Screening for phenylketonuria (PKU) –

laboratory methods

Péter Monostori

Phe & BH4 metabolism pathway

q-Dihydrobiopterin

During the hydroxylation of Phe by Phe hydroxylase (PAH) to form tyrosine (Tyr), tetrahydrobiopterin (BH4) is oxidized to a 4a-hydroxy-BH4 intermediate (when molecular oxygen and iron (Fe2+) are present).

Phe & BH4 metabolism pathway

Dihydropteridine reductase (DHPR)

Pterin-4α-carbinolamine dehydratase (PCD)

q-Dihydrobiopterin

This intermediate is subsequently regenerated back to BH4 via quinonoid (q)-dihydrobiopterin by the enzymes pterin-carbinolamie-4a-dehydratase (PCD) and by the NADH-dependent dihydropteridine reductase (DHPR).

Dihydropteridine reductase (DHPR)

Pterin-4α-carbinolamine dehydratase (PCD)

q-Dihydrobiopterin

60%

30%

5%

Phe & BH4 metabolism pathway

6-Pyruvoyl-tetrahydrobiopterin synthase (PTPS)

Sepiapterin reductase (SR)

GTP cyclohydrolase (GTPCH)

5%BH4 is synthesized from guanosine triphosphate (GTP) by three additional enzymes: GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetra-hydropterin synthase (PTPS), and sepiapterin reductase (SR). Mutations in genes coding enzymes for GTPCH, PCD, SR, DHPR and PTPS result in BH4 deficiency.

Which markers can be useful in the diagnosis of PKU and BH4 deficiency?

PKU:Phe (from DBS)Tyr (DBS)Phe/Tyr ratio (DBS)

BH4 deficiency:Neopterin, biopterin and pterin levels (urine, DBS)DHPR activity (DBS)Homovanillic acid (HVA) (liquor)5-Hydroxy-indoleacetic acid (5-HIAA) (liquor)

Definitions of positive/negative predictive value, sensitivity and specificitySensitivity

The proportion of affected subjects that have a positive test result

Specificity The proportion of unaffected subjects that have a negative

test resultPositive predictive value

The chance that a positive test result actually indicates an a ected individualff

The proportion of „real” positive samples within all positive results

Negative predictive value The chance that a negative test result actually excludes

the disorder The proportion of „real” negative samples within all

negative results

The beginnings…

1920: A child with developmental delay was born to American parents living in China. No one could help in finding the disease.

The mother wrote a book, describing the symptoms.

The child was later diagnosed as having classical PKU.

Overview of the methods for PKU screening – The Folling-test

1. In the 1930s: Asbjorn Folling: a mother noticed a strange smell of her mentally retarded child’s urine → Folling analyzed the urine with various tests including the ferric chloride test (for aromatic hydroxyl groups, such as those in ketones):

When ketones are present, urine develops a red-brown colour.This time the urine turned into dark-green.

Folling isolated a substance from the urine which was confirmed to be phenylpyruvate.

The Folling-test

not sensitive: usually positive at plasma Phe concentrations above 900 μM (dark green coloration)

not specific: a slightly altered color reaction may be indicative of other metabolic disorders/medication:

maple syrup disease (MSUD) tyrosinemia salicylates, L-DOPA metabolites…

traditionally, the reagent was dropped on the diaper of the baby

The ferric chloride test (for urine):

The Guthrie-method

2. From the 1960s: Robert Guthrie and Ada Susi developed a bacterial inhibition assay, suitable for the screening of PKU for the first time.

This assay monitors the growth of a mutant strain of Bacillus subtilis with a requirement for exogenous Phe for growth.DBS samples are placed onto agar plates containing mutant bacteria and an inhibitor.The sizes of the colonies are assessed after incubation.

The Guthrie-method – principals

The growth of Bacillus subtilis is inhibited by an appropriate amount of β-2-thienylalanine added to the agar.

This inhibition is reversed when a dried blood spot (DBS) containing the blood of a patient with PKU is placed on the agar → Phe in the blood permits the growth of bacteria around the DBS.

The test is positive if the diameter of the growth zone is between the 2 mg% (120 μM) and the 4 mg% (240 μM) standard points (marked).

The amount of growth is proportional to the level of Phe in the DBS.

The Guthrie-method

Standards: 2 4 8 16 32 (mg%)

The Guthrie-method

Control agar plate without β-2-thienylalanine inhibitor Rationale: antibiotic therapy can prevent the growth of

Bacillus subtilis, resulting in false-negative results A new blood sample is obtained if a zone with signs of

inhibited bacterial growth is found (marked)

The Guthrie-method – characteristics

inexpensive specific semiquantitative not very sensitive:

limit of detection ≈180-240 μM (=3-4 mg%)

Fluorimetric assays

3. From the 1960s: Fluorimetric assaysMcCaman and Robins (1962):

for the determinaton of Phe only principals: the reaction of Phe, ninhydrin and copper yields

a weakly fluorescent product the fluorescence is increased by the addition of a dipeptide,

L-leucyl-L-alanine

Wong, O’Flynn and Inouye (1964): modified the above method to measure Phe, and added

another method to determine Tyr

Ambrose, Ingerson, Garrettson and Cliung (1967): optimized the Phe-assay by changing several parameters

Fluorimetric assays – characteristics

quantitative automatization is possible sensitivity is good:

limit of detection may be as low as 6 μM (0.1 mg%)

not specific (other substances may also yield some degree of fluorescence)

