Background and proposal for a pilot EQA scheme for pterins enad Blau Division of Inborn Metabolic Diseases University Children's Hospital Heidelberg Germany
Tetrahydrobiopterin (BH 4 ) Brimstone butterfly Gowland Hopkins (1895) H 2 H O PTERO = Wing H H OH OH CH 3 Blau 2013
Biologically important pterins H 2 H 2 H 2 H H H O O O H H H OH OH OH OH O OH CH 3 CH 2 OH CH 3 Tetrahydrobiopterin eopterin Sepiapterin Blau 2013
Oxidation forms of BH4 H 2 H O H H 5,6,7,8-Tetrahydrobiopterin (BH4) Ox. q oder7,8-dihydrobiopterin (BH2) Ox. OH OH Biopterin (Bio) CH 3 Blau 2013
Regeneration of BH4 5,6,7,8-Tetrahydrobiopterin (BH4) DHPR H 2 H O q or 7,8-Dihydrobiopterin (BH2) Biopterin (Bio) H H Ox. Ox. OH OH CH 3 DHPR = Dihydropteridine reductase DHFR = Dihydrofolate reductase DHFR Blau 2013
Functions of tetrahydrobiopterin Cofactor for: Phenylalanine hydroxylase Phe Tyr Tyrosine hydroxylase Tyr L-Dopa Dopamin Tryptophan hydroxylase Trp 5-OH-Trp Serotonin itric oxide synthase (OS) Arg Cit + O. Glyceryl-ether monooxygenase Lipidether Glycerol + Aldehyd Blau 2013
Phenylalanine hydroxylating system Blau,., F. J. Van Spronsen and H. L. Levy (2010). "Phenylketonuria." Lancet 376: 1417-1427.
BH4 deficiencies DHPR q-dihydrobiopterin Guanosin triphosphate(gtp) Dihydroneopterin triphosphate 6-Pyruvoyl-tetrayhdropterin Tetrahydrobiopterin (BH4) PCD GTPCH PTPS SR Pterin-4acabinolamine Phenylalanine PAH with hyper-phe Tyrosine Total 699 patients Tyrosine TH BIODEFdb www.biopku.org L-Dopa TPH Tryptophan 5-OH-Tryphtophan without hyper-phe Blau 2013
Laboratory diagnosis of PKU and BH4 deficiencies Differential diagnosis 1. Quantitative analysis of Phe and Tyr in plasma (DBS) 2. Pterins (neopterin and biopterin) in DBS or urine 3. DHPR activity in DBS 4. BH 4 loading test Blau 2013
Laboratory diagnosis of PKU and BH4 deficiencies Pterins (neopterin and biopterin) in DBS or urine Every newborn with even slightly elevated blood Phe levels Before introducing the diet (at high blood Phe) CAVE: GTPCH deficiency may present with normal blood Phe in the BS Blau 2013
Laboratory diagnosis of PKU and BH4 deficiencies eopterin, biopterin and primapterin are markers for BH4 deficiency Light- and oxygen-sensitive In DBS pterins are stable (transport at RT) In urine pterins must be oxidized with MnO 2 Transport of native urine on dry ice (native urine) Blau 2013
Pterins in urine or DBS? URIE DBS Higher concentrations (µmol/l) eed for oxidation or shipping on dry ice Light and oxygen sensitive Creatinine Lower concentrations (nmol/l) Oxidized during drying and stable on filter paper Shipping at RT in envelope Hemoglobine
GTPCH deficiency Biopterin eopterin Guanosine triphosphate (GTP) GTP cyclohydrolase I Dihydropteridine reductase PTP synthase Sepiapterin reductase H2TP PTP Tetrahydrobiopterin (BH4) q-dihydrobiopterin Biopterin Phenylalanine +O 2 PCD Pterin- Tyrosine 4a-carbinolamine Primapterin M IX B Control P Phenylalanine hydroxylase B GTPCH def.
PTPS deficiency Biopterin eopterin Guanosine triphosphate (GTP) GTP cyclohydrolase I PTP synthase Sepiapterin reductase Dihydropteridine reductase H2TP PTP Tetrahydrobiopterin (BH4) q-dihydrobiopterin Biopterin Phenylalanine +O 2 PCD Pterin- Tyrosine 4a-carbinolamine Primapterin M IX B Control P M IX B PTPS def. Phenylalanine hydroxylase Blau 2013
DHPR deficiency eopterin Guanosine triphosphate (GTP) GTP cyclohydrolase I Dihydropteridine reductase PTP synthase Sepiapterin reductase H2TP PTP Tetrahydrobiopterin (BH4) q-dihydrobiopterin Biopterin Phenylalanine +O 2 PCD Pterin- Tyrosine 4a-carbinolamine Primapterin M IX B Control P M IX Phenylalanine hydroxylase B DHPR def. P Blau 2013
Aim of the EQA We plan to distribute urine and/or DBS samples twice a year. Samples will be obtained from patients with confirmed diagnosis and with known quantitative values for pterins and DHPR (tested in Heidelberg Metabolic Laboratories). Participating laboratory should be able to separate neopterin, monapterin, biopterin, primapterin, isoxanthopterin and pterin and to quantify total neopterin and biopterin. Blau 2013
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Samples DPT-1: DPT-2 Two urine samples (normal or pathologic) Two DBS samples (normal or pathologic) Two urine samples (normal or pathologic) Two DBS samples (normal or pathologic) Blau 2013
Investigations Quantitative results (urine): eopterin (total) Biopterin (total) Creatinine Quantitative results (DBS): eopterin (total) Biopterin (total) Dihydropteridine reductase activity (Hemoglobin) Diagnosis Blau 2013
Evaluation Test Samples Pterins* mmol/mol Creat *Cas Laboratory Blau 2013
ext steps Registration of potential participants Questionnaire (method details) Samples collection (Heidelberg) Samples processing and distribution (MCA Lab) Blau 2013
Acknowledgements Metabolic Laboratory Heidelberg Alex Anninos Brigitte Schmidt-Mader PD Dr. Jürgen Okun Streekziekenhuis Koningin Beatrix Winterswijk (MCA) Dr. Cas Weykamp