Screening for phenylketonuria PKU laboratory methods Pter Monostori

Screening for phenylketonuria (PKU) – laboratory methods Péter Monostori

Phe & BH 4 metabolism pathway During the hydroxylation of Phe by Phe hydroxylase (PAH) to form tyrosine (Tyr), tetrahydrobiopterin (BH 4) is oxidized to a 4 a-hydroxy-BH 4 intermediate (when molecular oxygen and iron (Fe 2+) are present). q-Dihydrobiopterin

Phe & BH 4 metabolism pathway This intermediate is subsequently regenerated back to BH 4 via quinonoid (q)-dihydrobiopterin by the enzymes pterin-carbinolamie 4 a-dehydratase (PCD) and by the NADH-dependent dihydropteridine reductase (DHPR). Dihydropteridine reductase (DHPR) q-Dihydrobiopterin Pterin-4α-carbinolamine dehydratase (PCD)

Phe & BH 4 metabolism pathway GTP cyclohydrolase (GTPCH) BH 4 is synthesized from guanosine triphosphate (GTP) by three additional enzymes: GTP cyclohydrolase I (GTPCH), 6 -pyruvoyltetra-hydropterin synthase (PTPS), and sepiapterin reductase (SR). Mutations in genes Dihydropteridine coding enzymes for reductase (DHPR) 30% GTPCH, PCD, SR, q-Dihydrobiopterin DHPR and PTPS result in BH 4 Pterin-4α-carbinolamine deficiency. dehydratase (PCD) 5% 5% 6 -Pyruvoyl-tetrahydrobiopterin synthase (PTPS) 60% Sepiapterin reductase (SR)

Which markers can be useful in the diagnosis of PKU and BH 4 deficiency? n PKU: n. Phe (from DBS) n. Tyr (DBS) n. Phe/Tyr ratio (DBS) n BH 4 deficiency: n. Neopterin, biopterin and pterin levels (urine, DBS) n. DHPR activity (DBS) n. Homovanillic acid (HVA) (liquor) n 5 -Hydroxy-indoleacetic acid (5 -HIAA) (liquor)

Definitions of positive/negative predictive value, sensitivity and specificity n. Sensitivity n The proportion of affected subjects that have a positive test result n. Specificity n The proportion of unaffected subjects that have a negative test result n. Positive predictive value n n The chance that a positive test result actually indicates an a�ected individual The proportion of „real” positive samples within all positive results n. Negative predictive value n n The chance that a negative test result actually excludes the disorder The proportion of „real” negative samples within all negative results

The beginnings… n 1920: A child with developmental delay was born to American parents living in China. No one could help in finding the disease. n The mother wrote a book, describing the symptoms. n The child was later diagnosed as having classical PKU.

Overview of the methods for PKU screening – The Folling-test n 1. In the 1930 s: 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): n. When ketones are present, urine develops a redbrown colour. n. This time the urine turned into dark-green. Folling isolated a substance from the urine which was confirmed to be phenylpyruvate.

The Folling-test n. The ferric chloride test (for urine): n 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: n maple syrup disease (MSUD) n tyrosinemia n salicylates, L-DOPA metabolites… traditionally, the reagent was dropped on the diaper of the baby

The Guthrie-method n 2. From the 1960 s: Robert Guthrie and Ada Susi developed a bacterial inhibition assay, suitable for the screening of PKU for the first time. n. This assay monitors the growth of a mutant strain of Bacillus subtilis with a requirement for exogenous Phe for growth. n. DBS samples are placed onto agar plates containing mutant bacteria and an inhibitor. n. The sizes of the colonies are assessed after incubation.

The Guthrie-method – principals n The growth of Bacillus subtilis is inhibited by an appropriate amount of β-2 -thienylalanine added to the agar. n 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. n 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). n 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 n Control agar plate without β-2 -thienylalanine inhibitor n Rationale: antibiotic therapy can prevent the growth of Bacillus subtilis, resulting in false-negative results n A new blood sample is obtained if a zone with signs of inhibited bacterial growth is found (marked)

The Guthrie-method – characteristics n inexpensive n specific n semiquantitative n not very sensitive: n limit of detection ≈180 -240 μM (=3 -4 mg%)

Fluorimetric assays n 3. From the 1960 s: Fluorimetric assays n. Mc. Caman 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 n. Wong, O’Flynn and Inouye (1964): § modified the above method to measure Phe, and added another method to determine Tyr n. Ambrose, Ingerson, Garrettson and Cliung (1967): § optimized the Phe-assay by changing several parameters

