Amino Acidss & Aminoacidopathies
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Transcript of Amino Acidss & Aminoacidopathies
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Amino Acids & Aminoacidopathies
16/5/2015
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• 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.
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• 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.
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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 α-carbon atom.
These carboxyl and amino groups are combined in peptide linkage.
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• The carbon is also bonded to a hydrogen atom and a larger side chain. The side chain is unique for each amino acid
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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.
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The 20 Amino acids are differ by their R group
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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
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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.
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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
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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
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Overview of amino acids metabolism
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Metabolism of amino acids
• Amino acids are then rapidly absorbed from the intestine into the blood and subsequently become part of the body’s 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
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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
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Non-polar amino acids
• Side chains consists entirely of carbon and hydrogen.
• Compounds composed of only carbon and hydrogen are nonpolar and hydrophobic.
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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)
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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.
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Chapter 9 19© 2011 Pearson Education, Inc.
When amino acids combine in a condensation reaction, the amide bond that is formed between them is called a peptide bond.
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• 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.
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Aminoacidopathies• class of rare inherited errors of metabolism in
which there is an enzyme defect that inhibits the body’s ability to metabolize certain amino acids
• The abnormalities may involve– Activity of specific enzymes– Metabolic pathway– Or membrane transport system for amino acids
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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
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Clinical Manifestation • Aminoaciduria • CNS dysfunction:
– Mental retardation– Seizure disorders– Behavioral disturbances
• Metabolic ketoacidosis• Occasionally:
– Liver disorders– Renal failure– Ocular lesions
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Aminoacidopathies
Neurological • PKU• Homocyctinuria• Hypertyrosinemia• Maple-syrup urine disease• Hyperglycenemia• Methyle melonic aciduria• Citrullinemia• Carnosinemia
Hypopigmentation• Brown –syndrome• Albinism• Chediac Higashi syndrome
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Phenyl Ketonuria (PKU)
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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
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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 µmole/l (upper limit 120 µmole/l)
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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
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Tests of PKU• principle of Guthrie test :
– Spores of organism Bacillus subtilisare incorporated into an agar plate containing β-2-thienylalanine (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.
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Tests of PKU• principle of Guthrie test :
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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
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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)
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Defect here causes Type I Tyrosinemia
Defect here causes alkaptonuria
Catabolic pathway for phenylalanine and tyrosine
Homogentisate dioxygenase
Fumarylacetoacetate hydrolase
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Albinism
• Autosomal recessive • Result from loss of TYROSINASE enzyme in
skin which converts tyr to DOPA and DOPA to melanin pigments
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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•
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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
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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
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• 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
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• 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
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• 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
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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
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Homocystin
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Homocystinuria
• Signs and complications:– Increased risk of blood clot (thrombosis)– Dislocation of eye lenses– Skeletal abnormality– Developmental delay– osteoporosis
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Laboratory tests for homocysteinuria
• Guthrie test (blood) : using L-methionine sulfoximine as the metabolic inhibitor
• Plasma methionine level – HPLC – GC& MS
• Urinary homocysteine:– HPLC
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Laboratory tests for homocysteinuria• Detection in urine : Cyanide-nitroprusside 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.
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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
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Cystinuria
• diagnosed by testing the urine for cystine using cyanide nitroprusside, which produces a red-purple color on reaction with sulfhydryl groups
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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
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Amino Acid analysis
• Urine samples : – Random for screening– 24 hr with thymol preservative for quantitative
analysis
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Amino Acid analysis
• Methods – TLC for screening– HPLC– MS/MS