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 ...... 164

Results ...... 167

Gut microbiota metabolism of L-carnitine produces γBB...... 167

γBB produces TMA/TMAO in a gut microbiota dependent manner .... 167

TMA formation occurs in the cecum and γBB is the dominant gut

microbiota product of L-carnitine gut microbiota metabolism...... 169

Metabolism of γBB by the gut microbiota to TMA/TMAO promotes

atherosclerosis ...... 169

Metabolism of γBB from L-carnitine is an inducible trait...... 170

5

γBB associates with a microbiome composition that differs from

TMA/TMAO formation ...... 171

TMAO production from γBB associates with microbiome

composition...... 172

Mice on a γBB diet have significant decreased liver expression of

Cyp7a1, but not Cyp27a1 ...... 172

Discussion ...... 173

Methods ...... 178

Materials and general procedures...... 178

Mouse challenge and atherosclerosis studies...... 179

Mouse microbiome studies...... 180

d9-γ-Butyrobetaine chloride preparation...... 182

Quantification of TMAO, TMA, a γBB, and L-carnitine ...... 183

In vitro mouse cecum study ...... 184

RNA preparation and real time PCR analysis...... 184

General Statistics...... 185

CHAPTER 6: γ-Butyrobetaine is a Gut Microbiota Dependent Product of L-

Carnitine...... 201

Introduction ...... 201

Results ...... 203

γBB associates with CVD prevalence ...... 203

6

γBB is associated with MACE, but not after TMAO adjustment ...... 204

γBB is produced from carnitine in a gut microbiota dependent manner

in humans...... 205

TMAO is the major gut microbiota metabolite of L-carnitine in

humans ...... 207

γBB does not associate with a omnivorous diet...... 207

Red meat is an exogenous source of γBB, but is found at lower

concentrations compared to carnitine...... 208

Discussion ...... 209

Methods ...... 212

Research subjects ...... 212

Human L-carnitine challenge test...... 214

Quantification of L-carnitine, γBB, and TMAO in plasma samples ..... 215

γ-Butyrobetaine quantification in meat samples ...... 215

General statistics...... 215

Chapter 7: Transcrotonobetaine, a Gut Microbiota Metabolite of Carnitine

Metabolism, Promotes Atherosclerosis...... 225

Introduction ...... 225

Results ...... 225

TC is a gut microbiota dependent product of L-carnitine ...... 225

TC is an abundant gut microbiota metabolite of L-carnitine in mice .. 226

7

TC produces both γ-butyrobetaine and TMA/TMAO in a gut microbiota

dependent manner...... 227

TC independently associates with cardiovascular disease, but not after

multivariate model adjustment with TMAO ...... 228

Dietary TC promotion of atherosclerosis is gut microbiota-dependent

manner...... 229

Discussion ...... 230

Methods ...... 233

Materials and general procedures...... 233

Research subjects ...... 234

Mouse challenge and atherosclerosis studies...... 234

d9-TC and native TC preparation...... 235

Quantification of TC, TMAO, TMA, γBB, and L-carnitine...... 236

In vitro mouse cecum study ...... 236

General Statistics...... 237

Chapter 8: Summary, Conclusions, and Future Directions...... 248

Clinical implications ...... 248

A hypothetical role for the gut microbiota and TMAO in other disease

states ...... 250

Summary ...... 250

REFERENCES...... 253

8

LIST OF TABLES Supp. Table 2-1 Characteristics of analyte m/z = 162 determined in LC/MS positive ion mode from plasma samples used in Validation and Learning cohorts (n = 150) of metabolomics study from Wang et. al., Nature, 2011 107

Supp. Table 2-2 Subject characteristics, demographics, and laboratory values in the whole cohort (n = 2595), and across quartiles of plasma carnitine 108

Supp. Table 2-3 Plasma levels of triglycerides, cholesterol, glucose, and insulin from mice on normal chow vs. carnitine supplemented diet 109

Supp. Table 2-4 Liver levels of triglycerides and total cholesterol in mice on normal chow versus carnitine supplemented diet 110

Supp. Table 2-5 Plasma levels of triglycerides, cholesterol, and glucose from mice on normal chow, carnitine, choline, and TMAO supplemented diets during the in vivo RCT studies 111

Table 4-1 Baseline characteristics 154

Table 4-2 Unadjusted and adjusted hazard ratio for risks of MACE at 3-years stratified by quartile levels of TMAO 155

Supp. Table 4-1 Baseline characteristics of cohort according to TMAO quartiles values expressed in mean ± standard deviation or median (interquartile range) 159

Table 5-1 Plasma and liver lipid levels in C57BL/6J, Apoe-/- female mice used in γBB atherosclerosis study 186

Table 6-1 Baseline clinical characteristics of n = 1445 Genebank subjects used in analyses with γBB 217

Table 6-2 Quantification of carnitine and γBB in beef, lamb, chicken, and perch samples 218

Table 7-1 Baseline clinical characteristics of n = 836 Genebank subjects used in analyses with TC 238

Table 7-2 Plasma levels of triglycerides, cholesterol, and glucose from mice on normal chow vs. transcrotonobetaine supplemented diet 239

9

LIST OF FIGURES Figure 1-1 Scheme of gut microbiota dependent metabolism of dietary PC and atherosclerosis 40

Figure 1-2 Metabolomics studies scheme and correlations 41

Figure 1-3 Production of TMAO from PC is gut flora dependent 42

Figure 1-4 Choline, TMAO and betaine are associated with CVD in humans 43

Figure 1-5 Dietary choline or TMAO enhances atherosclerosis 44

Figure 1-6 Hepatic FMOs associate with atherosclerosis 45

Figure 1-7 Dietary choline enhances atherosclerosis in a gut flora dependent manner 46

Figure 2-1 TMAO production from carnitine is a microbiota dependent process in humans 101

Figure 2-2 The formation of TMAO from ingested L-carnitine is negligible in vegans, and fecal microbiota composition associates with plasma TMAO concentrations 102

Figure 2-3 The metabolism of carnitine to TMAO is an inducible trait and associates with microbiota composition 103

Figure 2-4 Relation between plasma carnitine and CVD risks 104

Figure 2-5 Dietary carnitine accelerates atherosclerosis and inhibits reverse cholesterol transport in a microbiota dependent fashion 105

Figure 2-6 Effect of TMAO on cholesterol and sterol metabolism 106

Supp. Figure 2-1 Mass spectrometry analyses identify unknown plasma analyte at retention time of 5.1 min and m/z = 162 as carnitine 112

Supp. Figure 2-2 LC/MS/MS analysis of synthetic heavy isotope standard d9(trimethyl)carnitine spiked into human plasma sample confirms unknown peak at 5.10 min (m/z = 162) is carnitine 113

10

Supp. Figure 2-3 Standard curves for LC/MS/MS quantification of carnitine and d3-(methyl)-carnitine in plasma matrix 114

Supp. Figure 2-4 LC/MS/MS analyses of a subject’s 24 hr urine samples demonstrate an obligatory role for gut microbiota in production of TMAO from carnitine 115

Supp. Figure 2-5 Plasma levels of carnitine and TMAO following 116 carnitine challenge in a typical omnivorous subject

Supp. Figure 2-6 Plasma levels of carnitine and d3-carnitine following carnitine challenge (steak and d3-carnitine) in typical omnivore with frequent red meat dietary history and a vegan subject 117

Supp. Figure 2-7 Plasma levels of d3-carnitine following d3-carnitine challenge (no steak) in omnivorous (n = 5) versus vegan subjects (n = 5) 118

Supp. Figure 2-8 Human fecal microbiota taxa associate with plasma TMAO 119

Supp. Figure 2-9 Demonstration of an obligatory role of the commensal gut microbiota of mice in the production of TMA and TMAO from oral carnitine in germ-free and conventionalized mice 120

Supp. Figure 2-10 Demonstration of an obligatory role of commensal gut microbiota of mice in the production of TMA and TMAO from oral carnitine 121

Supp. Figure 2-11 Analysis of mouse plasma TMA and TMAO concentrations and gut microbiome composition can distinguish dietary status 122

Supp. Figure 2-12 Haematoxylin/eosin (H/E) and oil-red-O stained liver sections 123

Supp. Figure 2-13 Arginine transport in the presence of 100 µM trimethylamine-containing compounds 124

Supp. Figure 2-14 Expression levels of cholesterol synthesis enzymes, transporters, and inflammatory genes in the presence or absence of TMAO 125

Supp. Figure 2-15 Effect of TMAO on desmosterol levels in media of

11

cultured mouse peritoneal macrophages in the presence of increasing cholesterol and acetylated LDL (AcLDL) concentrations 126

Supp. Figure 2-16 Plasma concentrations of TMAO in mice undergoing in vivo reverse cholesterol transport studies 127

Supp. Figure 2-17 [14C] Cholesterol recovered from mice on normal chow vs. TMAO diet enrolled in in vivo reverse cholesterol transport studies 128

Supp. Figure 2-18 Effect of TMAO on mouse peritoneal macrophages 129

Supp. Figure 2-19 Effect of TMAO on cultured macrophage cholesterol efflux 130

Supp. Figure 2-20 Liver expression of cholesterol transporters in mice examined during reverse cholesterol transport studies 131

Supp. Figure 2-21 Western blot analysis of liver scavenger receptor B1 (Srb1) expression 132

Supp. Figure 2-22 Small intestines expression profile of bile acid transporters in mice 133

Supp. Figure 2-23 Small intestines expression profile of cholesterol transporters in mice 134

Figure 3-1 Carnitine supplementation can induce the gut microbiota 138

Figure 4-1 Human plasma levels of phosphatidylcholine Metabolites (TMAO, choline, betaine) after oral ingestion of two hard-boiled eggs and d9- Phosphatidylcholine before and after antibiotics 156

Figure 4-2 Kaplan-Meier estimates of long-term major adverse cardiac events, according to TMAO Quartiles 157

Figure 4-3 Pathways linking dietary phosphatidylcholine, intestinal microflora (gut flora), and incident adverse cardiovascular events 158

Supp. Figure 4-1 Human plasma levels of phosphatidylcholine metabolites (TMAO, choline, betaine) after oral ingestion of two hard-boiled eEggs and d9-

12

phosphatidylcholine before and after antibiotics 160

Supp. Figure 4-2 Human 24-hour urine levels of TMAO after oral ingestion of two hard-boiled eggs and d9- phosphatidylcholine before and after antibiotics 161

Supp. Figure 4-3 Risks of major adverse cardiac events (MACE) among patient subgroups, according to baseline TMAO levels 162

Figure 5-1 γBB is produced as a major gut microbiota metabolite of L-carnitine 187

Figure 5-2 γBB is produced from L-carnitine in a gut microbiota dependent manner 188

Figure 5-3 TMA/TMAO is a gut a microbiota dependent product of γBB metabolism 189

Figure 5-4 Confirmatory studies that TMA/TMAO is a gut a microbiota dependent product of γBB metabolism 190

Figure 5-5 γBB is the dominant gut microbiota metabolite of L- carnitine and is metabolized to TMA at a great equamolar capacity than L-carnitine 191

Figure 5-6 γBB promotes atherosclerosis in a gut microbiota dependent manner 192

Figure 5-7 Plasma trimethylamine concentrations of C57BL/6J, Apoe-/- female mice used in γBB atherosclerosis study 193

Figure 5-8 γBB production from L-carnitine is an inducible trait 194

Figure 5-9 γBB production from L-carnitine associates with microbiome composition 195

Figure 5-10 γBB production from L-carnitine and microbiome composition associate with dietary status 196

Figure 5-11 TMA/TMAO production from γBB associates with microbiome composition 197

Figure 5-12 TMAO production from γBB and microbiome composition associate with dietary status 198

Figure 5-13 Liver Expression of Bile acid enzymes 199

13

Figure 5-14 Scheme of endogenous and exogenous γBB production 200

Figure 6-1 Relationship between plasma γBB and CVD prevalence 219

Figure 6-2 Relationship between plasma γBB and CVD risks 220

Figure 6-3 Relationship between plasma γBB, plasma TMAO, and CVD risks 221

Figure 6-4 γBB production from carnitine is a gut microbiota dependent process in humans 222

Figure 6-5 TMAO is the major gut microbiota metabolite in human carnitine catabolism 223

Figure 6-6 The formation of γBB from ingested L-carnitine is similar in vegans and vegetarians compared to omnivores 224

Figure 7-1 Demonstration of an obligatory role of the commensal gut microbiota of mice in the production of TC from oral carnitine in germ-free and conventionalized mice 240

Figure 7-2 TC is the an abundant gut microbiota metabolite of L- carnitine 241

Figure 7-3 Proposed scheme of carnitine metabolism 242

Figure 7-4 Demonstration of an obligatory role of commensal gut microbiota of mice in the production of TMA,TMAO, and γ-butyrobetaine from oral TC challenge 243

Figure 7-5 Plasma TC is associated with MACE over a 3-year period 244

Figure 7-6 Plasma TC is not associated with MACE over a 3-year period after adjustment with other CVD risk factors in n = 836 subjects. 245

Figure 7-7 Dietary TC gut microbiota metabolism accelerates atherosclerosis 246

Figure 7-8 Plasma analytes from TC atherosclerosis study 247

14

Figure 8-1 Relationship of dietary trimethylamines, 252 atherosclerosis, and homocysteine formation

15

ACKNOWLEDGEMENTS

There are many people over the last several years that have been critical for my personal and professional development. I would like to thank first and foremost my wife, Kim, for her consummate support, advice, and patience through the trials and tribulations of the M.D./Ph.D. process. Thanks goes to my newborn son

Alden Scott Koeth for helping to bring perspective to this process. I would like to also thank my friends and family for their support.

I wish to acknowledge the mentors both informal and formal that have helped encourage and shape my scientific endeavors. Assistance provided by Hazen laboratory members, the Cleveland Clinic Cardiovascular Prevention Research

Laboratories, and members of the Lerner Research Institute was also greatly appreciated. Specifically, I would like to acknowledge Bruce S. Levison, Zeneng

Wang, and Jennifer Buffa for providing crucial training and scientific support.

Thanks also go all collaborators for helping to add valuable scientific insight and crucial experimental data to my studies. Thank you to the Department of

Pathology of Case Western Reserve University for support and the opportunity to pursue a Ph.D. I would like to offer my special thanks to my committee members,

Drs. George R. Dubyak, Jonathan D. Smith, and Clive R. Hamlin, and my committee chair, Dr. Alan D. Levine.

16

Thanks also go to the Cleveland Clinic Lerner College of Medicine and the

Medical Scientist Training Program of Case Western Reserve University for giving me this opportunity. Last, but certainly not least, I would like to acknowledge my graduate student mentor Dr. Stanley L. Hazen who challenged me both professionally and personally to develop skills essential to becoming a successful physician scientist.

17

Dietary Trimethylamines, the Gut Microbiota,

and Atherosclerosis

Abstract

by

ROBERT ALDEN KOETH

The gut microbiota has critical roles in mammalian physiological processes and has been increasingly recognized to be a culprit in disease pathogenesis. We recently identified a pathway that links the consumption of dietary phosphatidylcholine, the major dietary source of choline, the gut microbiota, and atherosclerosis. Choline, a trimethylamine compound, is metabolized by the gut microbiota to produce an intermediate compound known as trimethylamine

(TMA). TMA is oxidized by hepatic flavin monooxygenase 3 (FMO3) to form the proatherogenic metabolite trimethyl amine N-oxide (TMAO). The recognition that the gut mediated metabolism of choline to TMAO promoted atherosclerosis raised the possibility that carnitine, another dietary trimethylamine found in red meat, could contribute to TMAO formation. Studies in mice and humans confirm that the formation of TMAO from carnitine is gut microbiota dependent.

Interestingly, omnivorous subjects have a greater capacity to metabolize TMAO from carnitine than vegans/vegetarians and demonstrate significant differences in gut microbiota composition. Plasma TMAO levels are independently associated with prospective major adverse cardiovascular events (death, MI, stroke) and can

18

promote atherosclerosis by causing dysfunction in forward and reverse

cholesterol transport. Subsequent studies of carnitine metabolism by the gut

microbiota demonstrate the production of two other gut microbiota metabolites, γ-

butyrobetaine (γBB) and transcrotonobetaine (TC). γBB is the dominant gut

microbiota metabolite of carnitine in mice and is an intermediate in the gut

microbiota dependent metabolism of carnitine to TMAO. Remarkably, two

separate bacterial taxa in the gut microbiota associate with the two step

metabolism of carnitine to TMAO suggesting distinct populations in the gut

microbiota working in cooperation. Although humans also have the capacity to

generate TMAO from carnitine in a gut microbiota dependent manner, γBB is produced at a lesser amount than TMAO. Additionally, plasma γBB had no

association with dietary status (omnivore vs. vegan/vegetarian). This data suggests that in humans the direct metabolism of carnitine to TMA/TMAO is the

major gut microbiota mediated pathway of carnitine metabolism. Interestingly, a minor gut microbiota metabolite of carnitine, transcrotonobetaine, also promotes atherosclerosis in a gut microbiota dependent manner. Together these data provide a previously unrecognized link between the consumption of dietary trimethylamines, the gut microbiota, and atherosclerosis.

19

CHAPTER 1: Introduction to Dietary Trimethylamines, the Gut Microbiota,

and Atherosclerosis

Cardiovascular Disease and Atherosclerosis

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in

the developed world1 and atherosclerotic sequelae accounts for greater than

50% of all CVD deaths2. Atherosclerosis is a chronic systemic inflammatory

disease characterized by the evolution and accumulation of large lipid laden plaques in the artery wall. Myocardial infarction (MI) remains one of the most

deadly sequelae of atherosclerotic disease and is largely precipitated by rupture of the thin fibrous cap or endothelial cell erosion of the atherosclerotic lesion2.

The resulting thrombus formed within the coronary artery completely or partially

occludes the vessel causing ischemia and eventual death of the myocardium.

Cholesterol is the major culprit lipid associated with the progression of atherosclerosis. The foundation of this association is based on a combination of

a vast number of clinical outcome studies and cellular based studies

demonstrating the link between hypercholesterolemia and atheroma formation2-5.

Still today, measurement of cholesterol is the major mode of risk stratifying

subjects for CVD, and intervention in lipid metabolism with pharmacological

agents, like statins for example, remains a major preventive treatment. Overall,

atherosclerotic disease can be generally viewed as a balance between forward

and reverse cholesterol transport (RCT). Forward cholesterol transport is

20

characterized by the accumulation of lipid in cells, most notably, in the

macrophage. The precipitating event of atherosclerotic plaque formation involves

physiological stress to the vessel endothelium resulting in the adherence and

transmigration of macrophages into the vessel intima2. Within the intima

macrophages engulf large amounts of lipids (principally cholesterol) creating a

characteristic “foam cell.” Foam cells comprise the majority of fatty streaks, the earliest atherosclerotic lesions, which develop early in life. Indeed, a recent evaluation of coronary artery disease (CAD) in young adults and teenagers demonstrated a high prevalence of early atherosclerotic disease6. Foam cells are

not only involved in the initiation, but also the progression, and sequelae of

atherosclerotic disease suggesting a central role of the macrophage foam cell in atherosclerosis1,2,7.

RCT is defined as the net movement of cholesterol from peripheral sources to

the feces for elimination and is believed to be one of the major mechanisms by

which high density lipoprotein (HDL) mediates its antiatherogenic effect. The vast

majority of peripheral cells do not have the capacity to catabolize cholesterol

making transport the only major way to eliminate cholesterol from the cell8. RCT consists of movement of cholesterol through multiple compartments in the body.

Transport begins when cholesterol is made available to move from cellular sources (e.g. macrophage foam cells) to apolipoprotein A1 (apoA-1) containing molecules (native apoA-1 and HDL). Mature HDL interacts with scavenger receptor b1 (SR-B1) and is taken up by the liver for further metabolism. In the

21

canonical pathway, cholesterol is secreted into bile or metabolized into bile acids

for excretion into the gastrointestinal (GI) tract. Bile acids and cholesterol are

reabsorbed in an enteroheptic pathway for return to the liver or ultimately

eliminated in the feces. RCT was coined several decades ago by Glomset, but

more recently, there has been an increased recognition of the importance of the

RCT pathway in cholesterol metabolism9. This is large part due to the

development of an in vivo RCT assay by Rader and colleagues, and the

recognition that HDL function may be a better measure of atheroprotection than

absolute HDL cholesterol concentrations8,10. Indeed, studies of macrophage

RCT rates are associated atherosclerosis burden in mice11. Mice deficient in apoA-1, the major lipoprotein of HDL, impairs RCT and apoA-1 overexpression increases total RCT12,13. These data are consistent with the atheroprotective role

of apoA-1 and HDL8. Macrophage cholesterol transporters ATP-binding cassette

sub-family G member 1(ABCG1) and ATP binding cassette transporter A1

(ABCA1) are critical for removing cholesterol from macrophage foam cells and

the absence of these cholesterol transporters in macrophage foam cells impairs

RCT14. Finally, inflammation, a major contributor to the pathogenesis of

atherosclerosis, has been shown to impair the overall RCT pathway15. Together

these data suggest important roles for net forward and reverse cholesterol

transport in the atherosclerotic disease process.

