HAEM SYNTHESIS IN IDIOPATHIC SIDEROBLASTIC ANAEMIA Submitted by Russell Thomas Edwards for the award of Master of Science 1988 DECLARATIONS 1. I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree or diploma of a university or other institute of higher learning, except where due acknowledgement is made in the text RUSSELL THOMAS EDWARDS INDEX List of Figures and Tables Page 1 Acknowledgements Page2 Summary Page3 Terms and Abbreviations Page4 CHAPTER I Haem Synthesis 1.1 Introduction Page6 1.2 ALA Synthetase (EC 2.3.1.37) Page7 1.3 ALA Dehydratase (EC 4.2.1.24) Page 10 1.4 Hydroxymethylbilane Synthetase (EC 4.3.1.8) and Page 12 Uroporphyrinogen III Synthetase (EC 4.2.1.75) 1.S Uroporphyrinogen Decarboxylase (EC 4.1.1.37) Page 15 1.6 Coproporphyrinogen Oxidase (EC 1.3.3.3) Page 16 1.7 Protoporphyrinogen Oxidase Page 17 1.8 Ferrochelatase (EC 4.99.1.1) Page 17 1.9 Regulation of Haem Synthesis Page 18 1.10 Iron and Haem Synthesis Page22 1.11 Haem Regulation of Globin Synthesis Page23 1.12 Sideroblastic Anaemia (S.A.) Page24 1.13 Congenital Sideroblastic Anaemia Page25 1.14 Acquired Sideroblastic Anaemia Page26 1.1S Haem Synthesis in AISA Page 28 1.16 Statement of Objectives and the Problems to be Investigated Page31 CHAPTER II Materials and Methods 2.1 Chemicals Page 38 2.2 Dowex Page 38 2.3 Culture. R. Spheroides Page 39 2.4 Partial Purification of Hydroxymethylbilane Synthetase Page 39 2.S Specimen Preparation - ALA Synthetase Assay Page42 2.6 Specimen Preparation - Hydroxymethylbilane Synthetase Page42 2.7 Speciment Preparation -Uroporphyrinogen III Synthetase Assay Page43 2.8 [14CJ ALA Isolation (Ebert, et.al., 1970; Pasanen, et.al., 1980) Page43 2.9 Reticulocyted Rich Red Cell Sample Page43 2.10 Full Blood Count Page44 2.11 Reticulocyte Count Page44 2.12 Serum Iron and Iron Ringling Capacity Page44 2.13 Serum Ferritin Page44 2.14 Protoporphyrin Page45 2.1S Protein Page45 2.16 Statistics Page 45 CHAPTER Ill Evaluation and Modification of Enzyme Assays 3.1 ALA Synthetase Assay Page46 3.1.1 Introduction Page46 3.1.2 Incubation Conditions Page47 3.1.3 Reagent Blank Page48 3.1.4 Workup Page48 3.1.S Product Isolation & Measurement Page49 3.1.5.1 Chromatography Page49 3.1.5.2 Pyrrole Fonnation and Isolation Page49 3.1.5.3 Calculations Page49 3.2 Investigations of ALA Synthetase Assay Page 50 3. 2. 1 Purity of [2,3, 14c] Succinic Acid Page50 3.2.1.1 Ion Exchange Chromatography of Page 50 [2,3, 14q Succinic Acid and [4, l4q ALA 3. 2. 2 Composition of Incubation Mixture Page53 3.2.2.1 Variations in Reaction Mixture and Conditions Page53 3.2.2.2 Blocking Side Reactions Page 54 3.2.2.3 Variation in Succinate Concentration Page 58 3.2.2.4 Variation of Glycine Concentration Page 59 3. 2. 3 General Assay Conditions Page59 3.2.3.1 Evaluation of Storage Page59 3.2.3.2 Variation in Sonication Times Page61 3.2.3.3 Reagent Blank Page61 3.2.3.4 Incubation Times Page63 3.2.3.S Association of the Assay with Reticulocyte Numbers Page64 3. 2. 4 Isolation of [ 14c] ALA or Related Pyrrole Page66 3.2.4.1 Recovery of [4,14q ALA from Distillation Flasks Page66 3.2.4.2 Comparison of Two ALA Isolation Methods Page66 3.2.4.3 Recovery Page67 3. 2. 5 Nonnal Reference Range Page69 3-~. b D\ac.'-'SS,cn of f\\.,,F\ s-r~e.¼-0.&e. RSSQ.'1 ~'~ 3.3 Hydroxymethylbilane Synthetase Assay Page 70 3.3.1 Introduction Page 70 3.3.2 Assay Procedure Page 70 3.3.3 Calculations Page 71 3.4 Investigations of Hydroxymethylbilane Synthetase Assay Page 71 3.4.1 pH Dependence Page 71 3.4.2 Association Between the Assay Results and Reticulocyte Numbers Page 72 3.4.3 Time and Protein Dependence Page 73 3.4.4 Substrate Variation Page 73 3.4.S Recovery Page 76 3.4.6 Normal Reference Range Page 76 ii 3.5 Uroporphyrinogen m Synthetase Assay Page 77 3.5.1 Introduction Page 77 3.5.2 Assay Method Page 77 3.5.3 Calculations Page 78 3.6 Investigations of Uroporphyrinogen m Synthetase Assay Page 79 3.6.1 Sample Volume Page 79 3.6.2 Substrate Concentration Page 79 3.6.3 Hydroxymethylbilane Synthetase Concentration Page 79 3.6.4 Normal Reference Range Page 80 3.7 Conclusion Page 83 CHAPTER IV Results and Discussion - Acquired Idiopathic Sideroblastic Anaemia 4.1 Patients Tested Page 84 4.2 Test Results Page 85 4 . 2 . 1 Haematological Tests Page 85 4. 2. 2 Enzyme Assays Page 89 4.2.2.1 ALA Synthetase Activity Page 89 4.2.2.2 Hydroxymethylbilane Synthetase Activity Page89 4.2.2.3 Uroporphyrinogen m Synthetase Activity Page 89 4 . 