i VASCULAR BIOCHEMISTRY OF VITAMIN B12: EXPLORING THE RELATIONSHIP BETWEEN INTRACELLULAR COBALAMIN AND REDOX STATUS IN HUMAN AORTIC ENDOTHELIAL CELLS A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Edward Suarez Moreira May, 2010 i Dissertation written by Edward Suarez Moreira B.Sc., Universidad de la República, Uruguay 2002 Ph.D. Kent State University, 2010 Approved by _________________________________, Chair, Doctoral Dissertation Committee Nicola E. Brasch, Ph.D. _________________________________, Co-Adviser, Doctoral Dissertation Committee June Yun, Ph.D. _________________________________, Member, Doctoral Dissertation Committee William M. Chilian, Ph.D. _________________________________, Member, Doctoral Dissertation Committee Roger B. Gregory, Ph.D. _________________________________, Graduate Faculty Representative Eric M. Mintz, Ph.D. Accepted by _________________________________, Chair, School of Biomedical Sciences Robert V. Dorman, Ph.D. _________________________________, Dean, College of Arts and Sciences John R. D. Stalvey, Ph.D. ii iii TABLE OF CONTENTS LIST OF FIGURES ...................................................................................................... VII LIST OF TABLES ........................................................................................................... X LIST OF ABBREVIATIONS ........................................................................................ XI DEDICATION............................................................................................................... XV ACKNOWLEDGEMENTS ........................................................................................ XVI ABSTRACT .................................................................................................................. XIX CHAPTER 1 INTRODUCTION ..................................................................................... 1 1.1 Cobalamin Structure and Catalytic Function ............................................................ 1 1.1.1 Cobalamin Structure ........................................................................................ 2 1.1.2 Catalytic Function ........................................................................................... 6 1.2 Cobalamin Metabolism in Mammals ...................................................................... 10 1.2.1 Transport and Processing .............................................................................. 10 1.2.2 Cobalamin Deficiency and its Diagnosis ...................................................... 18 1.3 Cobalamin and Reactive Species ............................................................................ 20 1.3.1 Reactive Oxygen and Nitrogen Species ........................................................ 20 1.3.2 Cobalamin and Reactive Species .................................................................. 23 1.4 Cobalamin and Vascular Biology ........................................................................... 25 1.4.1 The Vascular Endothelium ............................................................................ 25 1.4.2 Oxidative Stress and Cardiovascular Disease ............................................... 26 1.4.3 Cobalamin and Hyperhomocysteinemia ....................................................... 28 1.5 Non-cofactor Functions of Cobalamin .................................................................... 30 iii 1.6 Aims ........................................................................................................................ 31 CHAPTER 2 SYNTHESIS AND CHARACTERIZATION OF BIOLOGICALLY RELEVANT COBALAMINS ............................................................................ 33 2.1 Introduction ............................................................................................................. 33 2.2 Experimental Section .............................................................................................. 37 2.2.1 Materials ........................................................................................................ 37 2.2.2 General Methods ........................................................................................... 37 2.2.3 Synthesis of Thiolatocobalamins .................................................................. 39 2.2.4 Synthesis of Non-thiolatocobalamins............................................................ 41 2.2.5 Kinetic Measurements ................................................................................... 44 2.3 Results ..................................................................................................................... 45 2.3.1 Synthesis and Characterization of D,L-Homocysteinylcobalamin, N-Acetyl- L-cysteinylcobalamin (Na+ salt) and 2-N-acetylamino-2-carbomethoxy-L- ethanethiolatocobalamin................................................................................ 45 2.3.2 Acid-catalyzed Decomposition of GSCbl and NACMECbl ......................... 46 2.3.3 Synthesis and Characterization of Sulfitocobalamin .................................... 50 2.4 Discussion ............................................................................................................... 52 CHAPTER 3 EXPLORING THE ROLE OF GLUTATHIONE IN COBALAMIN PROCESSING IN HUMAN AORTIC ENDOTHELIAL CELLS ................. 56 3.1 Introduction ............................................................................................................. 56 3.2 Experimental Section .............................................................................................. 60 3.3 Results ..................................................................................................................... 65 iv 3.3.1 Extraction and Identification of Intracellular Cbl Derivatives in HAEC ...... 65 3.3.2 Extraction and Identification of Intracellular Cbl Derivatives in HAEC in the Presence of a Free Ligand Trap..................................................................... 72 3.3.3 Determination of Intracellular Cobalamins in GSH-depleted HAEC ........... 81 3.4 Discussion ............................................................................................................... 85 CHAPTER 4 VITAMIN B12 PROTECTS AGAINST HOMOCYSTEINE- INDUCED CELL INJURY IN HUMAN AORTIC ENDOTHELIAL CELLS ............................................................................................................................... 90 4.1 Introduction ............................................................................................................. 90 4.2 Experimental Section .............................................................................................. 92 4.3 Results ..................................................................................................................... 94 4.3.1 Cobalamin Protection against Homocysteine-Induced Increase in ROS ...... 94 4.3.2 Subcellular Localization and Cobalamin Protection against Homocysteine- •- Induced O2 increase ..................................................................................... 97 4.3.3 Cobalamin Protection against Homocysteine-Induced Cell Death ............. 100 4.4 Discussion ............................................................................................................. 105 CHAPTER 5 VITAMIN B12 AND REDOX HOMEOSTASIS: COB(II)ALAMIN REACTS WITH SUPEROXIDE AT RATES APPROACHING SUPEROXIDE DISMUTASE ......................................................................... 111 5.1 Introduction ........................................................................................................... 111 5.2 Experimental Section ............................................................................................ 113 5.2.1 General Methods ......................................................................................... 113 v 5.2.2 Kinetic Measurements ................................................................................. 116 5.2.3 Intracellular Studies..................................................................................... 118 5.3 Results ................................................................................................................... 119 •- 5.3.1 In vitro Kinetic Studies on the Reaction between Cbl(II) and O2 ............. 119 •- 5.3.2 Cobalamin Protects against Intracellular O2 ............................................. 131 5.4 Discussion ............................................................................................................. 135 CHAPTER 6 SUMMARY AND FUTURE DIRECTIONS ...................................... 138 6.1 Summary ............................................................................................................... 138 6.2 Future Directions ................................................................................................... 139 6.2.1 Glutathionylcobalamin and Cobalamin Processing. ................................... 139 •- 6.2.2 Cobalamin as a Direct O2 Scavenger ........................................................ 140 6.2.3 Cobalamin as General Antioxidant. ............................................................ 141 REFERENCES .............................................................................................................. 143 vi LIST OF FIGURES CHAPTER 1 INTRODUCTION Figure 1.1 Structure of the three tetrapyrrolic macrocycles 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