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Electrochemical Measurement of Nitric Oxide from Biological Systems

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Electrochemical Measurement of Nitric Oxide from Biological Systems ELECTROCHEMICAL MEASUREMENT OF NITRIC OXIDE FROM BIOLOGICAL SYSTEMS Rebecca Anne Hunter A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry (Analytical Chemistry). Chapel Hill 2014 Approved by: Mark H. Schoenfisch Royce W. Murray James W. Jorgenson Bruce A. Cairns Robert Maile © 2014 Rebecca Anne Hunter ALL RIGHTS RESERVED ii ABSTRACT REBECCA ANNE HUNTER: Electrochemical Detection of Nitric Oxide from Biological Systems (Under the direction of Mark H. Schoenfisch) Nitric oxide (NO) is known to be involved in a number of physiological processes, including the immune response. As such, its role in severe infection and sepsis has been investigated, but previous measurement techniques have relied on complicated instrumentation or the quantification of NO byproducts (e.g., nitrate and nitrite). Herein, the fabrication of a microfluidic amperometric sensor for the direct detection of NO in whole blood is described. These sensors were used to evaluate the potential of NO and nitrosothiols (a stable transporter) as prognostic and/or diagnostic biomarkers for infection and sepsis. The microfluidic devices facilitated the selective electrochemical measurement of NO in small volumes of blood at the point-of-care, with adequate sensitivity and limits of detection achieved in buffer, wound fluid, and whole blood. A green (530 nm) light-emitting diode was coupled to the device to enable photolysis of S-nitrosothiol species with subsequent NO detection. While inefficient photolysis prevented the measurement of nitrosothiols in whole blood, detection in serum was achieved. A porcine model of sepsis permitted monitoring of temporal changes in NO and nitrosothiols throughout disease progression. While increases in NO were observed concurrently with other indicators (e.g., increased blood lactate and base deficit), the iii accumulation of nitrosothiols was observed hours prior to the onset of other symptoms, despite a dramatic drop in the circulating white blood cells that produce NO. A murine model of sepsis was utilized to understand the effects of bacterial virulence and immune suppression on NO during an infection. A non-lethal pneumonia with Pseudomonas aeruginosa resulted in elevated NO levels at 72 h that returned to baseline concentrations after 1 wk. A more virulent bacterium, Klebsiella pneumoniae, resulted in much greater increases in NO, reflecting its pathogenicity. Conversely, in a murine model of post-burn immune suppression and infection, blood NO concentrations remained unchanged relative to uninfected animals despite increased infection severity. Nitric oxide-selective microelectrodes were also used to study NO release at the single cell level, from both immune cells and neurons. Upregulation of carbon monoxide production by the macrophages was demonstrated to inhibit their ability to release NO following immune stimulation. Additionally, the concentration and kinetics of NO release from neurons were determined. iv This work is dedicated to my parents, who have always encouraged me to work hard and follow my dreams. v ACKNOWLEDGEMENTS The work presented within this dissertation would not have been possible without the assistance of numerous colleagues, collaborators, family members, and friends. My research advisor, Mark Schoenfisch, has been both a critic and a supporter throughout my graduate school career. With his encouragement, I’ve not only made it though the long winding road of graduate school, but have also grown immensely as a scientist and been able to achieve more than I ever imagined possible. Additionally, Professor Domenic Tiani has been an amazing mentor and allowed me to develop my teaching abilities to the greatest extent possible. His support allowed me to achieve my post-graduate school career goals. Finally, my numerous mentors from Hood College have been with me in spirit since the beginning of my graduate school career, and have never hesitated to offer me advice or encouragement. Current and former members of the Schoenfisch lab deserve special thanks, as they have not only been colleagues, but have also been some of the best of friends. In particular, Danielle Slomberg, Ahyeon Koh, Scott Nichols, and Roobert Soto helped make the “fun- geon” as thrilling as it could possibly be, especially on Friday afternoons. Those who have helped with overnight pig studies over the years (Benjamin Privett, Scott Nichols, and Micah Brown) hold a special place in my heart; you form a unique bond with a person when you can share in experience of handling pigs. Their willingness to help out allowed me to maintain my sanity (somewhat) during these experiments. I have also