Submitted by: Emma Jane Footitt Clinical and Molecular Genetics Unit Institute of Child Health University College London April 2012 Funded by Great Ormond Street Hospital Children’s Charity Submitted in application for the award of Doctor of Philosophy (Ph.D) 1 I, Emma Footitt confirm that the work presented in this thesis is my own. Where information has been derived from other sources I confirm that it has been indicated. Signed…………………………………. …..Date ………………………………… 2 Pyridoxal 5’-phosphate (PLP) is the active form of vitamin B6 in man where it functions as a cofactor for more than 140 enzyme catalysed reactions. Several inherited diseases characterised by seizures have been described which result in an intracellular deficiency of PLP; laboratory measurement of B6 forms an important element in the diagnosis and monitoring of these disorders. A review of PLP measured by HPLC in CSF from patients with neurological disorders showed that variance is greater than indicated by previous studies and the age-related reference limit was revised. This thesis also describes the metabolic disorders that may lead to PLP depletion and examines the relationship of CSF PLP to sulphite accumulation, medications and seizures in patient groups. B6 exists as six different vitamers and is catabolised to 4-pyridoxic acid for urinary excretion. An LC-MS/MS method was developed which could measure all vitameric forms in plasma. Its application to children with B6 responsive seizure disorders showed that patients with inborn errors of metabolism have characteristic B6 profiles which allow them to be differentiated from each other and control populations. PLP is the cofactor for aromatic L-amino acid decarboxylase (AADC) which catalyses the final step in serotonin biosynthesis. This thesis tested the hypothesis that hyperserotonaemia observed in some patients with autism is related to an abnormality in this pathway by investigating the relationship between plasma B6 vitamers, AADC activity and whole blood serotonin in a group of patients and controls. Plasma AADC activity was significantly reduced in autistic subjects; this is considered in the context of current biochemical and molecular understanding and its possible relevance to disease mechanisms is discussed. 3 Declaration 2 Abstract 3 Table of contents 4 List of tables 14 List of figures 18 Abbreviations 23 Acknowledgements 28 Chapter 1: Introduction 1.1 The metabolism, homeostasis and inborn errors of Vitamin B6 30 1.1.1 Chemical structure of B6 vitamers 30 1.1.2 Reactivity of pyridoxal 5 -phosphate 32 1.1.2.1 Reactions of pyridoxal 5’phosphatte with lysine residues and N-terminal amino acids of proteins 32 Enzymes 32 Albumin 32 Haemoglobin 33 1.1.2.2 Reactions of B6 vitamers with free radicals 33 1.1.2.3 Reactions catalysed by pyridoxal 5’-phosphate-enzymes 33 Reactions involving amino acids 33 1.1.2.4 Other pyridoxal 5’phosphate-catalysed reactions 35 1.1.3 Dietary sources of vitamin B6 35 1.1.4 Normal Physiology and Metabolic Pathways 36 1.1.4.1 Salvage Pathway 39 1.1.5 Homeostasis 40 1.1.6 Circadian rhythm of pyridoxal kinase and pyridoxal 5’-phosphate 41 4 1.1.7 Enzymes involved in interconversions of B6 