A History of the Gut Microbiota

The human microbiota consists of trillions of bacteria that form a complex

symbiotic relationship with the host. Anywhere from 500-1,000 different bacterial

22

species live in a human microbiome, and the total microbial cells are

quantitatively approximately 10 fold the total number of eukaryotic cells in the

host16,17. The importance of the gut was first recognized by Hippocrates who

noted that “All diseases begin in the gut” and that “death sits in the bowels”18. In

modern medicine, the focus has traditionally been on the pathologic invasion of

the gut by various bacteria and viruses. Indeed, at the turn of the 20th century diarrhea and gastroenteritis were the 3rd leading causes of death accounting for

almost 10% of all deaths in the United States19. The advent of antimicrobial

agents, development of vaccinations, improved nutrition, advancement of

epidemiology, and recognition of the importance of sanitation and hygiene theory

have effectively seen this and other infectious diseases be eclipsed by chronic

disease as the major challenge in modern medicine19. The emergence of chronic

disease and, more recently, the increased recognition for the capacity of the commensal human microbiota to influence human physiology has led to renewed

interest of the gut in human health and disease.

Location and Composition of the Gut Microbiota

The gut microbiota is heterogeneous in its composition and quantity throughout

the GI tract. Overall, the amount and biodiversity of bacteria per gram of content

in the gut increases from proximal to distal ends of the GI tract20. The relative lack of stable colonization of a microbiome in the proximal GI tract has been attributed to the pulsatile contractions of the small bowel and the harsh environmental conditions in the GI lumen (bile acids, HCl, and pancreatic

23

enzymes)16. In contrast, the large bowel contains a diversity of gut microbes culminating with 1011 to 1012 per gram of intestinal luminal contents20,21. The vast

majority of bacteria that inhabit the gut are anaerobes or facultative anaerobes

with anaerobes dominating overall22. Currently, anywhere from 300-1,000

different bacterial species are believed to colonize the human GI tract, but with

advances in sequence technology this number could mushroom. Indeed some

recent analyses have suggested that as many as 35,000 different species of

bacteria may in fact colonize the human GI tract23.

Humans have a sterile gut in utero and begin to acquire a microbiome at time of

birth. Passage through the vaginal canal exposes infants to both maternal vaginal and fecal flora initiating the development of the gut microbiome24,25. One

study suggested that the initial makeup of infant GI microbiota and maternal

vaginal flora are closely aligned immediately after vaginal delivery26. However,

this appears to be a transient establishment as infants quantitatively have 109

CFU/g feces of enteric bacteria established by the end of the first day of life that expands to 1011 CFU/g feces of enteric bacteria by the first month of age27. The initiation and establishment of the gut microbiome is the culmination of a complex interplay between a number of extrinsic and intrinsic factors including: mode of birth (delivery vs. cesarean section), maternal flora, host genetics, diet, exposure to antimicrobials, bile acids, peristalsis, drugs, host immunity, intestinal luminal pH, intermicrobial interactions, and the bacterial load in the environment24. As a result, the infant microbiome remains immature and greatly variable becoming

24

more similar in quantity and composition to an adult microbiome by the first year of age20.

Normal Functions of the Gut Microbiota

The gut microbiome has a mutualistic relationship with the mammalian host that has developed through millions of years of coevolution. The establishment of the microbiome from birth becomes critical for the normal maturation and development of GI structure and function. Insight into the importance of the gut microbiome and normal GI development has been demonstrated by mouse germ free (mice lacking any microbiome; GF) animals studies. Structurally, these animals have impaired peristalsis, a reduction in the villous capillary network, and decreased overall surface area20. GF animals characteristically develop physically enlarged cecums that can often predispose the animal to both reproductive and GI dysfunction20.

The GI tract is the largest exposed mucosal surface and contains the largest number of immunocompetent cells in the human body. The GI tract therefore serves as a critical component of the development and maintenance of a normal immune system16. Insights into the importance of the gut microbiota in the development of the immune system have been garnered by studies in gnotobiotic

(GF) rodents. GF mice have less lymphoid tissue, lower numbers of immunocompetent cells, decreased expression of immune receptors such as Toll like receptors (TLRs), and overall decreased circulating immunoglobulin

25

concentrations compared to conventional mice20,28. The subsequent rapid expansion and development of the GI immune system in GF mice upon exposure to luminal microbes suggests important roles of the gut microbiota in the development of both GI and systemic immunity20,28.

These defects most notably result in increased susceptibility to pathological infection20,28. Germ free guinea pigs challenged with the gram-negative enteric pathogen Shigella flexneri showed increased mortality when compared to conventional guinea pigs29. Additionally, infection of GF mice with Listeria monocytogenes resulted in decreased clearance, and infection with Salmonella enterica resulted in more severe gastroenteritis compared to conventional control mice30,31.

The microbiome also provides an important site for immune tolerance and modulation. Conventional mice challenged with oral ovalbumin antigen, for example, showed systemic tolerance to the same antigen for a 2-3 month period.

In contrast, Germ free experimental mice showed a loss of tolerance between only a 7-21 day period32. Additionally, gut mucosa epithelial cells constantly sample ingested and commensal microbiota antigen providing real time immunological adaptation to the environment by generation of cytokines and transmitting signals to submucosal inflammatory and immune cells33. These data together suggest an important role of the gut in immune host defense.

26

Not only does the gut microbiota aid in the development of the immune system, it

also provides a critical barrier function against invading pathogen microbes.

There are several mechanisms that establish the resident microbiome as a

barrier. Ostensibly, a barrier is established physically by the competitive

exclusion of pathogens and opportunistic microbes by growth. There is also tight control over nutrient exchange between the host and microbiome, thereby preventing excess available nutrients for opportunistic and/or pathogen establishment16. Although the exact molecular mechanisms are ill-defined,

numerous studies have demonstrated that human fecal bacterial species have

antimicrobial activities against specific invading enteric pathogens20.

Presumably, one of the major anti microbial mechanisms is widely produced

proteinacious substances known as bacteriocins16,20. Interestingly, bacteriocins

often utilize host proteases for both activation and degradation reinforcing the

mutualistic host-microbiome relationship16,20. Other mechanisms include bacterial

production of metabolites like lactic acid by Lactobacillus species that inhibit local

bacterial growth20. The gut microbiome also stimulates the host to synthesize

antimicrobial peptides (AMPs) like defensins, cathelicidins, and C-type lectins

that serve to prevent the gut microbiota from overgrowing and invading the

epithelial cell barrier, but also will serve as protection against pathogens20.

Finally, the intestinal microbiota helps repair damaged epithelial cells, maintain

tight junctions between epithelial cells, and maintain epithelial cells through

interaction with surface epithelial receptors and stimulation of signaling

cascades34,35.

27

Over an average human lifetime approximately 60 tons of food will pass through

the gastrointestinal tract implying an important relationship between the gut

microbiota and diet18. An increased recognition of the importance of the gut microbiota in energy harvest and metabolism has occurred over the last 10-15

years. Indeed, the gut microbiome has a critical role influencing nutrition, energy

harvest, and normal metabolism in mammals.

Humans are not able to metabolize most complex carbohydrates or plant

polysaccarrhides like cellulose, some starches, and xylan36. These products are

instead degraded by the gut microbiota into short chain fatty acids (SCFAs) such

as acetate36,37. SCFAs provide an energy source for the gut microbiota itself, the

colonic epithelium, and peripheral tissues. Additionally, SCFAs can influence

inflammation, wound healing, motility, and vessel vasoreactivity36. Recently, a

role for SCFA in normal protein homeostasis has been shown37. The most

abundant SCFA produced by the human microbiome, acetate, was demonstrated

to contribute to pools of acetyl-CoA, participate in lysine-ε-acetylation, and influence protein function36,38. Insights into the importance of the gut microbiota

in energy harvest are mostly garnered from studies in GF animals.

GF rodents produce less SCFAs and excrete 2 fold more calories in the feces

and urine compared to conventional mice39,40. This results in decreased

adiposity in germ free mice and the consumption of twice the caloric intake of

28

conventional mice40-42. Remarkably, germ free mice can normalize their adiposity after only 2 weeks post conventionalization36,41,42. Further support of the importance of the gut microbiota in energy harvest is demonstrated in studies of

ob/ob mice, a mouse model of obesity. ob/ob mice have more cecal SCFAs and less residual calories found in their feces43. Together these studies suggest that

the gut microbiota can influence energy harvest in mammals.

GF rodents are also noted to have dysfunctional lipid metabolism. Overall,

systemic cholesterol metabolism is reduced, but surprisingly GF mice develop

increased cholesterol content in the liver and excrete more cholesterol in feces44.

Moreover, GF rodents also have exhibited dysfunction in bile acid metabolism.

Primary bile acids are synthesized from cholesterol and excreted into the

intestinal lumen where the gut microbiota further metabolizes them into

secondary bile acids. The metabolism of bile acids by the gut microbiota allows

bypass of the normal mechanisms of reuptake and excretion into the feces36.

However, in GF animals the absence of bacteria allows for unmetabolized bile

acids to be taken up vastly expanding the bile acid pool size36. Additionally, both

primary and secondary bile acids function in signaling and regulation of normal host metabolism. Disruption of the normal metabolism of bile acids consequently could cause metabolic dysfunction36.

Many of the advances in understanding the role of the microbiota in normal

human metabolism have coincided with and largely been driven by the

29

development of sophisticated (e.g. 16s ribosomal RNA surveys) tools to

characterize the gut microbiome. As a result of these technological

advancements, there has been increased recognition that long term diets fundamentally alter and determine the composition of the gut microbiota. A study by Ley et al. demonstrated that interspecies analysis of carnivorous, omnivorous, and herbivore mammals reveal closely aligned gut microbiota compositions suggesting dietary patterns have a had a great influence on the coevolution of the mammals and the gut microbiome45. Indeed, a follow-up study showed that

gut microbiota composition significantly aligned with the dietary components total

protein, insoluble fiber, and carbohydrates46. Moreover, comparison of the gut

microbiota composition of children from rural Africa who predominantly consume

a carbohydrate and plant based diet and European children where fats and

protein constitute a larger part of the diet, show distinct differences47.

Overall, the human microbiome can be subdivided into five major known bacteria

phyla: Firmicutes, Bacteroides, Actinobacteria, Proteobacteria, and

Verrucomicorbia36. More recently more refined classification systems have

suggested subdividing major intermicrobial communities that work in a symbiotic

relationship. There has also been a suggestion that the human gut microbiome can be stratified into 3 major enterotypes primarily composed of the genera

Bacteroides, Prevotella, and Ruminococcus respectively47. Remarkably, these

enterotypes have also been found to associate with dietary habits (e.g.

Bacteroides with a high protein, carnivorous diet; Prevotella with a carbohydrate

30

based diet)48. Moreover, 10 day dietary interventions failed to significantly alter

the composition of the gut microbiota suggesting that only long term dietary

habits are important in determining its composition49. Mice also have an altered

microbiota based on diet. Mice consuming high fat diets, for example, have a gut

microbiota that have an increased composition of Firmicutes and

Proteobacteria50. These descriptive microbiota differences imply that the gut microbiome plays an important role in dietary metabolism.

The Relationship between the Gut Microbiota and Disease

Like disruption of other normal physiological processes in our body, dysfunction of the gut microbiome can contribute to pathological processes16,20,51. For

example, the pathogenesis of inflammatory bowel disease (IBD) has been partly

attributed to dysfunction in the interaction of host immunity with the gut

commensal microbiota1,5. Studies of the commensal gut microbiota have

demonstrated that certain genera of bacteria (e.g. Bacteroides) are associated

with the severity and presence of IBD16. IBD patients often have a greater mass

of commensal bacteria adhering to the epithelial cell layer and commensal

bacteria have been found to invade into the epithelial cell layer52. A role for bacteria has been further elaborated in mice by studies utilizing broad spectrum antibiotics that suppress the gut microbiota and result in decreased mucosal inflammation in rodents53,54. These observations were recapitulated in humans by

the demonstration that antibiotic treatment decreased mucosal inflammation in

subjects with IBD to a greater extent than systemic steroid treatment53,55. Host

31

immunity has also been implicated in the disease pathogenesis of IBD patients

who characteristically contain IgG against a wide range of commensal microbiota

species including relatively innocuous species56. The generation of IgG promotes

epithelial cell injury and inflammatory cascades that further damage the mucosal

intestinal barrier57. Moreover, an estimated 25% of patients with Crohn’s disease

have a loss of function mutation in the NOD2/CARD25 gene that is found

primarily in leukocytes and recognizes the muramyl dipeptide (MDP) moiety in

bacteria58. These data together suggest a role of intestinal microbiota in the

pathogenesis of IBD.

A role for the gut microbiota in colon cancer has also been implicated. There are

multiple studies demonstrating links between gut microbiota bacterial taxa and

colon cancer. Bacterial genera including Bacteroides, Clostridium, and

Bifodobacterium are associated with colon cancer; whereas species including

Lactobaccilus and Eubacterium are inversely associated59,60. The intestinal

microbiota may also play a role in facilitating the metabolism of dietary nutrients into carcinogenic products like N-nitroso compounds16. These observations were

confirmed by a recent study that demonstrated a direct relationship between the

gut microbiota and colon cancer61. An enterotoxigenic species Bacteroides

fragilis that can asymptomatically colonize the colons in a proportion of the

human population can secrete the Bacteroides fragilis toxin (BFT). BFT has been

known to cause human inflammatory diarrhea, but also can promote colon

cancer via a TH17 (subtype of CD4+ T cells)-dependent pathway61.

32

A more expansive role of the gut microbiota and disease pathogenesis has been implicated in syndromes that are associated with breakdown of the normal epithelial mucosa that allow bacteria to translocate across the mucosal barrier16.

Gut microbiota bacteria translocation can lead to adverse severe sequelae such as sepsis, toxemia, multisystem failure, or death16. Translocation is believed to be mediated by at least three major mechanisms which include bacterial overgrowth, gut immunological deficiencies, or increased permeability of the gut mucosa62. Many disease states such as multi-system organ failure, pancreatitis, liver cirrhosis, and intestinal obstruction have demonstrated evidence of invading gut microbiota bacteria into the intestinal wall16,62. A more recent notable example of immunological deficiency resulting in loss of the mucosal barrier derives from studies in HIV and pathogenic SIV infection. These studies show that chronic depletion of TH17 cells, a critical cell in the maintenance of the normal immunological gut mucosa barrier, of the GI mucosa are associated with progression of HIV pathogenesis63. Depletion of TH17 cells and the subsequent loss of the secreted proinflammatory cytokine interleukin-17 (IL-17) led to the ability of opportunistic infections to advance64. Presumably, the depletion of these cells may also allow for translocation of gut microbiota across the GI mucosa and adverse sequelae63.

High rates of positive culture of gut microbiota in mesenteric lymph nodes in diseases commonly associated with bacterial translocation such as IBD,

33

pancreatitis, or liver cirrhosis are expected. Indeed an estimated 16-40% of these

patients have positive cultures65. However, positive mesenteric lymph node

cultures are also typically found an estimated 5% of the population65. This observation demonstrates that bacterial translocation is a common occurrence and may have a role in less acutely insidious pathological processes such as

CVD or diabetes. Together, these data demonstrate a role of the gut microbiota in cancer, infectious and inflammatory disease states.

A relationship of the gut microbiota with complex metabolic diseases was first described by Gordon and colleagues that published seminal papers for the role of the gut microbiota and obesity. Most notably was a study where donation of an

“obese microbiota” (from ob/ob mice) to a germ free animal significantly increased total body fat when compared to parallel transplantation of GF mice with conventional mouse microbiota43. The mechanism of this effect is largely

attributed to an increase in energy harvest from the microbiota of ob/ob mice.

Indeed, the composition of the microbiota in mice fed a high fat diet is significantly different compared to mice on a chow diet 50,66. These data are

further supported by studies in lean and obese twins43. The obese individuals of

the twin pairs were more associated with decreased gut microbiota diversity, phyla differences, and altered bacterial metabolic pathways43. Overall, there have

been many reports demonstrating significant changes in the gut microbiota

composition in accordance with weight43,67,68. Interestingly, the gut microbiota composition in patients undergoing gastric bypass operations is also significantly

34

altered post-op36. The observation that these subjects are able to experience an antidiabetic effect even before significant weight loss occurs suggests that the

gut microbiota may also play a role in this direct effect36.

Gut Microbiota Mediated Metabolism of Phosphatidylcholine Promotes

Cardiovascular Disease a, 69

Recently, the role of the microbiota has been extended from complex metabolic diseases such as diabetes and obesity to atherosclerotic disease69. The gut

microbiota metabolism of phosphatidylcholine, the major source of dietary

choline, produces a noxious intermediate compound known as trimethylamine

(TMA) that is further metabolized by liver Flavin monooxygenase (FMO) to

TMAO thereby promoting atherosclerotic disease (Fig. 1-1).

The discovery of this pathway began with a search for novel pathways involved

in cardiovascular disease pathogenesis by using an unbiased metabolomic study

(Fig. 1-2). Small-analyte plasma profiles were acquired initially in a “Learning

Cohort” that consisted of subjects undergoing elective coronary angiography who

then experienced an ensuing major adverse cardiovascular event (MACE; MI,

stroke, or death) over a 3-year period and age and gender matched controls that

did not experience MACE. Direct comparison between diseased and control

subject profiles using liquid chromatography with on-line spectrometry (LC/MS)

demonstrated that 40 analytes out of >2,000 analyzed were associated with

a From Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease, v.472 Copyright © (2011) Nature Publishing group. Reprinted with permission. 35

cardiac risks69. A second “Validation Cohort” study was performed in which

small-analyte plasma profiles were acquired for a cohort of completely

independent subjects undergoing elective coronary angiography who also

experienced MACE over a 3-year period and age or gender matched controls

that did not experience MACE. Analysis of >2,000 possible analyses yielded

significant differences in 24. When comparing the Learning and Validation

cohorts 18 common unknown analytes significantly associated with cardiovascular disease (Fig. 1-2). Among these unknown analytes 3 had a common association with m/z 76, 104, 118 respectively suggesting they may be

part of a common biochemical pathway. Further structural studies confirmed the

identities of these unknowns as trimethyl amine N-oxide (TMAO; m/z 76), choline

(m/z 104), and betaine (m/z 118)69.

Remarkably, the metabolism of choline to TMAO was a gut microbiota mediated

process and suggested a role of the gut microbiota in atherosclerosis69,70. The major dietary source of choline is in the form a member of the phospholipid class of lipids, phosphatidylcholine (PC). Interestingly, whereas the other two major classes of lipids, sterols and triglycerides, have been associated with CVD, a role for phospholipids has not. These data suggested a link between dietary phospholipids, the gut microbiota, and atherosclerosis69.

Challenge of mice with heavy stable isotope labeled d9-phosphatidylcholine (d9-

PC) demonstrated production of both d9-TMAO and d9-betaine (Fig. 1-3).

36

Following suppression of the gut microbiota with oral broad spectrum antibiotics,

mice rechallenged with d9-PC showed complete absence of d9-TMAO

production, but still demonstrated production of d9-betaine.

Reconventionalization of mice shows reacquisition of the gut microbiota

production of d9-TMAO from d9-PC demonstrating an obligatory role of the gut

microbiota in TMAO production from d9-PC, but not betaine. These observations

were also confirmed in germ free mouse d9-PC challenges studies69.

Next confirmatory studies of the relationship between plasma choline, TMAO, and betaine discovered in the unbiased metabolomics approach were performed.

Quantification of plasma trimethylamines in n=1,876 sequential subjects undergoing elective coronary angiography at Cleveland Clinic showed increasing concentrations of choline, TMAO, and betaine were associated with increased, dose dependent, prevalence of CVD (Fig. 1-4). Moreover, adjustment for traditional risk factors with multivariate modeling demonstrates the PC metabolites are independently associated with CVD69.

Together these data raised the possibility that dietary supplementation of choline

and TMAO may promote atherosclerosis. Atherosclerosis-prone female

C57BL/6J, Apoe-/-mice were placed on increasing concentrations of dietary choline or TMAO at time of weaning (4 weeks of age) for 16 weeks before sacrifice. Quantification of aortic root plaque showed an increased amount of

atherosclerotic plaque at the aortic root of mice fed a trimethylamine diet

37

compared to chow controls (Fig. 1-5) despite no significant increases in plasma

lipid profiles or liver pathology69. Interestingly, plasma TMAO levels significantly

correlated with burden of plaque at the aortic root suggesting the terminal gut

microbiota dependent product, TMAO is responsible for promotion of

atherosclerosis. A similar trend was observed in humans when examining the

burden of atherosclerotic disease (defined by the presence of coronary artery

disease (CAD) in one, two, or three vessels) with plasma TMAO levels69.

