University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2012 Silec: A New Tool to Study Mitochondrial and Metabolic Disease Sankha Subhra Basu University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Analytical Chemistry Commons, Biochemistry Commons, and the Pharmacology Commons Recommended Citation Basu, Sankha Subhra, "Silec: A New Tool to Study Mitochondrial and Metabolic Disease" (2012). Publicly Accessible Penn Dissertations. 490. https://repository.upenn.edu/edissertations/490 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/490 For more information, please contact [email protected]. Silec: A New Tool to Study Mitochondrial and Metabolic Disease Abstract Coenzyme A (CoA) and CoA thioesters are central to numerous metabolic pathways. However, the inability to synthesize isotopically labeled CoA has limited analytical methods to measure these compounds. In this thesis, we developed Stable Isotope Labeling with Essential nutrients in Cell culture (SILEC), a method to generate isotopically labeled CoA derivatives by growing cells in a labeled CoA precursor, [13C15N]-pantothenate. Labeled CoA derivatives were used as internal standards in a stable isotope dilution liquid chromatography-mass spectrometry (LC-MS) method to measure changes in short chain acyl-CoA species. This assay was also adapted to include measurement of glutathione-CoA (CoASSG), a mixed disulfide increased in mitochondrial oxidative stress. Menadione significantly increased intracellular levels of CoASSG, and decreased levels of CoASH through the formation of CoA- menadione adduct. In addition, rotenone, an organic pesticide and potent complex I inhibitor, induced a dose-dependent decrease in succinyl-CoA and increase in beta-hydroxybutyryl-CoA (BHB-CoA) in multiple human cell lines, as well as inhibited glucose-derived acetyl-CoA and succinyl-CoA biosynthesis. This SILEC assay was further adapted for use in freshly isolated human platelets to assess metabolic changes in Friedreich's Ataxia (FA), an inherited mitochondrial disease caused by mutations in the frataxin gene. FA patients were found to have significantly decreased acetyl-CoA: succinyl-CoA ratio, consistent with an in vitro siRNA knockdown model of frataxin. Finally, platelets were demonstrated to serve as a powerful ex vivo metabolic and toxicologic challenge platform evidenced by treatment with propionate, rotenone and a number of stable isotope metabolic tracers. Together, these methods provide a paradigm for the discovery of novel biomarkers for metabolic and mitochondrial disease. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Pharmacology First Advisor Ian A. Blair Keywords Coenzyme A, Mass Spectrometry, Metabolism, Mitochondria, Stable Isotope Subject Categories Analytical Chemistry | Biochemistry | Pharmacology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/490 SILEC: A NEW TOOL TO STUDY MITOCHONDRIAL AND METABOLIC DISEASE Sankha S. Basu A DISSERTATION in Pharmacology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2012 Supervisor of Dissertation: Signature: _______________________ Ian A. Blair, PhD, A.N. Richards Professor of Pharmacology Graduate Group Chairperson: Signature: ______________________ Vladimir Muzykantov, MD, PhD, Professor of Pharmacology Dissertation Committee: Trevor Penning, PhD, Professor of Pharmacology Harry Ischiropoulos, PhD, Research Professor of Pediatrics Jeff Field, PhD, Professor of Pharmacology Alexander Whitehead, PhD, Professor of Pharmacology To my parents, whose sacrifices and inspiration made this possible. “Far better is it to dare mighty things, to win glorious triumphs, even though checkered by failure...than to rank with those poor spirits who neither enjoy nor suffer much, because they live in a gray twilight that knows not victory nor defeat.” -Theodore Roosevelt ii Acknowledgments First, I would like to thank my thesis advisor, Dr. Ian Blair, whose continual encouragement has laid the foundation for my future as an independent investigator. I would also like to thank Dr. Stacy Gelhaus and Dr. Clementina Mesaros, who have helped me transition from molecular biology to analytical chemistry. In addition, I would like to thank Dr. Angela Wehr and Matthew Macdonald, who have helped provide scientific and personal support throughout my time in the lab. I would like to extend special thanks to Christine Shwed, who has been integral in not only improving the research environment, but whose tireless efforts have enriched our lab experience. Lastly, I would also like to thank all former and current members of the Blair Lab, whose research has been integral to much