have been identified
that result from inborn errors of amino acid
metabolism.
• Because these disorders produce symptoms
early in life, newborns are routinely screened
for several common ones.
M. Zaharna Clin. Chem. 2009
 • Phenylketonuria (PKU)
• Tyrosinemia
• Alkaptonuria
• Maple syrup urine disease
• Isovaleric Acidemia
• Homocystinuria
• Cystinuria
M. Zaharna Clin. Chem. 2009
 Phenylketonuria (PKU)
• Phenylalanine hydroxylase is
an enzyme which converts
phenylalanine to tyrosine.
• A deficiency of this enzyme
leads to a buildup of
phenylalanine which results in
severe mental retardation.
• Phenylalanine accumulates and is metabolized
by alternate degradative pathway into
phenylpyruvic acid and others leading to mental
retardation.

M. Zaharna Clin. Chem. 2009

 • This condition, known
as phenylketonuria
(PKU), is an
autosomal recessive
inborn error of
metabolism.

M. Zaharna Clin. Chem. 2009

 Testing for PKU

• Testing is done either on the serum (Guthrie test) or on

the urine.
• Testing is not valid until the newborn has ingested an
ample amount of the amino acid phenylalanine, which is
found in human and cow’s milk.
• Two or three days of intake are usually sufficient for the
Guthrie test.
• Urine PKU testing is usually done after the infant is 4 to 6
weeks old.
• Normal blood phenylalanine level is about 1.2 - 3.4 mg/dl.
In PKU, levels may range from 6 to 80mg/dl, usually
greater than 30mg/dl

M. Zaharna Clin. Chem. 2009

 Guthrie bacterial inhibition assay

• Spores of B. Subtilis are incorporated into

an agar plate that contains 2-
thienylalanine, a metabolic antagonist to
B. Subtilis growth.
• A filter paper disk impregnated with blood
from the infant is placed on the agar.
• If the blood level of phenylalanine
exceeds a range of 2 – 4 mg/dl, the
phenylalanine counteracts the antagonist,
and the bacterial growth occurs.

M. Zaharna Clin. Chem. 2009

 Guthrie bacterial inhibition assay
• The infant must be at least 24h of
age to ensure adequate time for
enzyme and amino acid levels to
develop.
• The sample should be taken before
the administration of antibiotics or
transfusion of blood.
• Premature infants can show false-
positive results due to the
immaturity of the liver's enzyme
systems.
M. Zaharna Clin. Chem. 2009
 Microfluorometric assay
• For the direct measurement of phenylalanine in
dried blood filter discs. It yields quantitative
results, not affected by the presence of antibiotics.
• The procedure is based on the fluorescence of a
complex formed of phenylalanine-ninhydrin-
copper in the presence of a dipeptide "l-leucyl-l-
alanine".
• The procedure starts with extraction using
trichloroacetic acid, then a mixture of ninhydrin,
succinate, leucylalanine and copper tartarate is
added, the fluorescence of the complex is
measured using excitation/emission wavelengths
of 360nm and 530nm respectively.
M. Zaharna Clin. Chem. 2009
 Reference method
• The reference method for quantitative
serum phenylalanine is HPLC.
• The normal limits of serum
phenylalanine levels of full term normal
weight newborns range from 1.2 mg/dl
– 3.4 mg/dl.

M. Zaharna Clin. Chem. 2009

 Urine testing for phenylpyruvic
acid
• Used for diagnosis in questionable
cases and for monitoring of dietary
therapy.
• It involves the reaction of ferric chloride
with phenylpyruvic acid in urine to
produce a green color.

M. Zaharna Clin. Chem. 2009

 Prenatal diagnosis and detection
of carrier status

• In families with PKU, testing is now

available using DNA analysis.
• The test is based on revealing multiple
independent mutations at the
phenylalanine hydroxylase locus.

M. Zaharna Clin. Chem. 2009

 Tyrosinemia
• Tyrosinemia is a genetic disorder
characterized by elevated blood levels of the
amino acid tyrosine,
• Tyrosine is a building block of most proteins.
• Tyrosinemia is caused by the shortage
(deficiency) of one of the enzymes required
for the multistep process that breaks down
tyrosine.
• If untreated, tyrosine and its byproducts build
up in tissues and organs, which leads to
serious medical problems.
M. Zaharna Clin. Chem. 2009
 Tyrosinemia

• There are three types of tyrosinemia. Each

has distinctive symptoms and is caused by
the deficiency of a different enzyme.
• Type I tyrosinemia, the most severe form of
this disorder, is caused by a shortage of the
enzyme fumarylacetoacetate hydrolase.
• Type II tyrosinemia is caused by a deficiency
of the enzyme tyrosine aminotransferase.

