(M) Lec 5: Amino Acids (P1: Aminoacidopathies)

Question 1

• These are the building blocks of proteins (100-150 of these = proteins)
• The second most abundant NPN (second to urea)

Answer 1

Amino Acids

Question 2

Amino Acids are called as such because of what 2 groups in their composition attached to Carbon?

Answer 2

Basic = Amino group (nitrogen-containing)
Acid = Carboxyl group

Note: They only vary in their side chains (R-group)

Question 3

Conditions/disorders wherein an individual lacks certain enzymes which are important for the metabolism of amino acids

Answer 3

Aminoacidopathies (or elevated amino acid levels, in other words)

Question 4

These amino acids cannot be synthesized by the body and therefore comes from the proteins that we eat

Answer 4

Essential Amino Acids

Ex.: Histidine, Leucine, Isoleucine, Arginine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, and Valine

Question 5

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

Answer 5

Non-Essential Amino Acids

Question 6

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

Answer 6

Semi-Essential (Conditionally Essential)

Ex.: Arginine, Cysteine, Glutamine, Glycine, Proline, and Tyrosine

Question 7

• 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

Answer 7

Aminoacidopathies

Question 8

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)?

Answer 8

Phenylalanine will increase leading to Phenylketonuria

Question 9

Aminoacidopathies

If an individual lacks the tyrosine aminotransferase enzyme, what condition will the person manifest?

Answer 9

Hereditary Type 2 Tyrosinemia

Tyrosine cannot be converted to 4HPP

Question 10

Aminoacidopathies

Hereditary Type 3 Tyrosinemia occurs if there is a deficiency of what enzyme?

Answer 10

4HPP Dioxygenase

Question 11

Aminoacidopathies

Homogentisate (HGA) will be converted to Maleylacetoacetate (MAA) with the help of what enzyme (+ include the condition)?

Answer 11

HGA Oxidase (alkaptonuria)

Question 12

Aminoacidopathies

Maleylacetoacetate (MAA) will be converted to what using the MAA isomerase?

Answer 12

Fumarylacetoacetate (FAA)

Question 13

Aminoacidopathies

Fumarylacetoacetate (FAA) will be degraded into what 2 compounds?

Answer 13

1. Fumarate
2. Acetoacetate

Question 14

Aminoacidopathies

A lack of the FAA hydrolase produces what condition?

Answer 14

Hereditary Tyrosinemia Type 1

Most severe form

Question 15

Aminoacidopathies

A deficiency of phenylalanine hydroxylase (PAH) which catalyzes the conversion of phenylalanine to tyrosine

Answer 15

Phenylketonuria

Question 16

Aminoacidopathies

Phenylketonuria can also happen when there is a problem with what 2 substances? (aside from PAH)

Answer 16

1. Tetrahydrobiopterin
2. Dihydrobiopterin

Are co-enzymes (important for the reaction)

Question 17

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

Answer 17

Phenylketonuria

Question 18

Aminoacidopathies

A condition wherein tyrosine levels are decreased (important for oxidation, protein synthesis, catecholamine, thyroxine, and melanin-formation)

Answer 18

Phenylketonuria

Question 19

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

Answer 19

Guhtrie Test

Question 20

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

Answer 20

Microfluorimetric Assay

Question 21

Diagnosing Phenylketonuria

Familiarize yourself with the OTHER possible tests for phenylketonuria diagnosis (apart from the Guthrie Test and Microfluorimetric Assay)

Answer 21

1. GC/MS
2. Tandem Mass Spectrometry (the GOLD STANDARD)
3. DNA Analysis

Tandem Mass Spectrometry: Compares both phenylalanine and tyrosine concentrations

Question 22

Aminoacidopathies

• Inborn metabolic disorder of tyrosine catabolism (increased levels)
• Excretion of tyrosine and tyrosine catabolites in urine

Answer 22

Tyrosinemia

Question 23

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

Answer 23

Type 1

Question 24

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

Answer 24

Type 2

Question 25

# 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

Answer 25

Type 3 | A rare type

Question 26

# Diagnosing Tyrosinemia What tests can be employed?

Answer 26

1. Tandem MS 2. Mass Spectrometry

Question 27

# 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: 1. HGA accumulation and pigment 2. Dark Spots on the sclera 3. Deposition in cartilage 4. Arthritis-like degeneration

Answer 27

Alkaptonuria

Question 28

# Diagnosing Alkaptonuria During urinalysis, the urine (with homogentisic acid) of the patient turns black upon the addition of what compound?

Answer 28

Ferric Chloride (FeCl2)

Question 29

# 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

Answer 29

Maple Syrupe Urine Disorder

Question 30

# Diagnosing MSUD - A **neonatal** screening test - **B. subtilis** in a culture media with **4-azaleucine** as inhibitor - Positive result: (+) growth of B. subtilis

Answer 30

Modified Guthrie Test | Modified because a different inhibitor is used

Question 31

# 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

Answer 31

Microfluorimetric Assay

Question 32

# Diagnosing MSUD The gold standard test

Answer 32

Tandem MS

Question 33

# 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

Answer 33

Isovaleric Acidemia

Question 34

# Diagnosing Isovaleric Acidemia What is the gold standard and what is the common method? (2 answers)

Answer 34

Gold: Tandem MS Common: Chromatography

Question 35

# Aminoacidopathies If chromatography is the **common screening method** performed by laboratories, Tandem MS is done by who?

Answer 35

Reference Labs

Question 36

# 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)

Answer 36

Homocystinuria

Question 37

# Diagnosing Homocystinuria - B. subtilis in a culture media with **L-methionine sulfoximine** as an inhibitor

Answer 37

Modified Guthrie Test

Question 38

# 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

Answer 38

Tandem MS

Question 39

# Diagnosing Homocystinuria - This **confirmatory test** indicates that a methionine level **greater than 2 mg/dL** confirms a positive result

Answer 39

HPLC

Question 40

# Aminoacidopathies - Defect in the **amino acid transport system** (no enzyme deficiency) - Inadequate renal reabsorption of **cystine** → renal stones/crystals

Answer 40

Cystinuria

Question 41

# 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)

Answer 41

Urine Test

Question 42

# 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

Answer 42

Plasma

Question 43

# 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)

Answer 43

Urine

Question 44

# Amino Acid Analysis Familiarize with the 3 methods in analysis

Answer 44

1. Liquid Chromatography - **Method of Choice** 2. Ion Exchange Chromatography - for **quantification** 3. Mass Spectrometry