(M) Lec 5: Amino Acids (P1: Aminoacidopathies) Flashcards
- These are the building blocks of proteins (100-150 of these = proteins)
- The second most abundant NPN (second to urea)
Amino Acids
Amino Acids are called as such because of what 2 groups in their composition attached to Carbon?
Basic = Amino group (nitrogen-containing)
Acid = Carboxyl group
Note: They only vary in their side chains (R-group)
Conditions/disorders wherein an individual lacks certain enzymes which are important for the metabolism of amino acids
Aminoacidopathies (or elevated amino acid levels, in other words)
These amino acids cannot be synthesized by the body and therefore comes from the proteins that we eat
Essential Amino Acids
Ex.: Histidine, Leucine, Isoleucine, Arginine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, and Valine
These amino acids can be naturally synthesized by the body therefore dietary intake of these are not necessary
Ex.: Alanine, Asparagine, Aspartic Acid, Glutamic Acid, Selenocysteine, and Serine
Non-Essential Amino Acids
These amino acids are normally produced in a healthy individual however, there are infants who acquire inborn errors which manifest as aminoacidopathies leading to decreased amounts of this kind of amino acid
Semi-Essential (Conditionally Essential)
Ex.: Arginine, Cysteine, Glutamine, Glycine, Proline, and Tyrosine
- Refers to inherited errors of metabolism caused by:
1. A defective/deficient enzyme that inhibits the body’s ability to metabolize certain amino acids
2. Abnormalities that exist either in the activity of a specific enzyme in the pathway or membrane transport system
Aminoacidopathies
Aminoacidopathies
If an individual lacks the phenylalanine hydroxylase enzyme, what will happen to the phenylalanine levels in the body (+ include the name of the condition)?
Phenylalanine will increase leading to Phenylketonuria
Aminoacidopathies
If an individual lacks the tyrosine aminotransferase enzyme, what condition will the person manifest?
Hereditary Type 2 Tyrosinemia
Tyrosine cannot be converted to 4HPP
Aminoacidopathies
Hereditary Type 3 Tyrosinemia occurs if there is a deficiency of what enzyme?
4HPP Dioxygenase
Aminoacidopathies
Homogentisate (HGA) will be converted to Maleylacetoacetate (MAA) with the help of what enzyme (+ include the condition)?
HGA Oxidase (alkaptonuria)
Aminoacidopathies
Maleylacetoacetate (MAA) will be converted to what using the MAA isomerase?
Fumarylacetoacetate (FAA)
Aminoacidopathies
Fumarylacetoacetate (FAA) will be degraded into what 2 compounds?
- Fumarate
- Acetoacetate
Aminoacidopathies
A lack of the FAA hydrolase produces what condition?
Hereditary Tyrosinemia Type 1
Most severe form
Aminoacidopathies
A deficiency of phenylalanine hydroxylase (PAH) which catalyzes the conversion of phenylalanine to tyrosine
Phenylketonuria
Aminoacidopathies
Phenylketonuria can also happen when there is a problem with what 2 substances? (aside from PAH)
- Tetrahydrobiopterin
- Dihydrobiopterin
Are co-enzymes (important for the reaction)
Aminoacidopathies
Patients are usually:
1. Normal at birth but manifest mental retardation at the 6th month
2. Decreased skin pigmentation, eczema, and musty odor in blood and urine
Phenylketonuria
Aminoacidopathies
A condition wherein tyrosine levels are decreased (important for oxidation, protein synthesis, catecholamine, thyroxine, and melanin-formation)
Phenylketonuria
Diagnosing Phenylketonuria
- Principle: Semiquantitive bacterial inhibition assay
- Elevated phenylalanine levels facilitate the growth of B. subtilis in a culture medium with an inhibitor (beta-2-thienylalanine)
- The inhibitor is no longer effective against bacteria
- Can detect serum phenylalanine levels of 180-240 umol/L (3-4 mg/dL)
- Positive Result: (+) growth of B. subtilis (elevated phenylalanine)
- Prone to false (-) negative results
Guhtrie Test
Diagnosing Phenylketonuria
- Principle: Quantitative assay that directly measures phenylalanine in blood filter disks via immunofluorescence
- It yields quantitative results, is more adaptable to automation, and is not affected by the presence of antibiotics
- Based on fluorescence of a complex formed of phenylalanine-ninhydrin-copper in the presence of dipeptide
- Any positive results must be verified
Microfluorimetric Assay
Diagnosing Phenylketonuria
Familiarize yourself with the OTHER possible tests for phenylketonuria diagnosis (apart from the Guthrie Test and Microfluorimetric Assay)
- GC/MS
- Tandem Mass Spectrometry (the GOLD STANDARD)
- DNA Analysis
Tandem Mass Spectrometry: Compares both phenylalanine and tyrosine concentrations
Aminoacidopathies
- Inborn metabolic disorder of tyrosine catabolism (increased levels)
- Excretion of tyrosine and tyrosine catabolites in urine
Tyrosinemia
Types of Tyrosinemia
- Most severe kind; a deficiency of fumarylacetoacetate hydrolase
- Failure to thrive, diarrhea, vomiting, jaundice, cabbage-like odor, distended abdomen, swelling of legs, and increased predisposition for bleeding
- Can lead to liver and kidney failure (Fanconi Syndrome), affects CNS, and increases risk of cirrhosis or liver cancer
Type 1
Types of Tyrosinemia
- A deficiency of tyrosine aminotransferase
- Half are mentally retarded and have symptoms of excessive tearing, photophobia, eye pain and redness, and painful skin lesions on the palms and soles
Type 2
Types of Tyrosinemia
- A deficiency of 4-hydroxyphenylpyruvate dioxygenase (4HPP Dioxygenase)
- Clinical presentation includes mild mental retardation, seizures, and periodic loss of balance and coordination
Type 3
A rare type
Diagnosing Tyrosinemia
What tests can be employed?
