1. RESEARCH UPDATE IN PHENYLKETONURIA
Dr. Maureen Cleary
Great Ormond Street Hospital NHS Trust

2. Blood-brain barrier studies in PKU
Large Neutral amino acids
Essential fatty acids supplementation
Biopterin treatment
Ammonia lyase
Gene therapy

3. Blood-brain barrier studies

4. Phenylketonuria Monitor metabolic control by blood phe
Preferable to measure phe at site of action (brain) rather than point of delivery (blood)

5. 1H-Magnetic Resonance Spectroscopy
Nucleus is magnetic
Magnetic field causes all the magnetic nuclei to align themselves to the major axis of the field
A second magnetic field is applied
Nuclei tilted to a specific angle
When field removed they re-align themselves to the major axis of the magnetic field

6. Magnetic Resonance Spectroscopy
capable of identifying different molecules
Same nuclei eg protons experience different local magnetic fields
Give rise to different MR spectra
Area under peak proportional to concentration

7. NAA Cr
Cho

8. 1H-Magnetic Resonance Spectroscopy in PKU
Non-invasive assessment of changes in brain metabolism
Initial reports measure N-Acetyl-Aspartate, NA Choline, inositol, creatinine

10. 1H-Magnetic Resonance Spectroscopy in PKU
Normal NAA, choline, creatinine
Suggests no demyelination

11. PKU and Magnetic Resonance Spectroscopy (MRS)
Rabbit made hyperphe
MRS detected ‘phe’ peak
Intensity correlated with brain phe on postmortem
Correlated poorly with plasma phe

12. MRS and brain phe
1995
Detection and quantitation methodology of brain metabolites in patients with PKU

13. NAA Cr
Cho

14. MRS: normal
NAA
phe

15. MRS: PKU
phe
NAA

16. Measurement of phe Present in relatively small quantities
Cf NAA, choline
Need to use ‘difference spectroscopy’
i.e. subtract spectra from non PKU controls

17. MRS of brain phe studies in PKU
17 PKU (mean age 25.8 yrs)
10 healthy controls (25.3)
Early treated
6 off diet, 3 protein restricted, 8 on aa supp
(stopped 2 weeks pre-scan)
‘steady state’
(Rupp et al., 2001)

18. MRS results Control brain phe mean 0.05, sd 0.025
Blood versus brain linear relationship
Blood to brain phe: 4:1
Measurement error 0.03mol/kg ww

19. Blood- brain relationship
Pietz et al.,(1999)

20. Magnetic Resonance Spectroscopy
Weglage et al., 1998
two siblings aged 17 and 30 yrs early treated
R408W/R408W
IQ’s 90 and 77
oral load phe
max brain phe hrs post phe load
sib I blood 2448: brain 642 (IQ 90)
sib II blood 2316: brain 804 (IQ 77)

21. Magnetic Resonance Spectroscopy
Weglage et al., 1998
4 untreated adults
two IQ unobtainable ages 34 and 28 yrs
blood 1320,1211/ brain 650,670
two IQ 100 and 105 ages 33 and 31 yrs
blood 1200, 1210/ brain <200, <200

22. Only really explains unusual patients
Suggests
Intervariability of brain phe
Explains different outcomes
Only really explains unusual patients

23. MRS blood:brain Pietz et al., (1999) linear blood: brain 4:1
Moller et al.,(2000)
saturated at higher phe levels
Moats et al. (2000)
?? exponential

24. Blood-brain relationships
Pietz et al.,(1999)
Moller et al.,(2000)
Moats et al., (2000)

25. Large Neutral amino acids

26. LNAA and PKU
Large neutral amino acids compete for entry to brain with phenylalanine

27. Large neutral amino acids and PKU
Administer large quantities of LNAA and reduce phe entry to the brain

28. Large Neutral AminoAcids and PKU
What is the evidence that it should work?
Earlier studies (animals or functional testing)
Later studies (humans) using Magnetic Resonance Spectroscopy

29. LNAA and PKU
Cerebral protein synthesis reduced in hyperphe state in rats
Improves upon supplementing with LNAA
Binek-Singer & Johnson, 1982