had the opportunity to vi work with two talented undergraduate students, Simon Menaker and Karli Gast, who have continuously inspired me to be come a better teacher and mentor. Due in part to the interdisciplinary nature of my research, I’ve worked with numerous collaborators during my graduate school career. The study of nitric oxide release from snail neurons would not have been possible without our collaborators from the lab of Professor Vincent Rehder at Georgia State University, especially Liana Artinian who always provided us with a place to stay during our visits and even drove a tank full of snails up to our lab for further experiments. The microfluidic devices used to measure nitric oxide in blood were designed with the help of W. Hampton Henley in the lab of Professor J. Michael Ramsey at UNC. All of the porcine sepsis studies in swine were made possible by the lab of Dr. James Manning in the Department of Emergency Medicine at UNC, especially Shane McCurdy and Ben Ellington who endured the long and sometimes frustrating days and nights with me and two pigs. Work in the murine model of sepsis began with the lab of Dr. Craig Coopersmith at Emory University, thanks to the help of Dr. Jonathan McDunn. Studies in this model were able to continue at UNC in collaboration with the lab of Drs. Bruce Cairns and Robert Maile of the North Carolina Jaycee Burn Center and Department of Microbiology and Immunology. I owe special thanks to a fellow graduate student, Julie Malik, for her direct involvement with all of these experiments in the burn model of sepsis. Michelle Palacio, of Professor Virginia Miller’s lab at UNC, also assisted with some of these experiments. I am also particularly thankful to the medical doctors we collaborated with, Bruce Cairns and James Manning, for their valuable insight on clinical utility of nitric oxide measurements. Finally, I have to thank my family for all the support they have provided to me through the highs and lows of graduate school: my soon-to-be husband, Josh, for constantly vii supporting me—even from afar—and always being my closest confidant, and my parents, who have always been encouraging and forced me to keep going even when I was ready to quit. Most of all, thanks to all of you for putting up with my incredible grumpiness during those evening phone calls when you asked me how my day was… 1 1 PEARLS BEFORE SWINE © Stephan Pastis. Reprinted by permission of Universal Uclick for viii TABLE OF CONTENTS LIST OF TABLES ................................................................................................................. xiv LIST OF FIGURES ................................................................................................................ xv LIST OF ABBREVIATIONS AND SYMBOLS ................................................................ xviii CHAPTER 1. PHYSIOLOGICAL SIGNIFICANCE OF NITRIC OXIDE AND ITS MEASUREMENT WITHIN BIOLOGICAL SYSTEMS ....................................... 1 1.1 The ubiquitous roles of nitric oxide ............................................................................ 1 1.1.1 Nitric oxide in the immune system .................................................................... 2 1.1.2 Nitric oxide in the nervous and cardiovascular systems .................................... 3 1.2 Nitrosothiols as physiological transporters of nitric oxide ......................................... 4 1.3 Detection of nitric oxide and nitrosothiols .................................................................. 9 1.3.1 Spectroscopic detection of nitric oxide .............................................................. 9 1.3.2 Real-time amperometric detection of nitric oxide ........................................... 10 1.3.3 Detection of nitrosothiols ................................................................................. 16 1.4 Nitric oxide and nitrosothiols in sepsis ..................................................................... 21 1.4.1 Sepsis disease progression ............................................................................... 23 1.4.2 Clinical manifestations of sepsis ...................................................................... 26 1.4.3 Nitric oxide and nitrosothiols as prognostic/diagnostic biomarkers ................ 27 1.4.4 Animal models of sepsis .................................................................................. 28 1.5 Summary of dissertation research ............................................................................. 33 1.6 References ................................................................................................................. 35 ix CHAPTER 2: CHALLENGES OF NITRIC OXIDE MEASUREMENT FROM BIOLOGICAL SYSTEMS ........................................................................................
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