Hepatic FMO3 is the enzymatic source of TMAO production in humans and loss of function mutations in the gene coding for FMO3 protein results in “fish malodor syndrome” that is characterized by an individual’s inability to metabolize TMA, a noxious gas at room temperature that smells like rotting fish, to odorless

TMAO71,72. The end result for afflicted individuals is body odor that smells like

rotting fish that is made worse by consuming dietary sources rich in

trimethylamine containing compounds (e.g. dairy products and meats). The

involvement of endogenous FMO3 in TMAO production raised the possibility of

genetic regulatory involvement in atherosclerosis. Using integrative genetic

approaches the role of FMO3 expression and regulation was investigated in

murine atherosclerosis. Association studies between purified liver FMO3 mRNA from a F2 intercross between an atherosclerotic resistant mouse strain

(C3H/HeJ, Apoe-/-) and an atherosclerotic prone mouse (C57BL/6J, Apoe-/-) and atherosclerotic plaque burden showed a positive correlation (R = 0.29, P =

0.002). Moreover, hepatic FMO3 expression also had a significant positive

38

association with plasma TMAO levels and a negative association with mouse plasma HDL levels (R = 0.80, P < 0.001). In a next set of studies eQTL analysis was performed using mice from the same F2 intercross. A single nucleotide polymorphism on mouse chromosome1 that was in close proximity to the FMO3 gene, and that was simultaneously in a region that had previously been linked to atherosclerotic disease burden, showed a dose dependent relationship with atherosclerotic disease burden (Fig. 1-6). These data provide evidence of a relationship between FMO3 expression and atherosclerotic disease.

In a final set of studies, a role for the gut microbiota in dietary choline induction of atherosclerosis was determined. C57BL/6J, Apoe-/- male and female mice were placed on a choline supplemented or control diet in the presence or absence of broad spectrum antibiotics (used to suppress the gut microbiota) for 16 weeks post weaning. Quantification of atherosclerotic plaque at the aortic root of these mice showed a significant increase in plaque area compared to chow controls.

Importantly, this increase was also significant compared to mice supplemented with choline and with a concomitant suppressed gut microbiota. This confirms a gut flora dependence mechanism in choline induced atherosclerosis (Fig. 1-7).

These data link together a previously unrecognized pathway between dietary lipids (in the form of phosphatidylcholine), the gut microbiota, and atherosclerosis. Moreover, these data also suggested that other dietary trimethylamine containing compounds may contribute to TMAO formation and atherosclerotic disease.

39

Figure 1-1. Scheme of gut microbiota dependent metabolism of dietary PC and atherosclerosis. Choline, a trimethylamine species that is found in food principally as phosphatidylcholine (PC), is metabolized by commensal gut flora to form the noxious intermediate compound trimethylamine (TMA). TMA is further oxidized by flavin monooxygenases (FMOs) to form TMAO promoting the formation of atherosclerotic plaque69.

40

Figure 1-2. Metabolomics studies scheme and correlations. a. Scheme of unbiased metabolomic study that identified plasma analytes associated with CVD. b. Significant correlations between analytes m/z 76, 104, 118 from metabolomics studies suggested a common biochemical pathway69.

41

.

Figure 1-3. Production of TMAO from PC is gut flora dependent. LC/MS/MS plasma quantification of d9-choline, d9-TMAO, d9-betaine after gastric gavage of d9-DPPC in conventional mice, following suppression of the gut microbiota with broad spectrum antibiotics (3 weeks), and then following a reacquisition period (4 week housing with non-sterile mice (i.e. – “conventionalized”)). Data are presented as mean ± SE from 4 independent replicates. d9-TMAO production69.

42

Figure 1-4. Choline, TMAO and betaine are associated with CVD in humans. a-c. Logistic regression spline plots of the relationship between plasma analytes choline, TMAO, and betaine with cardiovascular disease (CVD) (with 95% CI) in n = 1876 subjects69.

43

Figure 1-5. Dietary choline or TMAO enhances atherosclerosis. C57BL/6J, Apoe-/- female mice at time of weaning (4 weeks) were placed on the respective choline diet, TMAO diet or a chow diet for 16 weeks. Atherosclerotic plaque was quantified at the aortic roots of mice at time of sacrifice69.

44

Figure 1-6. Hepatic FMOs associate with atherosclerosis. Association of the FMO3 genotype (SNP rs3689151) with both (C57BL/6J, Apoe-/-) and atherosclerosis resistant (C3H/HeJ, Apoe-/-) mice69.

45

Figure 1-7. Dietary choline enhances atherosclerosis in a gut flora dependent manner. C57BL/6J, Apoe-/- male and female mice at time of weaning (4 weeks) were placed on either a choline diet (1.0%) or a chow diet (0.08 % total choline) in the presence or absence of broad spectrum antibiotics (+ABS) in the drinking water for 16 weeks. Atherosclerotic plaque was quantified at the aortic roots of mice at time of sacrifice69.

46

CHAPTER 2b: Intestinal Microbiota Metabolism of L-Carnitine, a Nutrient in 73 Red Meat, Promotes Atherosclerosis

Authors: Robert A. Koeth, Zeneng Wang, Bruce S. Levison, Jennifer A. Buffa,

Elin Org, Brendan T. Sheehy, Earl B. Britt, Xiaoming Fu, Yuping Wu, Lin Li,

Jonathan D. Smith, Joseph A. DiDonato, Jun Chen, Hongzhe Li, Gary D. Wu,

James D. Lewis, Manya Warrier, J. Mark Brown, Ronald M. Krauss, W. H. Wilson

Tang, Frederic D. Bushman, Aldons J. Lusis, and Stanley L. Hazen

Abstract

Intestinal microbiota (i.e. "gut flora") - metabolism of choline/phosphatidylcholine

produces trimethylamine (TMA), which is further metabolized to a proatherogenic

species, trimethylamine-N-oxide (TMAO)69. Herein we demonstrate that gut microbiota metabolism of dietary L-carnitine, a trimethylamine abundant in red meat, also produces TMA, TMAO and accelerates atherosclerosis. Omnivorous

subjects to a far greater extent than vegans/vegetarians are shown to produce

TMAO following ingestion of L-carnitine through a gut microbiota dependent

mechanism. Specific bacterial taxa in human feces are shown to associate with

both plasma TMAO and omnivore versus vegan/vegetarian status. Plasma L-

carnitine levels in sequential stable subjects undergoing cardiac evaluation

(n>2,500) predict increased risks for both prevalent cardiovascular disease

(CVD) and incident major adverse cardiac events (MI, stroke or death), but only

among subjects with concurrently high TMAO levels. Chronic dietary L-carnitine

supplementation in mice is shown to significantly alter cecal microbial

b From Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis, Copyright © (2013) Nature Publishing group. Reprinted with permission 47

composition, augment synthesis of TMA/TMAO by over 10-fold, and increase

aortic root lesion area, but not following suppression of intestinal microbiota.

Dietary supplementation of TMAO in mice, or either carnitine or choline in mice with intact but not suppressed intestinal flora, significantly reduced reverse cholesterol transport in vivo. Gut microbiota may thus participate in the well-

established link between increased red meat consumption and CVD risk.

Introduction

Cardiovascular disease (CVD) remains the leading cause of morbidity and

mortality in western societies. The high-frequency consumption of meat products

in the developed world is linked to cardiovascular disease risk, presumably due

to the large content of saturated fats and cholesterol found in these foods74,75.

However, a recent meta-analysis of prospective cohort studies showed no

association between dietary saturated fat intake and CVD, prompting the

suggestion that other environmental exposures linked to increased dietary meat

consumption are responsible76. In fact, the suspicion that the cholesterol and

saturated fat content of red meat may not be sufficiently high to account for

observed risks has long stimulated the investigation of alternative sources of

disease-promoting exposures that accompany dietary meat ingestion, such as

the high content of salt or heterocyclic compounds generated during cooking77,78.

Of note, to date, such studies have largely focused on the biochemical content of

meat itself before or following processing, and have yet to address the impact of

48

our commensal intestinal microbiota (i.e. gut flora) and their participation in

modifying the diet-host interaction.

Trillions of bacteria populate our digestive system, exceeding by approximately

an order of magnitude the total number of cells in our body. Our gut microbiota

has been linked to intestinal health, immune function, bioactivation of critical

nutrients and vitamins, and more recently, complex disease phenotypes such as

obesity and insulin resistance43,79,80. We recently reported a novel pathway in

both humans and murine models of atherosclerosis linking gut microbiota

metabolism of dietary choline to CVD pathogenesis69. Choline, a trimethylamine

containing compound and part of the head group of phosphatidylcholine (PC), the

major dietary source of choline, is metabolized by the action of gut microbiota to

produce an intermediate gaseous compound known as trimethylamine (TMA)

(Fig. 2-1a). TMA is rapidly further oxidized by one or more hepatic flavin

monooxygenases (FMO) to form the metabolite trimethyl amine N-oxide (TMAO),

which was shown to be proatherogenic. Atherosclerotic prone apolipoprotein E-/- mice treated with a diet supplemented with either choline or the downstream metabolite, TMAO, demonstrated enhanced aortic root atherosclerotic burden. In

contrast, germ-free mice, or animals with suppressed intestinal microbiota

through use of oral broad spectrum antibiotics, failed to make both TMA and

TMAO following ingestion of either phosphatidylcholine or choline, and showed

no increase in atherosclerosis from a high choline diet. Further, integrative

genetics studies demonstrate the FMO gene cluster on chromosome 1 as an

49

atherosclerosis susceptibility locus in the rodent model. Finally, plasma levels of

TMAO and choline in subjects were associated with CVD risks69. These results

collectively indicated both an obligatory role for gut microbiota in the production

of TMAO from dietary choline and phosphatidylcholine, and that elevated levels

of TMAO are mechanistically linked to accelerated atherosclerotic heart disease

in rodent models and humans. The findings further raise the possibility that other

dietary nutrients that possess a similar trimethylamine structure may also

contribute to TMAO formation via gut microbiota, and consequently, accelerated

atherosclerosis. How TMAO is mechanistically linked to development of

accelerated atherosclerosis and which specific microbial species contribute to

TMAO formation remain unknown.

L-carnitine is an abundant dietary nutrient in red meat that contains a trimethylamine structure similar to choline (Fig. 2-1a). A hydrophilic quaternary

amino acid, its name is derived from Latin “carnis”, meaning flesh. While

ingestion of L-carnitine from diet is a major source of the compound in

omnivores, the amino acid is also endogenously produced in mammals from

lysine, and serves an essential function in the transport of fatty acids from the cell

cytoplasm to the mitochondrial compartment81,82. Recent changes in dietary

habits in industrialized societies have included a tremendous growth in L-

carnitine supplementation as a food or drink additive, particularly in many power

or energy drinks, or nutritional supplements aimed at increasing muscle mass.

Over the past few years L-carnitine also has become a common supplement

50

added to commercial beverages including coffees/espresso, flavored

vitamin/water drinks, and other beverages widely consumed by the public.

Whether there is a potential health risk for such pervasive and rapidly growing

nutritional supplement practices has not been considered, much less explored.

Herein we examine the gut microbiota-dependent metabolism of L-carnitine to produce TMAO in both rodents and humans (omnivore vs. vegans). Through a combination of isotope tracer studies, large scale clinical studies and animal model investigations employing both germ-free mice and mice with intact and suppressed intestinal microbiota, we demonstrate a role for gut microbiota metabolism of L-carnitine in atherosclerosis pathogenesis in the appropriate dietary setting (high carnitine ingestion). In addition to the upregulation of macrophage scavenger receptors potentially contributing to enhanced "forward

cholesterol transport"69, we further show that TMAO, and its dietary precursors

choline and carnitine, suppress reverse cholesterol transport through gut

microbiota dependent mechanisms in vivo. Finally, we define microbial taxa in

humans and murine models associated with both TMAO production and dietary carnitine ingestion, and show dynamic microbial compositional changes that occur with carnitine supplementation, and consequent marked enhancement in

TMAO synthetic capacity in vivo.

Results

Metabolomic studies link L-carnitine with CVD

51

Given the similarity in structure between L-carnitine and choline (Fig. 2-1a) we hypothesized that dietary ingestion of L-carnitine in humans, like choline and phosphatidylcholine, might produce TMA and TMAO in a gut microbiota dependent fashion, and be associated with atherosclerosis risk in humans. To test this we initially examined data from our recently published unbiased small molecule metabolomics analyses of plasma analytes and CVD risks69. An analyte

with identical molecular weight to L-carnitine (mass to charge ratio (m/z) 162)

was not in the top tier of analytes that met the stringent P value cutoff for

association with CVD after Bonferroni adjustment for multiple comparisons in

both the initial Learning Cohort and the subsequent Validation Cohort69.

However, a hypothesis-driven examination of the data using less stringent criteria

(no adjustment for multiple testing) did reveal an analyte with m/z 162 that showed a tendency toward positive association with major adverse cardiovascular events over a 3 year period in the combined cohort of patients

(unadjusted Hazard Ratio 2.63; 95% CI(1.03-6.75); P = 0.04)(Supplementary

Table 2-1). In further studies we were able to confirm the identity of the plasma

analyte as L-carnitine by using multiple approaches, including demonstration of

identical retention time under multiple chromatographic conditions during LC/MS

analysis, identical collision-induced dissociation (CID) mass spectrum with that of

an authenticate standard L-carnitine, and co-elution of multiple characteristic

precursor → product ion transitions in a plasma sample spiked with synthetic

stable isotope labeled (d9-trimethyl)-carnitine standard (Supplementary Figs. 2-

1, 2-2). Unbiased metabolomics data as performed69 are semi-quantitative in

52

nature; however, they are hypothesis generating, and thus suggested that

plasma levels of L-carnitine may associate with CVD risks. For all subsequent

studies we therefore developed and used quantitative stable isotope dilution

LC/MS/MS methods for measuring endogenous L-carnitine using a synthetic

isotopologue of L-carnitine (d9-(trimethyl)-L-carnitine) as internal standard

(Supplementary Fig. 2-3).

Gut microbiota plays an obligatory role in forming TMAO from L-carnitine in humans

The participation of gut microbiota in TMAO production from dietary L-carnitine in humans has not yet been shown. We therefore first sought to test the ability of human micro microbiota to help produce TMAO from ingested L-carnitine by developing a human “L-carnitine challenge test”. Since the bioavailability of dietary L-carnitine within an endogenous food source is reported to be substantially higher (estimated 4-fold) than the bioavailability of carnitine supplements83, in initial subjects (omnivores), the L-carnitine challenge test

incorporated a major source of dietary L-carnitine (8 ounce sirloin steak,

corresponding to an estimated 180 mg L-carnitine)84,85 and a capsule containing

250 mg of a heavy isotope labeled L-carnitine (synthetic d3-(methyl)-L-carnitine).

At baseline (Visit 1), post-prandial increases in d3-TMAO and d3-L-carnitine in

plasma were readily detected, and 24 hour urine collections also revealed d3-

TMAO (Fig. 2-1b-e; Supplementary Fig. 2-4, 2-5. Data shown in all panels of

Fig. 2-1 and Supplementary Fig. 2-4 are tracings from a representative

53

omnivorous subject, of n=5 studied with complete serial blood draws post carnitine challenge). As previously observed 69, endogenous (non-labeled)

fasting plasma TMAO levels showed wide variation in levels at baseline among

subjects, suggesting wide inter-individual variations exist in gut microbiota capacity to generate TMAO (see below). In most subjects examined, despite clear increases in plasma d3-carnitine and d3-TMAO over time, post prandial changes in endogenous (non-labeled) carnitine and TMAO were modest

(Supplementary Fig. 2-5), consistent with a total body (and intravascular) pool of natural abundance carnitine and TMAO that are relatively vast in relation to the amount of carnitine ingested and TMAO produced from the carnitine challenge.

To examine the potential contribution of gut microbiota to TMAO formation from

dietary L-carnitine, volunteers were then placed on oral broad spectrum

antibiotics to suppress intestinal microbiota for a week as described under

Methods, and then another baseline sample collected, and repeat L-carnitine

challenge performed (Visit 2). A remarkable complete suppression of measurable endogenous TMAO at baseline in both plasma and urine were noted (Fig. 2-1b- e; Supplementary Fig. 2-5). Moreover, in every subject examined with carnitine challenge following the course of oral antibiotics, virtually no detectable formation of either native or d3-labeled TMAO was observed in post prandial plasma or 24 hour urine samples, demonstrating that TMAO production from dietary L-carnitine in subjects has an obligatory role for gut microbiota(Supplementary Fig. 2-4). In contrast, both d3-L-carnitine and unlabeled L-carnitine were readily detected

54

following their ingestion during carnitine challenge, and showed little change in

the overall time course for post prandial changes in levels observed before (Visit

1) versus after antibiotic treatment (Visit 2; Fig. 2-1e, Supplementary Fig. 2-5).

After discontinuation of antibiotics, subjects were invited back for a third visit after at least another three weeks. Examination of baseline and post L-carnitine challenge plasma and urine samples again showed TMAO and d3-TMAO formation in both plasma and urine, consistent with intestinal re-colonization (Fig.

2-1b-e; Supplementary Fig. 2-4, 2-5). Collectively, these data clearly show that

TMAO production from dietary L-carnitine in humans is gut microbiota

dependent.

Vegans and vegetarians produce substantially less TMAO from dietary L-

carnitine

As noted above, the capacity to produce native and d3-labeled TMAO following

native and d3-L-carnitine ingestion was variable among individuals. A post-hoc

nutritional survey performed amongst the volunteers suggested that the

antecedent dietary habits (red meat consumption) may influence the capacity to

generate TMAO from L-carnitine. To test this prospectively, we examined TMAO

and d3-TMAO production following the same L-carnitine challenge, first in a long

term (>5 years) vegan who consented to the carnitine challenge (including both

steak and d3-(methyl)-carnitine consumption). Figure 2-2a illustrates results from

carnitine challenge in this vegan volunteer who was willing to ingest steak as part

of the carnitine challenge. Also shown for comparison are data from a single

55

omnivore with reported common (near daily) dietary consumption of red meat.

Post-prandially we noted that the omnivorous subject with common red meat consumption showed both an increase in TMAO and d3-TMAO levels in sequential plasma measurements (Fig. 2-2a), and in a 24 hour urine collection sample (Fig. 2-2b). In contrast, the vegan subject showed nominal fasting plasma and urine TMAO levels at baseline, and virtually no capacity to generate d3-TMAO or TMAO in plasma after the carnitine challenge, with approximately

1000-fold less d3-TMAO produced from the same oral d3-L-carnitine load compared to the representative omnivore (Fig. 2-2a,b). The vegan subject also had lower fasting plasma levels of L-carnitine compared to the omnivorous subject (Supplementary Fig. 2-6). To confirm and extend these findings we examined additional vegans/vegetarians (n=23) and omnivorous subjects (n=51).

Fasting baseline TMAO levels were significantly lower among vegan/vegetarian subjects compared to omnivores (Fig. 2-2c). In a subset of these individuals an oral d3(methyl)-carnitine challenge (but with no steak) was performed, confirming that long term (> 1 year) vegan/vegetarians have markedly reduced synthetic capacity to produce TMAO from oral carnitine (Fig. 2-2c,d). Interestingly, vegan/vegetarians challenged with d3-carnitine also had significantly more post- challenge plasma d3-carnitine compared to omnivorous subjects

(Supplementary Fig. 2-7), a result that may reflect decreased intestinal microbial metabolism of carnitine prior to absorption.

56

Plasma TMAO levels significantly associate with specific human gut

microbial taxa

Dietary habits (e.g. vegan/vegetarian versus omnivore/carnivore) are associated

with significant alterations in intestinal microbiota composition and function45,46,86.

We therefore examined fecal samples from both vegans/vegetarians (n=23) and

omnivores (n=30) for analyses of the gene encoding for bacterial 16S ribosomal

RNA, and in parallel, plasma TMAO, and carnitine and choline levels were

quantified by stable isotope dilution LC/MS/MS. Global analysis of taxa

proportions by combining both the weighted and unweighted Unifrac distances

using PeranovaG revealed significant associations with plasma TMAO levels

(P=0.03), but not plasma carnitine (P= 0.77) or choline (P =0.74) levels. Several

bacterial taxa remained significantly associated with plasma TMAO levels after

false discovery rate (FDR) adjustment for multiple comparisons (Supplementary

Fig. 2-8). When subjects were classified into previously reported enterotypes49 based upon fecal microbial composition, individuals with an enterotype characterized by enriched proportions of the genus Prevotella (n=4) demonstrated higher (p<0.05) plasma TMAO levels than subjects with an enterotype notable for enrichment of Bacteroides (n=49) genus (Fig. 2-2e).

Examination of the proportion of specific bacterial genera and subject TMAO levels revealed several taxa (genus level) that simultaneously were significantly associated with both vegan/vegetarian versus omnivore status, and plasma

TMAO levels (Fig. 2-2f).

57

TMAO production from dietary L-carnitine is an inducible trait

Analyses of data from vegan/vegetarians versus omnivores thus far suggested

that preceding dietary habits modulate gut microbiota composition, and the

synthetic capacity to ultimately produce TMAO from dietary L-carnitine may be

highly adaptable. We next investigated the ability of chronic dietary L-carnitine to induce gut flora-dependent production of TMA and TMAO in the murine model.

