Amino Acids &

Aminoacidopathies
16/5/2015

 Amino acids are the building blocks of proteins,

which have structural and functional properties.
Proteins are polymers of amino acids covalently
bonded in specific sequences.
When amino acids are covalently linked to one
another, this chain can twist and fold to form a
unique three-dimensional structure that has a
specific function.

 There are 20 commonly occurring amino

acids that make up proteins, and the order
of amino acids in proteins determines its
structure and biological function.

Amino Acid Structure

Each amino acid (except for proline) has:
1. A carboxyl group (-COO-) .
2. Protonated amino group (-NH3+) .
3. Side chain ("R-group") bonded to the carbon atom.
These carboxyl and amino groups are combined
in peptide linkage.

 The carbon is also bonded to a

hydrogen atom and a larger side chain.
The side chain is unique for each amino
acid

Amino Acids have two enantiomers

The L-amino acids are the building blocks for

proteins. Some D-amino acids occur in nature, but
not in proteins.

The 20 Amino acids are differ by

their R group

organic compounds represented in the

structures of different amino acids
1. Alkanes
2. Aromatics
3. Thioethers
4. Alcohols
5. Phenols
6. Thiols
7. Amides
8. Carboxylic acids
9. Amines

Essential amino acids

There are 10 amino acids that are
essential amino acids because they
cannot be synthesized in the human body
and must be obtained in the diet.
Two of these amino acids, arginine and
histidine, are essential in children, but not
adults.

Essential amino acids

Arginine (Arg) : amine , cell division , wound
healing
Histidine (His) : basic (imidazole),
Isoleucine (Ile): branched-chain, healing muscle cells
Leucine (Leu) : branched-chain, healing muscle cells
Lysine (Lys)
Methionine (Met): initiatetranslation of messenger
RNA
Phenylalanine (Phe): non polar , used by brain
Threonine (Thr)
Tryptophan (Trp)
Valine (Val): branched-chain, healing muscle cells

Metabolism of amino acids

Essential amino acids should be
provided by diet
Other amino acid synthesized by the
body or from essential amino acids
Tyrosine produced from
phenylalanine
Dietary proteins are completely
digested into amino acids by
proteolytic enzymes

Overview of amino acids

metabolism

Metabolism of amino acids

Amino acids are then rapidly absorbed from
the intestine into the blood and
subsequently become part of the bodys
pool of amino acids
Amino acids are also released by the
normal breakdown of body proteins
The primary purpose of amino acids is for
the synthesis of body proteins
synthesis of nonprotein nitrogen-containing
compounds such as purines, pyrimidines,
porphyrins, creatine

Classification of amino acids

based on the side chains:
1. Nonpolar amino acids
2. Polar amino acids, which are further
divided into:
Neutral amino acids
Acidic amino acids
Basic amino acids

Non-polar amino acids

Side chains consists entirely of carbon and
hydrogen.
Compounds composed of only carbon and
hydrogen are nonpolar and hydrophobic.

Polar amino acids

Contain functional groups, such as hydroxyl (
OH) and amide (CONH2).
Side chains can form hydrogen bonds with
water.

Hydrophilic (An exception is cysteine,

which does not form hydrogen bonds)

Protein formation
When two amino acids condense, a dipeptide is
formed.
The carboxylate ion (COO-) of one amino acid
reacts with the protonated amine (NH3+) of a
second amino acid.
A water molecule is lost and an amide functional
group is formed. An amide bond is formed
between the two amino acids.

When amino acids combine in a condensation

reaction, the amide bond that is formed between
them is called a peptide bond.

Chapter 9

19

2011 Pearson
Education, Inc.

 In this dipeptide, alanine is called the

N-terminus because it has an
unreacted
-amino group.
Valine is called the C-terminus because it
has an unreacted -carboxylate group.