- Tandem MS
- Mass Spectrometry
Aminoacidopathies
- A secondary deficiency of homogentisate oxidase (HGA is increased and will not be coverted to Maleylacetoacetate)
- Urine turns brownish-black when mixed with air due to an accumulation of homogentisic acid (HGA) in the urine (when oxidized produces a dark pigment)
- Onchronosis - pigmentations on different parts of the body such as:
- HGA accumulation and pigment
- Dark Spots on the sclera
- Deposition in cartilage
- Arthritis-like degeneration
Alkaptonuria
Diagnosing Alkaptonuria
During urinalysis, the urine (with homogentisic acid) of the patient turns black upon the addition of what compound?
Ferric Chloride (FeCl2)
Aminoacidopathies
- Burnt sugar odor in the urine, breath, and skin
- Deficiency of α-ketoacid decarboxylase
- There is an accumulation of branched chain amino acids (leucine, isoleucine, and valine)
- Normal at Birth → Lethargy, MR, and CNS symptoms
Maple Syrupe Urine Disorder
Diagnosing MSUD
- A neonatal screening test
- B. subtilis in a culture media with 4-azaleucine as inhibitor
- Positive result: (+) growth of B. subtilis
Modified Guthrie Test
Modified because a different inhibitor is used
Diagnosing MSUD
- 3 branched-chain amino acids uses filter paper specimen treated with a solvent mixture of methanol and acetone to denature the hemoglobin
- Leucine dehydrogenase is added for the fluorescence of NADH
- Leucine level above 4 mg/dL is indicative of MSUD
Microfluorimetric Assay
Diagnosing MSUD
The gold standard test
Tandem MS
Aminoacidopathies
- Deficiency of isovaleryl-CoA dehydrogenase (preventing normal metabolism of leucine ONLY)
- Sweaty feet odor and brain damage
- Mild to moderate ketonuria, metabolic acidosis,
hyperammonemia, thrombocytopenia, and neutropenia
Isovaleric Acidemia
Diagnosing Isovaleric Acidemia
What is the gold standard and what is the common method? (2 answers)
Gold: Tandem MS
Common: Chromatography
Aminoacidopathies
If chromatography is the common screening method performed by laboratories, Tandem MS is done by who?
Reference Labs
Aminoacidopathies
- Deficiency of cystathionine-βsynthetase (necessary for the metabolism of methionine, that results in elevated plasma and urine levels of methionine and its precursor, homocysteine)
- Multisystemic connective tissue disorder (muscles and bones)
Homocystinuria
Diagnosing Homocystinuria
- B. subtilis in a culture media with L-methionine sulfoximine as an inhibitor
Modified Guthrie Test
Diagnosing Homocystinuria
This screening test detects elevations in urinary total homocysteine which can be measured in high testing volumes and provide a rapid turn around by using liquid chromatography electrospray
Tandem MS
Diagnosing Homocystinuria
- This confirmatory test indicates that a methionine level greater than 2 mg/dL confirms a positive result
HPLC
Aminoacidopathies
- Defect in the amino acid transport system (no enzyme deficiency)
- Inadequate renal reabsorption of cystine → renal stones/crystals
Cystinuria
Diagnosing Cystinuria
- Testing the urine for cystine using cyanide nitroprusside which produces a red-purple color upon reaction with sulfhydryl groups
- False positive results as a result of homocysteine must be ruled out (Homocysteine, Lysine, Arginine, Ornithine)
Urine Test
Amino Acid Analysis
Identify the sample:
- 6-8 hour fasting period (to remove the dietary effects of amino acids)
- Must come from a heparin tube and be separated immediately with no blood cells
Plasma
Amino Acid Analysis
Identify the sample:
- A random specimen is allowed for screening purposes
- For quantification purposes, a 24-hour period of collecting this sample is preserved with thymol or organic solvent
- Amniotic fluid may also be analyzed (for inborn errors)
Urine
Amino Acid Analysis
Familiarize with the 3 methods in analysis
- Liquid Chromatography - Method of Choice
- Ion Exchange Chromatography - for quantification
- Mass Spectrometry
