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Metabolomic Assessment of Dietary Interventions in Obesity by Capillary Electrophoresis Mass Spectrometry

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Metabolomic Assessment of Dietary Interventions in Obesity by Capillary Electrophoresis Mass Spectrometry Metabolomic Assessment of Dietary Interventions in Obesity by Capillary Electrophoresis Mass Spectrometry Metabolomic Assessment of Dietary Interventions in Obesity by Capillary Electrophoresis Mass Spectrometry By Karen Phoebe Lam, H.B.Sc., M.Sc. A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy McMaster University © Copyright by Karen Phoebe Lam, July 2018 Doctor of Philosophy (2018) McMaster University (Department of Chemisty & Chemical Biology) Hamilton, Ontario TITLE: Metabolomic Assessment of Dietary Interventions in Obesity by Capillary Electrophoresis Mass Spectrometry AUTHOR: Karen Phoebe Lam, H.B.Sc. (University of Toronto), M.Sc. (McMaster University) SUPERVISOR: Professor Philip Britz-McKibbin NUMBER OF PAGES: xxix, 347 ii Abstract Capillary electrophoresis mass spectrometry (CE-MS) is a versatile instrumental method for metabolomics, which allows for comprehensive metabolite profiling of volume-limited biological specimens in order to better understand the molecular mechanisms associated with chronic diseases, including an alarming epidemic of obesity worldwide. Multiplexed CE separations enable high-throughput metabolite screening with quality assurance to prevent false discoveries when combined with rigorous method validation, robust experimental designs, complementary statistical methods, and high-resolution tandem mass spectrometry (MS/MS) for unknown metabolite identification. In this thesis, multiplexed CE-MS technology is applied for both targeted and untargeted metabolite profiling of various biological fluids, including covalently bound thiol-protein conjugates, as well as free circulating metabolites in serum and plasma, and excreted/bio-transformed compounds in urine due to complex host-gut microflora co-metabolism. This work was applied to characterize aberrant metabolic responses of obese subjects in response to dietary challenges, and measure the benefits of dietary interventions that reduce adiposity without deleterious muscle loss. Chapter 2 presents, a simple, sensitive yet robust analytical protocol to expand metabolome coverage in CE-MS for the discovery of labile protein thiols in human plasma using a rapid chemical derivatization method based on N-tert-butylmaleimide (NTBM). Chapter 3 describes targeted metabolite profiling of serum and plasma to investigate the differential metabolic responses between healthy and unhealthy obese individuals before and after consumption of a standardized high-caloric meal, respectively. Chapter 4 of this thesis describes an untargeted metabolite profiling strategy for urine using multisegment-injection (MSI)-CE-MS for elucidating the effects iii of protein supplementation following a short-term dietary weight-loss intervention study. This work revealed six urinary metabolites that were classified as top-ranking treatment response biomarkers useful for discriminating between subjects consuming carbohydrate (control), soy, and whey supplemented diets. In summary, this thesis demonstrated the successful implementation of multiplexed CE-MS technology for biomarker discovery in nutritional-based metabolomic studies as required for more effective treatment and prevention of obesity for innovations in public health. iv Acknowledgements This thesis would not have been possible without the collective contributions of the many people whom I have encountered throughout the course of my graduate research. First, I would like to sincerely thank my supervisor, Prof. Philip Britz-McKibbin, who welcomed me into his research group and introduced me to the wonderful world of CE. He has provided me with many opportunities throughout my graduate studies, in which I have gained a lot of invaluable hands-on training and experience. He has guided my research journey with unparalleled optimism and an unforgettable adventure-seeking spirit, in the same way that he has guided me and fellow group members through rugged treks to find zen (aka, “the waterfall”) during our memorable annual Britz-group lab hikes. I would also like to express my appreciation to supervisory committee members, Prof. Murray Potter, Prof. Jose Moran-Mirabal and the late Prof. Brian McCarry, as well as Kirk Green from the McMaster Regional Centre for Mass Spectroscopy, for their insightful questions, advice, support, encouragement, and technical assistance over the years. Additionally, I would like to thank my collaborators, Prof. David Mutch (University of Guelph) and Prof. Stuart Phillips (Department of Kinesiology), for their contributions and involvement in the various studies described in this thesis, aside from the stimulating discussions during our project-update meetings. The cordial lab atmosphere, collaborative group dynamics, and sense of camaraderie amongst the Britz group members have played an important role in enhancing my v graduate student experience and cannot be overstated. I would