Aminoacidopathies
class of rare inherited errors of
metabolism in which there is an enzyme
defect that inhibits the bodys ability to
metabolize certain amino acids
The abnormalities may involve
Activity of specific enzymes
Metabolic pathway
Or membrane transport system for amino
acids

Aminoacidopathies
These defects leads to accumulation of
amino acids , its precursors or by products
Excessive accumulation in blood or tissues
leads to physical symptoms of the disease

Clinical Manifestation
Aminoaciduria
CNS dysfunction:
Mental retardation
Seizure disorders
Behavioral disturbances

Metabolic ketoacidosis
Occasionally:
Liver disorders
Renal failure
Ocular lesions

Aminoacidopathies
PKU
Homocyctinuria
Hypertyrosinemia
Neurological
Maple-syrup urine
disease
Hyperglycenemia
Methyle melonic
aciduria
Citrullinemia
Carnosinemia

Hypopigmentation

Brown syndrome
Albinism
Chediac Higashi
syndrome

Phenyl Ketonuria (PKU)

Phenyleketoneuria
autosomal recessive disorder
Incidence rate 1 in 14000
Common compared to other
aminoacidopathy
Deficiency of phenylanaline
hydroxylase
Phenylalanine is metabolized
via alterative pathway leading
to accumulation phenyle
pyruvic acid (deamination)
Accumulate in blood and urine
and gives musty odor to it

Phenyleketoneuria
Phenyl pyruvic acid is neurotoxic and in
undiagnosed newborn can cause severe
mental retardation due to brain damage,
which starts in 2nd and 3rd week post birth.
Early detection allows diet control therapy
low in phenylalanine up to the age of 5-6yrs
until normal metabolism develops, however
low IQ levels have been reported after
termination of special diet.
Phenylalanine > 1200 mole/l (upper limit
120 mole/l)

Tests of PKU
Detection of phe (Screening ):
Ferric chloride test
Guthrie test : bacterial inhibition assay for
phenylalanine that uses the ability of
phenylalanine to facilitate bacterial growth
in a culture medium with an inhibitor
(Bacillus subtilis and -2-thienylalanine).
Automated methods

Plasma level estimation (Confirmatory or

reference method)
Chromatography
Fluoremetric methods

Tests of PKU
principle of Guthrie test :
Spores of organism Bacillus subtilisare
incorporated into an agar plate containing -2thienylalanine (a metabolic antagonist to B.
Subtilis).
A filter paper disk, impregnated with dried
blood sample is placed onto the agar
If the blood phenylalanine level is higher than
2.5 mg/dl, the phenylalanine counteracts the
antagonist and the bacteria grows.
The sample should be collected prior to
administration of antibiotics or transfusion of
blood or blood products.

Tests of PKU
principle of Guthrie test :

Tests of PKU
Genetic pre-natal diagnosis of
PKU:
detection of carrier status in families
with PKU using DNA analysis
PKU is a complex polygenic disorder and
results in multiple independent
mutations at the PAH locus
Screening for mutations using PAH gene
probes

2. ALCAPTONURIA

AKU

It is rare autosomal recessive

inherited genetic disorder of
phenylalanine and tyrosine
metabolism

defect in the HGD gene (Homogentisic

acid oxidase deficiency)

the body unable to properly break down

certain amino acids (tyrosine and
phenylalanine)

homogentisic acid accumulates in the

skin and other body tissues
BLACK URINE
BLACK NAILS (OCHRONOSIS), SKIN
BLACK JOINT CARTILAGE (SEVERE
ARTHRITIS)

Catabolic pathway for phenylalanine and

tyrosine

Defect here
causes
alkaptonuria

Defect here
causes Type I
Tyrosinemia

Homogentisate
dioxygenase

Fumarylacetoacetate
hydrolase

Albinism
Autosomal recessive
Result from loss of TYROSINASE
enzyme in skin which converts tyr to
DOPA and DOPA to melanin pigments

