1. Measuring phenylalanine R Neil Dalton The WellChild Trust Laboratory King’s College, London Evelina Children’s Hospital at Guy’s Hospital NSPKU, Skipton, 26 th February 2004

2. Measuring phenylalanine Phenylalanine The bad boy of phenylketonuria?

3. Measuring phenylalanine Phenylalanine is an essential amino acid One 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

4. Measuring phenylalanine phenylalanine 3D structure

5. Measuring phenylalanine phenylalanine structure

6. 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?

7. Measuring phenylalanine Guthrie bacterial inhibition assay

8. Measuring phenylalanine Thin layer chromatography

9. Measuring phenylalanine spotting the samples

10. Measuring phenylalanine chromatography

11. Measuring phenylalanine methods  fluorimetry  enzymatic (PAL, PDH)  high pressure liquid chromatography Electrospray mass spectrometry-mass spectrometry

12. Measuring phenylalanine Electrospray (John Fenn)

13. Measuring phenylalanine Electrospray Ionisation

18. Measuring phenylalanine mass spectrometry-mass spectrometry DETECTORDETECTOR MS1 Collision Cell MS2 IONS Q1 Q2 Q3

19. Measuring phenylalanine product ion scan DETECTORDETECTOR 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

20. Measuring phenylalanine product ion scan

21. Measuring phenylalanine fragmentation

22. Measuring phenylalanine DETECTORDETECTOR 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

23. Measuring phenylalanine optimal phenylalanine control

24. Checking in l Samples posted in are received at the laboratory and are logged in to the computer.

25. Punching out spot l A special machine punches out the spots

26. Measuring phenylalanine Sample Preparation  Punch 3mm diameter blood spot into deep-well (2ml) microtitre plate  Add 150  l 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

27. Mixing and shaking l Samples are then put onto plates and mixed.

28. Measuring phenylalanine Sample delivery & MSMS acquisition  Mobile phase acetonitrile/water (1:1) with formic acid (0.025%)  Flow rate50  l/min  Sample volume2  l  Acquisition time55s  Cycle time66s  Precursor product pairs phenylalanine (166/120) d5-phenylalanine (171/125) tyrosine (182/136)d4-tyrosine (186/140)

29. Measuring phenylalanine calibration

31. Measuring phenylalanine poor phenylalanine control

32. results l Sample results are then checked by the laboratory staff.

33. Lab team l Our hard working biochemistry team.

34. Reported results l Our dietitians have both a phoned and faxed report of all the day’s phenylalanine samples received

35. Phoning parents l 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.

36. 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

37. Acknowledgements Charles Turner Sue Bird The WellChild Trust Charitable Foundation Guy’s & St Thomas’ Hospitals Guy’s and St Thomas’ NHS Trust