1. A Window upon the Brain Analysis of CSF Neurotransmitters Cofactors and Vitamins John Earl Australasian Neurochemistry Laboratory Clinical Biochemistry Department The Children’s Hospital at Westmead Sydney, NSW, Australia AACB ASM Perth 27 th October 2010 NSW/ACT AACB 17 th May 2011

2. CSF Neurotransmitters by HPLC Measurement Catecholamines Catecholamines Tyrosine pathway metabolites Tyrosine pathway metabolites Tryptophan pathway metabolites Tryptophan pathway metabolites Dopamine metabolites Dopamine metabolites Serotonin metabolites Serotonin metabolites Pteridines Pteridines 5-methyltetrahydrofolate 5-methyltetrahydrofolate Histamine & metabolites Histamine & metabolites Amino Acids Amino Acids Kynurenine pathway Kynurenine pathway B6 Vitamers B6 Vitamers Trace level analysis of unstable compounds Protect from light, Store frozen -40 o C Australasian Neurochemistry Laboratory Clinical Biochemistry Department The Children’s Hospital at Westmead Sydney, NSW, Australia

3. Neurochemistry Laboratory Cerebrospinal Fluid (CSF) Collected in 5 tubes with various preservatives 0.5ml CSF added to each tube Immediately frozen. Transport on dry ice Instructions / Details on Internet http://www.cs.nsw.gov.au/csls/handbook/FactSheetView.asp?Number=45 Spot Urine (frozen) Sample Collection

4. Amperometric Detector Coulometric Detector Array

5. Single Detector Array Detector

12. Tyrosine Hydroxylase Deficiency L-DOPA -> Dopamine -> Noradrenaline -> Adrenaline Deficiency of brain dopamine and noradrenaline. Responds to L-DOPA therapy

13. Pteridines XanthopterinNeopterinMonapterinBiopterin7-Biopterin

14. Central Inflammatory Disease Normal Brain Pterin Profile CNS Inflammatory Disease High levels of xanthopterinneopterin,monapterin

15. CNS Inflammatory Disease CSF Neopterin Monapterin CSF Neopterin Monapterin (<30nM) (<4nM) (<30nM) (<4nM) Aicardi Goutieres disease23129 HIE31928 ADEM10221 Rasmussen’s encephalitis13621 Cat scratch encephalitis14617 Rotavirus encephalitis134 9 Batten disease 7910 Acute cerebellar ataxia 8714 Sydenham’s chorea 49 5 Development regression/ ataxia 58 9 Demyelinating disease 59 5 CSF Neopterin in Paediatric Neurology. A marker of active CNS inflammation Dale Cr, Brilot F, Fagan E, Earl J Develop Med Child Neurol 2009; 51(4): 317-323

16. Almost miraculous (awakenings) but transient response to L-DOPA in a group of patients with Encephalitis Lethargica

17. Progression of Encephalitis Lethargica Ref Ranges: Neopterin4 – 30nM Biopterin 25 – 40nM

18. Progression of Encephalitis Lethargica Ref Ranges: HVA > 0.34 5-HIAA > 0.10

19. Encephalitis lethargica syndrome: 20 new cases and evidence of basal ganglia autoimmunity. Russell C. Dale, Andrew J. Church, Robert A. H. Surtees, Andrew J. Lees, Jane E. Adcock, Brian Harding, Brian G. R. Neville and Gavin Giovannoni Brain (2004), 127, 21±33 Autoimmune process targeting the NMDA receptor.

20. Infantile Spasms Normal CSF HVA, 5-HIAA and pterins 56% Low HVA, 5-HIAA, Biopterin44% * Raised CSF Neopterin 1% 10 years - 65 cases for Neurotransmitter testing * Increasing evidence for Brain structural abnormalities including defective ARX Homeobox gene

21. B6 Vitamers (pyridoxal phosphate) Pyridoxic acid PyridoxalPyridoxinePyridoxamine

22. Pyridoxal 5-Phosphate L-DOPA 5-HTP Glutamate Aromatic Amino Acid Glutamate Aromatic Amino Acid Glutamate Decarboxylase Decarboxylase Decarboxylase Decarboxylase (P5P) (P5P) DopamineSerotonin GABA

23. Neonatal Seizures ? PNPO MRI: White matter high intensity signals Newborn boy developed seizures at 3 weeks (atypical). No response to pyridoxine Good response after pyridoxal phosphate. Provisional Diagnosis: Pyridox(am)ine Oxidase Deficiency – PNPO

24. Neonatal Seizures ? PNPO CSF B6 Vitamers CSF B6 Vitamers PNPO PNPO Seizure Seizure Unknown * PNPO PNPO Seizure Seizure Unknown * P5P P5P P5P Px P5P P5P P5P P5P Px P5P Pyridoxal 2570 720 6096 1403 1694 Pyridoxine 3470 996 9109 4841 28 Pyridoxamine 1910 622 74 26 nd Pyridoxic Acid 120 12 1543 66 145 Pyridoxic AL 1170 36 1056 489 470 * Diagnosis: Unlikely to be PNPO Deficiency

25. Neonatal Seizures ? PNPO CSF Neurotransmitter Analysis Male: 32 days of age. Neopterin 101.6 * ( < 30nmol/L) Monapterin 15.5 ( < 8 nmol/L) HVA 520(540 – 1142 nmol/L) 5-HIAA 400(380 – 1030 nmol/L) 5-MTHF 178.8(60 -180 nmol/L) * Provisional Diagnosis: CNS Inflammatory Disease

26. Neonatal Seizures CNS Inflammatory Disease -> Late Onset neonatal seizures -> MRI: White Matter Hyperintensities Masking Therapeutic Trials with Pyridoxine & Pyridoxal Phosphate

27. CSF 5-Methyl-tetrahydrofolate

30. A Window upon the Brain Metabolic Diseases Deficiency Diseases Inflammatory Diseases Neurodegenerative Diseases Structural diseases

31. Improvement in detection and identification of Brain Diseases by cerebrospinal fluid analysis. by cerebrospinal fluid analysis. Improve monitoring and treatment of Brain Diseases Towards evidence based Neurology and Neuropharmacology “Chemical Imaging of the Brain” - Identify chemical markers which arise from different brain regions. A window upon the Brain