vitamers 42 1.1.7.1 Phosphatases 43 Alkaline phosphatases 43 Tissue non-specific alkaline phosphatase 44 Pyridoxal 5’-phosphate phosphatase 45 PHOSPHO2 45 1.1.7.2 Pyridoxal kinase 45 1.1.7.3 Pyridox(am)ine 5’-phosphate oxidase (PNPO) 46 1.1.8 Enzymes involved in catabolism of B6 vitamers 47 1.1.8.1 Aldehyde oxidase 47 + 1.1.8.2 NAD -dependent aldehyde dehydrogenase 48 1.1.9 Vitamin B6 deficiency states 48 1.1.10 Biochemical assessment of vitamin B6 status 49 1.1.10.1 Indirect Methods 49 1.1.10.2 Direct Methods 50 1.1.11 Vitamin B6 requirements 50 1.1.12 Variation of vitamin B6 metabolism with age 51 1.1.13 Inherited disorders of B6 metabolism 53 1.1.13.1 Antiquitin deficiency (pyridoxine dependent epilepsy, PDE) 58 1.1.13.2 Pyridoxamine 5’-phosphate oxidase (PNPO) deficiency 61 1.1.13.3 Other pyridoxal 5’-phosphate responsive patients 63 1.1.13.4 Hypophosphatasia 64 1.1.13.5 Hyperphosphatasia 64 1.1.13.6 Hyperprolinaemia type II 65 1.1.14 Drugs and Vitamin B6 66 1.1.14.1 Isoniazid (Isonicotinic acid hydrazide) 67 1.1.14.2 L-Dopa 67 1.1.14.3 Methylxanthines including theophylline 68 5 1.1.14.4 Anticonvulsants 68 1.1.15 Pyridoxine and PLP toxicity 69 1.1.15.1 Acute toxicity of pyridoxine 69 1.1.15.2 Chronic toxicity of pyridoxine 69 1.1.15.3 Toxicity of pyridoxal 5’-phosphate 71 1.2 Serotonin metabolism and function 71 1.2.1 Biosynthetic pathway of serotonin 71 1.2.1.1 Tryptophan hydroxylase 1 and 2 (TPH1 and TPH2) 72 1.2.1.2 Aromatic L-amino acid decarboxylase (AADC) 73 1.2.2 Synthesis of melatonin from serotonin 74 1.2.3 Metabolism, storage and mechanisms of action of serotonin 74 1.2.4 Catabolism of serotonin 77 1.2.5 Physiological functions of serotonin 78 1.2.5.1 Neurological and behavioural 79 1.2.5.2 Gastro-intestinal tract 79 1.2.5.3 Cardiovascular system 80 1.2.5.4 Genito-urinary 81 1.2.5.5 Glucose homeostasis 81 1.2.6 Inherited disorders associated with an abnormality of serotonin metabolism 81 1.2.7 Carcinoid syndrome 83 1.2.8 Serotonin abnormalities in autism 84 1.3 Autism 85 1.3.1 Diagnosis of autism 85 1.3.2 Epidemiology of autism 86 1.3.3 Associated medical problems 87 1.3.4 Treatment 88 6 1.3.5 Genetic risk for autism 89 1.3.6 Neuropathology and neuroanatomy 92 1.4 Summary and Aims 93 Chapter 2: Materials and Methods 2.1 Materials 96 2.2 Biochemistry Methods 97 2.2.1 Measurement of PLP in plasma and CSF by reverse phase HPLC 97 2.2.2 Measurement of B6 vitamers and 4-pyridoxic acid by HPLC linked tandem mass spectrometry (LC-MS/MS) 98 2.2.2.1 Sample collection and preparation 98 2.2.2.2 Determination of B6 vitamers and 4- pyridoxic acid 98 2.2.2.3 Quantification of B6 vitamers and 4-pyridoxic acid 101 2.2.3 Aromatic L-amino acid decarboxylase (AADC) activity assay in plasma 101 2.2.3.1 Principle 101 2.2.3.2 Method for L-dopa decarboxylation 102 2.2.3.3 HPLC-ECD for detection of dopamine 102 2.2.3.4 Data analysis and calculation of enzyme activity 103 2.2.4 Whole blood serotonin 104 2.2.4.1 Principle 104 2.4.4.2 Specimen collection, handling and storage 104 2.4.4.3 HPLC conditions and instrumentation 104 2.4.4.4 Preparation of working standard 104 2.4.4.5 Preparation and analysis of subject samples and quality control (QC) 105 2.4.4.6 