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RESEARCH UPDATE IN PHENYLKETONURIA Dr. Maureen Cleary Great Ormond Street Hospital NHS Trust Blood-brain barrier studies in PKU • • • • • • Blood-brain barrier studies in PKU Large Neutral amino acids Essential fatty acids supplementation Biopterin treatment Ammonia lyase Gene therapy Blood-brain barrier studies Phenylketonuria • Monitor metabolic control by blood phe • Preferable to measure phe at site of action (brain) rather than point of delivery (blood) 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 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 NAA Cr Cho 1H-Magnetic Resonance Spectroscopy in PKU • Non-invasive assessment of changes in brain metabolism • Initial reports measure N-Acetyl-Aspartate, NA Choline, inositol, creatinine 1H-Magnetic Resonance Spectroscopy in PKU • Normal NAA, choline, creatinine • Suggests no demyelination 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 MRS and brain phe • 1995 – Detection and quantitation methodology of brain metabolites in patients with PKU NAA Cr Cho MRS: normal NAA phe MRS: PKU NAA phe Measurement of phe • Present in relatively small quantities – Cf NAA, choline – Need to use ‘difference spectroscopy’ – i.e. subtract spectra from non PKU controls 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) 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 Blood- brain relationship Pietz et al.,(1999) 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 12-23 hrs post phe load • sib I blood 2448: brain 642 (IQ 90) • sib II blood 2316: brain 804 (IQ 77) 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 • Suggests – Intervariability of brain phe – Explains different outcomes • Only really explains unusual patients 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 Blood-brain relationships Pietz et al.,(1999) Moller et al.,(2000) Moats et al., (2000) Large Neutral amino acids LNAA and PKU • Large neutral amino acids compete for entry to brain with phenylalanine Large neutral amino acids and PKU • Administer large quantities of LNAA and reduce phe entry to the brain 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 LNAA and PKU • Cerebral protein synthesis reduced in hyperphe state in rats • Improves upon supplementing with LNAA – Binek-Singer & Johnson, 1982 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 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 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 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 LNAA study • Brain phe after oral phe load – mean preload – mean post load 6 hrs – mean post load 12 hrs 252 344 377 • Brain phe after oral phe + LNAA – mean preload – mean post load 6 hrs – mean post load 12 hrs 226 235 210 Further considerations • Is MRS sufficiently robust tool for intervention studies? • What are the relationships between BB phe entry and actual brain tissue phe levels? LNAA, MRS and MOUSE • PAHENU-2 mouse model – 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) 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’ 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 Essential fatty acids in PKU 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? 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 PKU and biopterin PKU • Phenylalanine Tyrosine Biopterin metabolism Biopterin responsive PKU • Should we be treating some patients with biopterin? 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 Biopterin in PKU • Recent observation that biopterin may benefit phe hydroxylase deficient patients 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 Biopterin and PKU • Suggest loading test in all patients • However newborn failed loading test patients have subsequently been found to be responsive 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 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 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 PKU and Gene therapy 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 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