like to thank past and present lab members in the Britz group for, not only sharing their advice, ideas and providing assistance when needed, but also, for their individual contributions to creating a warm, supportive, and friendly working atmosphere: Lisa D’Agostino, Naomi Kuehnbaum, Meera Shanmuganathan, Alicia DiBattista, Nadine Wellington, Adriana Macedo, Michelle Saoi, Mai Yamamoto, Jennifer Wild, Stellena Mathiaparanam, Ritchie Ly, Sandi Azab, and Biban Gill. Outside of the lab environment, I am very grateful for all the friends that I have made at McMaster, in addition to my long-time friends from Toronto, as well as those living out-of-province. Thank you for all the fun times we had together, whether it be going for a stroll or a bike ride; enjoying a movie, bowling or board game night; going out for a nice dinner or being invited for potluck or a delicious home-cooked meal; indulging in desserts; or even, as simple as a telephone call or text message. And of course, thank you for always rooting for me. All these gestures mean so much to me and I am very blessed to have your friendship. Special thanks go to my friend, Fan Fei, for her kind support and graciously offering her time and expertise to help me with particular revisions of this thesis. My parents, Connie and Joseph, my brother, Eugene, and all other family members have played a significant role in my life and I would not be here without them. I thank them all for their unconditional love, sacrifices, patience, and continuous support throughout this journey. In addition, I thank my Mom for giving me the gift of music, along with its natural healing powers; I thank my Dad for teaching me patience, forgiveness, and the art of prayer; and I thank my brother for making me feel valued and important in this world as an older sister. As well, I also thank Patrick Wilson and his parents, Anna and Robert, for their care, kindness, love, guidance, and encouragement. In particular, I express my deepest heartfelt gratitude to Patrick, who has been by my side for most of this adventure and especially, at the most vi difficult times. He has guided and supported me tremendously in the completion of this journey in more ways than words can ever describe. He has selflessly devoted countless hours, days, weeks, and months of his personal time and energy to my cause, including assisting with multivariate data analysis and completing multiple rounds of meticulous typesetting, formatting, and editing of this entire thesis. Most importantly, he has encouraged and emboldened me to reach the finish line of this pursuit with his abundant love and immense support – and by firmly believing in me, much more than I believe in myself. Finally, I thank God for giving me endurance and personal strength, and for everyone in my life that has helped me to be where I am today. vii This thesis is dedicated to three very special people in my life: My 90-year-old energetic grandmother, Chiu Lai Har, a recent breast cancer survivor whose love, support, courage, and fearless determination have inspired me to complete this work; My long-time mentor and friend of 20 years, Toronto-based pianist and performer Peter Longworth, whom I will always remember as being a major influence in my life and on my music as a pianist, up until his sudden passing at an early age due to kidney cancer on June 26, 2018; and My late Godfather, Vincent Lam, who passed away on February 1, 2018 after a heroic, yet painful, battle with lymphoma. He celebrated life up until his last day and was the one to witness my first steps as a toddler and would later inspire me to pursue and develop a passion for the sciences. viii Table of Contents Front Matter i Abstract . iii Acknowledgements . .v List of Figures . xiv List of Tables . xviii List of Acronyms & Abbreviations . xxi 1 Defining Metabolomics and its Role in Health and Nutrition 1 1.1 Overview of Metabolomics . .1 1.2 Instrumental Methods in Metabolomics . .5 1.3 Principles of CE . .7 1.4 Interfacing CE to MS . 12 1.5 Metabolomics in Nutrition and Dietary Intervention Studies . 16 1.6 Functional and Compositional Differences Between Biofluids . 19 1.7 Pre-Analytical Challenges in the Collection, Handling, and Storage of Biofluids . 21 1.7.1 Sample Collection and Handling . 21 1.7.2 Sample Storage . 24 1.7.3 Sample Pretreatment . 25 ix 1.8 Pretreatment of Data . 26 1.8.1 Data Normalization, Batch Correction, and Quality Controls . 26 1.8.2 Data Transformation . 28 1.8.3 Data Scaling . 30 1.9 Dealing with Massive Data Using Multivariate Statistical Techniques 31 1.9.1 Statistical Data Mining . 31 1.9.2 Variable Selection and Filtering . 34 1.9.3 Comparison Between Multiple Groups . 39 1.10 Thesis Motivation, Objectives, and Contributions . 43 2 Development of a Novel CE-MS Method for the Discovery of Protein- Bound Thiols 47 2.1 Thiol Detection Enhancement by Chemical Derivatization with Malei- mide . 48 2.1.1 Introduction . 48 2.1.2 Materials and Methods . 52 2.1.3 Results & Discussion . 55 2.1.4 Conclusions . 64 2.2 Characterization of Protein-Bound Thiols in Plasma . 66 2.2.1 Introduction . 67 2.2.2 Materials and Methods .
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