Enzymatic colorimetric assays

4. From the 1980s: Enzymatic colorimetric assaysWendel, Hummel and Langenbeck (1989):

for the measurement of Phe using L-phenylalanine dehydrogenase, NAD and a chlorophore

Campbell et al. (1992): modified the method to reach greater specificity (lower

cross-reactivity with Tyr)

Enzymatic colorimetric assays – characteristics

quantitative automatization is possible sensitivity is acceptable:

the limit of detection is about 43 μM (0.7 mg%) (higher than that of the fluorescence assay)

specific

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

5. From the 1990s: liquid chromatography-tandem mass spectrometry (LC-MS/MS)

allows the simultaneous measurement of a number of disorders of amino acid, organic acid and fatty acid metabolism, including PKUdeuterated internal standards are usedderivatization with butanol-acetyl chloride is employedselected ratios of the amino acids (or acylcarnitines) are used to help the evaluation

LC-MS/MS assays – characteristics

quantitative automated rapid very sensitive: lower than 1 μM (0.07 mg%) very specific the false-positive rate is the lowest,

highlighting the advantage of using the Phe/Tyr ratio (Tyr levels are simultaneously measured):

example: parenteral amino acid supplementation or too much blood on the filter paper: Phe ↑, but Phe/Tyr is normal → PKU can be excluded

PKU screening – Blood sampling

primary sample: blood spots dried on filter paper (DBS)

stability of DBS: ≈10 days at room temperature for amino acids (≈7 days for acylcarnitines)

problems associated with blood sampling:inappropriate timinginappropriate techniquedelayed deliveryinsufficient data on the patient/parent

Problems with blood sampling (DBS)

a) Inappropriate timing of blood sampling (rule: 48-72 h of age; earlier: < 5 days)

the catabolic state associated with birth is the main trigger of most amino acid (incl. Phe) and acylcarnitine elevations in the first few days of life (and not feeding)(this is not true for galactosaemia and some other disorders)delayed blood sampling may cause false-negative results

Problems with blood sampling (DBS)

b) Inappropriate technique of blood samplingis mainly responsible for the SD of the MS/MS method

insufficient bloodexcess bloodDBS has not dried

SafeNOT safe

Safe

Problems with blood sampling (DBS)

c) Delayed delivery of samples

d) Insufficient data on the child/parentabout drugs, parenteral feeding/glucose/middle-chain triglycerides given to the newborncontact address and telephone number of the parent

The diagnostic value of Phe assays

a positive screening result in a Phe assay is generally sufficient to conclude that some form of hyperphenylalaninemia (PKU, transient hyperphenylalaninemia or BH4 deficiency) is present

confirmation by means of genetic testing or gas chromatography-mass spectrometry (GC/MS) is not essential

Analysis of PKU with (GC/MS): urine samples

Plus: 4-hydroxy-phenyllactate, 4-hydroxy-phenylpyruvate, mandelic acid

The diagnostic value of Phe assays (continued)

PKU and BH4 deficiency cannot be distinguished from each other by Phe levels plus Phe/Tyr ratios

for the differential diagnosis of BH4 deficiency:BH4 loading test, pterin profile analysis (from urine or DBS), dihydropteridine reductase (DHPR) activity measurement (from DBS) should be performed

Dihydropteridine reductase (DHPR)

Pterin-4α-carbinolamine dehydratase (PCD)

q-Dihydrobiopterin

60%

30%

5%

Phe & BH4 metabolism pathway

6-Pyruvoyl-tetrahydrobiopterin synthase (PTPS)

Sepiapterin reductase (SR)

GTP cyclohydrolase (GTPCH)

5%Mutations in genes coding enzymes for BH4 synthesis (GTPCH, PCD, SR), and BH4 recycling (PTPS, DHPR) result in BH4 deficiency.

Diagnosis of BH4 deficiency

1. BH4 loading testuseful in all forms of BH4 deficiencysingle Phe dose plus a single BH4 dose 3 h later

blood sampling: -3; 0; 4; 8; 12; 16; 24 h(if basal Phe level is low (e.g. < 360 μM), a 24 h Phe loading test may be performed prior to the BH4 test)

Diagnosis of BH4 deficiency

2. Analysis of pterins with HPLC plus fluorescent or MS/MS detectionLevels of neopterin, biopterin and pterin are measured from urine or DBS.Chromatographic separation is needed. Identifies variants: 65-70% of cases.

3. DHPR activity measurementPrimary sample: DBSIdentifies a single variant: 30-35% of cases.

Pterin levels and DHPR activity in variants of BH4 deficiency

Phe (plasma)

Biopterin (urine)

Neopterin (urine)

DHPR activity (blood)

Homovanillic acid (HVA,

liquor)

5-hydroxy-indoleacetic acid (5-HIAA, liquor)

GTPCH1 (recessive)

N

GTPCH1 (dominant)

N N ( in liquor)

N ( in liquor)

N N/

PTPS N

PCD N/ primapterin

N N N

DHPR N

SR N N ( in liquor)

N ( liquor sepiapterin)

N

Summary

The ideal method for PKU screening is sensitive, specific, rapid and reliable.

Of the numerous techniques for the measurement of Phe, the Guthrie method, fluorimetric and enzymatic colorimetric assays, and LC-MS/MS (the most recent technique) are used widely for screening purposes.

For the differential diagnosis of hyperphenylalaninemias, BH4 loading test, pterin profile analysis, or measurement of DHPR activity can be performed.

Thank you for your attention!