Quantification of whole blood serotonin in subject samples and QC 105 7 2.2.5 Measurement of 5’-hydroxyindolacetic acid (5-HIAA), homovanillic acid (HVA), 5-methyltetrahydrofolate (5-MTHF) and pterins in cerebrospinal fluid (CSF) by HPLC 105 2.2.5.1 Specimen collection 106 2.2.5.2 Summary of HPLC methodology 106 2.3 Molecular biology methods 107 2.3.1 Extraction of genomic DNA from whole blood and fibroblasts 107 2.3.1.1 Principle 107 2.3.1.2 Method for whole blood and fibroblasts 107 2.3.2 Amplification of genomic DNA of targeted genes by the Polymerase Chain Reaction (PCR) 108 2.3.2.1 PCR conditions 108 2.3.2.2 Analysis of PCR products by agarose gel electrophoresis 109 Materials 109 2.3.3 Sequencing 110 2.3.3.1 Purification of PCR products 110 Principle 110 Method 110 2.3.3.2 Sequencing of PCR products using the Sanger method 110 Principle 110 Method 111 2.3.3.3 DNA precipitation of sequencing reaction 111 Principle 111 Method 112 Chapter 3: Factors affecting the concentration of pyridoxal 5'- phosphate in cerebrospinal fluid 3.1 Introduction 114 3.2 Methods 116 8 3.2.1 Patients and sample collection 116 3.2.2 Biochemical Analysis 118 3.2.3 Molecular Genetic Analysis - Sequencing of the sulphite oxidase gene (SUOX, ENSG00000139531) 118 3.2.4 Statistical Analysis 119 3.2.5 Survey of Patients with Low CSF PLP 120 3.3 Results 120 3.3.1 Effect of Age 122 3.3.2 Effect of seizures, anti-epileptic drugs (AED) and L-Dopa 127 3.3.3 Relationship between PLP and 5-MTHF in CSF 129 3.3.4 Relationship between PLP and BH4 in CSF 130 3.3.5 Survey of patients with CSF PLP below the revised lower reference limit 131 3.3.6 Plasma: CSF PLP ratio 135 3.4 Discussion 136 3.4.1 Epilepsy/seizure disorders 137 3.4.2 Medications 137 3.4.2.1 Antiepileptic drugs (AED) 137 3.4.2.2 L-DOPA 138 3.4.3 Correlation between CSF concentrations of PLP and 5-MTHF 140 3.4.4 Correlation between CSF concentrations of PLP and tetrahydrobiopterin (BH4) 140 3.4.5 CSF PLP in disorders of sulphite accumulation 140 3.4.6 Neonatal seizure disorder with increased plasma: CSF PLP ratio 145 3.4.7 Nutrition 145 3.5 Summary 146 9 Chapter 4: Measurement of plasma B6 vitamers profiles in children with epilepsy using a LC-MS/MS method 4.1 Introduction 148 4.1.1 Laboratory measurement of the B6 vitamers 148 4.1.2 Liquid chromatography mass spectrometry 149 4.1.3 High pressure liquid chromatography (HPLC) 150 4.1.4 Electrospray ionisation 150 4.1.5 Quadrupole analysers 151 4.1.6 Considerations for the measurement of B6 vitamers in plasma 151 4.2 Materials and Method Development 152 4.2.1 Chemical reagents 152 4.2.2 Sample collection & preparation 155 4.2.3 High Pressure Liquid chromatography 155 4.2.3.1 HPLC column selection 155 4.2.3.2 Optimisation of the mobile phase 156 4.2.4 Electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) 159 4.2.4.1 Quantification of B6 vitamers and pyridoxic acid 163 4.3 Results 164 4.3.1 Calibration curves and linearity 164 4.3.2 Precision studies: Intra- and inter-batch coefficient of variance and recovery studies 168 4.3.3 Indicators of accuracy 169 4.3.4 Study of matrix effects 170 4.3.5 Stability of standards and plasma samples 170 4.3.6 Application of the