2 • 3 Red Cell Protoporphyrin Levels Page90 4.3 Association of Enzyme Assays With Each Other and Page95 With Protoporphyrin Levels 4.4 Discussion Page98 CHAPTER V Concluding Discussion Page 102 F"u.r-tne.r- \C'\uee+~aT\ot'\S ~rdi,it Literature Cited ~ac:hhoC'\Q\ \....1\e.rQ-4-ure CH-ed. iii LIST OF FIGURES AND TABLES TABLES Table 1 Page29 Table 2 Page41 Table 3 Page 57 Table4 Page63 Table 5 Page68 Table6 Page68 Table 7 Page 72 Table 8 Page 76 Table9 Page 85 Table 10 Page 86 Table 11 Page 87 Table 12 Page 88 Table 13 Page 88 Table 14 Page91 Table 15 Page93 Table 16 Page93 Table 17 Page94 Table 18 Page94 FIGURES Figure 1 Page 34 Figure 2 Page 34 Figure 3 Page 35 Figure 4 Page 36 Figure 5 Page 37 Figure 6 Page 37 Figure 7 Page 51 Figure 8 Page 52 Figure 9 Page 55 Figure 10 Page 56 Figure 11 Page60 Figure 12 Page 62 Figure 13 Page65 Figure 14 Page 74 Figure 15 Page 75 Figure 16 Page81 Figure 17 Page 81 Figure 18 Page 82 Figure 19 Page92 Figure 20 Page96 Figure 21 Page96 Figure 22 Page97 1 ACKNOWLEDGEMENTS I would like to acknowledge and thank Professor R. Pitney for his assistance in the choice of topic and area of investigation and Associate Professor K. Rienits for his great assistance and guidance during the performance and analysis of the material contained in this thesis. I would also like to thank the St. George Hospital for the use of their laboratories and for their provision of reagents and Dr. V. Poulos of Royal Prince Alfred Hospital for allowing me to perform protoporphyrin assays in his laboratory. In addition I must thank D. K. Phadke and Dr. A. Manoharran for allowing me access to their patients. 2 SUMMARY Patients suffering from acquired idiopathic sideroblastic anaemia (AISA) are thought to have a defect in the synthesis of the haem moiety of haemoglobin. The cause of this defect is thought to be the reduced activity, in erythroid tissue, of the first enzyme of the haem synthetic chain - ALA synthetase. This thesis has investigated haem synthesis in AISA patients by measuring the activities of three haem synthetic enzymes, measuring the levels of one of the products of the haem synthetic chain, non-haem protoporphryin, and measuring the levels of iron and ferritin in the plasma of AISA patients. It was thought that this approach would provide a coherent interpretation of haem synthetic activity in AISA patients. The ALA synthetase activity was measured in peripheral reticulocytes using an improved and potentially more specific method and the mean activity was found to be significantly reduced but, considerable overlap was found with the levels of activity in normals. The mean activities in peripheral red cells of hydroxymethylbilane synthetase and uroporphyrinogen ill synthetase were found to be increased as were the levels of non-haem protoporphyrin and zinc protoporphyrin. The mean plasma transferrin saturation and mean plasma ferritin levels were also shown to be significantly increased. Significant correlations were obtained between the activities of hydroxymethylbilane synthetase and uroporphyrinogen III synthetase and between the activities of both of these enzymes and total non-haem protoporphyrin levels. No significant association could be found between the reduced ALA synthetase activity and the hydroxymethylbilane synthetase activity, the uroporphyrinogen III synthetase activity or the total non-haem protoporphryin levels. No significant correlation was found between the plasma transferrin saturation and non-haem protoporphyrin levels. 3 These data have been interpreted as indicating that despite a decrease in the mean ALA synthetase activity, there is an increased flux through the haem synthetic chain in patients with AISA. This increased flux is considered to cause the accumulation of one of the;"1t,...cJ.·,ta of the haem synthe sis - non-haem protoporphyrin. 4 TERMS AND ABBREVIATIONS In addition to the standard scientific terms and abbreviations, the following have been used throughout this thesis; AISA = acquired idiopathic sideroblastic anaemia ALA = aminolaevulinic acid EDTA = ethylenediamine tetra-acetic acid GTP = guanosine triphosphate PBG = potphobilinogen SD = standard deviation TCA = trichloroacetic acid. 5 CHAPTER I HAEM SYNTHESIS 1.1 INTRODUCTION The haem molecules play a major role in life and form the prosthetic groups of a diverse group of haemoproteins whose functions are varied. One of these haemoproteins, haemoglobin, whose major role is the transport of oxygen, has been shown to have an apparent defect in the synthesis of the haem moiety in a group of diseases called the Sideroblastic Anaemias.