AP - Phenylketonuria

Aminoacidopathies

class of inherited errors of metabolism

Phenylketonuria

caused by phenylalanine hydroxylase (PAH) deficiency - [PAH K U]

Phenylalanine hydroxylase (PAH) deficiency

disruption in the normal conversion of phenylalanine to tyrosine

greater than 1,200 umol/L

Without the enzyme: phenylalanine levels are usually

UL - 120 umol/L (2mg/dL)

as high as 2.4 mM/L

What is the level of PKU in newborn? How about blood level of untreated classic PKU?

Musty Odor

What is the urine odor of PKU?

Partial deficiency of PAH (mild PKU)

What deficiency of PKU is present if the phenylalanine level is between 600-1,200 umol/L

Non-PKU mild hyperphenylalaninemia

What deficiency of PKU is present if the phenylalanine level is between 180-600 umol/L

Mental retardation and microcephaly

Patients with PKU may experience:

Tetrahydrobiopterin (BH4)

is a co-factor in the enzymatic hydroxylation of phenylalanine, tryptophan and tyrosine

Co-Factor

a non-protein chemical/entity that must bind to a particular enzyme before a reaction occurs

Kuvan (sapropterin dihydrochloride)

First US FDA approved drug to help manage PKU, it helps by increasing the activity of the enzyme in particular of the phenylalanine hydroxylase enzyme

Permanent Blue-Green

What coloration can be detected In PKU when the urine is treated with Ferric Chloride reagent?

Guthrie Test / Guthrie Bacterial Inhibition Test

It is a test in PKU that is semiquantitative, bacterial inhibition assay (bacterial growth in culture even with presence of inhibitor)

180-240 umol/L (3-4 mg/dL)

What is the sensitivity of Guthrie Test?

1. Collect the newborns blood

2. Punch out a small disk

3. Prepare the agar plate

4. Place the blood disk on the agar

5. Phenylalanine moves into the agar (high amount diffuses)

6. The inhibition is overcome (extra phenylalanine counteracts the inhibitory effect of b-2-thienylalanine)

7. Inhibitor cant stop the bacteria, Bacillus subtilis can grow around the disk)

8. Interpret

• Bacterial growth → high phenylalanine → possible PKU

• Little or no growth → phenylalanine is not elevated → PKU is less likely.

Step by Step of Guthrie Test.

B-2-thienylalanine

What is the inhibitor of Bacillus subtilis?

Permanent Blue-Green

What colorization the Ferric Chloride Tube Test produce?

1. Place 1mL of urine in a tube

2. Slowly add 5 drops of 10% Ferric Chloride

3. Observe color for permanent blue-green color

Ferric Chloride tube test Step

Microfluorometric assay

Direct measurement of phenylalanine in dried blood filter disks; quantitative method

Fluorescence of a complex formed of phenylalanine-ninhydrin-copper in the presence of a dipeptide.

What is the principle of Microfluorometric assay?

High-Performance Liquid Chromatography (HPLC)

Reference method for quantitative serum phenylalanine. Verify positive result in screening test.

Tandem Mass Spectrometry (MS/MS)

Used in screening for inherited disorders in newborns

Silylation

It increase the stability of analyte and improve the gas chromatography behavior

N,Obis(trimethylsilyl)-trifluoroacetamide

What is the silylating reagent used?

microwave irradiation

Amino acids are extracted from neonatal blood samples and rapidly derived with N,Obis(trimethylsilyl)-trifluoroacetamide under

gas chromatography - mass spectrometry

What does GC/MS means