Evolving Methods for the Measurement of …pku.vegss.si/old_page/Publikacije/espku2004/espku2004...1...
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EvolvingEvolving MethodsMethods forfor thethe MeasurementMeasurement of of PhenylalaninePhenylalanine ––
A vital A vital diagnosticdiagnostic markermarker and and indicatorindicator forfor followfollow--upup in in casescases of of hyperphenylalaninemiahyperphenylalaninemia and and tetrahydrobiopterintetrahydrobiopterin
deficiencydeficiency
Dr. Zoltan LukacsDepartment of Pediatrics, University Hospital Hamburg-Eppendorf,
Germany
PhenylketonuriaPhenylketonuria
Dr. Z. Lukacs - University Hospital Hamburg
Hyperphenylalaninemia
Phenylketonuria „atypical Phenylketonuria“BH4-deficiency
common feature: elevated phenylalanine concentration
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BiochemistryBiochemistry
Dr. Z. Lukacs - University Hospital Hamburg
COO-
+H3N
Phe
COO-
+H3N
TyrBH4 BH2 OH
PAH / O2 Transaminase
αααα-Ketogluterate
COO-
O
OH
Dioxygenase Ascorbate / O2
-OOC
OH
OH
Homogentisate
Dioxygenase
O2
-OOCO O COO-
4-Maleylacetoacetate
-OOCO O
4-Fumarylacetoacetate
COO-
Dr. Z. Lukacs - University Hospital Hamburg
Old Old MethodsMethods
Fellow citizens, we cannot escape historyAbraham Lincoln, Annual message to congress, Dec. 1862
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FeClFeCl33 in in urineurine
Dr. Z. Lukacs - University Hospital Hamburg
purpleSalicylates
GreenAcetaminophen
Effervescent blue-greenAlkaptonuria/homogentisic acid
Greenish grayMSUD/ branched chain ketoacids
Rapidly fading greenTyrosinemia/p-hydroxyphenylpyruvic acid
GreenPKU/phenylpyruvate
ColorDisease/Analyte
unreliable
example fast
but
TheThe „Guthrie Test“„Guthrie Test“
Dr. Z. Lukacs - University Hospital Hamburg
Dried blood : - is easily transported- is reasonably stable- allows easy handling- can be stored
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BacterialBacterial Inhibition Inhibition AssayAssay (BIA)(BIA)
Dr. Z. Lukacs - University Hospital Hamburg
Dr. R. Guthrie
•Phenylalanine-free agar•B. subtilis spores• β-2-thienylalanine (inhibitor)
Dried blood spots areput on the agar.
Areas of intensegrowth indicatehigh phenylalanineconcentrations
Guthrie R, Susi A. Pediatrics 1963; 32: 338-43
Dr. Z. Lukacs - University Hospital Hamburg
ChromatographyChromatography
eluent
samplestart
plate
paper lid
Principle of thin-layer chromatography example
Cholesterol
lipid extract from urine
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Paper/TLC Paper/TLC ChromatographyChromatography
Dr. Z. Lukacs - University Hospital Hamburg
1- Phenylketonuria6- Severe Tyrosinemia
Scriver CR, Davies E, Cullen AM The Lancet 1964; II: 230-32.
•Plasma or dried bloodis spotted on paper
•Chromatograms weredeveloped overnight
•stained with ninhydrin-isatin
Limit of detection (phe):6-8 mg/dL
New New MethodsMethods
Dr. Z. Lukacs - University Hospital Hamburg
We are learning to fly
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Dr. Z. Lukacs - University Hospital Hamburg
UV/VIS UV/VIS SpectroscopySpectroscopy
Principle of UV/VISSpectroscopy
Wav
elen
gth
Light sourceFilter Sample Detector Display
Phenylalanine + H2O Phenylpyruvate + NH4+
NAD+ NADH/H+
Electron acceptor (reduced) Electron acceptor (oxidized)
Tetrazolium Formazan
Dr. Z. Lukacs - University Hospital Hamburg
UV/VIS UV/VIS SpectroscopySpectroscopy IIII
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Dr. Z. Lukacs - University Hospital Hamburg
UV/VIS UV/VIS SpectroscopySpectroscopy IIIIII
- 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.
