measuring phenylalanine r neil dalton the wellchild trust laboratory king’s college, london...
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Measuring phenylalanine
R Neil DaltonThe WellChild Trust Laboratory
King’s College, London Evelina Children’s Hospital at Guy’s Hospital
NSPKU, Skipton, 26th February 2004
Measuring phenylalanine
Phenylalanine
The bad boy of phenylketonuria?
Measuring phenylalanine
Phenylalanine is an essential amino acidOne of the building blocks of all proteins
Essential that the blood level of phenylalanine is maintained at an optimum concentration
Optimum concentration range relatively wide, but in phenylalanine hydroxylase deficiency needs to be
routinely monitored
Measuring phenylalaninephenylalanine 3D structure
Measuring phenylalaninephenylalanine structure
Measuring phenylalanine
Collection of the blood spot is the most important step in obtaining an accurate measurement of
phenylalanine in the blood
How do we actually measure it?
Measuring phenylalanineGuthrie bacterial inhibition assay
Measuring phenylalanineThin layer chromatography
Measuring phenylalaninespotting the samples
Measuring phenylalaninechromatography
Measuring phenylalaninemethods
fluorimetry enzymatic (PAL, PDH) high pressure liquid chromatography
Electrospray mass spectrometry-mass spectrometry
Measuring phenylalanine Electrospray (John Fenn)
Measuring phenylalanineElectrospray Ionisation
Measuring phenylalanineElectrospray Ionisation
Measuring phenylalanineElectrospray Ionisation
Measuring phenylalanineElectrospray Ionisation
Measuring phenylalanineElectrospray Ionisation
Measuring phenylalaninemass spectrometry-mass spectrometry
DETECTOR
MS1 Collision Cell MS2
IONS
Q1 Q2 Q3
Measuring phenylalanineproduct ion scan
DETECTOR
Phe+ Collision Cell MS2
IONS
Q1 Q2 Q3
Q1 - selection of phenylalanine ion
Q2 – fragment the phenylalanine ions
Q3 – look for fragments of the phenylalanine
Measuring phenylalanine product ion scan
Measuring phenylalanine fragmentation
Measuring phenylalanine
DETECTOR
Phe+ Collision Cell x+IONS
Q1 Q2 Q3
Q1 - selection of phenylalanine ion m/z 166
Q2 – fragmentation of phenylalanine ion
Q3 - selection of specific fragment ion m/z 120
Measuring phenylalanine optimal phenylalanine control
Checking in
Samples posted in are received at the laboratory and are logged in to the computer.
Punching out spot
A special machine punches out the spots
Measuring phenylalanine Sample Preparation
Punch 3mm diameter blood spot into deep-well (2ml) microtitre plate
Add 150l methanol/water (83:17) containing d5-phe and d4-tyr (5.6mmol/l)
Seal plate with lid and mix for 20min at 300 cycles/min
Place on Gilson autosampler at RT for injection
Mixing and shaking
Samples are then put onto plates and mixed.
Measuring phenylalanine Sample delivery & MSMS acquisition
Mobile phaseacetonitrile/water (1:1) with formic acid (0.025%)
Flow rate 50l/min Sample volume 2l Acquisition time 55s Cycle time 66s
Precursor product pairsphenylalanine (166/120) d5-phenylalanine (171/125)
tyrosine (182/136) d4-tyrosine (186/140)
Measuring phenylalanine calibration
Spiked blood spots run as unknowns (3/level/day)Blood spots from normal subjects (39-82/subject/day)
Data shown as median & range
0
200
400
600
800
1000
1200
1400
1600
02-05-1999 07-05-1999 12-05-1999 17-05-1999 22-05-1999 27-05-1999 01-06-1999 06-06-1999
Date
Phe
mol
/l
1200
600
400
300
200
240 cutoff
480 cutoff
Subject 3
Subject 2
Subject 1
Measuring phenylalaninepoor phenylalanine control
results
Sample results are then checked by the laboratory staff.
Lab team
Our hard working biochemistry team.
Reported results
Our dietitians have both a phoned and faxed report of all the day’s phenylalanine samples received
Phoning parents
With all the results available, the dietitian phones the families, discusses both the child’s well being dietary changes if needed and often a bit of a social chit-chat.
Measuring phenylalanine
A multi-disciplinary team of parents/individual, health care professionals, and leading edge
analytical technology are involved in maintaining optimal blood phenylalanine
Collection of the blood spot is the most important step in obtaining an accurate measurement of
phenylalanine in the blood
Acknowledgements
Charles Turner
Sue Bird
The WellChild Trust
Charitable Foundation Guy’s & St Thomas’ Hospitals
Guy’s and St Thomas’ NHS Trust