AVENANTHRAMIDE SUPPLEMENTATION IN YOUNG AND OLDER WOMEN: PROTECTION AGAINST ECCENTRIC EXERCISE-INDUCED INFLAMMATION AND OXIDATIVE STRESS by RYAN THOMAS KOENIG A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Kinesiology) UNIVERSITY OF WISCONSIN – MADISON 2012 Date of final oral examination: 05/17/12 The dissertation is approved by the following members of the Final Oral Committee: Li Li Ji Gary Diffee William Schrage Kirk Parkin Mitchell Wise i ACKNOWLEDGMENTS In the summer of 2005, I was working on the grounds crew of the Saint Vincent Cemetery in Latrobe, Pennsylvania. I had just graduated college and been rejected by a dozen or so medical schools. With little idea of what I was getting into, I started investigating graduate schools and came across the name Li Li Ji. We spoke on the phone long distance and found that our interests overlapped, and before long I was on a trip to Madison, Wisconsin—a place I assumed was locked in ice for at least ten months a year. I met Dr. Ji, toured the Ji Lab, landed an apartment, and discovered that Madison was not only ice-free in July but also a place I wanted to be. Just like that, I was on my way to starting the research that now culminates in the following pages. I need to thank Dr. Ji for giving me this chance. He has provided ceaseless advice and guidance to go with the resources that made this work possible. For the final months of this journey, we have both been estranged from Madison, but he has still been as accessible as when I used to knock on his office door. I am so grateful for his help, especially during this time that I have been away from Madison when it would have been easy to stray from the path. I also need to express my gratitude to Jon Dickman, who is going for the grand slam of degrees from the UW while at the same time remaining a close friend and incredible lab mate. If Jon was my right hand in the Ji Lab, Choung-Hun Kang was my left, and I thank them both for the endless hours of assistance they provided. Dr. Mitchell Wise introduced me to avenanthramide, and none of this would have been possible without his guidance. He also served on my thesis committee along with Drs. Diffee, Schrage, and Parkin, whose insight and advice were critical in the work presented here. ii Finally, without the love and support of my wife Natalie, I could never have made it this far. Without her as my foundation, I would have tumbled from these lofty heights I chose to climb. Without her arms to fall into at the end of the day, the long hours would never have paid off. And, of course, without her I wouldn’t have my two boys, Arthur and Felix, who make every single day a great one. It’s been a long trip from the Saint Vincent Cemetery, and I got a lot of help from some very special people along the way. iii CONTENTS ACKNOWLEDGMENTS ............................................................................................................... i CONTENTS ................................................................................................................................. iiiii TABLES AND FIGURES ............................................................................................................ iix ABBREVIATIONS ....................................................................................................................... xi INTRODUCTION .......................................................................................................................... 1 PURPOSES AND HYPOTHESES................................................................................................. 5 REVIEW OF THE LITERATURE ................................................................................................ 6 1. Reactive Oxygen Species ........................................................................................................ 6 A. Mitochondrial source ......................................................................................................... 6 B. Antioxidant defense............................................................................................................ 6 C. Neutrophil source ............................................................................................................... 7 2. Inflammation ........................................................................................................................... 7 A. Role in disease ................................................................................................................... 8 B. Cytokines ............................................................................................................................ 8 C. Eccentric Contraction ....................................................................................................... 11 3. Aging..................................................................................................................................... 15 A. Role of ROS and inflammation ........................................................................................ 15 B. Estrogens and Aging ........................................................................................................ 17 4. Avenanthramides .................................................................................................................. 18 iv A. Introduction ...................................................................................................................... 18 B. Antioxidant Capacity........................................................................................................ 19 C. Antioxidant Activity in Rats ............................................................................................ 22 D. Bioavailability in Rodents ................................................................................................ 23 F. Anti-Inflammatory Action ................................................................................................ 26 G. Nitric Oxide and SMC Proliferation ................................................................................ 27 H. NFκB Signaling ............................................................................................................... 29 I. Colon Cancer Prevention ................................................................................................... 30 STUDY 1: Effect of AVA Supplementation on Eccentric Exercise-induced Inflammation and Oxidative Stress in Young Women............................................................................................... 31 Abstract ..................................................................................................................................... 32 Introduction ............................................................................................................................... 34 Methods..................................................................................................................................... 35 A. Subjects ............................................................................................................................ 35 B. Study Visits ...................................................................................................................... 36 C. Dietary Supplementation .................................................................................................. 38 D. Downhill Running ............................................................................................................ 39 E. Blood sample collection and preparation ......................................................................... 39 F. Biological Measurements ................................................................................................. 40 1. ELISA…………………………………………………………………………………40 v a. Inflammatory markers…………………………………………………………40 b. NFκB………………………………………………………………………….41 2. HPLC………………………………………………………………………………….41 a. Plasma glutathione…………………………………………………………….41 b. Avenanthramide concentration………………………………………………..42 3. Spectrophotometric assays…………………………………………………………….43 a. Plasma TAC…………………………………………………………………...43 b. Plasma creatine kinase………………………………………………………...43 c. Erythrocyte superoxide dismutase…………………………………………….44 d. Erythrocyte glutathione peroxidase…………………………………………...44 e. Hemoglobin……………………………………………………………………45 4. Neutrophil respiratory burst…………………………………………………………...45 5. Pain and soreness ratings……………………………………………………………...46 G. Statistical Analysis ........................................................................................................... 46 Results ....................................................................................................................................... 49 A. Participant Data ................................................................................................................ 49 B. Muscle damage caused by (DR)....................................................................................... 49 C. Inflammatory Markers...................................................................................................... 49 D. NFκB ................................................................................................................................ 54 E. Plasma Total Antioxidant Capacity .................................................................................. 54 F. Erythrocyte Antioxidant Enzymes ...................................................................................