Identification of Biomarker Responses in Humans Under Experimentally Induced Zinc Depletion
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IDENTIFICATION OF BIOMARKER RESPONSES IN HUMANS UNDER EXPERIMENTALLY INDUCED ZINC DEPLETION By MOON-SUHN RYU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011 1 © 2011 Moon-Suhn Ryu 2 To my parents, Ji-Chul Ryu and Soon-Young Park, and my grandmother, Jung-Seo Seo 3 ACKNOWLEDGMENTS The achievements from the past years in my life as a doctoral student would not exist without the presence of the support and love from my family far way in South Korea. I thank my parents for their love and belief in me which have been the greatest motivation for me to pursue accomplishments and success with my best efforts. I also render my gratefulness to my younger sister, Jin-Suhn Ryu, for being the best supporter in all aspects of my life. Additionally, I thank everyone with whom I shared the moments in the lab, especially, Dr. Juan P. Liuzzi, Dr. Louis A. Lichten, Dr. Liang Guo, Dr. Shou- mei Chang, Tolunay Beker Aydemir, for their invaluable advices and help. The excitement from our findings was always the greatest motivation for me to stay in progress. I also thank Gregory Guthrie, Alyssa Maki, Luisa Rios and Vanessa Da Silva for their cheer and support, particularly, during the last year of my doctoral research. I thank Meena Shankar and the staff members at the General Clinical Research Center for their advices and assistance during the phases of study design and implementation of the dietary regimen. Finally, I fully render my gratefulness to my supervisory committee members, Dr. Robert J. Cousins, Dr. Bobbi Langkamp-Henken, Dr. James F. Collins and Dr. Nancy D. Denslow, for being the best mentors and role-models for my career in the field of nutrition and molecular biology. Their advice has been the key for the development of my insights in research, and will last long in my minds. Especially, I thank Dr. Robert J. Cousins for allowing me to substantiate my thoughts and ideas by research, and for encouraging me to continue my journey in the field of discovery by science. I will truly miss the times under his guidance at the University of Florida, and will continue my best with pride of being a previous member of the Cousins lab. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 15 CHAPTER 1 INTRODUCTION .................................................................................................... 17 Zinc Biology ............................................................................................................ 17 Regulation of Zinc Homeostasis ............................................................................. 18 Whole Body Zinc .............................................................................................. 18 Cellular Zinc ..................................................................................................... 20 Dietary Zinc Deficiency ........................................................................................... 21 Biomarker Studies of Dietary Zinc Deficiency ......................................................... 23 Blood MT and Zinc Transporters ...................................................................... 24 Cellular Zinc Concentrations ............................................................................ 25 Other Potential Zinc Indices ............................................................................. 26 Advances in Techniques for Transcriptome Analysis ............................................. 29 Microarray Analysis .......................................................................................... 29 RNA Sequencing (RNA-Seq) ........................................................................... 30 Sampling and Processing of Biospecimens for Transcriptome Profiling ................. 31 Study Aims .............................................................................................................. 32 2 MATERIALS AND METHODS ................................................................................ 34 Human Subjects ..................................................................................................... 34 Acute Dietary Zinc Depletion .................................................................................. 34 Sample Collection ................................................................................................... 35 Preparation of Buccal RNA ..................................................................................... 36 Processing of Blood Samples ................................................................................. 37 Measures of Serum Zinc Concentrations ................................................................ 38 Isolation and Processing of Blood Cell RNA ........................................................... 39 RNA Amplification, Hybridization, and Microarray Analysis .................................... 39 Microarray Data Analysis ........................................................................................ 40 Real-Time Quantitative PCR ................................................................................... 41 Western Analysis of RBC Zinc Transporters ........................................................... 42 Immunoprecipitation and Mass Spectrometry ......................................................... 43 5 Whole Blood Cytokine Assay .................................................................................. 44 Enzyme-Linked Immunosorbent Assay................................................................... 44 Isolation and Quantitation of Serum MicroRNA ...................................................... 45 Statistical Analysis .................................................................................................. 46 3 IDENTIFICATION OF ZINC-RESPONSIVE GENE TRANSCRIPTS IN BUCCAL AND BLOOD CELLS .............................................................................................. 53 Introductory Remarks.............................................................................................. 53 Results .................................................................................................................... 55 Experimental Zinc-Depletion in Human Subjects ............................................. 55 Effects of Zinc-Depletion on Zinc-Related Transcript Levels ............................ 56 Whole Blood RNA Processing and Quality Assessment for Microarray Analysis ......................................................................................................... 57 Global Effect of Zinc-Depletion on Whole Blood Transcriptome ....................... 58 Gene Transcripts with Potential of being Biomarkers of Zinc Deficiency .......... 61 Discussion .............................................................................................................. 62 4 EFFECTS OF ACUTE DIETARY ZINC DEPLETION ON WHOLE BLOOD CYTOKINE RELEASE INDUCED BY EX VIVO IMMUNOCHALLENGES ............ 100 Introductory Remarks............................................................................................ 100 Results .................................................................................................................. 102 Inflammatory Cytokine Production by Whole Blood ........................................ 102 Zinc Transporter Transcript Levels ................................................................. 103 Discussion ............................................................................................................ 103 5 SERUM MICRORNA AS BIOMARKERS OF DIETARY ZINC STATUS ............... 114 Introductory Remarks............................................................................................ 114 Results .................................................................................................................. 115 Discussion ............................................................................................................ 116 6 ERYTHROCYTE MEMBRANE ZINC TRANSPORTERS AND DEMATIN LEVELS IN HUMANS UNDER SHORT-TERM DIETARY ZINC RESTRICTION .. 124 Introductory Remarks............................................................................................ 124 Results .................................................................................................................. 125 Erythrocyte Zinc Transporter Expression during Low Zinc Intake .................. 125 Identification of Dematin as a Zinc-Responsive Erythrocyte Membrane Protein ......................................................................................................... 125 Discussion ............................................................................................................ 127 7 CONCLUSIONS AND FUTURE DIRECTIONS .................................................... 138 APPENDIX A SCREENING QUESTIONNAIRE