Pilot LC/MS/MS studies first confirmed the presence of L-carnitine in plasma of conventional C57BL/6J and atherosclerosis-prone C57BL/6J, Apoe-/-mice on normal chow diet, which contains no L-carnitine per manufacturer (carnitine content of chow diet was also confirmed by LC/MS/MS analyses, data not shown). To confirm that mouse intestinal microbiota could produce TMA and

TMAO from dietary L-carnitine, we also examined germ-free mice and observed no detectable plasma d3-(methyl)TMA or d3-(methyl)TMAO following oral

(gastric gavage) d3-(methyl)carnitine challenge, but acquisition of capacity to produce both d3-(methyl)TMA and d3-(methyl)TMAO following oral (gastric gavage) d3-(methyl)carnitine after a several week period in conventional cages to allow for microbial colonization (i.e. “conventionalization”) (Supplementary

Fig. 2-9). Parallel studies with conventional C57BL/6J, Apoe-/- mice that were

placed on a cocktail of oral broad spectrum antibiotics previously shown to

suppress intestinal microflora35,69 showed similar results as the germ-free mice

(i.e., complete suppression of both TMA and TMAO formation; Supplementary

Fig. 2-10), confirming in the mouse model an obligatory role for gut flora in both

TMA and TMAO production from dietary L-carnitine. To examine the impact of

58

dietary L-carnitine on inducibility of TMA and TMAO production from intestinal

microbiota, we compared the pre- and post-prandial plasma profile of C57BL/6J,

Apoe-/- mice on normal chow diet versus a diet supplemented in L-carnitine for

15 weeks. The production of both d3-(methyl)TMA and d3-(methyl)TMAO

following oral ingestion (gastric gavage) of d3-(methyl)carnitine was induced by

approximately 10-fold in mice on the L-carnitine supplemented diet compared to normal chow diet fed controls (Fig. 2-3a). Further, plasma post-prandial d3-

(methyl)carnitine levels in mice in the carnitine supplemented diet arm were significantly lower than that observed in mice on the carnitine free diet (normal chow), consistent with enhanced gut flora-dependent catabolism prior to absorption in the carnitine supplemented mice.

TMA / TMAO production associates with specific mouse gut microbial taxa

The marked effect of chronic dietary carnitine on enhanced TMA and TMAO production from a carnitine challenge (d3-(methyl)carnitine by gavage) suggested that carnitine supplementation may have significantly altered intestinal microbial composition with enrichment of taxa better suited for TMA production from carnitine. To test this we first identified the cecum as the segment of the entire intestinal tract of mice that shows the highest synthetic capacity to form TMA from carnitine (data not shown). We then sequenced 16S rRNA gene amplicons from cecum of mice on either normal chow (n=10) or carnitine supplemented diet

(n=11) and in parallel, quantified plasma levels of TMA and TMAO using stable

isotope dilution LC/MS/MS (Fig. 2-3b,c). Global analyses of individual taxa

59

proportions reveals that in general, microbial genera that show increased

proportions coincident with increased plasma levels of TMA also tend to show increased proportions coincident with plasma TMAO levels. Several bacterial taxa remained significantly associated with plasma TMA and/or TMAO levels after false discovery rate (FDR) adjustment for multiple comparisons (Fig. 2-3b).

Further analyses examining the proportion of specific bacterial genera and mouse plasma TMA and TMAO levels revealed several taxa that significantly segregate with both mouse dietary groups and are associated with plasma TMA or TMA levels (Fig. 2-3c; Supplementary Fig. 2-11). Interestingly, a direct comparison of genera identified in humans versus mice that significantly associated with plasma TMAO levels failed to identify common genera, consistent with prior reports that microbes identified from the distal gut of the mouse represent genera that are typically not detected in humans45,68.

Plasma levels of L-carnitine associate with CVD

We next investigated the relationship of fasting plasma levels of L-carnitine with

CVD risks in an independent large cohort of stable subjects (n=2,595)

undergoing elective cardiac evaluation. Patient demographics, laboratory values, and clinical characteristics are provided in Supplementary Table 2-2. A

significant dose – dependent association between L-carnitine levels and risk of

prevalent coronary artery disease (CAD), peripheral artery disease (PAD), and

overall CVD was noted (Fig. 2-4a-c). Moreover, the association of plasma L- carnitine levels with CAD, PAD and CVD remained significant following

60

adjustments for traditional CVD risk factors, including age, sex, history of diabetes mellitus, smoking, systolic blood pressure, and lipoproteins/lipids. In further analyses, plasma levels of L-carnitine were observed to be increased in subjects with significant (≥ 50% stenosis) angiographic evidence of CAD, regardless of the extent (e.g. single versus multi-vessel) of CAD, as revealed by diagnostic cardiac catheterization (Fig. 2-4d). Next, the relationship between baseline fasting plasma levels of L-carnitine and incident (3 year) risk for major adverse cardiac events (MACE = composite of death, MI, stroke, and revascularization) was examined. Elevated levels of L-carnitine (4th quartile) remained an independent predictor of MACE even after adjusting for traditional

CVD risk factors (Fig. 2-4e). After further adjustment for both TMAO and a larger number of comorbidities that might be known at time of presentation (extent of

CAD, ejection fraction, medications, and estimated renal function), the significant relationship between carnitine and MACE risk was completely attenuated (Model

2) (Fig. 2-4e). Notably, the significant association between carnitine and incident cardiovascular event risks was observed in Cox regression models after multivariate adjustment, but only among those subjects with concurrent high plasma TMAO levels (Fig. 2-4f). Thus, while plasma levels of carnitine appear to be associated with prevalent and incident cardiovascular risks, the present results are consistent with TMAO, and not the dietary precursor carnitine, which serves as the primary driver of the association with cardiovascular risks (i.e. it is

TMAO that may be the pro-atherogenic species).

61

Dietary L-carnitine in mice promotes atherosclerosis in a gut microbiota dependent manner

We therefore next sought to investigate whether dietary L-carnitine had any impact on the extent of atherosclerosis in the presence vs. absence of TMAO formation in animal models. C57BL/6J, Apoe-/- mice were initially fed normal chow diet versus the same diet supplemented with L-carnitine from time of weaning. Aortic root atherosclerotic plaque quantification revealed approximately a doubling in disease burden compared to normal chow fed animals (Fig. 2-5a, b). Importantly, parallel studies in mice placed on oral antibiotic cocktail to suppress intestinal microflora showed marked reductions in plasma TMA and

TMAO levels (Fig. 2-5c), as well as complete inhibition of the dietary L-carnitine- dependent increase in atherosclerotic lesion burden (Fig. 2-5b). Analysis of plasma revealed that the increase in atherosclerotic plaque burden noted with dietary L-carnitine supplementation occurs in the absence of significant pro- atherogenic changes in plasma lipids, lipoproteins, glucose, or insulin levels; moreover, both biochemical and histological analyses of livers in the mice failed to demonstrate significant steatosis (Supplementary Table 2-3, 2-4;

Supplementary Fig. 2-12). Quantification of plasma levels of L-carnitine in the mice revealed a significant increase in the L-carnitine fed animals versus the normal chow fed controls (Fig. 2-5c). Interestingly, an even higher increase in plasma L-carnitine levels was noted in mice supplemented with L-carnitine on the antibiotic arm of the study, which failed to show enhanced atherosclerosis. These results parallel what was observed with mice on carnitine supplemented diet

62

following the d3-(methyl)carnitine challenge (Fig. 2-3a), and are consistent with a

major role for gut microbiota in the catabolism of dietary L-carnitine in the setting of chronic carnitine ingestion. They also suggest that it is not L-carnitine itself, but a down-stream (presumably) gut flora-dependent metabolite that promotes the

increased atherosclerosis burden (Fig. 2-5c). Consistent with this hypothesis,

whereas plasma levels of L-carnitine in the mice had no significant association

with atherosclerotic disease burden (R=0.09(Spearman), P=0.59), plasma levels of both of its gut microbiota-dependent monitored metabolites, TMA (R=0.30,

P<0.01) and TMAO (R=0.45, P<0.01), showed significant dose dependent

associations with atherosclerotic plaque burden.

Gut microbiota dependent formation of TMAO inhibits reverse cholesterol

transport

In recent studies we showed that TMAO can promote macrophage cholesterol

accumulation in vivo in a gut microbiota dependent manner by increasing surface

expression of scavenger receptors CD36 and SRA169 , receptors previously

shown to participate in atherosclerosis in murine models87,88. In an effort to identify additional mechanisms through which TMAO may promote a pro- atherosclerotic phenotype, additional experiments were performed. We first noted that TMAO and its trimethylamine nutrient precursors are all quaternary amines, and thus have the potential to compete with the amino acid arginine for cellular uptake via cationic amino acid transporters. TMAO might thus hypothetically limit arginine bioavailability and hence, nitric oxide synthesis,

63

under conditions of elevated plasma TMAO and its dietary precursors. However,

direct testing of this hypothesis in bovine aortic endothelial cells through

competition studies using [14C]arginine and (patho)physiologically relevant levels

of TMAO and other trimethylamine containing compounds demonstrated no significant decrease in [14C]arginine transport (Supplementary Fig 2-13).

Taking note of the enhanced cholesterol accumulation within macrophages

recovered from mice in the presence of either dietary (directly) or gut microbiota- generated TMAO69, we decided to next focus on the impact of TMAO on various

aspects of cholesterol metabolism in vivo. Taking a "black box" approach, one

can in general envision three non-exclusive mechanisms through which

cholesterol can accumulate within cells of the artery wall such as a peripheral

macrophage: (i) enhanced rate of flux in (as noted above, a mechanism already

shown to occur with TMAO-induced increased surface levels of macrophage

scavenger receptors SRA1 and CD36) 69; (ii) enhanced synthesis; or (iii)

diminished rate of flux out. To test whether TMAO might alter the canonical down

regulation of cholesterol biosynthesis genes attendant with macrophage

cholesterol loading5, several different sources of macrophages were loaded with

cholesterol and suppression of cholesterol synthesis related genes and LDL

receptor was confirmed. Concomitant addition of TMAO to media at

physiologically relevant concentrations (corresponding to those observed in the top 1 percentile in patient plasma), however, failed to alter mRNA levels of the

LDL receptor or cholesterol synthesis genes (Supplementary Fig. 2-14). Parallel

64

studies examining desmosterol levels and macrophage inflammatory gene

expression in the presence vs. absence of cholesterol loading, processes

recently linked89, failed to show any effect of TMAO within tissue culture media

(Supplemental Figs. 2-14, 2-15).

Turning our attention next to potential mechanisms of cholesterol removal from

macrophages (i.e. diminished rates of cholesterol efflux), we sought to test the

hypothesis that dietary sources of TMAO inhibit reverse cholesterol transport

(RCT) in vivo using an adaptation of the model system first described by Rader and colleagues90. Mice were placed on normal chow or diets supplemented with

either carnitine or choline. After several weeks of diet, [14C]cholesterol-loaded

peripheral macrophages were injected subcutaneously into the different groups

of mice and RCT quantified by counting fecal radiolabel cholesterol, as described

under Methods. Remarkably, a significant (~30%, P<0.05) decrease in RCT was

observed in mice on either the choline or carnitine supplemented diets compared

to normal chow controls (Fig. 2-5d, left panel). Furthermore, suppression of gut microbiota (and plasma TMAO levels) with oral broad spectrum antibiotics completely blocked the diet-dependent (for both choline and carnitine) suppression of RCT in vivo (Fig. 2-5d, middle panel), suggesting that a gut flora-generated product (e.g. TMAO) inhibits RCT in vivo (Supplementary Fig.

2-16). To test this hypothesis, in a separate series of studies mice were placed on either normal chow versus a diet supplemented with TMAO, and after several weeks, [14C]cholesterol-loaded peripheral macrophages were injected

65

subcutaneously for RCT quantification. Analyses of fecal [14C]sterol levels

showed significant reduction in mice on the TMAO containing diet (35% decrease

relative to normal chow, P<0.05) (Fig. 2-5d, right panel). Further examination of plasma, liver and bile compartments in the normal chow versus TMAO supplemented diet mice demonstrated significant reduction in [14C]cholesterol

recovered within plasma of the TMAO fed mice (16%, p<0.05), but no significant

changes in counts recovered within the liver or bile (Supplementary Fig. 2-17).

TMAO promotes significant alterations in cholesterol and sterol

metabolism in multiple compartments in vivo

In an effort to better understand potential molecular mechanism(s) through which

TMAO reduces RCT in vivo, we examined candidate genes and biological

processes in multiple compartments (i.e. macrophage, plasma, liver, intestine)

known to participate in cholesterol/sterol metabolism and RCT. Mouse peritoneal

macrophages recovered from C57BL/6J mice were exposed to TMAO in vitro

and mRNA levels of cholesterol transporters ATP-binding cassette, sub-family A

(ABC1), member 1 (Abca1), scavenger receptor class B, member 1(Srb1) and

ATP-binding cassette, sub-family G, member 1 (Abcg1) were examined. Modest

but statistically significant increases in expression of Abca1 and Abcg1 were

noted (P < 0.05) (Supplementary Fig. 2-18). Parallel examination of plasma

recovered from both groups of mice showed no significant differences in total

cholesterol and HDL cholesterol concentrations (Supplementary Table 2-5). To

assess the potential biological significance of the modest TMAO-induced

66

changes in macrophage cholesterol transporter mRNA levels observed, parallel studies were performed quantifying cholesterol efflux from [14C]cholesterol-

loaded macrophages cultured in media in the absence vs. presence of TMAO.

Modest but statistically significant increases in cholesterol efflux to apolipoprotein

A1, but not to HDL, as cholesterol acceptor were noted in the macrophages cultured in vitro in the presence of TMAO (Supplementary Fig. 2-19).

Collectively, these results show that TMAO promotes modest changes in

macrophage cholesterol transporter expression and function both in vitro and in

vivo; however, the directionality of the modest changes induced by TMAO are

opposite to what one would expect inasmuch as an increase in these

transporters cannot account for the observed significant global reductions in RCT

in vivo induced by TMAO.

In parallel studies, we examined the expression levels of cholesterol transporters

(i.e. Sr-b1, Abca1, Abcg1, Abcg5, Abcg8, and Shp) within mouse liver between

the normal chow vs. TMAO dietary groups. No significant differences were noted

(Supplementary Fig. 2-20, 2-21). In contrast, liver expression of the key bile

acid synthetic enzymes Cyp7a1 and Cyp27a1 showed significant reductions in

mice supplemented with dietary TMAO (p<0.05 each; Fig. 2-5e;). Interestingly,

dietary supplementation of TMAO did not decrease expression of Shp, an

upstream regulator of Cyp7a1, suggesting another upstream target for TMAO

(Supplementary Fig. 2-20). Bile acid transporters in the liver (e.g. Oatp1, Oatp4,

Bsep, Mrp2, Ephx1/mEH, and Ntcp) also showed a dietary TMAO-induced

67

decrease in expression (p<0.05 each; Fig. 2-5f). Despite these TMAO-induced

changes in mouse liver, no significant differences in bile acid transporter

expression in the gut were noted between dietary groups (Supplementary Fig.

2-22). Taken together, these data suggest that the gut flora dependent metabolite TMAO fosters significant alterations in a major pathway for cholesterol

elimination from the body, the bile acid synthetic pathway. To confirm these

findings, the total bile acid pool size was examined. Mice supplemented with

TMAO showed significant decreases in the total bile acid pool size (Fig. 2-6a).

Dietary supplementation with TMAO also markedly reduced expression of both

intestinal cholesterol transporters Npc1L1 (transports cholesterol into enterocyte

from the gut lumen91; and Abcg5/8 (transports cholesterol out of enterocyte into

gut lumen91; (Supplementary Figure 2-23). Previous studies with either Cyp7a1

or Cyp27a1 null mice have demonstrated a reduction in cholesterol absorption. In

separate studies, dietary TMAO supplementation similarly promoted a reduction

(26%, P <0 .01) in total cholesterol absorption (Fig. 2-6b).

Discussion

L-carnitine has been studied for more than a century since its initial discovery in

1905 from muscle extracts92. Although eukaryotic organisms can endogenously

produce L-carnitine, only prokaryotic organisms have known metabolic pathways

that can catabolize L-carnitine82. While a role for gut microbiota in TMAO

production from dietary carnitine has been suggested from studies in rats, and

TMAO production from several dietary trimethylamine containing compounds has

68

been suggested in humans, a role for gut microbiota in production of TMAO from dietary L-carnitine in humans has not yet been demonstrated93-95. The present

studies reveal an obligatory role of gut flora in the production of TMAO from

orally ingested L-carnitine in humans (Fig. 2-6c). They also reveal an additional

potential nutritional basis in the pathogenesis of CVD that involves dietary L-

carnitine, an abundant nutrient in meat, the intestinal microbial community, and

production of the recently identified pro-atherosclerotic down-stream metabolite,

TMAO. Finally, they show that the gut flora dependent metabolite, TMAO,

impacts multiple distinct compartments and processes involving cholesterol and

sterol metabolism in vivo, with net increase in atherosclerosis in vivo through a

combination of both enhanced forward cholesterol transport into macrophages

and reduced reverse cholesterol transport (Fig. 2-6c).

The present studies also suggest a mechanistic rationale for the observed

relationship between dietary red meat ingestion and accelerated atherosclerosis.

Although L-carnitine is endogenously produced in all mammals, consuming foods

rich in L-carnitine (predominantly red meat and to a lesser extent dairy products)

can significantly increase fasting human L-carnitine plasma levels96. Meats and

full fat dairy products are abundant foods in the Western diet and excess

consumption of these is commonly cited as a major contributor to CVD morbidity

and death worldwide. Moreover, numerous studies have suggested a decrease

in atherosclerotic disease risk in vegan/vegetarian individuals when compared to

omnivorous subjects97-99. Together, L-carnitine and choline containing lipids can

69

constitute up to 2%84,85,100 of these foods, suggesting that gut flora dependent

production of TMAO may have a significant contributory role in the pathogenesis of atherosclerosis, particularly in omnivorous subjects.

Despite the elaboration of this new diet - gut microbiota- host interaction as it relates to CVD pathogenesis and TMAO formation, the molecular mechanism(s) accounting for how TMAO promotes acceleration of atherosclerosis in vivo are only partially illuminated. As shown in the present studies, one potential mechanism is through reduction in RCT. Both dietary carnitine and choline each promoted a significant reduction in RCT in vivo, but only in the presence of intact intestinal microbiota when TMAO was produced (Fig. 2-5b). Importantly, suppression of intestinal microbiota and TMAO production completely eliminated the diet-dependent inhibition in RCT with both choline and carnitine supplementation, and dietary supplementation with TMAO directly promoted a similar ~30-35% reduction in RCT in vivo. These results are thus consistent with a gut microbiota dependent mechanism in the setting of specific dietary exposures (such as a diet rich in carnitine and total choline) whereby generation of TMAO impairs RCT in vivo and contributes to a pro-atherosclerotic phenotype.

One additional mechanism through which TMAO may contribute to accelerated atherosclerosis is by influencing macrophage cholesterol metabolism, leading to cholesterol deposition and foam cell formation, since macrophages from TMAO supplemented mice also demonstrate significant increases in scavenger receptor expression (SRA1 and CD36) 69 (Fig. 2-6c). Within the macrophage, TMAO does

70

not appear to alter desmosterol levels, cholesterol biosynthetic enzyme expression levels, or LDL receptor expression levels, and thus does not appear to directly impact either the regulation of cholesterol biosynthetic and uptake pathways initially reported by Brown and Goldstein4,5, or the more recently described regulatory role of desmosterol by Glass and colleagues in integrating macrophage lipid metabolism and inflammatory gene responses89. Within the liver, a consistent finding observed to be associated with elevated TMAO levels is decreased bile acid pool size and altered composition, as well as reduction in key bile acid synthesis and transport proteins (Figs. 2-5, 2-6). However, whether these changes contribute to the reductions in RCT in vivo that accompany TMAO supplementation are unclear. They are consistent with reports that human genetic variants in the Cyp7a1 gene, the major bile acid synthetic enzyme and rate limiting step in the catabolism of cholesterol, are associated with reduced bile acid synthesis, elevated plasma cholesterol levels refractory to statin therapy, decreased bile acid secretion into the intestines and enhanced atherosclerosis101-103. Further, up- (as opposed to down-) regulation of Cyp7a1 is reported to lead to an expansion of the bile acid pool size, increased RCT, and reduced atherosclerotic plaque in susceptible mice104-106. Moreover, an overall increase in bile acid secretion via alternative mechanisms has been reported to be associated with reduced atherosclerosis and an increase in reverse cholesterol transport105. Within the intestines, TMAO again was associated with marked changes in cholesterol metabolism (Fig. 2-6), but the significant reductions in cholesterol absorption observed, while consistent with the reduction

71

in intestinal Npc1L1107 (and hepatic Cyp7a1 and Cyp27a1108,109), cannot explain

the reproducible reduction in RCT observed in mice supplemented with TMAO.

Thus, the molecular mechanisms through which the gut microbiota → TMAO pathway inhibits RCT in vivo are not entirely clear, and whether there exist additional mechanisms through which TMAO exerts a pro-atherosclerotic effect remains to be determined. Finally, it is not known whether TMAO interacts with a specific receptor(s) directly to promote the many observed biological effects, or

whether it acts to alter signaling pathways indirectly by altering protein

conformation (i.e., via allosteric effects). A small quaternary amine with some

aliphatic character, TMAO is reportedly capable of directly inducing

conformational changes in proteins, including both stabilization of protein folding,

and functioning as a small molecule protein chaperone110,111. It is also of interest

that recent studies show that TMA can influence signal transduction by direct interaction with a family of G protein-coupled receptors112,113. It is thus

conceivable that TMAO may potentially alter a multitude of signaling pathways

without directly acting at a “TMAO receptor”.