M. Zaharna Clin. Chem. 2009

 Alkaptonuria
• It is due the deficiency of homogentistate
oxidase in the tyrosine catabolic pathway.
• Accumulation of homogentisic acid in urine
causes its darkening upon exposure to a
atmosphere.
• Alkaptonuric patients have no immediate
problems, but later high levels of
homogentisic acid gradually accumulate in
connective tissue, causing generalized
pigmentation of these tissues and an arthritis
like degeneration.

M. Zaharna Clin. Chem. 2009

M. Zaharna Clin. Chem. 2009
 Maple Syrup Urine Disease
"MSUD"
• Maple syrup urine disease is an inherited disorder
in which the body is unable to process certain
amino acids properly. The condition gets its name
from the distinctive sweet odor of affected infants'
urine.
• Mutations in 4 genes cause maple syrup urine
disease.
• These four genes provide instructions for making
proteins that work together as a complex.
• The protein complex is essential for breaking
down the amino acids leucine, isoleucine, and
valine, which are present in many kinds of food

M. Zaharna Clin. Chem. 2009

 • As a result, these amino acids and their
byproducts build up in the body.
• Because high levels of these substances are
toxic to the brain and other organs, their
accumulation leads to the serious medical
problems.

M. Zaharna Clin. Chem. 2009

 Tests
• A modified Guthrie test is used for neonatal
screening. The metabolic inhibitor of B. Subtilis is 4-
azaleucine.
• Positive test for MUSD, elevated level of leucine
from a filter paper disc impregnated with infants
blood will overcome the inhibitor and bacterial
growth occurs.
• Confirmed diagnosis is based on finding increased
levels of the three amino acids in plasma and urine
with leucine being in highest concentration.
• A leucine level above 4 mg/dl is indicative of MUSD.
• MUSD can be diagnosed prenatally by measuring
the decarboxylase enzyme concentration in cells
cultured from amniotic fluid.

M. Zaharna Clin. Chem. 2009

 Isovaleric Acidemia

• It results from a deficiency of the

enzyme isovaleryl-COA
dehydrogenase in the degradative
pathway of leucine.
• The elevated isovalerylglycine levels
can be identified by chromatography.

M. Zaharna Clin. Chem. 2009

 Homocystinuria
• Homocysteine is an intermediate amino acid
in the synthesis of cysteine from
menthionine.
• Homocystinuria is caused by the impaired
activity of the enzyme cystathionine -
synthase which results in elevated plasma
and urine levels of homocysteine and
methionine.
• Newborns show no abnormalities, physical
defects develop gradually with age.
• Clinical findings in late childhood include
thrombosis, osteoporosis, dislocated eye
lenses due to the lack of cysteine synthesis
which is essential for collagen formation.
M. Zaharna Clin. Chem. 2009
M. Zaharna Clin. Chem. 2009
 • Neonatal screening with a Guthrie test
using L-methionine sulfoximine as the
metabolic inhibitor. Increased plasma
methionine levels from affected infants
will result in bacterial growth.
• Elevations in urinary homocystine can
be detected by the cyanide-
nitroprusside spot test.

M. Zaharna Clin. Chem. 2009

 Cystinuria
• It is caused by a defect in the amino acid transport
system rather than a metabolic enzyme deficiency.
• Normally, amino acids are free filtered by the glomerulus
and then actively reabsorbed in the proximal renal
tubules.
• In cystinuria, there is a 20-30 fold increase in the urinary
excretion of cystine due to a genetic defect in the renal
resorptive mechanism.
• Because cystine is relatively insoluble, it tends to
precipitate in the kidney tubules and form urinary calculi.
• Cystinuria can be tested by cyanide-nitroprusside test.

M. Zaharna Clin. Chem. 2009

 Amino Acid Analysis
• Blood samples drawn after 6-8 h fasting to
avoid the effect of absorbed amino acids
originating from dietary proteins.
• The sample is collected in heparin and the
plasma is promptly removed from the cells
"as cells contain higher concentration of
amino acids", hemolysis should be avoided
for the same reason.
• Deproteinization within 30 min of collection,
analysis should be performed immediately or
the sample should be stored at -20OC to
-40OC.

M. Zaharna Clin. Chem. 2009

 • Urinary amino acid analysis can be
performed on a random specimen for
screening purposes, but for quantitation, a
24 h urine preserved with thymol or organic
solvent is required. Amniotic fluid also may
be analyzed.
• The method of choice is the two-dimensional
chromatography, the amino acids are
allowed to migrate along one solvent front,
and then the chromatogram is rotated 90O
and a second solvent migration occurs.

M. Zaharna Clin. Chem. 2009

 • The chromatogram is visualized by
staining with ninhydrin, which gives a
blue color with most amino acids.
• Confirmatory test for an amino acid
disorder include separation and
quantitation by cation-exchange
chromatography using a gradient buffer
elution.
• HPLC reversed-phase system
equipped with fluorescence detection is
another choice.
M. Zaharna Clin. Chem. 2009