HYDROGEN SULFIDE BIOMARKERS AND VITAMIN B-6 STATUS By BARBARA NEAL DERATT A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2016 © 2016 Barbara DeRatt To all my family and friends ACKNOWLEDGMENTS I have been so lucky to have such a great support system during my time as a doctoral student. I would like to thank my mentor, Dr. Jess Gregory for guiding me in my research project. I am forever thankful for all you taught me and I aspire to be a scientist of your caliber one day. I would also like to thank my committee members, Dr. Harry Sitren, Dr. Susan Percival and Dr. Yueh-Yun Chi for their input on my project and support through the many meetings and exams. I would also like to thank my lab members and friends, Dr. Luisa Rios-Avila, Dr. Lili Huang, and Maria Ralat, for their positive influence and advice. My family and friends have also been a blessing through these past 5 years. I would like to thank my parents and my sister Jamie for always listening to my problems and pushing me to finish. I would also like to thank my sister Lindsey for being the best roommate/ chemistry advisor I know. To all my friends, who have listening to me complain and helped with any of my scientific questions or to give me emotional support, I am so indebted to you. And finally, I have to thank Duke Lemmens for being an amazing boyfriend during the hardest part of my doctoral degree. You always supported me and wanted to listen to everything I was doing even though you had no idea what I was talking about. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ........................................................................................... 10 ABSTRACT ................................................................................................................... 14 CHAPTER 1 LITERATURE REVIEW .......................................................................................... 16 Vitamin B-6 ............................................................................................................. 16 Hydrogen Sulfide .................................................................................................... 22 One Carbon Metabolism ......................................................................................... 26 Transsulfuration Pathway ....................................................................................... 27 Cardiovascular Disease .......................................................................................... 29 Study Aims and Objectives ..................................................................................... 30 2 KINETIC STUDIES OF LANTHIONINE AND HOMOLANTHIONINE IN CELL CULTURE ............................................................................................................... 35 Introduction ............................................................................................................. 35 Materials and Methods............................................................................................ 36 Materials ........................................................................................................... 36 Primary Human Hepatocytes ............................................................................ 36 HepG2 Cell Culture .......................................................................................... 37 Analytical Methods ........................................................................................... 37 Isotopically Labelled H2S Biomarker Synthesis and Purification ...................... 39 In-Vivo Kinetics of the Transsulfuration Pathway ............................................. 39 Statistical Analysis ............................................................................................ 40 Results .................................................................................................................... 41 Vitamin B-6 Analysis in Cell Culture Experiments ............................................ 41 PAG Inhibition Affects In-Vivo Kinetics and Concentrations Similarly in Primary Human Hepatocytes and HepG2 Cells ............................................ 41 HepG2 Amino-thiol Concentrations Differ Slightly by Treatment Group After Incubation with Labeled Lanthionine and Homolanthionine .......................... 45 Discussion .............................................................................................................. 46 3 EFFECT OF SHORT-TERM, MARGINAL VITAMIN B-6 DEFICIENCY ON THE CONCENTRATION OF H2S BIOMARKERS, LANTHIONINE AND HOMOLANTIHONINE, IN HEALTHY HUMAN SUBJECTS .................................... 57 5 Abstract ................................................................................................................... 57 Introduction ............................................................................................................. 58 Material and Methods ............................................................................................. 60 Human Vitamin B-6 Restriction Protocols......................................................... 60 Analytical Methods ........................................................................................... 61 Statistical Analyses .......................................................................................... 62 Results .................................................................................................................... 62 Effect of Vitamin B-6 Restriction Protocol ......................................................... 62 H2S Biomarkers in Healthy Human Subjects .................................................... 63 Relationships Among Lanthionine, Homolanthionine, Total Homocysteine, Total Cysteine and Functional Indicators of Vitamin B-6 Deficiency ............. 64 Discussion .............................................................................................................. 65 4 METABOLIC CONSEQUENCES OF PLASMA CYSTATHIONINE ELEVATION IN SUBJECTS WITH SUSPECTED CORONARY ARTERY DISEASE .................. 72 Introduction ............................................................................................................. 72 Materials and Methods............................................................................................ 73 Human Subjects ............................................................................................... 73 Analytical Methods ........................................................................................... 74 Statistical Analyses .......................................................................................... 75 Results .................................................................................................................... 75 Targeted Metabolomics .................................................................................... 75 LCMS Analysis ................................................................................................. 77 NMR Analysis ................................................................................................... 78 Discussion .............................................................................................................. 79 5 CONCLUSIONS AND FUTURE DIRECTIONS .................................................... 106 APPENDIX A ISOTOPICALLY LABELED LANTHIONINE AND HOMOLANTHIONINE SYNTHESIS ......................................................................................................... 111 B PAG CONCENTRATION STUDY ......................................................................... 113 REFERENCES ............................................................................................................ 116 BIOGRAPHICAL SKETCH .......................................................................................... 130 6 LIST OF TABLES Table page 2-1 Isotopic enrichments of methionine and cysteine in media in primary human hepatocytes and HepG2 cells. ............................................................................ 47 2-2 Molar equivalent concentrations of H2S from reactions in the transsulfuration pathway of primary human hepatocytes. ............................................................ 48 2-3 Molar equivalent concentrations of H2S from reactions in the transsulfuration pathway of HepG2 cells ...................................................................................... 48 3-1 Concentrations of amino acid and vitamin B-6 functional biomarkers in preprandial human plasma before and after 28-d dietary vitamin B-6 restriction ............................................................................................................ 68 4-1 Baseline characteristics of 80 subjects from previously conducted Norwegian trials .................................................................................................................... 82 4-2 Concentrations of selected plasma variables in 80 subjects from previously conducted Norwegian trials (NORCAD) ............................................................