Analysis of Neurotransmitters. Simon Heales

Transcription

1 Analysis of Neurotransmitters Simon Heales

2 Tyrosine O 2 L-Dopa Dopamine HVA Tryptophan 5-HTP PLP Serotonin 5-HIAA Phenylalanine Tyrosine BH4 qbh2 BH2

3 CSF Sample Requirements Tube 1 Tube 2 Tube 3 0.5ml HVA & 5-HIAA 0.5ml 5-MTHF/PLP 1.0ml Pterins (DTE/DETAPAC) Collect at bedside and freeze immediately (not the form!) Date of Birth, Sample Date, Medication essential

4 GTP GTP cyclohydrolase Dihydroneopterin Triphosphate P 3 Dihydroneopterin -VE Pyruvoyl tetrahydroptein synthase 6-Pyruvoyltetrahydropterin Aldose reductase /Sepiapterin reductase Tetrahydrobiopterin

5 BH4 Salvage Tyr BH4 PCD + DHPR L-Dopa qbh2 BH2 NAD + NADH PCD = pterin carbinolamine dehydratase DHPR = dihydropteridine reductase

6 Data Capture Data Capture E 1 E 2 BH 2 B Em 440nm Eluent Ex 360 nm BH 4 qbh 2 BH 4 NH 2 N Waste DTE +OX - Red 900 mv

7 nmol/l Metabolite Age (years) Mean Range BH Adult BH2 ALL NH2 ALL Pediatr Res (1993) 34, 10-14

8 Disorders of BH4 metabolism With Hyperphenylalaninemia GTP cyclohydrolase I (GTPCH) deficiency; Phe = umol/l 6-Pyruvoyl-tetrahydropterin synthase (PTPS) deficiency; Phe = umol/l Dihydropteridine reductase (DHPR) deficiency; Phe = umol/l Pterin-4a-carbinolamine dehydratase (PCD) deficiency; Phe = umol/l Without hyperphenylalaninemia Sepiapterin reductase deficiency (SR). Dopa-responsive dystonia (DRD) due to GTPCH deficiency;

9 Sepiapterin Reductase Deficiency GTP Dihydroneopterin Triphosphate 6-Pyruvoyltetrahydropterin Tetrahydrobiopterin

10 Sepiapterin Reductase Deficiency Sex; Male. Dob; 31/12/1987. Sample; 09/05/2003. Dystonia responsive to L-DOPA. No hyperphenylalaninaemia. DHPR normal. HVA: 23 ( nmol/l) 5-HIAA: 2 ( nmol/l) BH4: 11 (9-39 nmol/l) BH2: 64 ( nmol/l) Total Neopterin: 19 (7-65 nmol/l)

11 GTP Dihydroneopterin Triphosphate 6-Pyruvoyltetrahydropterin BH2 DHFR -Liver Tetrahydrobiopterin

12 Tyrosine O 2 L-Dopa Dopamine HVA Tryptophan 5-HTP PLP Serotonin 5-HIAA Phenylalanine Tyrosine BH4 qbh2 BH2

13 Electrochemical Detection Data Capture Eluent Waste E1 50 mv E2 420 mv

14 nmol/l Metabolite Age (years) Mean Range HVA Adult HIAA Adult Pediatr Res (1993) 34, 10-14

15 Pilot External QC

16 CSF in house QC scheme 5-HIAA nmol/l HVA nmol/l

17 DHPR Deficiency Response to Treatment HVA 5-HIAA nmol/l Age (Years)

18 Other Neurotransmitter Disorders Tyrosine Hydroxylase Deficiency Dopamine Transporter Defect Aromatic Amino Acid Decarboxyalse Deficiency

19 Tyrosine Hydroxylase Deficiency Tyr Dopa Dopamine HVA Parkinsonian, ptosis, drooling, myoclonic jerks, severe head lag and trunkal hypotonia. L-Dopa marked and sustained improvement in hypokinesia and parkinsonian symptoms. Identified from CSF analysis; Normal pterin & 5-HIAA concentration. Very low HVA. Mutation analysis also available.