One of the more remarkable finding of the present studies is the magnitude with which long term preceding dietary habits impacts TMAO synthetic capacity in

both humans (i.e. vegan/vegetarian vs. omnivore) and mice (normal chow vs.

chronic carnitine supplementation). Microbial composition analyses from both

humans (fecal) and mice (cecal) revealed specific taxa that segregated with both

preceding dietary status and plasma TMAO levels. Recent studies have shown

72

that significant global changes in gut microbial composition, or "enterotype" (i.e.

the clustering of microbial communities), are associated with long-term dietary

changes49, and indeed, we observed that plasma TMAO levels were significantly

different within subjects segregated according to prior reported enterotypes (Fig.

2-2e). Using a combination of studies involving germ-free mice, as well as in both

humans and mice before vs. following suppression of intestinal microflora using a cocktail of poorly absorbed antibiotics, an obligatory role for gut microbiota in

TMAO formation from dietary carnitine was shown. The marked differences observed in TMAO production following an "L-carnitine challenge" within omnivore versus vegan subjects (Fig. 2-2) is striking, consistent with the observed differences in microbial community composition. Recent reports have shown significant differences in microbial communities among vegetarians and vegans versus those who commonly consume animal proteins in their diet114. Of

note, we observed a significant increase in baseline plasma TMAO

concentrations in what historically was called enterotype 2 (Prevotella), a

relatively rare enterotype that previously in one study was associated with low

animal fat and protein consumption49. Notably, in our study, 3 of the 4 individuals

classified into enterotype 2 are self-identified omnivores suggesting more

complexity in the human gut microbiome perhaps than anticipated with only a few

enterotypes. Indeed, other studies have demonstrated variable results in

associating human bacterial genera, including Bacteroides and Prevotella, to

omnivorous and vegetarian eating habits86,115. This complexity is no doubt in part

attributed to the fact that there are many species within any genus and distinct

73

species within the same genus may have different capacity to use carnitine as a fuel and form TMA. Indeed, prior studies have suggested that multiple bacterial

strains can metabolize carnitine in culture116, and by analogy, comparison of

distinct species within the genus Clostridium reveals some that are capable and

others not of using choline as the sole source of carbon and nitrogen in

culture117. A search of the Prevotella genus, for example, reveals ~250 known

species (NCBI search 09-24-2012). The present studies, coupled with the

demonstration of both inducibility of enhanced L-carnitine metabolism and TMAO

production with antecedent L-carnitine feeding, and the association of bacterial

taxa that associate with a carnitine enriched diet (Figs 2-2, 2-3), suggests that

multiple “proatherogenic” (i.e. TMA/TMAO producing) species, likely exist.

Consistent with this supposition, others have reported that several bacterial

phylotypes are associated with a history of atherosclerosis, and that the human

gut flora biodiversity may at least in part be influenced by carnivorous eating

habits45,49,118.

The association observed between plasma carnitine levels and both prevalent

and incident cardiovascular risks further supports the potential physiological

importance of the carnitine → gut microbiota → TMA/TMAO → atherosclerosis

pathway. Of note, the association remained significant even following

adjustments for traditional cardiovascular risk factors and comorbidities. The

significance of this association was only attenuated (becoming completely non-

significant) following addition of plasma TMAO levels to the model. These

74

findings are consistent with the proposed mechanism whereby the association of

carnitine with atherosclerotic cardiovascular disease risks is mediated via the gut

microbiota metabolite TMAO, and not the dietary nutrient carnitine itself. Further,

we are tempted to speculate that similar to the increased sensitivity observed with use of an oral glucose tolerance test versus a fasting plasma glucose level in the diagnosis of diabetes, it is possible that use of a provocative challenge test involving a defined oral load of isotope labeled L-carnitine alone or in combination with other trimethylamine precursor nutrients like phosphatidylcholine or choline, has a greater potential to identify those at increased risk for cardiovascular disease over fasting plasma TMAO levels alone. A provocative oral challenge test following isotope labeled L-carnitine administration may also better allow one to characterize and identify microbial communities most likely to promote disease. There are several reports of specific intestinal anaerobic and aerobic prokaryotic bacterial species that can utilize L- carnitine as a carbon nitrogen source81,82,119. Based upon the present studies,

one might speculate that a microbial composition that has adapted to produce

more TMA/TMAO may equip the host with greater potential to develop enhanced atherosclerotic disease burden in the setting of a diet rich in trimethylamine containing nutrients. It logically follows, but remains to be proven, that development of a prebiotic or probiotic intervention that alters microbial compositions associated with enhanced TMAO production may serve as an alternative therapy for the treatment or prevention of atherosclerotic disease.

75

L-carnitine has indispensable roles in animal metabolism. It is essential in the import of activated long chain fatty acids from the cytoplasm into mitochondria for

β-oxidation. It also participates in the transport of intermediate and short chain organic acids from peroxisomes into mitochondria, functions as a reservoir of activated acetyl groups, and impacts upon crucial steps of intermediate

metabolism81,82. As a consequence of these critical roles, L-carnitine

supplementation has been widely studied and therefore merits some comment.

There are case reports of L-carnitine supplementation showing benefit in terms of

symptomatic improvement for individuals with inherited primary and acquired

secondary L-carnitine deficiency syndromes83. L-Carnitine has also been

suggested for treatment of subjects with end stage renal disease undergoing

hemodialysis, as they commonly acquire a secondary L-carnitine deficiency that

may participate in several dialysis-related symptoms including muscle weakness

and diminished exercise capacity. While some of these studies have shown

improvement following supplementation, others have yielded conflicting results,

possibly in part because of heterogeneity in the route of administration of L-

carnitine amongst other factors120,121. Oral treatment with L-carnitine (1gram) in

end stage renal disease patients undergoing hemodialysis over a brief period has

been shown to raise plasma L-carnitine (pre-dialysis) to normal levels, but with

accompanying substantial increases in plasma TMAO to supraphysiological

levels. A broader potential therapeutic scope for L-carnitine and two related

metabolites, acetyl-L-carnitine and propionyl-L-carnitine, has also been explored

in the treatment of acute ischemic events including myocardial infarction and

76

stroke, as well as for chronic treatment of a multitude of cardio-metabolic

disorders like PAD, congestive heart failure and diabetes121. Here too, results

from studies are conflicting. One potential explanation for the discrepant findings

of various intervention studies may be explained in part by variations in the route

of administration and the length of time of L-carnitine dosing. Many studies have provided one of the L-carnitines over short intervals of treatment, and often in part via parenteral route, bypassing the gut flora. The obligatory role of the gut flora in the promotion of TMAO production and atherosclerotic disease

enhancement observed in the present studies likely also explains the apparent

contradictory report from Sayed-Ahmed et al. that showed intraperitoneal

administration of L-carnitine reduced atherosclerotic lesion in the

hypercholesterolemic rabbit model through unclear mechanisms122. There are

also a number of studies showing long term treatment with Mildronate, an

inhibitor of L-carnitine synthesis, can both reduce atherosclerosis and promote

cardio-protective effects123,124. Carnitine metabolism is clearly complex, and

administration of the L-carnitine vs. acetyl or proprionyl carnitine forms may not

elicit the same responses.

Discovery of a link between oral carnitine ingestion and cardiovascular disease

risks has broad health related implications. The results of the present studies

underscore the need for further examination of the safety of chronic oral L-

carnitine supplementation. They also argue for careful attention to route of

administration when designing and comparing carnitine intervention

77

studies/strategies. Lastly, the present studies raise the possibility that chronic ingestion of high amounts of carnitine through either supplements and/or carnivorous eating habits may under some conditions prime our gut microbiota to become proatherogenic. Further studies on the long term health impact of increased levels of carnitine ingestion are needed.

Acknowledgements

We thank L. Kerchenski and C. Stevenson for assistance in performing the clinical studies; A. Pratt, S. Neale, M. Pepoy, and B. Sullivan for technical assistance with human specimen processing and routine clinical diagnostic testing; E. Klipfell, F. McNally, and M. Berk for technical assistance, and the subjects who consented to participate in these studies. Mass Spectrometry instrumentation used was housed within the Cleveland Clinic Mass Spectrometry

Facility with partial support through a Center of Innovation by AB SCIEX. Germ free animals used were obtained from the University of North Carolina

Gnotobiotic Facility, which is supported by P30-DK034987-25-28 and P40-

RR018603-06-08.

Methods

Materials and general procedures

C57BL/6J, Apoe–/– and C57BL/6J mice were obtained from Jackson

Laboratories. All animal studies were performed under approval of the Animal

Research Committee of the Cleveland Clinic. Mouse plasma total cholesterol and

78

triglycerides, and human fasting lipid profiles, glucose, creatinine, and high

sensitivity C-reactive protein levels were assayed using the Abbott ARCHITECT

platform model ci8200 (Abbott Diagnostics, Abbott Park, IL). Mouse HDL

cholesterol was determined enzymatically (Stan bio, Houston, TX) from mouse

plasma HDL isolated using density ultracentrifugation. Mouse plasma insulin

measurements were performed using the Mercodia Mouse Insulin Elisa Kit

(Uppsala, Sweden). Human plasma MPO levels were measured using the US

Food and Drug Administration-cleared CardioMPO test (Cleveland Heart Lab,

Inc., Cleveland, OH). Liver triglyceride content was quantified by GPO reagent

(Pointe Scientific, Canton, MI) and normalized to liver weight in grams as

described125. Liver cholesterol was quantified in liver homoginates in which

coprostanol (Steraloids, Inc, Newport, RI) was added as an internal standard,

lipids extracted by the Folch method (chloroform:methanol (2:1, v/v)), and then

cholesterol quantified as its trimethylsilane (TMS) derivative (Sylon HTP, Sigma-

Aldrich, Sigma St. Louis, MO) by GC/MS (Agilent 5973N model, Santa Clara CA)

on a DB-1 column (12m x 0.2 mm diameter x 0.250um film thickness)126,127.

Research subjects

Two cohorts of subjects were used in the present studies. The first group of volunteers had extensive dietary questioning, and stool, plasma and urine collection. A subset of subjects with stool collected also underwent oral carnitine challenge testing (n = 5 omnivores and n = 5 vegans), consisting of d3(methyl)carnitine (250 mg within a veggie capsule). Where indicated,

79

additional omnivores, and an individual vegan, also underwent carnitine

challenge testing with combined ingestion of the synthetic d3-carnitine capsule

(250 mg) and an 8 ounce steak (within 10 minutes). Male and female volunteers

gave written informed consent and included individuals of at least 18 years of

age. Volunteers were excluded if they were pregnant, had chronic illness

(including a known history of heart failure, renal failure, pulmonary disease,

gastrointestinal disorders, or hematologic diseases), an active infection, received

antibiotics within 2 months of study enrollment, used any over the counter or

prescriptive probiotic or bowel cleansing preparation within the past 2 months,

ingested yogurt within the past 7 days, or had undergone bariatric or other

intestinal (e.g. gall bladder removal, bowel resection) surgery.

The second cohort of subjects (n = 2,595) were from GeneBank, a

research registry of sequential consenting stable subjects undergoing elective

cardiac evaluation and who were subsequently followed longitudinally for incident

cardiovascular disease (CVD) outcomes69,128. Patients with a recent (< 4 weeks) clinical history of myocardial infarction or elevated troponin I (> 0.03 md dl–1) at

enrollment were excluded from the study. CVD was clinically defined as having a

previous history of coronary artery disease (CAD), peripheral artery disease

(PAD), and/or cerebral vascular disease (history of a transient ischemic attack or

cerebrovascular accident), history of revascularization (coronary artery bypass

graft, angioplasty, or stent) or significant angiographic evidence of CAD (≥50%

stenosis) in at least one major coronary artery. Subjects with CAD included

patients with diagnoses of stable or unstable angina, myocardial infarction,

80

history of coronary revascularization, or angiographic evidence of ≥50% stenosis

of one or more major coronary arteries. PAD was defined as subjects having

clinical evidence of extra-coronary atherosclerosis. Medications were

documented by patient interview and chart review. All subjects gave written

informed consent. The Institutional Review Board of the Cleveland Clinic

approved all study protocols.

General Statistics

The Student’s t-test or the Wilcoxon Rank-Sum test for continuous variables was

used for two-group comparison. The analysis of variance (ANOVA, if normally

distributed) or Kruskal-Wallis test (if not normally distributed) was used for

multiple group comparisons of continuous variables and a Chi-square test was

used for categorical variables. Odds ratios for cardiac phenotypes (CAD, PAD,

and CVD) and corresponding 95% confidence intervals were calculated using

logistic regression models. Kaplan–Meier analysis with Cox proportional hazards

regression was used for time-to-event analysis to determine Hazard ratio (HR)

and 95% confidence intervals (95%CI) for adverse cardiac events (death, MI,

stroke, and revascularization). Adjustments were made for individual traditional

cardiac risk factors (age, gender, diabetes mellitus, systolic blood pressure,

former or current cigarette smoking, low-density lipoprotein cholesterol, high-

density lipoprotein cholesterol), extent of CAD, left ventricular ejection fraction,

history of MI, baseline medications (aspirin, statins, β-blockers, and ACE inhibitors), and renal function by estimated creatinine clearance. A robust

81

Hotelling T2 test was used to examine the difference in the proportion of specific

bacterial genera along with subject TMAO levels between the different dietary

groups. All data was analyzed using R software version 2.15 and Prism

(Graphpad Software, San Diego, CA).

Metabolomics study

In a previous study we reported results from a metabolomics study in which small

molecule analytes were sought that associated with cardiovascular risks69. The metabolomics study design used a two stage screening strategy. In the first phase, totally unbiased metabolomics studies were performed on randomly selected plasma samples from a Learning Cohort generated from Genebank subjects that was comprised of 50 cases, defined as those that experienced a major adverse cardiovascular event (defined as non-fatal myocardial infarction, stroke, or death) in the 3 year period following enrollment, versus age- and gender-matched controls (n = 50) that did not experience an event. A second phase (Validation Cohort) of unbiased metabolomics analyses were performed on an independent (non-overlapping) cohort of cases (n = 25) and age- and gender-matched controls (n = 25) using identical inclusion / exclusion criteria1.

Analytes were only known for their m/z and retention time, with identities unknown. Analytes considered of interest in the metabolomics studies were selected based on the following criteria: (i) the unknown analyte had a significant difference (P < 0.05) between cases and controls in the Learning and Validation

Cohorts after a two sided Bonferroni adjusted t-test; (ii) the unknown analyte had

82

a significant (P < 0.05) dose-response relationship between analyte peak area

and major adverse cardiovascular event risk using an unadjusted log-rank test of

trend; and (iii) to facilitate future quantification and structural identification efforts,

analytes had to have a signal-to-noise ratio of 5:1 in at least 75% of subjects

within cases and controls of both the Learning and Validation Cohorts as

previously described69.

While an ion with m/z of 162 and retention time identical to carnitine was

not among the top analytes identified in the above metabolomics studies, we

attempted for the present studies to examine the original data, this time using

less stringent criterion, with the hypothesis-generated focus of examining just the

single ion that had chromatographic and mass spectral characteristics observed

identical with standard L-carnitine: namely, m/z = 162 and appropriate retention time. Examination of Supplementary Table 2-1 shows that an analyte with appropriate m/z and retention time in the Learning and Validation Cohorts was observed that failed to meet significance using the originally used stringent criteria (the more strict Bonferroni adjusted p value). However, reexamination of the combined Learning and Validation cohorts (n = 75 cases, n = 75 controls)

without adjustment for multiple testing (since only one analyte here was being

screened for) showed the unknown analyte giving rise to an ion at m/z = 162 with

retention time identical to carnitine was associated with atherosclerotic disease

outcomes.

Identification of L-carnitine and d9-carnitine preparation

83

Matching CID spectra of the unknown metabolite of interest with a precursor ion

at m/z = 162 and authentic L-carnitine standard were examined using a Cohesive

Technologies Aria HPLC interfaced to a AB Sciex API 5000 triple quadrupole mass spectrometer (Applied Biosystems) in positive ion mode by a method described previously69. Mouse and human samples were also spiked with

synthetic d9(trimethyl)-carnitine as internal standard. Samples were analyzed

using a similar system as above except a Shimadzu (Columbia, MD) dual

gradient HPLC system was interfaced to the AB Sciex API 5000 tandem mass

spectrometer. Multiple distinct parent → product ion transitions specific for the

natural abundance and d9-isotopologue of carnitine were monitored

simultaneously in the spiked sample, to determine if multiple characteristic MRM

channels for each isotopologue of carnitine were present and co- chromatographed.

Synthesis of the d9-carnitine standard for the above experiment, and for use as internal standard in stable isotope dilution LC/MS/MS analyses of carnitine and synthetic d3-carnitine following a carnitine challenge, were prepared and characterized as follows: First, 3-hydroxy-4-aminobutyric acid

(Chem-Impex Intl.) was dissolved in methanol and reacted with d3-methyl iodide

(Cambridge Isotope Labs, Boston, MA) in the presence of potassium hydrogen carbonate to give d9-carnitine, as per Chen and Benoiton129. The d9-L-carnitine

was isolated by passing the reaction mixture directly over a silica gel column

rinsing with additional methanol, and then eluting the heavy isotope labeled L-

carnitine with 30% v/v water in methanol. The product was dried via azeotropic

84

distillation of absolute ethanol and subsequently recrystallized from ethanol and acetone. The white to off-white crystalline product was dried over P2O5 in vacuo and stored refrigerated. TLC on silica gel eluted with methanol plus 0.2%v/v formic acid visualized by iodine staining showed one spot with the same Rf as L- carnitine. The mass spectrum of the compound dissolved in 50% v/v methanol / water (5 mM formic acid) to a concentration of 50 µg ml–1 exhibited a base peak at m/z = 171 in the positive ion mode corresponding to [M]+. CID fragments peaks were observed at m/z = 111,103, 85, 69, 57, and 43. Mass spectral fragmentation patterns and m/z ratios are consistent with the L-carnitine except for those fragment ions that contain the trimethylammonium group; these ions exhibit fragments 9 atomic mass units (amu) higher than the corresponding signals from L-carnitine due to the incorporation of 9 deuterium atoms on the methyl groups attached to the nitrogen.

Quantification of TMAO, TMA, and L-carnitine

Stable isotope dilution LC/MS/MS was used to quantify TMAO, TMA and L- carnitine from acidified plasma samples in positive MRM mode. Precursor → product ion transitions at m/z 76 to 58, m/z 162 to 60 and m/z 60 to 44 were used for TMAO, L-carnitine, and TMA respectively. As internal standards, d9(trimethyl)TMAO (d9-TMAO), d9(trimethyl)carnitine (d9-carnitine), and d9(trimethyl)TMA (d9-TMA) were added to mouse and human plasma samples for their respective native compounds. Increasing concentrations of L-carnitine,

TMA and TMAO standards with a fixed amount of internal standard were added

85

to human control plasma to generate calibration curves for determining plasma concentrations of each analyte, using methods similar in approach to that previously described69, with samples run on an AB Sciex API 5000 triple quadrupole mass spectrometer.

Human microbiota analyses

Stool samples were stored at – 80 oC and DNA for the gene encoding 16SrRNA was isolated using the MoBio PowerSoil kit (Carlsbad, CA) according to the manufacturer's instructions. DNA samples were amplified using V1-V2 region primers targeting bacterial 16S genes and sequenced using 454/Roche Titanium technology. Sequence reads from this study are available from the Sequence

Read Archive (CaFE: SRX037803, SRX021237, SRX021236, SRX020772,

SRX020771, SRX020588, SRX020587, SRX020379, SRX020378

(metagenomic). COMBO: SRX020773, SRX020770). Overall association between TMAO measurements and microbiome compositions was assessed using PermanovaG130 by combining both the weighted and unweighted UniFrac distances. Associations between TMAO measurements and individual taxa proportions were assessed by Spearman's rank correlation test. False discovery rate (FDR) control based on the Benjamini–Hochberg procedure was used to account for multiple comparisons when evaluating these associations. Each of the samples was assigned to an enterotype category based on their microbiome distances (Jensen-Shannon distance) to the medoids of the enterotype clusters as defined in the COMBO data131. Association between enterotypes and TMAO

86

level was assessed by Wilcoxon rank sum test. Student’s t-test was used to test the difference in means of TMAO level between omnivores and vegans. A robust

Hotelling T2 test 132 was used to examine the association between both the proportion of specific bacterial taxa and TMAO levels in groups using R software version 2.15.