calibration curve normal distribution
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FluorometryFluorometry II
Light Source
Filter
Fluorescence
Sample Filter Detector Display
Principle of Fluorometry
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Dr. Z. Lukacs - University Hospital Hamburg
FluorometryFluorometry IIII
O
O
OH
OH+
O
O
OH
H
Hydrindantin
CHO
+ + NH3 +H+ + CO2+
COOH
H2N
X – 390 nmE – 486 nm
LOD : 12 µµµµMcut-off: 151 µµµµM
Enhancer: L-leucyl-L-alanin
Dr. Z. Lukacs - University Hospital Hamburg
FluorometryFluorometry IIIIII
-more sensitive than UV/VIS-Allows follow-up of patients
Hill JB et al. Clin Chem 1965; 11: 541-46
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HPLC HPLC ChromatographyChromatography
Advantage: multi-analyteDisadvantage: OPA-derivatization
pre-column has to be replaced frequentlyVollmer et al. Anal Biochem 1990; 189: 115-21
normal PKU
Dr. Z. Lukacs - University Hospital Hamburg
Ion Exchange Ion Exchange ChromatographyChromatography
TyrPhe
IXC - chromatogram
Reference Methodfor
amino acids
Advantage: most amino acids quantitatively analyzedDisadvantage: takes long (> 2 h)
derivatization necessaryunreliable equipment
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Dr. Z. Lukacs - University Hospital Hamburg
Tandem Tandem MassMass SpectrometrySpectrometry
Advantage: multi-analytePhe/Tyr-Ratiofast and reliable
Disadvantage: expensive equipmenthighly trained staff
Screening Center North (Germany): ca. 40000 samples/aca. 4 new PKU cases/a
Follow-up of Hyperphenylalaninemias: 280 PKU patients2 patients with BH4-deficiency
who are monitored regularly in Hamburg
Dr. Z. Lukacs - University Hospital Hamburg
SpecialisedSpecialised MethodsMethods
giving you the whole picture...
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Dr. Z. Lukacs - University Hospital Hamburg
BiosensorBiosensor
Linear range: 20-150 µMLimit of detection : 5 µMTime for 1 measurement : 1 minElectrode life: 11 d
Phe
Huang T et al. Anal Chem 1998; 70: 991-7.
1. Phe PhenylpyruvatePheDH
NAD+ NADH/H+
2. Salicylate Catechol
Tyrosinase
o-Quinone
2e-
Eappl=-50 mV
Dr. Z. Lukacs - University Hospital Hamburg
MR MR SpectroscopySpectroscopy
Phe Phe
Blood Brain
Single cases of untreated PKU with normal intelligence reportedlow Phe in brain was detected by MR spectroscopy
Möller H. et al. Eur J Pediatr 2000; 159 (Suppl 2): S121-5.
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Dr. Z. Lukacs - University Hospital Hamburg
Isotope Isotope labeledlabeled PhenylalaninePhenylalanine
L[1-13C]Phenylalanine 13CO2
Analyzed by IRMS
Treacy E.P. et al. Pediatr Res 1997; 42: 430-5.
Dr. Z. Lukacs - University Hospital Hamburg
BHBH44 loadingloading testtest
200
600
1000
1400
6 am 12 pm 6 pm 12 am 6 am
without BH4 with BH4
Difference > 30%
Blo
odPh
e[µ
mol
/L]
Time
PKU-Patient
Phenylalanine measurement to evaluate BH4-responsiveness
Steinfeld R., Kohlschütter A., Zschocke J., Lindner M., Ullrich K., Lukacs Z.Eur. J. Ped. 2002; 161: 403-5.
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Dr. Z. Lukacs - University Hospital Hamburg
BHBH44 as a as a treatmenttreatment optionoption
0
100
200
300
400
500
600
700
800
0 20 40 60 80 100
Blo
odPh
e[µ
mol
/L]
Time [weeks]
Patient 1 under BH4 treatment
Steinfeld R., Kohlschütter A, Ullrich K, Lukacs Z. J Inherit Metab Dis 2004; 27: 449-53.
Dr. Z. Lukacs - University Hospital Hamburg
ConclusionConclusion
•Early screening tests provided little accuracy
•Current methods allow neonatal screening andvalid follow-up of patients under treatment
•Scientific research with collaboration betweenchemists, biologists and medical doctors helps to reveal new strategies for monitoring and treatmentof phenylketonuria and BH4-deficiency
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