of the work presented in this thesis. I would like to acknowledge a number of research collaborators. I would like to extend a special thanks to Eric Deutsch and Dr. David Lynch for their help on the frataxin and Friedreich’s Ataxia studies, along with all volunteers who have provided platelets for these studies. I would like to thank Dr. Alec Schmaier, for his guidance in the platelet studies, Andrew Worth, for his help in developing the LC-MS organic acid method and Ellyn Boukus for her help with mass isotopomer distribution analysis. I would like to thank Dr. Ronald Carnemolla from Vladimir Muzykantov’s lab for helping with the SILEC insect cell culture experiments, and Dr. Michael Schupp from Mitch Lazar’s lab for providing mouse liver for the SILEC tissue experiments. Finally, I would like to thank Margie Maronski and Dr. Mark Dichter for providing cortical rat neurons for the rotenone studies. iii I would like to thank my thesis committee members, Dr. Trevor Penning, Dr. Harry Ischiropoulos, Dr. Steve Whitehead and Dr. Jeff Field for their insights and guidance. In particular, I would like to thank Dr. Penning who served as my committee chair as well as a mentor in the field of toxicology. I would also like to thank Dr. Skip Brass and Maggie Krall for their support in providing a tremendous environment to grow as a physician-scientist. I would like to thank Barbara Zolotorow for her help in organizing the training grant, as well as the National Institutes of Health (NIH) for funding which made this research possible. Finally, I would like to thank my friends and family who have been tremendously supportive and patient during the graduate school process. iv Abstract SILEC: A NEW TOOL TO STUDY MITOCHONDRIAL AND METABOLIC DISEASE Sankha S. Basu Ian A. Blair, PhD Coenzyme A (CoA) and CoA thioesters are central to numerous metabolic pathways. However, the inability to synthesize isotopically labeled CoA has limited analytical methods to measure these compounds. In this thesis, we developed Stable Isotope Labeling with Essential nutrients in Cell culture (SILEC), a method to generate isotopically labeled CoA derivatives by growing cells in a labeled CoA precursor, 13 15 [ C3 N]-pantothenate. Labeled CoA derivatives were used as internal standards in a stable isotope dilution liquid chromatography-mass spectrometry (LC-MS) method to measure changes in short chain acyl-CoA species. This assay was also adapted to include measurement of glutathione-CoA (CoASSG), a mixed disulfide increased in mitochondrial oxidative stress. Menadione significantly increased intracellular levels of CoASSG, and decreased levels of CoASH through the formation of CoA-menadione adduct. In addition, rotenone, an organic pesticide and potent complex I inhibitor, induced a dose-dependent decrease in succinyl-CoA and increase in β-hydroxybutyryl- CoA (BHB-CoA) in multiple human cell lines, as well as inhibited glucose-derived acetyl-CoA and succinyl-CoA biosynthesis. This SILEC assay was further adapted for v use in freshly isolated human platelets to assess metabolic changes in Friedreich’s Ataxia (FA), an inherited mitochondrial disease caused by mutations in the frataxin gene. FA patients were found to have significantly decreased acetyl-CoA: succinyl-CoA ratio, consistent with in vitro siRNA knockdown model of frataxin. Finally, platelets were demonstrated to serve as a powerful ex vivo metabolic and toxicologic challenge platform evidenced by treatment with propionate, rotenone and a number of stable isotope metabolic tracers. Together, these methods provide a paradigm for the discovery of novel biomarkers for metabolic and mitochondrial disease. vi Table of Contents Dedication……………………………………………………………………..…….……ii Acknowledgments…………………………………………………………….…….……iii Abstract…………………………………………………………….…….……..…….…...v Table of Contents ………………………………………………………………..……....vii List of Tables ……………………………………………………..………………….…viii List of Figures………………………………………..…………………………………...ix Abbreviations………………………………………..………………………………..…..xi Chapter 1. Introduction: The metabolic role of coenzyme A (CoA) and developments in the analytical measurement of CoA derivatives……………………..….………….…1 Chapter 2. The development of Stable Isotope Labeling by Essential Nutrients in Cell Culture (SILEC) for preparation of labeled coenzyme A and its thioesters……...…15 Chapter 3. A practical protocol for the generation and application of SILEC mass spectrometry standards to biological samples……………………………………..…38 Chapter 4. Development of an LC-MS method to measure CoASSG and its relevance to mitochondrial oxidative stress…………………………………….…………………63