Evolving Methods for the Measurement of …pku.vegss.si/old_page/Publikacije/espku2004/espku2004...1...

1

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

2

BiochemistryBiochemistry


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-


Old Old MethodsMethods

Fellow citizens, we cannot escape historyAbraham Lincoln, Annual message to congress, Dec. 1862

3

FeClFeCl33 in in urineurine


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“


Dried blood : - is easily transported- is reasonably stable- allows easy handling- can be stored

4

BacterialBacterial Inhibition Inhibition AssayAssay (BIA)(BIA)


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


ChromatographyChromatography

eluent

samplestart

plate

paper lid

Principle of thin-layer chromatography example

Cholesterol

lipid extract from urine

5

Paper/TLC Paper/TLC ChromatographyChromatography


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


We are learning to fly

6


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


UV/VIS UV/VIS SpectroscopySpectroscopy IIII

7


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


FluorometryFluorometry II

Light Source

Filter

Fluorescence

Sample Filter Detector Display

Principle of Fluorometry

8


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


FluorometryFluorometry IIIIII

-more sensitive than UV/VIS-Allows follow-up of patients

Hill JB et al. Clin Chem 1965; 11: 541-46

9


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


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

10


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


SpecialisedSpecialised MethodsMethods

giving you the whole picture...

11


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


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.

12


Isotope Isotope labeledlabeled PhenylalaninePhenylalanine

L[1-13C]Phenylalanine 13CO2

Analyzed by IRMS

Treacy E.P. et al. Pediatr Res 1997; 42: 430-5.


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.

13


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.


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

14

Thank youfor your

attention !

Now it is up to you to choose

Evolving Methods for the Measurement of …pku.vegss.si/old_page/Publikacije/espku2004/espku2004...1...

Documents

Transcript of Evolving Methods for the Measurement of …pku.vegss.si/old_page/Publikacije/espku2004/espku2004...1...