Evolving Methods for the Measurement of Phenylalanine A vital diagnostic marker and indicator for follow-up in cases of hyperphenylalaninemia and tetrahydrobiopterin deficiency Dr. Zoltan Lukacs Department of Pediatrics, University Hospital Hamburg-Eppendorf, Germany Phenylketonuria Hyperphenylalaninemia Phenylketonuria atypical Phenylketonuria BH 4 -deficiency common feature: elevated phenylalanine concentration 1
Biochemistry + H 3 N COO - PAH / O 2 + H 3 N COO - Transaminase O COO - α-ketogluterate Phe BH 4 BH 2 OH Tyr OH Dioxygenase Ascorbate / O 2 O O - OOC COO - 4-Fumarylacetoacetate - OOC O O COO - 4-Maleylacetoacetate - OOC OH Dioxygenase O 2 OH Homogentisate Old Methods Fellow citizens, we cannot escape history Abraham Lincoln, Annual message to congress, Dec. 1862 2
FeCl 3 in urine Disease/Analyte PKU/phenylpyruvate Tyrosinemia/p-hydroxyphenylpyruvic acid MSUD/ branched chain ketoacids Alkaptonuria/homogentisic acid Acetaminophen Salicylates Color Green Rapidly fading green Greenish gray Effervescent blue-green Green purple example fast but unreliable The Guthrie Test Dried blood : - is easily transported - is reasonably stable - allows easy handling - can be stored 3
Bacterial Inhibition Assay (BIA) Phenylalanine-free agar B. subtilis spores β-2-thienylalanine (inhibitor) Dr. R. Guthrie Dried blood spots are put on the agar. Areas of intense growth indicate high phenylalanine concentrations Guthrie R, Susi A. Pediatrics 1963; 32: 338-43 Chromatography paper lid plate Cholesterol start sample eluent Principle of thin-layer chromatography example lipid extract from urine 4
Plasma or dried blood is spotted on paper Chromatograms were developed overnight stained with ninhydrinisatin Paper/TLC Chromatography Limit of detection (phe): 6-8 mg/dl 1- Phenylketonuria 6- Severe Tyrosinemia Scriver CR, Davies E, Cullen AM The Lancet 1964; II: 230-32. New Methods We are learning to fly 5
UV/VIS Spectroscopy Wavelength Principle of UV/VIS Spectroscopy Light source Filter Sample Detector Display UV/VIS Spectroscopy II Phenylalanine + H 2 O Phenylpyruvate + NH 4 + NAD + NADH/H + Electron acceptor (reduced) Electron acceptor (oxidized) Tetrazolium Formazan 6
UV/VIS Spectroscopy III calibration curve normal distribution - Interference of tyrosine reduced at ph 10.8 - CV: within-run 4.2%; between-run 10.4% - Limit of detection: 43 µm Wendel U et al. Clin Chim Acta 1990; 192: 165-70. Fluorometry I Principle of Fluorometry Light Source Filter Fluorescence Sample Filter Detector Display 7
Fluorometry II O O H 2 N OH OH + COOH CHO O OH + + + NH H 3 +H + + CO 2 O Hydrindantin Enhancer: L-leucyl-L-alanin X 390 nm E 486 nm LOD : 12 µm cut-off: 151 µm Fluorometry III -more sensitive than UV/VIS -Allows follow-up of patients Hill JB et al. Clin Chem 1965; 11: 541-46 8
HPLC Chromatography normal PKU Advantage: multi-analyte Disadvantage: OPA-derivatization pre-column has to be replaced frequently Vollmer et al. Anal Biochem 1990; 189: 115-21 IXC - chromatogram Ion Exchange Chromatography Tyr Phe Reference Method for amino acids Advantage: most amino acids quantitatively analyzed Disadvantage: takes long (> 2 h) derivatization necessary unreliable equipment 9
Tandem Mass Spectrometry Advantage: multi-analyte Phe/Tyr-Ratio fast and reliable Disadvantage: expensive equipment highly trained staff Screening Center North (Germany): ca. 40000 samples/a ca. 4 new PKU cases/a Follow-up of Hyperphenylalaninemias: 280 PKU patients 2 patients with BH 4 -deficiency who are monitored regularly in Hamburg Specialised Methods giving you the whole picture... 10
PheDH 1. Phe Phenylpyruvate Biosensor NAD + NADH/H + 2. Salicylate Catechol Phe 2e - Tyrosinase o-quinone E appl =-50 mv Linear range: 20-150 µm Limit of detection : 5 µm Time for 1 measurement : 1 min Electrode life: 11 d Huang T et al. Anal Chem 1998; 70: 991-7. MR Spectroscopy Phe Blood Phe Brain Single cases of untreated PKU with normal intelligence reported low Phe in brain was detected by MR spectroscopy Möller H. et al. Eur J Pediatr 2000; 159 (Suppl 2): S121-5. 11
Isotope labeled Phenylalanine L[1-13 C]Phenylalanine 13 CO 2 Analyzed by IRMS Treacy E.P. et al. Pediatr Res 1997; 42: 430-5. BH 4 loading test Blood Phe [µmol/l] 1400 1000 600 200 PKU-Patient without BH 4 with BH 4 Difference > 30% 6 am 12 pm 6 pm 12 am 6 am Time Phenylalanine measurement to evaluate BH 4 -responsiveness Steinfeld R., Kohlschütter A., Zschocke J., Lindner M., Ullrich K., Lukacs Z. Eur. J. Ped. 2002; 161: 403-5. 12
BH 4 as a treatment option 800 700 Patient 1 under BH 4 treatment Blood Phe [µmol/l] 600 500 400 300 200 100 0 0 20 40 60 80 100 Time [weeks] Steinfeld R., Kohlschütter A, Ullrich K, Lukacs Z. J Inherit Metab Dis 2004; 27: 449-53. Conclusion Early screening tests provided little accuracy Current methods allow neonatal screening and valid follow-up of patients under treatment Scientific research with collaboration between chemists, biologists and medical doctors helps to reveal new strategies for monitoring and treatment of phenylketonuria and BH 4 -deficiency 13
Now it is up to you to choose Thank you for your attention! 14