30. LNAA and PKU: effect of supplements on brain amino acids in animals
Rats phe hydroxylase inhibited
Phe load
Phe load + LNAA
LNAA group had lower brain phe and similar blood phe
Andersen & Avins, 1976

31. LNAA and cerebral function in PKU
Valine, isoleucine and leucine supplements
Reduced brain and CSF phe in rats
Six patients with pku improved neuropsych performance whilst taking VIL
Berry et al., 1985

32. LNAA, PKU and MRS Later studies using MRS in humans One study
Pietz et al., (1999)
Six adults
Loading with oral phe 100mg/kg
Loading with oral phe plus LNAA
EEG testing

33. LNAA, PKU and MRS: Pietz et al. (1999)
Rise in brain phe occurred after loading
This rise blocked when LNAA taken with phe load
EEG spectra abnormalities not seen when LNAA ingested

34. LNAA study Brain phe after oral phe load
mean preload
mean post load 6 hrs 344
mean post load 12 hrs 377
Brain phe after oral phe + LNAA
mean preload
mean post load 6 hrs 235
mean post load 12 hrs 210

35. Further considerations
Is MRS sufficiently robust tool for intervention studies?
What are the relationships between BB phe entry and actual brain tissue phe levels?

36. LNAA, MRS and MOUSE PAHENU-2 mouse model (only two mice in each group)
0.5g/kg or 1.0 g/kg PreKUnil
Reduction in blood phe and brain phe
Spectroscopy on homogenized mouse brain
BCAT activity increased on LNAA
(only two mice in each group)
Matalon et al, (2003)

37. Conclusions
MRS can define a peak which is markedly elevated in individuals with PKU compared to normal spectra
MRS can show reduction in this peak when interventions occur such as LNAA application
MRS can show some unusual individuals who have low brain phe and are ‘protected’

38. Conclusions Blood:brain barrier relationship not clear
Extent of inter-individual variability not clear
Safety of long term LNAA not proven
To use the technique in dynamic studies need clarity of these changes through the day

39. Essential fatty acids in PKU

40. Essential fatty acids in PKU
Diet low in animal protein
low intake alpha-linolenic acid
low docoshexanoic acid
importance in brain cell membrane
Infant aminoacid formulae can be supplemented with PUFA’s
Should children’s formulae also be supplemented?

41. Essential fatty acids in PKU
AA product supplemented with fatty acids
Children had higher levels of DHA than unsupplemented group
Considered more palatable than unsupplemented formula

42. PKU and biopterin

43. PKU
Phenylalanine Tyrosine
Biopterin metabolism

44. Biopterin responsive PKU
Should we be treating some patients with biopterin?

45. Role of biopterin in PKU
Biopterin co-factor for phe hydroxylase
Inborn errors of biopterin detected by PKU screening programme
On biopterin those patient usually no longer need phe restriction

46. Biopterin in PKU
Recent observation that biopterin may benefit phe hydroxylase deficient patients

47. Biopterin in PKU Hyperphe rather than classical PKU
Mutations with residual activity
Frequently (but not exclusively) missense mutations within the coding region for the catalytic domain

49. Biopterin and PKU Suggest loading test in all patients
However newborn failed loading test patients have subsequently been found to be responsive

50. Biopterin and pku Cost of diet v. cost of biopterin Who would benefit?
Does it benefit those with severe PKU?
Is it safe in pregnancy?
Trial later this year 2004

51. Alternative therapies
Ammonia lyase therapy
Recombinant phenylalanine ammonia lyase
converts phe to trans-cinnamic acid in the gut
reduces plasma phe by approx 50% in PKU mouse

52. New therapies Ammonia lyase treatment may be useful
needs further studies to test safety
may still need some diet
may be many years before available

53. PKU and Gene therapy

54. Alternative therapies
GENE THERAPY
Adv/RSV-hPAH infused into portal vein of PAHenu2 mice
phe levels normalised with sufficient dose
comparable to 10-20% enzyme activity
successful only in short term
could not be duplicated due to immune response to vector

55. Conclusions Research is fairly active in PKU
Biopterin trial will find some individuals with milder PKU who may benefit form Biopterin treatment
Enzyme treatment is underway