University of Iowa Iowa Research Online Theses and Dissertations Spring 2016 Novel NAD+ metabolomic technologies and their applications to Nicotinamide Riboside interventions Samuel A.J. Trammell University of Iowa Follow this and additional works at: https://ir.uiowa.edu/etd Part of the Genetics Commons Copyright 2016 Samuel AJ Trammell This dissertation is available at Iowa Research Online: https://ir.uiowa.edu/etd/3203 Recommended Citation Trammell, Samuel A.J.. "Novel NAD+ metabolomic technologies and their applications to Nicotinamide Riboside interventions." PhD (Doctor of Philosophy) thesis, University of Iowa, 2016. https://doi.org/10.17077/etd.mk206led Follow this and additional works at: https://ir.uiowa.edu/etd Part of the Genetics Commons NOVEL NAD + METABOLOMIC TECHNOLOGIES AND THEIR APPLICATIONS TO NICOTINAMIDE RIBOSIDE INTERVENTIONS by Samuel A.J. Trammell A thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Genetics in the Graduate College of The University of Iowa May 2016 Thesis Supervisor: Professor Charles Brenner Copyright by Samuel A.J. Trammell 2016 All Rights Reserved Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL ____________________________ PH.D. THESIS _________________ This is to certify that the Ph.D. thesis of Samuel A.J. Trammell has been approved by the Examining Committee for the thesis requirement for the Doctor of Philosophy degree in Genetics at the May 2016 graduation. Thesis Committee: ____________________________________________ Charles Brenner, Thesis Supervisor ____________________________________________ Diane C. Slusarski ____________________________________________ Mary E. Wilson ____________________________________________ Michael E. Wright ____________________________________________ Robert C. Piper ACKNOWLEDGEMENTS I must give the greatest thanks to current and former members of the Brenner laboratory for being a constant supportive yet critical force in my thesis work. My mentor, Dr. Charles Brenner, was, is, and always will be a voice of optimism and encouragement that propelled my work forward. Dr. Brenner allowed me to work independently but was always there as a much needed critical, centering voice during the whole of my thesis. Recounting the contributions of all members of the Brenner laboratory is impossible due to my inability to properly measure the extreme aid and friendship each person provided. However, I would like to specifically thank Dr. Rebecca Fagan for the light, absurd, and humorous conversations had as co-workers and more importantly as friends. I would like to acknowledge former laboratory mates Dr. Szu-Chieh Mei, her husband Dr. Bokuan Wu, and Dr. Jennifer Bolyston for making the laboratory a fun and interesting place in which to work and for their insightful commentary into my work. To my current co-workers, thank you for continuing to contribute to the special milieu that is the Brenner laboratory. I could never properly thank Dr. Lynn Teesch and Mr. Vic Parcell enough. Dr. Teesch quickly became an unofficial advisor throughout my time working in the High Resolution Mass Spectrometry Facility on all things related to the operation and use of mass spectrometry. Conversations with Mr. Parcell varied from the deeply technical to the most mirthful. Both provided constant expertise and support that continually reinvigorated my passion for science and undoubtedly helped me through the more difficult portions of my time here at the University of Iowa. I would like to thank Drs. Diane Slusarski, Marry Wilson, Michael Wright, and Rob Piper for first agreeing to serve on my committee and then for the contributions they have made to my growth as a scientist and to my thesis. ii Finally, I would like to thank the many friends outside of my field and the University that I have met in Iowa City. I cannot imagine the person I would be today without their company. iii ABSTRACT Nicotinamide adenine dinucleotide (NAD +) is a cofactor in hydride transfer reactions and consumed substrate of several classes of glycohydrolyitc enzymes, including sirtuins. NAD +, its biosynthetic intermediates, breakdown products, and related nucleotides (the NAD metabolome) is altered in many metabolic disorders, such as aging and obesity. Supplementation with the novel NAD + precursor, nicotinamide riboside (NR), ameliorates these alterations and opposes systemic metabolic dysfunctions in rodent models. Based on the hypothesis that perturbations of the NAD metabolome are both a symptom and cause of metabolic disease, accurate assessment of the abundance of these metabolites is expected to provide insight into the biology of diseases and the mechanism of action of NR in promoting metabolic health. Current quantitative methods, such as HPLC, lack specificity and sensitivity to detect distinct alterations to the NAD metabolome. In this thesis, I developed novel sensitive, accurate, robust liquid chromatography mass spectrometry methodologies to quantify the NAD metabolome and applied these methods to determine the effects of disease states and NR supplementation on NAD + metabolism. My investigations indicate that NR robustly increases the NAD metabolome, especially NAD + in a manner kinetically different than any other NAD + precursor. I provide the first evidence of effective NAD + supplementation from NR in a healthy, 52 year old human male, suggesting the metabolic promoting qualities of NR uncovered in rodent studies are translatable to humans. During my investigation of NR supplementation, my work establishes an unexpected robust, dramatic increase in deamino–NAD +, NAAD, directly from NR, which I argue could serve as an accessible biomarker for efficacious NAD + supplementation and the effect of disease upon the NAD metabolome. Lastly, I further establish NR as a general therapeutic against metabolic disorder by detailing its ability to oppose aspects of chronic alcoholism and diabetes mellitus. iv PUBLIC ABSTRACT A century ago in the United States, a disease known as Pellagra ravaged areas mainly subsisting on maize. This disease was detrimental to quality of life and in some instances proved fatal. At the time, this disease was considered a major public health problem and many grant initiatives were announced to identify the cause of the disease and develop an effective treatment. Through these efforts, Pellagra was shown to be a non-infectious disease caused by a diet of maize and lard. It was cured by drinking milk and eating more animal meat. These efforts essentially eliminated the disease from high income nations. Further investigation identified the B3 vitamins commonly referred to as niacin as the anti-Pellagra components of milk and animal meat. Today, obesity, diabetes, and heart disease are prevalent in the US and areas around the world. These diseases are a new public health crisis resulting in the loss of billions of dollars and a decreased quality of life and lifespan. As it was a hundred years ago, public funds are now directed to identify effective treatments to counter these prevalent and devastating diseases. Work generously funded by the public has identified the most recently discovered B 3 vitamin, nicotinamide riboside, as a health promoting compound that could treat these diseases. The goal of my thesis was to develop improved tools to answer how this vitamin works in times of health and disease. In so doing, my work further establishes this novel B 3 vitamin as a health promoting compound and describes clinically relevant technologies to assess its effectiveness in future human trials. v TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................... xi LIST OF FIGURES .................................................................................................................. xiii LIST OF ABBREVIATIONS ....................................................................................................... xv CHAPTER 1 ............................................................................................................................... 1 INTRODUCTION .................................................................................................................... 1 1.1 Significance of NAD + and Description of the Need for Improved Technologies for Its Measurement ...................................................................................................................... 1 1.2 NAD + Transactions ....................................................................................................... 3 1.3 Thesis Goals ................................................................................................................. 8 1.4 Figure ..........................................................................................................................11 CHAPTER 2 ..............................................................................................................................12 NAD METABOLOME ANALYSIS VIA LIQUID CHROMATOGRAPHY MASS SPECTROMETRY.................................................................................................................12 2.1 Quantitative NAD + Metabolomics .................................................................................12 Optimized Extraction ......................................................................................................12 Optimized Internal Standards .........................................................................................14