Mouse microbiota analysis

Microbial community composition in mouse cecal contents was assessed by pyrosequencing 16S rRNA genes derived from the mice of normal chow diet (n =

11) and L-carnitine diet (n = 13). DNA was isolated using the MoBio PowerSoil

DNA Isolation Kit (Carlsbad, CA). The V4 region of the 16S ribosomal DNA gene was amplified using bar-coded fusion primers (F515/R806) with the 454 A

Titanium sequencing adapter. The barcoded primers were achieved following the protocol described by Hamady et al133. Sample preparation was performed similarly to that described by Costello et al.134. Each sample was amplified in triplicate, combined in equal amounts and cleaned using the PCR clean-up kit

(Mo Bio, Carlsbad, CA). Cleaned amplicons were quantified using Picogreen dsDNA reagent (Invitrogen, Grand Island, NY) before sequencing using 454 GS

FLX titanium chemistry at the EnGenCore Facility at the University of South

Carolina. The raw data from the 454 pyrosequencing machine were first processed through a quality filter that removed sequence reads that did not meet the quality criteria. Sequences were removed if they were shorter than 200 nucleotides, longer than 1,000 nucleotides, contained primer mismatches,

87

ambiguous bases, uncorrectable barcodes, or homopolymer runs in excess of six bases. The remaining sequences were analyzed using the open source software package Quantitative Insights Into Microbial Ecology (QIIME135,136). A total of

11519 quality filtered reads were obtained from 23 samples (1 sample was removed due to low number of sequences). Individual reads that passed filtering were distributed to each sample based on bar-code sequences. De-multiplexed sequences were assigned to operational taxonomic units (OTUs) using UCLUST with a threshold of 97% pair-wise identity. Representative sequences were selected and BLASTed against a reference Greengenes reference database. For each resulting OTU, a representative sequences were selected by choosing the most abundant sequence from the original post-quality filtered sequence collection. The taxonomic composition was assigned to the representative sequence of each OTU using Ribosomal Database Project (RDP) Classifier

2.0.1137 The relative abundances of bacteria at each taxonomic level (e.g., phylum, class, order, family and genus) were computed for each mouse. For tree-based analyses, a single representative sequence for each OTU was aligned using PyNAST138, then a phylogenetic tree was built using FastTree. The phylogenetic tree was used to measure the β-diversity (using unweighted

UniFrac) of samples139. Two-way ANOVAs were conducted to evaluate the effects of diet with P values corrected for multiple comparisons. Spearman correlations between relative abundance of gut microbiota and TMA and TMAO levels and association testing were performed in R. False discovery rates (FDR) of the multiple comparisons were estimated for each taxon based on the P-

88

values resulted from correlation estimates. A robust Hotelling T2 test 132 was used

to examine the association between both the proportion of specific bacterial taxa

and mouse plasma TMA/TMAO levels in groups using R software version 2.15.

Aortic root lesion quantification

Apolipoprotein E knockout mice on C57BL/6J background (C57BL/6J, Apoe–/–)

were weaned at 28 days of age and placed on a standard chow control diet

(Teklad 2018). L-carnitine was introduced into the diet by supplementing mouse drinking water with 1.3% L-carnitine (Chem-Impex Intl.), 1.3% L-carnitine and antibiotics, or antibiotics respectively. The antibiotic cocktail dissolved in mouse drinking water has previously been shown to suppress commensal gut microbiota, and included 0.1% Ampicillin sodium salt (Fisher Scientific), 0.1%

Metronidazole, 0.05% Vancomycin (Chem Impex Intl.), and 0.1% Neomycin sulfate (Gibco) 35,69. Mice were anaesthetized with Ketamine/Xylazine before

terminal bleeding by cardiac puncture to collect blood. Mouse hearts were fixed

and stored in 10% neutral buffered formalin before being frozen in OCT for sectioning. Aortic root slides were stained with Oil-red-O and counterstained with

Haematoxylin. The aortic root atherosclerotic lesion area was quantified as the

mean of sequential sections of 6 microns approximately 100 microns apart69.

Human L-carnitine challenge test and d3-L-carnitine preparation

Consented adult men and women fasted overnight (12 hours) before performing the "L-carnitine challenge test", which involved baseline blood and spot urine

89

collection, and then oral ingestion (T = 0 at time of initial ingestion) of a veggie

caps capsules containing 250 mg of a stable isotope labeled d3-L-carnitne

(under Investigational New Drug exemption). Where indicated, for a subset of

subjects, the carnitine challenge also included a natural source of L-carnitine (an

8 ounce sirloin steak cooked medium on a George Forman Grill) in a 10 minute

period concurrent with taking the capsule containing the d3-carnitine. After

combined ingestion of the steak and d3-L-carnitine, sequential venous serial

blood draws were performed at respective time points, and a 24 hour urine

collection was performed. An ensuing 1 week treatment period of oral antibiotics

(Metronidazole 500 mg bid, Ciprofloxacin 500 mg bid) was given to suppress

intestinal microbiota that use carnitine to form TMA and TMAO before repeating

the L-carnitine challenge. After at least 3 weeks off of all antibiotics to allow

reacquisition of intestinal microbiota, a third and final L-carnitine challenge test

was performed. Dietary habits (vegan vs ominivore) were determined using a

questionnaire assessment of dietary L-carnitine intake, similar to that conducted

by the Atherosclerotic Risk in Community (ARIC) study140.

Synthesis of d3-L-carnitine for carnitine challenge tests was prepared and

characterized as follows: L-Norcarnitine (3-hydroxy-4-dimethylaminobutyric acid)

was prepared from L-carnitine (Chem Impex International, Woodale, IL) with

thiophenol (Sigma Aldrich Milwaukee, WI) in N,N-dimethylaminoethanol (Sigma

Aldrich Milwaukee, WI) and subsequently converted to its sodium salt with sodium hydroxide by the method of Colucci, et. al.141. Sodium L-norcarnitine was

90

recrystallized three times from ethanol and 3 volumes of ethyl acetate prior to the

subsequent conversion to d3-L-carnitine. TLC on silica gel eluted with methanol

plus 0.2%v/v formic acid visualized by iodine staining showed one major spot

–1 with a higher Rf (> 0.1) than L-carnitine. 600MHz 1H-NMR (10 mg ml in D2O): δ

2.1ppm (singlet, 6H), δ 2.2ppm (complex multiplet, 3H), δ 2.3ppm (complex multiplet, 1H) δ 4.0ppm (complex multiplet, 1H). The mass spectrum of the compound dissolved in 50% v/v, methanol/water (5 mM formic acid) to a concentration of 50 µg ml–1 exhibited a base peak at m/z = 148 in the positive ion

mode corresponding to [M+H]+. CID fragments peaks were observed at m/z =

130,112, 94, 88, 85, 84(base) 82, 71, 69, 58, 57, 56, and 43. Sodium L-

norcarnitine was dissolved in methanol and reacted with d3-methyl iodide

(Cambridge Isotope Labs, Boston, MA) in the presence of potassium hydrogen

carbonate to give d3-L-carnitine as per Chen and Benoiton129. The d3-L-carnitine

was isolated by passing the reaction mixture directly over a silica gel column

rinsing with additional methanol and then eluting the heavy isotope labeled L-

carnitine with 30% v/v water in methanol. The product was dried via azeotropic

distillation of absolute ethanol and subsequently recrystallized from ethanol and

acetone. The white to off-white crystalline product was dried over P2O5 in vacuo

and stored refrigerated. Upon analysis, the d3-L-carnitine was found to be > 98%

pure by LC/MS, NMR and TLC. TLC on silica gel eluted with methanol plus

0.2%v/v, formic acid visualized by iodine staining shows one spot with the same

1 –1 Rf as L-carnitine. 600MHz H-NMR (10 mg ml in D2O): δ 2.3ppm (complex

multiplet 2H), δ 3.1ppm (singlet 6H), δ 3.3ppm (complex multiplet, 2H) δ 4.5ppm

91

(complex multiplet, 1H), which is consistent with the spectrum obtained for L-

carnitine under the same conditions and concentration except for the singlet peak

at 3.1 ppm corresponding to 9 protons on the trimethylammonium group on L-

carnitine integrates for 6 protons (three protons less) due to the incorporation of 3

deuterium atoms on one of the methyl amino groups in this compound. The only

impurity peaks observed corresponded to residual ethanol and acetone in the

product (integrated area less than 1% of total), and these were removed by

13 placement in vacuum dessicator. C-NMR (10 mg ml in D2O): δ 64.3ppm

(multiplet, 1C), δ 70.2ppm (multiplet, 1C), δ 54.2ppm (muliplet, 2C), δ 43.1ppm

(multiplet, 1C), δ 179.0ppm (singlet 1C). The mass spectrum of the compound

dissolved in 50% v/v, methanol/water (5 mM formic acid) to a concentration of 50

µg ml–1 exhibits a base peak at m/z = 165 in the positive ion mode,

corresponding to [M]+. CID fragments peaks were observed at m/z = 105,103,

85, 63, 57, and 43. Mass spectral fragmentation patterns and m/z ratios are

consistent with the L-carnitine except for those fragment ions that contain the

trimethylammonium group; these ions exhibit fragments 3 atomic mass units

(amu) higher than the corresponding signals from L-carnitine due to the

incorporation of 3 deuterium atoms on one of the methyl groups attached to the

nitrogen.

Germ-free mice and conventionalization studies

10-week-old female Swiss Webster germ-free mice (SWGF) underwent gastric

gavage with the indicated isotopologues of L-carnitine (see below for details of L-

92

carnitine challenge) immediately following removal from the germ-free

microisolator shipper. After performing the L-carnitine challenge, germ-free mice

were conventionalized by being housed in cages with non-sterile C57BL/6J

female mice. Approximately 4 weeks later, the L-carnitine challenge was

repeated on the conventionalized Swiss Webster mice. Quantification of natural

abundance and isotope labeled carnitine, TMA and TMAO in mouse plasma was performed using stable isotope dilution LC/MS/MS as described above.

Metabolic challenges in mice

C57BL/6J female or C57BL/6J, Apoe–/– female mice were provided via gastric

gavage d3-L-carnitine (150 µl of 150 mM stock) d3-L-carnitine (synthetically

prepared as above) dissolved in water using a 1.5-inch 20-gauge intubation

needle. Plasma was collected from the saphenous vein at baseline and at the indicated time points. C57BL/6J, Apoe–/– female mice were used in the study

examining the inducibility of microbiota to generate TMA and TMAO following

carnitine feeding. For these studies, animals were placed on an L-carnitine

supplemented diet (1.3% L-carnitine in mouse drinking water) for 10 weeks.

Quantification of natural abundance and isotope labeled forms of carnitine, TMA and TMAO in mouse plasma was performed using stable isotope dilution

LC/MS/MS as described above.

Preparation of bone marrow derived macrophages for reverse cholesterol

transport studies

93

Femur bone marrow from C57BL/6J mice was collected and cultured in PFA

bags (Welch Fluorocarbon, Dover, NH) with RPMI-640 supplemented with L-cell conditioned media, β-mecaptoethanol, penicillin/streptomycin, and glutamine for

6 days. Each PFA bag of bone marrow derived macrophages were then loaded

with 40 µCi [14C] cholesterol preincubated with carbamylated LDL for 48 hours.

Carbamylated LDL was prepared as described previously128. At the end of 48

hours bone marrow derived macrophages were collected for injection into

reverse cholesterol transport mice.

Reverse cholesterol transport studies

Adult (> 8 weeks of age) C57BL/6J, Apoe–/– female mice were placed on diets for 4 weeks prior to beginning of reverse cholesterol transport experiments. Mice were individually placed into single ventilated cages with wire rack inserts

(Ancare, Spring Valley, Illinois) for a 24-48 hour acclimatization period. Mice

were injected subcutaneously in the back with 300ul of labeled bone marrow

derived macrophages as described above. Feces were collected every 24 hours,

processed, and analyzed by a modified method previously described142. Briefly, each 24 hour feces collection was extracted with 3:2 chloroform/methanol and back extracted with 1:5 0.88% KCl. The organic phase was collected dried, dissolved in scintillation fluid, and counted on a Beckman Coulter LS6500 liquid scintillation counter. Total 72 hour reverse cholesterol transport studies were calculated as a sum of each 24 hour period. Percent reverse cholesterol transport is expressed as the percentage of [14C] DPM recovered from feces

94

versus [14C] counts injected into each mouse. At the end of the 72 hour period

animals were fasted for 3 hours and then sacrificed for collection of blood, liver,

bile, and intestine. [14C] was counted in aliquots of plasma and bile dissolved in scintillation fluid and counted on a Beckman Coulter LS6500 liquid scintillation counter. [14C] was quantified in liver by extraction with 3:2 chloroform methanol

and back extraction with 2:5 0.88% KCl. Both the aqueous and organic phases

were dried, dissolved in scintillation fluid, and counted on a Beckman Coulter

LS6500 liquid scintillation counter. The percent injected was calculated as the

percentage of [14C] DPM recovered from feces versus [14C] counts injected into

each mouse normalized by liver weight analyzed.

Cholesterol absorption studies

Cholesterol absorption experiments were performed as previously described143.

Briefly, adult (> 8 weeks of age) C57BL/6J, Apoe–/– female mice were placed on

the indicated diets for 4 weeks prior to beginning of cholesterol absorption

experiments. Mice were individually placed into single ventilated cages with wire

rack inserts (Ancare, Spring Valley, Illinois) for a 24-48 hour acclimatization

period. Animals were fasted 4 hours before gavage with olive oil supplemented

with [14C] cholesterol/ [3H] β-sitostanol. Feces were collected over a 24 hour period. Feces samples and cholesterol absorption rates were calculated as previously described143. Briefly, feces were extracted with 3:2

chloroform/methanol and back extracted with 1:5 0.88% KCl. The organic phase

was collected dried, dissolved in scintillation fluid, and counted on a Beckman

95

Coulter LS6500 liquid scintillation counter. The percent cholesterol absorption was calculated as the ratio of ([14C] DPM in the feces: [3H] β-sitostanol) / the ratio of [14C] DPM: [3H] β-sitostanol gavaged subtracted from 1.

Bile acid pool size and composition

Total bile acid pool size was determined in female C57BL/6J, Apoe–/– as the total bile acid content of the combined small intestine, gallbladder, and liver, which were extracted together in ethanol with Nor-Deoxycholate (Steraloids

Newport, RI) added as an internal standard. The extracts were filtered (Whatman paper #2), dried and resuspended in water. The samples were then passed through a C18 column (Sigma St. Louis, MO) and eluted with methanol. The eluted samples were again dried down and resuspended in methanol. A portion of this was subjected to HPLC using Waters Symmetry C18 column (4.6 × 250 mm No. WAT054275, Waters Corp., Milford, MA) and a mobile phase consisting of methanol: acetonitrile: water (53:23:24) with 30 mM ammonium acetate, pH

4.91, at a flow rate of 0.7 ml min–1. Bile acids were detected by evaporative light spray detector (Alltech ELSD 800, nitrogen at 3 bar, drift tube temperature 400C) and identified by comparing their respective retention times to those of valid standards (Taurocholate and Tauro-β-muricholate from Steraloids (Newport, RI);

Taurodeoxycholate and Taurochenodeoxycholate from Sigma (St. Louis, MO);

Tauroursodeoxycholate from Calbiochem (San Diego, CA). For quantitation, peak areas were integrated using software Chromperfect Spirit (Justice laboratory software, Denville, NJ) and bile acid pool size was expressed as

96

µmol/100 g body weight (bw) after correcting for procedural losses with nor-

deoxycholate.

Cholesterol efflux studies

RAW 264.7 mouse macrophages were cultured in a 48 well plate. Macrophages

were labeled with cholesterol using 1 µCi ml–1 [3H] cholesterol preincubated with

AcLDL for 24 hours. In wells examining Abca1 dependent efflux, Abca1 was

induced with 0.3 mM 8Br-cAMP as previously described144. Cells were washed

and chased with serum free media containing 8Br-cAMP and 10 µg ml–1 (final)

human APOA1 for 6 hours (for pretreated wells) or isolated human HDL (50 µg

protein ml–1 final) in serum free media. Media was counted directly using

Beckman Coulter LS6500 liquid scintillation counter. Cells were washed and

extracted with 3:2 hexane:isopropanol. Dried extracts were then counted using a

Beckman Coulter LS6500 liquid scintillation counter. Total Cholesterol efflux was

determined as total media DPM/ (total media DPM and Total extract DPM).

Abca1 efflux was determined as the difference between cholesterol efflux in the

presence of 8Br-cAMP compared to the absence of 8Br-cAMP.

Effect of TMAO on macrophage cholesterol biosynthesis, inflammatory

genes, and desmosterol levels

The effect of cholesterol loading on macrophage cholesterol biosynthetic and

inflammation genes, LDL receptor expression levels, and desmosterol levels,

were performed by a modified method as previously described89. Briefly, mouse peritoneal macrophages (MPMs) were thioglycollate elicited 4 days prior to

97

harvest and were subsequently cultured in RPMI 1640 supplemented with 10%

FCS and penicillin/streptomycin overnight. MPMs were then lipoprotein starved in

RPMI 1640 supplemented with 10% lipoprotein deficient serum (LPDS) and

penicillin/streptomycin for 24 hours and then further cultured in the same media

for 18 hours in the presence of increasing cholesterol, AcLDL concentrations or

vehicle (carrier for AcLDL and cholesterol (Sigma, St. Louis, MO) with (+) or without (–) 300 µM TMAO dehydrate (Sigma, St. Louis, MO)). AcLDL was prepared as previously described145.

RNA was prepared and analyzed as described below. Desmosterol in the

cholesterol loading studies was quantified by stable isotope dilution GC/MS

analysis. Briefly, desmosterol was extracted from 400 µl medium by 1 ml

isopropanol/hexane/2 M acetic acid (40/10/1, vol/vol/vol) with 100 ml of 10 mg

ml–1 deuterated internal standard, cholesterol-2,2,3,4,4,6-d6 (Sigma) in

isopropanol added beforehand. After adding 1 ml hexane, the mixture was

vortexed and spun down, desmosterol and cholesterol-2,2,3,4,4,6-d6 were

extracted to the hexane layer. The medium was re-extracted by the addition of 1

ml hexane, followed by vortexing and centrifugation. The hexane layer was

collected and combined with the previous hexane extract. The extract was dried

under N2. 50 ml Sylon™ HTP (HMDS+TMCS+Pyridine, 3:1:9) (Supelco) was

added to the dried desmosterol preparative and trimethylsilyl (TMS) ethers were

achieved in 1 hour at 90 oC. Calibration curves were prepared using varying desmosterol levels and a fixed amount of stable isotope-labeled internal

98

standard, d6(2,2,3,4,4,6) cholesterol undergoing derivatization to TMS ethers. 1

ml of the TMS ethers was injected onto a 6890/5973 GC/MS equipped with an

automatic liquid sampler (Agilent Technolgies) using the positive ion chemical

ionization mode with methane as the reagent gas. The source temperature was

set at 230 °C. The electron energy was 240 eV, and the emission current was

300 µA. The cholesterol TMS ethers were separated on a J&W Scientific

(Folsom, CA) DB-1 column (20 m, 0.18 mm inner diameter, 0.18-µm film thickness). The injector and the transfer line temperatures were maintained at

250 °C. The initial GC oven temperature was set at 230 °C and increased at 20

°C/min to 270 °C then increased at 4 °C/min to 300 °C. The GC chromatograms extracted at m/z = 327 and 335 corresponding to desmosterol and cholesterol-

2,2,3,4,4,6-d6, were extracted and the peak area were integrated, respectively.

RNA preparation and real time PCR analysis

RNA was first purified from tissue (macrophage, liver, or gut) using the animal

tissue protocol from the Qiagen Rneasy mini kit. Small bowel used for RNA

purification was sectioned sequentially in 5 equal segments from the duodenum

to illeum before RNA preparation. Purified total RNA and random primers were

used to synthesize first strand cDNA using the High Capacity cDNA Reverse

Transcription Kit (Applied Biosystems, Foster City, CA) reverse transcription

protocol. Quantitative real-time PCR was performed using Taqman qRT-PCR

probes (Applied Biosystems, Foster City, CA) and normalized to tissue β-Actin by

99

the ∆∆CT method using StepOne Software v2.1 (Applied Biosystems, Foster City,

CA).