Fluorimetric assays – characteristics n quantitative n automatization is possible n sensitivity is good: nlimit of detection may be as low as 6 μM (0. 1 mg%) n not specific (other substances may also yield some degree of fluorescence)

Enzymatic colorimetric assays n 4. From the 1980 s: Enzymatic colorimetric assays n. Wendel, Hummel and Langenbeck (1989): § for the measurement of Phe using L-phenylalanine dehydrogenase, NAD and a chlorophore n. Campbell et al. (1992): § modified the method to reach greater specificity (lower cross-reactivity with Tyr)

Enzymatic colorimetric assays – characteristics n quantitative n automatization is possible n sensitivity is acceptable: nthe limit of detection is about 43 μM (0. 7 mg%) (higher than that of the fluorescence assay) n specific

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) n 5. From the 1990 s: 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 PKU ndeuterated internal standards are used nderivatization with butanol-acetyl chloride is employed nselected ratios of the amino acids (or acylcarnitines) are used to help the evaluation n

LC-MS/MS assays – characteristics n quantitative n automated n rapid n very sensitive: lower than 1 μM (0. 07 mg%) n very specific n the false-positive rate is the lowest, highlighting the advantage of using the Phe/Tyr ratio (Tyr levels are simultaneously measured): nexample: 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 n primary sample: blood spots dried on filter paper (DBS) n stability of DBS: n≈10 days at room temperature for amino acids (≈7 days for acylcarnitines) n problems associated with blood sampling: ninappropriate timing ninappropriate technique ndelayed delivery ninsufficient data on the patient/parent

Problems with blood sampling (DBS) n a) Inappropriate timing of blood sampling (rule: 48 -72 h of age; earlier: < 5 days) nthe 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) n(this is not true for galactosaemia and some other disorders) ndelayed blood sampling may cause false-negative results

Problems with blood sampling (DBS) n b) Inappropriate technique of blood sampling nis mainly responsible for the SD of the MS/MS method insufficient blood nexcess blood n. DBS has not dried n Safe NOT safe Safe

Problems with blood sampling (DBS) n c) Delayed delivery of samples n d) Insufficient data on the child/parent nabout drugs, parenteral feeding/glucose/middlechain triglycerides given to the newborn ncontact address and telephone number of the parent

The diagnostic value of Phe assays n a positive screening result in a Phe assay is generally sufficient to conclude that some form of hyperphenylalaninemia (PKU, transient hyperphenylalaninemia or BH 4 deficiency) is present n 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) n PKU and BH 4 deficiency cannot be distinguished from each other by Phe levels plus Phe/Tyr ratios n for the differential diagnosis of BH 4 deficiency: n. BH 4 loading test, npterin profile analysis (from urine or DBS), ndihydropteridine reductase (DHPR) activity measurement (from DBS) should be performed

Phe & BH 4 metabolism pathway GTP cyclohydrolase (GTPCH) Mutations in genes coding enzymes for BH 4 synthesis (GTPCH, PCD, SR), and BH 4 recycling (PTPS, DHPR) result in BH 4 deficiency. 5% 6 -Pyruvoyl-tetrahydrobiopterin synthase (PTPS) 60% Sepiapterin reductase (SR) Dihydropteridine reductase (DHPR) 30% q-Dihydrobiopterin Pterin-4α-carbinolamine dehydratase (PCD) 5%

Diagnosis of BH 4 deficiency n 1. BH 4 loading test nuseful in all forms of BH 4 deficiency nsingle Phe dose plus a single BH 4 dose 3 h later nblood sampling: -3; 0; 4; 8; 12; 16; 24 h n(if basal Phe level is low (e. g. < 360 μM), a 24 h Phe loading test may be performed prior to the BH 4 test)

Diagnosis of BH 4 deficiency n 2. Analysis of pterins with HPLC plus fluorescent or MS/MS detection n. Levels of neopterin, biopterin and pterin are measured from urine or DBS. n. Chromatographic separation is needed. n. Identifies variants: 65 -70% of cases. n 3. DHPR activity measurement n. Primary sample: DBS n. Identifies a single variant: 30 -35% of cases.

Pterin levels and DHPR activity in variants of BH 4 deficiency Phe (plasma) Biopterin (urine) Neopterin (urine) DHPR activity (blood) Homovanillic acid (HVA, liquor) 5 -hydroxyindoleacetic acid (5 -HIAA, liquor) GTPCH 1 (recessive) N GTPCH 1 (dominant) N 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 n The ideal method for PKU screening is sensitive, specific, rapid and reliable. n 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. n For the differential diagnosis of hyperphenylalaninemias, BH 4 loading test, pterin profile analysis, or measurement of DHPR activity can be performed.

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