Measuring phenylalanine R Neil Dalton The WellChild Trust Laboratory King’s College, London...

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