100

Figure 2-1. TMAO production from carnitine is a microbiota dependent process in humans. (a) Structure of carnitine and scheme of carnitine and choline metabolism to TMAO. L-Carnitine and choline (are both dietary trimethylamines that can be metabolized by microbiota to TMA. TMA is then further oxidized to TMAO by flavin monooxygenases (FMOs). (b) Scheme of human carnitine challenge test. After a 12 hour overnight fast, subjects received a capsule of d3-carnitine (250 mg) alone, or in some cases (as in data for subject shown) also an 8 ounce steak (estimated 180 mg L- carnitine), whereupon serial plasma and 24h urine collection was obtained for TMA and TMAO analyses. After a weeklong regimen of oral broad spectrum antibiotics to suppress the intestinal microbiota, the challenge was repeated (Visit 2), and then again a final third time after a ≥ three week period to permit repopulation of intestinal microbiota (Visit 3). Data shown in (panels c-e) are from a representative omnivorous subject who underwent carnitine challenge. Data is organized to vertically correspond with the indicated visit schedule above (Visit 1, 2 or 3). (c,d) LC/MS/MS chromatograms of plasma TMAO or d3-TMAO in an omnivorous subject using specific precursor → product ion transitions indicated at T = 8 hour time point for each respective visit. (e) Stable isotope dilution LC/MS/MS time course measurements stable isotope (d3) labeled TMAO and carnitine, in plasma collected from sequential venous blood draws at noted times.73

101

Figure 2-2. The formation of TMAO from ingested L-carnitine is negligible in vegans, and fecal microbiota composition associates with plasma TMAO concentrations. (a-b) Data from a vegan in the carnitine challenge consisting of co-administration of 250 mg d3-carnitine and an 8 ounce sirloin steak, and a representative omnivore. (a) Plasma TMAO and d3-TMAO were quantified post carnitine challenge, and in a (b) 24 hour urine collection. (c) Baseline fasting plasma concentrations of (n = 26) vegans and vegetarians and (n = 51) omnivores. Boxes represent the 25th, 50th, and 75th percentile and whiskers represent the 5th and 95th percentile. (d) Plasma d3-TMAO levels in male and female (n = 5) vegan/ vegetarian versus (n = 5) omnivores participating in a d3-carnitine (250 mg) challenge. P value shown is for comparison between area under the curve (AUC) of groups using Wilcoxon non- parametric test. (e) Baseline plasma concentrations of TMAO associates with Enterotype 2 (Prevotella) subjects with a characterized gut enterotype. (f) Plasma TMAO concentrations (x axes) and the proportion of taxonomic operational units (OTUs, Y axes) were determined as described in Methods. Subjects were grouped as vegan/vegetarian (n = 23) or omnivore (n = 30). P value shown is for comparisons between dietary groups using a robust Hotelling T2 test.73

102

Figure 2-3. The metabolism of carnitine to TMAO is an inducible trait and associates with microbiota composition. (a) d3-Carnitine challenge of mice on either a carnitine supplemented diet (1.3%) at 10 weeks and age versus age-matched normal chow controls. Plasma d3-TMA and d3- TMAO were measured at the indicated times following d3-carnitine administration by oral gavage using stable isotope dilution LC/MS/MS. Data points represents mean ± SE of 4 replicates per group. (b) Correlation heat map demonstrating the association between the indicated microbiota taxonomic genera and TMA and TMAO levels (all reported as mean ±SE in µM) of mice grouped by dietary status (chow, n = 10 (TMA,1.3±0.4; TMAO, 17±1.9); and carnitine, n = 11 (TMA, 50±16; TMAO, 114±16). Red denotes a positive association, blue a negative association, and white no association. A single asterisk indicates a significant false discovery rate adjusted (FDR) association of P ≤ 0.1 and a double asterisk indicates a significant FDR adjusted association of P ≤ 0.01. (c) Plasma TMAO and TMA concentrations were determined by stable isotope dilution LC/MS/MS (x axes) and the proportion of taxonomic operational units (OTUs, Y axes) were determined. 73

103

Figure 2-4. Relation between plasma carnitine and CVD risks. (a-c) Forrest plots of odds ratio of CAD, PAD, and CVD and quartiles of carnitine before (closed circles) and after (open circles) logistic regression adjustments with traditional cardiovascular risk factors including age, sex, history of diabetes mellitus, smoking, systolic blood pressure, low density lipoprotein cholesterol, and high density lipoprotein cholesterol. Bars represent 95% confidence intervals. (d) Relationship of fasting plasma carnitine levels and angiographic evidence of CAD. Boxes represent the 25th, 50th, and 75th percentile of plasma carnitine and the whiskers represent the 10th and 90th percentile. The Kruskal- Wallis test was used to assess the degree of coronary vessel disease on L-carnitine levels. (e) Forrest plot of hazard ratio of MACE (death, non fatal-MI, stroke, and revascularization) and quartiles of carnitine unadjusted (closed circles), and after adjusting for traditional cardiovascular risk factors (open circles), or traditional cardiac risk factors plus creatinine clearance, history of MI, history of CAD, burden of CAD (one, two, or three vessel disease), left ventricular ejection fraction, baseline medications (ACE inhibitors, statins, β-blockers, and aspirin) and TMAO levels (open squares). Bars represent 95% confidence intervals. (f) Kaplan Meier plot (graph) and hazard ratios with 95% confidence intervals for unadjusted model, or following adjustments for traditional risk factors as in panel e. Median levels of carnitine (46.8 µM) and TMAO (4.6 µM) within the cohort were used to stratify subjects as ‘high’ (≥ median) or ‘low’ (< median) concentrations. 73 104

Figure 2-5. Dietary carnitine accelerates atherosclerosis and inhibits reverse cholesterol transport in a microbiota dependent fashion. (a) Representative Oil-red-O stained (counterstained with hematoxylin) aortic roots of 19 week old C57BL/6J, Apoe–/– female mice on the indicated diets in the presence versus absence of antibiotics (ABS). (b) Quantification of mouse aortic root plaque lesion area of 19 week-old C57BL/6J, Apoe–/– female mice on respective diets. (c) Carnitine, TMA, and TMAO were determined using stable isotope dilution LC/MS/MS analysis of plasma recovered from mice at time of sacrifice. (d) Reverse cholesterol transport (RCT) in female C57BL/6J, Apoe–/– mice on normal chow versus diet supplemented with either carnitine or choline, as well as following suppression of microbiota using cocktail of antibiotics (+ ABS). Also shown are RCT results in female C57BL/6J, Apoe–/– mice on normal chow versus diet supplemented with TMAO. (e,f) Relative mRNA levels (to β-actin) of mouse liver candidate genes involved in bile acid synthesis or transport. Ephx1, epoxide hydrolase 1, microsomal. 73

105

Figure 2-6. Effect of TMAO on cholesterol and sterol metabolism. Measurement of (a) total bile acid pool size and composition, as well as (b) cholesterol absorption in adult female (> 8 weeks of age) C57BL/6J, Apoe–/– mice on normal chow diet versus diet supplemented with TMAO for 4 weeks. (c) Summary scheme outlining pathway for microbiota participation in atherosclerosis via metabolism of dietary carnitine and choline forming TMA and TMAO, as well as the impact of TMAO on cholesterol and sterol metabolism in macrophages, liver and intestines. FMOs, flavin monooxygenases; TMA, trimethylamine; TMAO, trimethylamine-N-oxide; OST-α, solute carrier family 51, alpha subunit; ASBT, solute carrier family 10, member 2. 73

106

SUPPLEMENTARY MATERIAL

Supplementary Table 2-1: Characteristics of analyte m/z = 162 determined in LC/MS positive ion mode from plasma samples used in Validation and Learning cohorts (n = 150) of metabolomics study from Wang et. al., Nature, 2011. Plasma samples used in the metabolomics study described in Wang et al69 were from GeneBank, a large clinical repository of patients undergoing elective diagnostic cardiac evaluation. The original study utilized a Learning cohort of 50 cases (randomly selected GeneBank subjects who experienced death, non-fatal MI, or stroke in the ensuing 3 year follow up period) and 50 age and gender matched controls (subjects with no ensuing history of death, non-fatal MI or stroke in the 3 year period after enrollment). Peaks within LC chromatograms from the metabolomics analyses that exceeded a signal to noise ratio of greater than 5 were integrated. Bonferroni adjusted two sided T-tests were calculated and adjusted – logP value > 1.3 were considered significant. An odds ratio (OR) between the highest and lowest quartile was calculated for each unknown analyte. Only analytes with 95% confidence intervals not crossing unity were considered significant. Additionally, Cochran-Armitage trend tests across the quartiles were performed with P < 0.05 being considered significant. A similar analysis was performed in a non-overlapping Validation cohort consisting of 25 additional cases and controls from GeneBank. In both Learning and Validation cohorts only 18 plasma analytes met this strict set of validation criterion, and an analyte with m/z =162 (same as carnitine) was not among them 1. Results for an analyte with m/z = 162 and retention time similar to that of authentic L-carnitine in the Learning and Validation cohorts are shown. In a new hypothesis-generated analysis that did not adjust for multiple sampling (since only an analyte was being examined) and that used the combined data set (Learning + Validation cohorts, n = 75 cases and n = 75 age-gender matched controls), the plasma analyte with m/z = 162 and retention time identical to carnitine was significantly associated with cardiovascular risks (bottom table, P = 0.04). These results suggested that plasma levels of an analyte with m/z = 162, perhaps L-carnitine, may be associated with cardiovascular risks.73

107

Carnitine Quartiles P Patient Characteristics Whole cohort Q1 Q2 Q3 Q4

< 31.7 µM 31.7 - 37.8 37.9 - 45.2 µM > 45.2 µM (n = 2595) (n = 649) µM (n = 649) (n = 650) (n = 647)

Age (years) 62 (54-71) 63 (54-72) 62(54-71) 63(54-71) 61 (53-71) < 0.01

Male (%) 70 54 69 76 80 < 0.01

Smoking (%) 69 61 67 71 77 < 0.01

Diabetes mellitus (%) 28 27 26 27 31 0.20

Hypertension (%) 72 69 72 73 75 0.06

Hyperlipidemia (%) 85 81 86 87 88 < 0.01

Prior CAD (%) 74 65 73 75 83 < 0.01

CAD (%) 78 70 76 80 85 < 0.01

PAD (%) 22 22 19 21 26 0.01

CVD (%) 80 71 78 82 88 < 0.01

BMI (kg/m2) 29 (25-33) 28 (24-31) 29 (2-32) 29 (25-32) 29 (26-34) < 0.01

LDL cholesterol (mg dl–1) 96 (78-117) 94 (74-111) 100 (80-122) 97 (80-117) 96 (77-117) < 0.01

HDL cholesterol (mg dl–1) 34( 28-41) 35 (30-43) 34 (28-41) 32 (27-39) 32 (27-38) < 0.01

Total cholesterol (mg dl–1) 160 (139-188) 159 (138-184) 164 (140- 160 (139-188) 161 (139-188) < 0.01 194) Triglycerides (mg dl–1) 117 (85-167) 103 (76-148) 110 (84-159) 124 (88-170) 129 (96-192) < 0.01

hsCRP (mg l–1) 2.3 (1.0-5.4) 2.3 (1.0-5.5) 2.1 (1.0-5.0) 2.2 (1.0-5.0) 2.5 (1.1-6.0) < 0.01

MPO (pmol l–1) 113 (76-230) 122 (75-267) 109 (73-205) 110 (76-215) 114 (79-234) < 0.01

eGFR (ml min/1.73/m2) 83 (70-96) 86 (73-99) 85 (73-98) 83 (70-95) 79 (64-93) < 0.01

Carnitine (µM) 38 (32-45) 28 (25-30) 35 (33-36) 41 (39-43) 51 (48-56) < 0.01

Baseline medications (%)

ACE inhibitors 51 45 51 51 57 < 0.01

Beta-blockers 67 62 63 68 73 < 0.01

Statin 63 58 64 65 64 0.06

Aspirin 76 75 77 76 76 0.82

Supplementary Table 2-2: Subject characteristics, demographics, and laboratory values in the whole cohort (n = 2595), and across quartiles of plasma carnitine. Values are expressed in mean ± SD for normally distributed variables, or median (interquartile range) for non-normally distributed variables. The P value represents a Kruskal Wallis test for continuous variables and Chi-square test for categorical variables across quartiles of carnitine. Abbreviations: ACE, angiotensin converting enzyme; ATP III, Adult Treatment Panel III guidelines; BMI, body mass index; CAD, coronary artery disease; CVD, cardiovascular disease; cTnI = cardiac Troponin I; HDL, high-density lipoprotein; hsCRP, high-sensitivity C-reactive protein; LDL, low-density lipoprotein; MPO, myeloperoxidase; PAD, peripheral artery disease.73

108

Supplementary Table 2-3: Plasma levels of triglycerides, cholesterol, glucose, and insulin from mice on normal chow vs. carnitine supplemented diet. C57BL/6J, Apoe–/– female mice at time of weaning were placed on the indicated diets until time of sacrifice for aortic root quantification of atherosclerosis (19 weeks of age). Parallel groups of animals were also provided an antibiotics cocktail in drinking water as described under Methods. Lipid profiles, glucose, and insulin levels shown were determined in plasma isolated at time of organ harvest at conclusion of study. Data shown are mean ± SD for each of the indicated feeding groups. Student t-test comparisons are between chow and carnitine (1.3%) supplemented diets with the noted antibiotic (ABS) treatment status.73

109

Supplementary Table 2-4: Liver levels of triglycerides and total cholesterol in mice on normal chow versus carnitine supplemented diet. Liver was harvested from female C57BL/6J, Apoe–/– mice on the indicated diets at time of sacrifice for aorta harvest for aortic root quantification (19 weeks of age and 15 weeks on diets). Liver was homogenized and the content of triglycerides and total cholesterol determined as described under Methods. Data are presented as mean ± SD for each of the indicated groups of mice. A student t-test comparison was performed between chow and carnitine groups on or off a cocktail of oral broad spectrum antibiotics (+ ABS) as described in Methods. No significant increases in liver lipid levels were noted in the carnitine supplemented mice compared to the respective chow controls.73

110

Supplementary Table 2-5: Plasma levels of triglycerides, cholesterol, and glucose from mice on normal chow, carnitine, choline, and TMAO supplemented diets during the in vivo RCT studies. C57BL/6J, Apoe–/– female mice were enrolled in two separate studies to quantify in vivo reverse cholesterol transport (RCT) by placement on the indicated diets at time of weaning ("TMAO RCT" study, and "Carnitine and Choline RCT" study). Following 4 weeks of diet, [14C]cholesterol loaded macrophages were injected subcutaneously, and in vivo RCT quantified as described under Methods. Lipid profiles and glucose levels shown were determined in plasma isolated at time of organ harvest at conclusion of study (72h post injection of [14C]cholesterol loaded macrophages). Data shown are mean ± SD for each of the indicated dietary groups.73

111

Supplementary Figure 2-1: Mass spectrometry analyses identify unknown plasma analyte at retention time of 5.1 min and m/z = 162 as carnitine. a) Extracted ion chromatograms at m/z = 162 from human plasma sample (top), and authentic L-carnitine standard (bottom). Identical retention times under multiple chromatographic conditions during LC/MS analysis were demonstrated for analyte m/z =162 and L-carnitine standard. b) Collision-induced dissociation (CID) spectra from 5.10 min peak in human plasma and L-carnitine standard. This data demonstrate that the analyte at 5.10 min with m/z = 162 from human plasma possesses identical CID mass spectrum and retention time to authenticate synthetic L-carnitine standard.73

112

Supplementary Figure 2-2. LC/MS/MS analysis of synthetic heavy isotope standard d9(trimethyl)carnitine spiked into human plasma sample confirms unknown peak at 5.10 min (m/z = 162) is carnitine. a) Human plasma was spiked with synthetic d9(trimethyl)-carnitine. The sample was then analyzed by LC/MS/MS using multiple distinct precursor → product ion transitions in multiple reaction monitoring (MRM) mode that are characteristic for L-carnitine and its d9(trimethyl)-isotopologue. Note that multiple characteristic precursor → product transitions demonstrate identical retention times for both the plasma analyte with m/z = 162, and synthetic d9(trimethyl)-carnitine standard. b) Precursor → product ion transitions were determined from CID spectra of both authentic L-carnitine and synthetic d9(trimethyl)carnitine. Insets: Shown are proposed fragmentation overlay on structure from positive ion electrospray analyses of L-carnitine and d9- carnitine.73

113

Supplementary Figure 2-3. Standard curves for LC/MS/MS quantification of carnitine and d3-(methyl)-carnitine in plasma matrix. We used synthetic d9(trimethyl)carnitine as internal standard to quantify d3(methyl)carnitine, and natural abundance carnitine isotopologues in plasma recovered from mice and humans following carnitine challenge. To generate standard curves for each isotopologue in plasma matrix, a fixed amount of d9-(trimethyl)carnitine as an internal standard was added to dialyzed human plasma, and increasing concentrations of L-carnitine (a) and synthetic d3-(methyl)carnitine (b) were spiked into the samples. Plasma proteins were precipitated with a methanol at 0°C. Aliquots of the supernatant solution were analyzed by LC with on-line tandem mass spectrometry using electrospray ionization in positive ion mode on an AB SCIEX 5000 triple quadrupole mass spectrometer. Unique precursor → product ion transitions were selected for carnitine and its d3- and d9- isotopologues. Areas of peaks from multiple reaction monitoring (MRM) were divided by the peak area from m/z transition 171 → 69 from d9-carnitine. Standard curves of peak area ratio versus known concentrations are plotted on the same axis for carnitine (a) and d3-carnitine (b). For quantification, precursor → product transitions of 162 → 60 and 165 → 63 were typically used to measure carnitine and d3-carnitine, respectively, and if needed, alternative indicated transitions used to confirm results.73

114

Supplementary Figure 2-4. LC/MS/MS analyses of a subject’s 24 hr urine samples demonstrate an obligatory role for gut microbiota in production of TMAO from carnitine. (a) Scheme of overall study. There were 3 visits where carnitine challenge (following overnight fast, ingestion of carnitine in form of 8 oz steak (where indicated) and 250 mg d3-(methyl) carnitine) occurred with serial plasma and 24h urine collection. Visit 1 served as baseline. Subjects then took a cocktail of oral antibiotics for 1 week as described in Methods to suppress intestinal microbiota, and repeat carnitine challenge was performed at Visit 2. A third and final Visit was performed after at least 1 month of being off of antibiotics. (b) Data shown are chromatographic peaks from analysis of urine samples (aliquot of 24 hour collections) from a typical omnivorous subject (from n > 10 who underwent carnitine challenge and had complete serial blood draws performed) following carnitine challenge at the indicated visit shown above. The top row of chromatograms is from LC/MS/MS analyses of the indicated precursor → product transition specific for TMAO, and the bottom panel represents similar analyses using precursor → product transitions specific for d3-TMAO. Note that TMAO and d3-TMAO are readily detected at Visit 1 and 3 after d3-carnitine ingestion, but not Visit 2 where intestinal microbiota is suppressed by oral broad spectrum antibiotics, consistent with a requirement for gut microbiota involvement in both TMA and TMAO formation. Data is organized to vertically correspond with the indicated visit schedule above (Visit 1, 2 or 3).73

115

Supplementary Figure 2-5. Plasma levels of carnitine and TMAO following carnitine challenge in a typical omnivorous subject. (a) Scheme of human carnitine challenge test. After an overnight fast, subjects were challenged with a capsule of d3- carnitine (250 mg) alone and with an 8 ounce steak (estimated 180 mg L-carnitine). This was followed with serial plasma and a 24h urine collection for TMAO and carnitine analyses. Visit 2 occurred after a weeklong regimen of oral broad spectrum antibiotics to suppress the intestinal microflora. The challenge was repeated a third time after a ≥ three week period off antibiotics (Visit 3). Data shown (b) are from stable isotope dilution LC/MS/MS time course measurements of natural abundance TMAO and carnitine in plasma collected from sequential venous blood draws at noted times from a representative omnivorous subject of n > 10 who underwent carnitine challenge. Data is organized to vertically correspond with the indicated visit schedule above (Visit 1, 2 or 3).73

116

Supplementary Figure 2-6. Plasma levels of carnitine and d3-carnitine following carnitine challenge (steak and d3-carnitine) in typical omnivore with frequent red meat dietary history and a vegan subject. Plasma was isolated at baseline (T = 0) and the indicated times points following carnitine challenge (8-ounce steak + 250 mg of d3-carnitine) in an omnivore who reported near daily consumption of red meat, and in the one vegan subject who agreed to consume 8 ounces of steak with the d3-carnitine. Plasma levels of endogenous (natural abundance) carnitine (left panel) and the d3- carnitine isotopologue (right panel) were determined by stable isotope dilution LC/MS/MS analysis using synthetic d9-carnitine as internal standard as described in Methods. The data shown for natural abundance carnitine are typical for the omnivore, where nominal changes in plasma levels are noted following consumption of a steak, but increases from typically relatively lower levels (for vegans/vegetarians) are noted in the vegan subject shown. Substantial increases in the isotope labeled d3-carnitine were found in both vegan and omnivore alike. Also note the greater extent of increase in d3- carntine within the vegan observed compared to the omnivore following ingestion of the d3-carnitine containing capsule, consistent with more intestinal microbiota-mediated catabolism of the d3-carnitine in the omnivore, blunting the amount of carnitine absorbed relative to that observed in the vegan.73

117

Supplementary Figure 2-7. Plasma levels of d3-carnitine following d3-carnitine challenge (no steak) in omnivorous (n = 5) versus vegan subjects (n = 5). Similar studies to that shown in Supplementary Figure 2-6 where carnitine challenge did not include ingestion of steak, but only d3-carnitine (250 mg) in a capsule. Plasma was isolated at baseline (T = 0) and the indicated times points following d3-carnitine ingestion in both omnivorous (n = 5) and vegan (n = 5) subjects. Plasma levels of d3- carnitine were determined by stable isotope dilution LC/MS/MS analysis using synthetic d9-carnitine as internal standard as described in Methods. Statistical analysis was performed by a Wilcoxon rank-sums test between the mean area under the curve between subjects grouped by omnivorous versus vegan status. A significant increase in plasma d3-carnitine occurs in both vegan and omnivore alike over baseline values, but to a greater extent in vegans, following ingestion of the d3-carnitine containing capsule (P < 0.05). This is consistent with more intestinal microbiota-mediated catabolism of the d3-carnitine in the omnivore, blunting the amount absorbed relative to that observed in vegans. * P < 0.05 for difference between vegan and omnivore subjects.73