20 Tyrosine Hydoxylase Deficiency Sex; Male. Dob; 17/05/2007. Sample; 27/02/2008 HVA: <10 ( nmol/l) 5-HIAA: 137 ( nmol/l) BH4: 36 (19-56 nmol/l) BH2: 8 ( nmol/l) Total Neopterin: 9 (7-65 nmol/l) Serum Prolactin 706 ( mu/ml)

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22 Increased Dopamine Turnover First female child of consanguineous parents. 36 week gestation. Feeding difficulties from birth. 6 months reduced movements and failure to achieve milestones. 9 months able to smile but general paucity of movements. Rigidity of all limbs suggestive of dopamine deficiency. Left convergent squint but no abnormal eye movements detected. HVA: 1704 ( nmol/l) 5-HIAA: 250 ( nmol/l) Pterin profile and 5-MTHF status unremarkable Elevated urinary HVA Serum Prolactin; 915 (<500 mu/ml)

23 Dopamine Transporter Defect

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25 DAT Deficiency CSF HVA 1,873 1,704 1,135 ( nmol/L) 5-HIAA ( nmol/l) HVA:5-HIAA ( ) Urine HVA:Cr (2-15umol/mmol) Serum Prolactin ( mu/ml)

26 In Vivo Evidence of Loss of Function in DAT CONTROL PATIENT 3

27 Aromatic Amino Acid Decarboxylase Deficiency Tyr L-Dopa Dopamine HVA PLP Trp 5-HTP Serotonin 5-HIAA Clinical features resemble those of recessive BH4 deficiency; hypotonia, occulogyric crises, ptosis and paucity of spontaneous movement. Can be fatal Urine: Vanillactic acid CSF: Low HVA + 5-HIAA, 5 but normal pterin profile and accumulation of 3-O-methyldopa.. Enzymatic analysis possible on plasma. Treatment; B6, MAOI & dopamine agonists.

28 Control AADC Def

29 Vanillactic acid 3-Methyldopa Tyrosine Tryptophan BH4 L-Dopa 5-HTP AADC PLP Dopamine Serotonin HVA 5-HIAA

30 Aromatic Amino Acid Decarboxylase Deficiency Male. Dob; 28/08/2007. Sample; 10/01/2008 Floppy, episodes of dystonia, developmental delay HVA 47 ( nmol/l) 5-HIAA 14 ( nmol/l) 3-Methyldopa 1170 (<300 nmol/l) PLP 32 (23-87 nmol/l) Serum Prolactin 900 ( mu/ml) Plasma AADC Activity 0.7 ( pmol/min/ml)

31 Vanillactic acid 3-Methyldopa Tyrosine Tryptophan BH4 L-Dopa 5-HTP AADC PLP Dopamine Serotonin HVA 5-HIAA

32 Vitamin B 6 Pyridoxine-5 - phosphate CH 2 OH Pyridoxamine-5 - phosphate CH 2 NH 2 HO CH 2 OPO 3 H 2 HO CH 2 OPO 3 H 2 H 3 C N H 3 C N Pyridoxal-5 - phosphate PNPO CHO PNPO HO H 3 C N CH 2 OPO 3 H 2 PNPO = Pyridox(am)ine-5 -oxidase

33 PNPO Deficiency Neonatal epileptic encephalopathy Fetal distress, prenatal seizures, low Apgar Pseudo AADC deficiency Not consistent Glycine & Threonine Not consistent Vanillactate excretion Consistent?

34 PNPO Deficiency

35 CSF (PLP) PLP (nmol/l) Age (Years) Mol. Genet. Metab. 94. (2008)

36 CSF 5-MTHF Deficiency DHPR deficiency MTHFR deficiency AADC deficiency 3-Phosphoglycerate dehydrogenase def Rett syndrome Aicardi Goutieres Mitochondrial disorders L-dopa treatment Methotrexate Anticonvulsants Steroids Co-trimoxazole Cerebral Folate Deficiency - Neurological syndrome associated with low CSF 5-MTHF and normal peripheral folate.

37 Control CSF MTHFR Def CSF Food Chemistry, (1995), 53, HPLC +Fluorescence (290, 358 nm)

38 QC8 5-MTHF nmol/l Run Number

39 CSF 5-MTHF Deficiency & Mitochondrial Disorders 5-MTHF F. 15 yrs 29 ( nmol/l) M. 9 yrs 5 ( nmol/l) M. 8 yrs 44 ( nmol/l) F. 2 yrs 17 ( nmol/l) F. 6 yrs 7 ( nmol/l)

40 CSF 5-Methyltetrahydrofolate DHPR deficiency Long term L-dopa administration L-DOPA COMT 3-Methyldopa 10 year old female Dopamine GTP cyclohydrolase deficiency 62 ( nmol/l)

41 Secondary Causes Hypoxia Neurodegeneration Epilepsy Mitochondrial Disease Gaucher Drugs Sample Processing