Tyrosinemia
Tyrosine is an amino acid which is found in
most animal and plant proteins. The
metabolism of tyrosine in humans takes
place primarily in the liver
Hereditary tyrosinemia is a genetic
inborn error of metabolism associated
with severe liver disease in infancy.
The disease is inherited in an
autosomal recessive fashion.
Tyrosine catabolism defect
characterized by the excretion of tyrosine
and tyrosine catabolites in urine

Tyrosinemia
Type I tyrosinemia : caused by an absence
of
the
enzyme
fumarylacetoacetate
hydrolase (FAH)
most severe form of this aminoacidopathy
found in about 1 in 100,000 births
lead to liver and kidney failure

Type II tyrosinemia:
deficiency of the enzyme tyrosine
aminotransferase
occurs in fewer than 1 in 250,000 births
mentally retarded ,excessive tearing and
photophobia

Tyrosinemia
Diagnosis :
Elevation of tyrosine in plasma and
urine
MS/MS chromatography

Treatment :
Low protein diet
Drugs that inhibits maleylacetoacetic
acid and fumarylacetoacetic acid
formation

 results from an absence or greatly

reduced activity of the enzyme
branched-chain -ketoacid
decarboxylase
Normal metabolism of 3 amino acids
inhibited
MSUD is an autosomal recessive
genetic inherited disorder
1 in 150,000 births in the general
population

 characteristic maple syrup or burnt sugar

odor of the urine, breath, and skin
accumulation of the branched-chain amino
acids and their corresponding ketoacids in the
blood, urine, and cerebrospinal fluid (CSF).
Within a week, develop lethargy, vomiting,
lack of appetite, and signs of failure to thrive
severe mental retardation, seizures, acidosis,
and hypoglycemia
Can lead to death

 Detection and screening:

modified Guthrie test
The metabolic inhibitor to B. subtilis,
includedin the growth media, is 4-azaleucine

Microfluorometric assay for the three

branched-chain amino

Homocystinuria
Autosomal recessive disorder
Incidence : 1 in 200,000 birth
Deficiency y of the enzyme
cystathionine- synthetase, necessary
for the metabolism of the methionine
amino acid, that results in elevated plasma
and urine levels of methionine and of the
precursor homocysteine
Urinary exertion of homocystein is >
300 mg/24 hrs

Homocystin

Homocystinuria
Signs and complications:
Increased risk of blood clot
(thrombosis)
Dislocation of eye lenses
Skeletal abnormality
Developmental delay
osteoporosis

Laboratory tests for

homocysteinuria
Guthrie test (blood) : using Lmethionine sulfoximine as the
metabolic inhibitor
Plasma methionine level
HPLC
GC& MS

Urinary homocysteine:
HPLC

Laboratory tests for

homocysteinuria

Detection in urine : Cyanidenitroprusside spot test

cyanide-nitroprusside is added to urine

sample
High levels of homocysteine will be detected
by the development of purple-red colour
urinary cysteine might interfere in this test
to give false +ve.
Confirmation by adding silver, homocysteine
is reduced but not cysteine thus allowing
homocysteine to react with nitro-prusside to
produce reddish color.

Cystinuria
Autosomal recessive defect that is caused by
a defect in the amino acid transport system
rather than a metabolic enzyme deficiency
inadequate reabsorption of cystine during
the filtering process in the kidneys
Cystine
precipitates out of the urine and forms stones
in the kidneys,ureters, or bladder

Cystinuria
diagnosed by testing the urine for
cystine using cyanide nitroprusside,
which produces a red-purple color on
reaction with sulfhydryl groups

Amino Acid analysis

Samples
Plasma , urine and amnoitic fluid

Fasting sample 6 to 8 hrs

Heparin
Separate plasma as soon as possible
Contamination with platelets and WBCs
should be prevented
Heamolysis affect the results
Immediately analysed or sample should be
frozen -20
Deproteinization within 30 mins

Amino Acid analysis

Urine samples :
Random for screening
24 hr with thymol preservative for
quantitative analysis

Amino Acid analysis

Methods
TLC for screening
HPLC
MS/MS