Lecture 3 - Proteins, Amino Acids, Inborn Errors

PKU:

1) What is the Etiology?

2) What are the consequences/effects?

1) Etiology:

• Mutation in PAH gene that encodes for the phenylalanine hydroxylase enzyme

2) Effects:

• Significant brain damage due to accumulation of metabolites such as:

  • Phenylpyruvic acid, phenylpyruvate, and phenyllactic acid

• Progressive mental retardation in children

• Fetal microcephaly

• Causes musty or mousy odor in urine

PKU Testing:

1) How early should testing be done?

2) How does the Guthrie Test work?

3) In UA, what test is correlated?

4) What type of assay is used for direct measurement?

5) What is used for confirmation?

1) Typically, newborn screen spot test performed in all states at day 3 after birth

2) spores of Bacillus subtilis are inoculated into agar that contains B2-thienylalanine that inhibits growth; however, if increased phenylalanine can inhibit the B2-thienylalanine which will result in spore growth

3) Phenistix (ferric chloride reaction) positive with blue-gray to green reaction

4) Microfluorometric assay

5) Confirmed with HPLC and Tandem MS

Tyrosinemia:

1) What occurs in this condition?

2) What are the Types of genetic defect?

3) What is included in the Lab Testing?

1) Excess tyrosine in blood that causes overflow in urine, resulting cabbage-like odor

2) 3 Types:

• Type I → mutation in FAH gene [most severe, sever liver disease, cirrhosis, liver cancer risk later in life]

• Type II → mutation in TAT gene [impaired mental development, painful skin lesions, photophobia]

• Type III → mutation in HPD gene [rare, impaired mental development, seizures, loss of balance]

3) Lab Testing:

• Elevated tyrosine and succinylacetone in blood; tyrosine crystals in acidic urine that are fine silky needles

• Nitrosonaphthol test positive red color

• MS/MS or quantitative assay for confirmation

Alkaptonuria:

1) What is the Etiology?

2) How are patients affected?

3) What is included in the Lab Testing?

1) mutation in HGD gene which encodes the enzyme homogentisate oxidase to metabolize homogentistic acid in the phe/tyr pathway

2) Effects:

• Develop dark blue brown to black pigmentation in cartilage and connective tissue

• Arthritis

• Urine darkens (even black) upon standing at room temp or alkaline pH

3) Lab testing:

• Ferric Chloride positive in urine with transient deep blue

• GC and MS/MS confirm

MSUD:

1) What is the Etiology?

2) What are clinical features?

1) Absence of extremely low activity of a complex of enzymes known as Branched-chain alpha-ketoacid dehydrogenase (BCKD)

• Defective metabolism of leucine, isoleucine, valine

2) Clinical Features:

• Maple syrup or burnt sugar smell of urine,sweat, and breath

• Severe neonatal vomiting, acidosis, seizures, stupor, failure to thrive, mental retardation, even death

MUSD Lab Diagnosis:

1) How does Modified Guthrie Test work?

2) How is Microfluorometric Assay work?

3) What is done for prenatal diagnosis?

1) The agar contains 4-azaleucine to inhibit growth of Bacillus subtilis; but in the presence of branched-chain amino acids from newborn blood disk the bacteria will grow

2) Microfluorometric Assay:

• Solvent mixture of methanol + acetone is added onto filter paper with specimen to denature Hb

• Leucine dehydrogenase is added

• Fluorescence of NADH is measured at 450 nm with excitation at 360 nm

• Leucine of greater than 4 g/dL is indicative of MSUD

3) decarboxylase enzyme concentration in cells cultured from amniotic fluid

Homocystinuria:

1) What is the Etiology?

2) What are the Clinical Features?

3) What is included in the Lab Diagnosis?

1) Caused by mutation in the CBS gene that encodes for the enzyme cystathionine beta-synthase, resulting in elevated levels of homocysteine and methionine

2) no clinical effects until late childhood with vascular thrombosis, mental retardation, and ocular damage

3) Lab Diagnosis:

• Modified Guthrie with L-methionine sulfoximine as inhibitor

• HPLC and MS/MS confirm

• Methionine concentration of greater than 2 mg/dL is indicative

What occurs in Cystinuria?

• Defective reabsorption of cystine and dibasic amino acids arginine, lysine, and ornithine by renal tubular cells

• Renal stones develop

  • Cystine crystals in urine

What occurs in Cystinosis?

• True inborn error of metabolism

• Increased cystine in cornea, bone marrow, lymph nodes, and internal organs

• Fanconi Syndrome develops with renal failure

What are the Sample Requirements for Amino Acid Analysis?

• Collect Blood at least 6-8 hours fasting to avoid effects of absorbed amino acids from diet

• Avoid contamination

• No Hemolysis

• Deproteinization should be performed within 30 minutes of sample collection and analyze immediately

  • Otherwise, store sample at -20°C to -40°C

• Urine random collection for screening but 24-hour specimen preserved with thymol or organic solvents for quantitation