DIETARY TRIMETHYLAMINES, THE GUT MICROBIOTA, AND ATHEROSCLEROSIS By ROBERT ALDEN KOETH Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Adviser: Stanley L. Hazen, M.D., Ph.D. Department of Pathology CASE WESTERN RESERVE UNIVERSITY August, 2013 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Robert Alden Koeth candidate for the Ph.D. degree *. (signed) Alan D. Levine, Ph.D. (chair of the committee) Stanley L. Hazen, M.D., Ph.D. Jonathan D. Smith, Ph.D. George R. Dubyak, Ph.D. Clive R. Hamlin, Ph.D. (date) 04/16/2013 *We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS LIST OF TABLES ................................................................................................ 9 LIST OF FIGURES............................................................................................. 10 ACKNOWLEDGEMENTS .................................................................................. 16 ABSTRACT........................................................................................................ 18 CHAPTER1: Introduction to Dietary Trimethylamines, the Gut Microbiota, and Atherosclerosis ......................................................................................... 20 Cardiovascular Disease and Atherosclerosis............................................. 20 A History of the Gut Microbiota ................................................................... 22 Location and Composition of the Gut Microbiota ...................................... 23 Normal Functions of the Gut Microbiota..................................................... 25 The Relationship Between the Gut Microbiota and Disease ..................... 31 Gut Microbiota Mediated Metabolism of Phosphatidylcholine Promotes Cardiovascular Disease................................................................................ 35 CHAPTER 2: Intestinal Microbiota Metabolism of L-Carnitine, a Nutrient in Red Meat, Promotes Atherosclerosis ............................................................. 47 Authors .......................................................................................................... 47 Abstract.......................................................................................................... 47 Introduction ................................................................................................... 48 Results ........................................................................................................... 51 Metabolomic studies link L-carnitine with CVD ...................................... 51 1 Gut microbiota plays an obligatory role in forming TMAO from L- carnitine in humans ................................................................................... 53 Vegans and vegetarians produce substantially less TMAO from dietary L-carnitine .................................................................................................. 55 Plasma TMAO levels significantly associate with specific human gut microbial taxa............................................................................................. 57 TMAO production from dietary L-carnitine is an inducible trait ............ 58 TMA / TMAO production associates with specific mouse gut microbial taxa.............................................................................................................. 59 Plasma levels of L-carnitine associate with CVD.................................... 60 Dietary L-carnitine in mice promotes atherosclerosis in a gut microbiota dependent manner.................................................................. 62 Gut microbiota dependent formation of TMAO inhibits reverse cholesterol transport ................................................................................. 63 TMAO promotes significant alterations in cholesterol and sterol metabolism in multiple compartments in vivo ........................................ 66 Discussion ..................................................................................................... 68 Acknowledgements....................................................................................... 78 Methods ......................................................................................................... 78 Materials and general procedures............................................................ 78 Research subjects ..................................................................................... 79 2 General statistics....................................................................................... 81 Metabolomics study .................................................................................. 82 Identification of L-carnitine and d9-carnitine preparation...................... 83 Quantification of TMAO, TMA, and L-carnitine........................................ 85 Human microbiota analyses ..................................................................... 86 Mouse microbiota analysis ....................................................................... 87 Aortic root lesion quantification............................................................... 89 Human L-carnitine challenge test and d3-L-carnitine preparation ........ 89 Germ-free mice and conventionalization studies ................................... 92 Metabolic challenges in mice ................................................................... 93 Preparation of bone marrow derived macrophages for reverse cholesterol transport studies.................................................................... 93 Reverse cholesterol transport studies..................................................... 94 Cholesterol absorption studies ................................................................ 95 Bile acid pool size and composition ........................................................ 96 Cholesterol efflux studies ......................................................................... 97 Effect of TMAO on macrophage cholesterol biosynthesis, inflammatory genes, and desmosterol levels................................................................. 97 RNA preparation and real time PCR analysis.......................................... 99 3 CHAPTER 3: Carnitine, a Nutrient Found in Red Meat and a Frequent Additive by the Nutritional Supplement Industry, Can Induce the Human Gut Microbiota to Produce Proatherogenic TMAO...................................... 135 Authors ........................................................................................................ 135 Intro .............................................................................................................. 135 Methods ....................................................................................................... 135 Results ......................................................................................................... 136 Comment...................................................................................................... 136 CHAPTER 4: Intestinal Microbial Metabolism of Phosphatidylcholine and Cardiac Risk.................................................................................................... 139 Authors ........................................................................................................ 139 Abstract........................................................................................................ 139 Introduction ................................................................................................. 140 Results ......................................................................................................... 141 Role of intestinal microbiota in metabolism of dietary phosphatidylcholine ................................................................................ 141 Correlation of plasma levels of trimethylamine-N-oxide with major adverse cardiovascular events............................................................... 143 Correlation of trimethylamine-N-oxide levels with risk in low-risk subgroups ................................................................................................ 145 Discussion ................................................................................................... 145 4 Acknowledgements..................................................................................... 149 Methods ....................................................................................................... 149 Study patients and design ...................................................................... 149 Dietary phosphatidylcholine challenge ................................................. 151 Measurements of choline metabolites ................................................... 152 Statistical analysis for the clinical outcomes study ........................... 152 CHAPTER 5: Intestinal Microbiota Metabolism of L-Carnitine, a Nutrient in Red Meat, Produces TMAO Via Generation of an Intermediate Gut Microbiota Metabolite γ-Butyrobetaine ........................................................ 163 Authors ........................................................................................................ 163 Abstract........................................................................................................ 163 Introduction 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