118

Supplementary Figure 2-8. Human fecal microbiota taxa associate with plasma TMAO. Human fecal samples were collected from vegan/vegetarians (n = 23) and omnivores (n = 30) and microbiota gene encoding for 16S rRNA was analyzed as described under Methods. Associations between plasma TMAO and taxa proportions were assessed as described under Methods. False discovery rate (FDR) control based on the Benjamini–Hochberg procedure was used to account for multiple comparisons. Asterisked taxa met a FDR adjusted P value < 0.1. Further details of the preparation and analysis of human fecal samples can be found in Methods.73

119

Supplementary Figure 2-9. Demonstration of an obligatory role of the commensal gut microbiota of mice in the production of TMA and TMAO from oral carnitine in germ-free and conventionalized mice. d3-Carnitine challenge (oral gavage of d3- carnitine) in germ-free female Swiss Webster mice before and after ensuing conventionalization (≥ 3 weeks in conventional cages with conventional mice). Each point represents mean ± SE of 4 independent replicates. Plasma levels of d3-carnitine, d3-TMAO and d3-TMA were determined by stable isotope dilution LC/MS/MS analysis using synthetic d9-(trimethyl)carnitine, d9-(trimethyl)TMA, and d9-(trimethyl)TMAO as internal standards. Note that there is an obligatory role for gut microbiota in generation of TMA and TMAO from orally ingested carnitine, as reflected by the absence of these metabolites in the germ-free mice, but their formation within the conventionalized mice.73

120

Supplementary Figure 2-10. Demonstration of an obligatory role of commensal gut microbiota of mice in the production of TMA and TMAO from oral carnitine. Left panel - C57BL/6J, Apoe–/– female mice (n = 5) in conventional cages were given oral d3- carnitine via gavage at T = 0, and then serial blood draws were obtained at the indicated times. Plasma levels of d3-carnitine, d3-TMAO and d3-TMA were determined by stable isotope dilution LC/MS/MS analysis using synthetic d9-(trimethyl)carnitine, d9- (trimethyl)TMA, and d9-(trimethyl)TMAO as internal standards. Middle panel - Mice were then treated with a cocktail of oral broad spectrum antibiotics to suppress intestinal microbiota as described in Methods. Repeat gastric gavage with d3-carnitine was performed, and serial testing of plasma for quantification of d3-carnitine, d3-TMA and d3-TMAO levels were determined. Right panel - Antibiotics were stopped and mice allowed to reacquire (≥ 3 weeks) their intestinal microbiota in conventional cages. Repeat gastric gavage with d3-carnitine was performed, and d3-carntine and its metabolites d3-TMA and d3-TMAO were then quantified by LC/MS/MS in serial plasma samples. Results shown are mean ± SE for 5 animals.73

121

Supplementary Figure 2-11. Analysis of mouse plasma TMA and TMAO concentrations and gut microbiome composition can distinguish dietary status. C57BL/6J, Apoe–/– female mice were maintained either on normal chow (n = 10) or a carnitine supplemented (1.3%) diet (n = 11) as described under Methods. At sacrifice, blood and intestines were harvested, microbial DNA for the gene encoding 16S-rRNA was isolated from cecal contents, and microbiota composition analyzed as described under Methods. Plasma TMAO and TMA concentrations were determined by stable isotope dilution LC/MS/MS (plotted on x axes) and the proportion of taxonomic operational units of indicated taxa (OTUs, plotted on Y axes). Analyses and P values shown are for comparisons between dietary groups, and were determined as described in Methods.73

122

Supplementary Figure 2-12. Haematoxylin/eosin (H/E) and oil-red-O stained liver sections. Representative liver sections from female C57BL/6J, Apoe–/– mice used in atherosclerosis study on the indicated diets collected at time of aorta harvest (19 weeks of age and 15 weeks on diets). Liver was stained by H/E (left column) or oil-red-O and counterstained with Haematoxylin (right column). Mice on these diets exhibit no obvious hepatosteatosis or other pathology. As a positive control for comparison showing fatty liver, C57BL/6J mice fed a high fat diet (16 weeks of age and 6 weeks on the diet) is shown in bottom row.73

123

Supplementary Figure 2-13. Arginine transport in the presence of 100 µM trimethylamine-containing compounds. Bovine aortic endothelial cells (BAEC) were incubated in DMEM medium supplemented with glutamine, 10% FCS and penicillin/streptomycin and with 100 µM of the indicated trimethylamine-containing cationic compounds. BAEC cell arginine uptake studies were performed in Krebs– Henseleit buffer by the addition of 50 µM L-[3H] arginine (1 µCi ml–1). The samples were incubated for 30 min at 37°C and chased with cold 10 mM L-Arg. After washing with Krebs–Henseleit, the samples were solubilized with 0.1 M NaOH, transferred into plastic liquid scintillation vials and mixed with 4 ml scintillation fluid prior to counting in a Beckman Coulter LS6500 liquid scintillation counter. Data represented mean ± SE from 6 independent replicates. No significant reduction in arginine uptake is noted, suggesting TMAO, carnitine and choline, cationic amino acids, do not compete with arginine for uptake into BAEC.73

124

Supplementary Figure 2-14. Expression levels of cholesterol synthesis enzymes, transporters, and inflammatory genes in the presence or absence of TMAO. Elicited mouse peritoneal macrophages (MPMs) were cultured in RPMI 1640 supplemented with 10% FCS and penicillin/streptomycin overnight. MPMs were then lipoprotein starved in RPMI 1640 supplemented with 10% LPDS and penicillin/streptomycin for 24 hours and then further cultured in the same media for 18 hours in the presence of increasing cholesterol or AcLDL concentrations or vehicle (carrier for AcLDL and cholesterol) with (+) or without (–) 300 µM TMAO (the upper 1% of plasma levels of TMAO noted in the cohort examined in the present study). RNA was then purified, cDNA amplified, and relative (to β-actin) expression of the indicated genes quantified by RT-PCR as described in Methods. Data are expressed as the mean ± SE of n = 3 replicates. Differences between conditions + versus – TMAO were evaluated using a student’s t-test. Note that no consistent significant effects on candidate gene expression within MPMs in the presence or absence of TMAO are noted. Hmgcr, 3- hydroxy-3-methylglutaryl-Coenzyme A reductase; Srebp2, sterol regulatory element binding factor 2; Ldlr, low density lipoprotein receptor; Dhcr24, 24-dehydrocholesterol reductase; Cxcl9, chemokine (C-X-C motif) ligand 9; Cxcl10, chemokine (C-X-C motif) ligand 10.73

125

Supplementary Figure 2-15. Effect of TMAO on desmosterol levels in media of cultured mouse peritoneal macrophages in the presence of increasing cholesterol and acetylated LDL (AcLDL) concentrations. Elicited mouse peritoneal macrophages (MPMs) were cultured in RPMI 1640 supplemented with 10% FCS and penicillin/streptomycin overnight. MPMs were then lipoprotein starved in RPMI 1640 supplemented with 10% LPDS and penicillin/streptomycin for 24 hours and then further cultured in the same media for 18 hours in the presence of increasing cholesterol and AcLDL concentrations or vehicle (carrier for AcLDL and cholesterol) with (+) or without (– ) 300 µM TMAO. Media was harvested and the content of desmosterol was determined as described under Methods. Data represented mean ± SE from 3 independent replicates.73

126

Supplementary Figure 2-16. Plasma concentrations of TMAO in mice undergoing in vivo reverse cholesterol transport studies. Adult (> 8 weeks of age) C57BL/6J, Apoe–/– female mice were placed on normal chow or either carnitine (1.3%) or choline (1.3%) supplemented diets. Where indicated, some groups of mice also had addition of a cocktail of antibiotics to their drinking water as described under Methods throughout the duration of the dietary feeding period and RCT study. TMAO concentration was determined by stable isotope dilution LC/MS/MS analysis of plasma recovered from mice at time of sacrifice in the reverse cholesterol transport studies. A Wilcoxon non- parametric test was used to assess the difference in plasma TMAO between animal diets. Data shown are mean ± SE.73

127

Supplementary Figure 2-17. [14C] Cholesterol recovered from mice on normal chow vs. TMAO diet enrolled in in vivo reverse cholesterol transport studies. Adult (> 8 weeks of age) C57BL/6J, Apoe–/– female mice were placed on either normal chow or a TMAO (0.12%) supplemented diet for 4 weeks before performing in vivo reverse cholesterol transport studies as described under Methods. Mice were sacrificed 72 hours post injection with [14C]cholesterol-loaded bone marrow-derived macrophages and counts within plasma, liver, and bile were determined as described in Methods. Results shown are mean ± SE.73

128

Supplementary Figure 2-18. Effect of TMAO on mouse peritoneal macrophages. Thioglycollate elicited mouse peritoneal macrophages (MPMs) from C56Bl/6J mice were cultured in RPMI media supplemented with 5% lipoprotein deficient serum, glutamine, and penicillin and streptomycin. MPMs were then further incubated in the same media for an additional 20 hours with the indicated levels of TMAO. RNA was then purified, cDNA amplified, and relative (to β-actin) expression of the indicated genes quantified by RT-PCR as described in Methods. Data are expressed as the mean ± SE.73

129

Supplementary Figure 2-19. Effect of TMAO on cultured macrophage cholesterol efflux. RAW264.7 macrophages were cultured in DMEM media supplemented with 10% FBS and penicillin/streptomycin until 75% confluence. Cells were then further incubated in DMEM media supplemented with 12.5 g l–1glucose, 200 mM glutamine, 1.25 g l–1 BSA, and penicillin/streptomycin + or – cyclic AMP (for Abca1 expression induction) for an additional 16 hours with the indicated levels of TMAO. Abca1-dependent and total cholesterol efflux were then determined using lipid free isolated human apolipoprotein A1 (APOA1), or isolated human HDL, as cholesterol acceptor, as described in Methods. Data are expressed as the mean and ± SD of replicates (n = 4). A student’s t-test was used to assess the relative increase in cholesterol efflux relative to a PBS (no exposure) control. While a statistically significant increase in Abca1-dependent cholesterol efflux in macrophages exposed to TMAO is noted (P < 0.01), the biological significance is unclear given the modest level of the effect, even at the highest levels of TMAO used.73

130

Supplementary Figure 2-20. Liver expression of cholesterol transporters in mice examined during reverse cholesterol transport studies. Livers from C57BL/6J, Apoe–/– female mice on the indicated diets in the reverse cholesterol transport experiments were collected at time of sacrifice. The relative expression levels of the indicated genes were determined by RT-PCR as described in Methods. Data are presented as mean ± SE.73

131

Supplementary Figure 2-21. Western blot analysis of liver scavenger receptor B1 (Srb1) expression. Female C57BL/6J, Apoe–/– mice were placed on either normal chow or diet supplemented with TMAO (0.12%) at time of weaning, and then lever harvested at time of sacrifice (20 weeks of age). Mouse liver lysate (30 µg protein) was run on SDS PAGE and then transferred to PVDF membrane. The membranes were probed with antibodies against Srb1 (Novus, Littleton, CO) and β-actin (Sigma, St. Louise, MO), and intensity of bands quantified by densitometry using ImagePro Plus software. Data are expressed as means ± SE.73

132

Supplementary Figure 2-22. Small intestines expression profile of bile acid transporters in mice. Intestines from C57BL/6J, Apoe–/– female mice on the indicated diets enrolled in the in vivo reverse cholesterol transport experiments were harvest at completion of the study en-block. The small intestines were resected, extended lengthwise, and divided into 5ths. Tissue RNA was isolated from each segment and relative expression (to β-actin) of the indicated genes determined by RT-PCR as described in Methods. Data are expressed as mean ± SE. Note that there is no statistically significant differences noted in the expression pattern of the monitored genes along the length of the small intestines when comparing the pattern in chow vs. TMAO dietary groups of animals, as assessed by ANOVA. Ost-α; solute carrier family 51, alpha subunit; Asbt, solute carrier family 10, member 2.73

133

Supplementary Figure 2-23. Small intestines expression profile of cholesterol transporters in mice. Intestines from C57BL/6J, Apoe–/– female mice on the indicated diets enrolled in the in vivo reverse cholesterol transport experiments were harvest at completion of the study en-block. The small intestines were resected, extended lengthwise, and divided into 5ths. Tissue RNA was isolated from each segment and relative expression (to β actin) of the indicated genes determined by RT-PCR as described in Methods. Data are expressed as mean ± SE. P values for differences in the distribution of expression patterns of the monitored genes along the length of the small intestines when comparing the chow vs. TMAO dietary groups of animals were assessed by ANOVA.73

134

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

Authors: Robert A. Koeth, Bruce S. Levison, PhD, Zeneng Wang, PhD, Jill

Gregory, Stanley L. Hazen, MD, PhD

Intro: The pathogenesis of cardiovascular disease has been linked to gut flora

metabolism of carnitine to trimethylamine N-oxide (TMAO)73. Dietary production of TMAO promotes atherosclerosis and plasma concentrations independently associate with cardiovascular disease69,73. Dietary carnitine is principally found

in red meat and recently has become a frequent additive to the multi-billion dollar

energy drink industry. We recently reported that omnivorous subject gut

microbiota has a greater capacity to metabolize carnitine to TMAO compared to

vegan/vegetarians, and mice placed on a chronic carnitine diet also had an

increased capacity to metabolize carnitine to TMAO73. This raised the possibility

that chronic carnitine ingestion in humans can induce the gut microbiota to

produce the atherogenic gut microbiota metabolite TMAO.

Methods: Volunteer subjects with no history of chronic disease, recent infection,

or recent antibiotic/probiotic use were enrolled to perform an oral carnitine

challenge (250 mg d3-carnitine synthesized as previously described73. Subjects

were then placed on oral carnitine supplement (500 mg daily (L-Carnitine

capsules) and rechallenged in 2 follow-up visits. The interval time between the

baseline visit and visit 1 was 1 month and 2-3 months from the baseline visit to

135

visit 2. TMAO/d3-TMAO measurements were quantified in sequential venous blood draw plasma by stable isotope dilution LC/MS/MS at the indicated times as

described73. 2-way ANOVA analysis was performed on composite study of d3-

carnitine challenge and a 1-way ANOVA was performed on the baseline TMAO

plasma levels. The P values represent overall differences between groups.

Results: Quantification of d3-TMAO in plasma from serial venous blood draws

demonstrates an increase in d3-TMAO production post carnitine challenge at

each subsequent visit (Fig. 3-1a). We noted great variability in the both the

kinetics and capacity of individual gut microbiota to metabolize d3-carnitine.

However, increases in d3-TMAO production from all five individual gut microbiota

studied were noted at subsequent visits and the composite analysis of all

subjects challenged revealed a significant increase in the gut microbiota capacity

to metabolize d3-carnitine (Fig. 3-1a). Baseline plasma measurements of TMAO

at each visit also revealed significant increases in fasting TMAO levels that are

comparable to concentrations in mice supplemented with dietary carnitine with

accelerated atherosclerosis (Fig. 3-1b)73 . Remarkably, the dosage subjects

received in this study is comparable to the mass of carnitine in energy drinks

found on today’s market and the total content of carnitine found in a 8 ounce

steak84.

Comment: These data demonstrate that chronic carnitine supplementation can increase the capacity of the gut microbiota to produce TMAO. The important physiologic role of carnitine in fatty acid metabolism has led to the pervasive belief (and use by the nutritional supplement industry) that oral consumption of

136

carnitine is beneficial in energy expenditure, when, in fact, there is no compelling evidence suggesting any enhancement in healthy individuals. This is particularly concerning as the energy drink industry frequently adds carnitine to beverages and markets to adolescents and young adults146. These studies demonstrate that frequent consumption of dietary carnitine can induce gut flora capacity to produce TMAO and may be priming our gut flora to become proatherogenic at an alarmingly young age.

137

Figure 3-1. Carnitine supplementation can induce the gut microbiota. a) Composite plasma tracings of d3-TMAO in sequential venous blood draws post oral d3-carnitine challenge in n=5 subjects at baseline, visit 1 (V1), and visit 2 (V2). 2-way ANOVA analysis reveals that plasma d3-TMAO production is significantly higher after carnitine supplementation. Points represent means + SE at T=0, 2, 4, 6, 8, and 24 hours. b) Baseline plasma TMAO measurements of subjects in d3-carnitine challenge. Bars represent means + SE. One way-ANOVA analysis was used to assess differences between groups.

138

CHAPTER 4c,d: Intestinal Microbial Metabolism of Phosphatidylcholine and Cardiac Risk147 Authors: W. H. Wilson Tang, MD, Zeneng Wang, PhD, Bruce S. Levison, PhD,

Robert A. Koeth, Earl B. Britt, MD, Xiaoming Fu, Yuping Wu, PhD, Stanley L.

Hazen, MD, PhD

Abstract

Background: Recent animal studies show a mechanistic link between intestinal

microbial metabolism of the choline moiety in dietary phosphatidylcholine and

coronary artery disease pathogenesis via production of a pro-atherosclerotic

metabolite, trimethylamine-N-oxide. In this study we investigated the relationship

between intestinal microbiota-dependent metabolism of dietary

phosphatidylcholine, trimethylamine-N-oxide levels, and adverse cardiac events

in humans.

Methods: We quantified plasma trimethylamine-N-oxide, choline, betaine, and

urine trimethylamine-N-oxide levels by liquid chromatography with online tandem

mass spectrometry following phosphatidylcholine challenge (ingestion of stable

isotope (d9)-labeled phosphatidylcholine and two hard-boiled eggs) in healthy

individuals before and following intestinal microflora suppression with oral broad-

spectrum antibiotics. We further examined the relationship between fasting

plasma levels of trimethylamine-N-oxide and incident major adverse cardiac

c Reproduced with permission from (Tang, W.H., et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med 368, 1575-1584 (2013).), Copyright Massachusetts Medical Society. d This chapter was drafted for submission to NEJM with my being the fourth author. After a joint writing effort, lead by the primary author Dr. Wilson Tang, the final version was agreed upon, as it appears as Chapter 4 in this dissertation. I wish to thank Dr. Wilson Tang, Dr. Stanley Hazen, and my coauthors (listed above) for their contributions. 139

events (death, myocardial infarction, or stroke) over 3-year follow-up in 4,007

stable patients undergoing elective coronary angiography.

Results: Time-dependent increases in levels of both trimethylamine-N-oxide and its d9 isotopologue, as well as other choline metabolites, were detected following phosphatidylcholine challenge. Plasma levels of trimethylamine-N-oxide were markedly suppressed following antibiotics, and reappeared after cessation of antibiotics. Higher levels of plasma trimethylamine-N-oxide were associated with increased risk of major adverse cardiovascular events (Hazard ratio for highest versus lowest quartile, 2.5; 95% confidence interval 2.0-3.2; p<0.001). Elevated trimethylamine-N-oxide levels predicted risk of major adverse cardiovascular events following adjustments for traditional risk factors (p<0.001), as well as in lower-risk subgroups.

Conclusion: Trimethylamine-N-oxide production from dietary phosphatidylcholine in humans is dependent on metabolism by the intestinal microbiota. Higher trimethylamine-N-oxide levels are associated with higher risk of incident major adverse cardiovascular events.

Introduction

The phospholipid phosphatidylcholine (lecithin) is the major dietary source of choline, a semi-essential nutrient that is part of the B-complex vitamin family148,149. Choline has various metabolic roles ranging from its essential

involvement in lipid metabolism and cell membrane structure, to serving as a

precursor for synthesis of the neurotransmitter acetylcholine. Choline and some

140

of its metabolites, like betaine, can also serve as a source of methyl groups that

are required for proper metabolism of certain amino acids, such as homocysteine

and methionine150.

There is a growing awareness that intestinal microbial organisms,

collectively termed "microbiota", participate in global metabolism of their host42,151,152. We recently demonstrated a potential role of a complex phosphatidylcholine/choline metabolism pathway involving gut microbiota in

contributing to the pathogenesis of atherosclerotic coronary artery disease in

animal models69. We also reported an association between history of prevalent

cardiovascular disease and elevated fasting plasma levels of trimethylamine-N-

oxide, an intestinal microbiota-dependent metabolite of the choline headgroup of

phosphatidylcholine that is excreted in the urine69,153-157. Herein, we examine the

relationship between oral intake of phosphatidylcholine and the involvement of

the intestinal microbiota in formation of trimethylamine-N-oxide in humans. We

also further examine the relationship between fasting plasma levels of

trimethylamine-N-oxide and long-term risk for occurrence of incident major

adverse cardiac events.

Results

Role of intestinal microbiota in metabolism of dietary phosphatidylcholine

For the 40 participants in the phosphatidylcholine challenge study, plasma levels

of trimethylamine-N-oxide are shown in Fig. 4-1, and plasma levels of choline

and betaine in Supplementary Fig. 4-1. Endogenous (non-labeled)

141

trimethylamine-N-oxide (Fig. 4-1c), choline, and betaine (Supplementary Fig. 4-

1c) were present in fasting plasma at baseline. Both trimethylamine-N-oxide and d9-trimethylamine-N-oxide were readily detected in plasma following the dietary phosphatidylcholine challenge at Visit 1 (Fig. 4-1a,b, left panels). Time- dependent increases in both the natural isotopes (Fig. 4-1c, left panel) and d9- tracer forms (Fig. 4-1d, left panel) of trimethylamine-N-oxide were also observed postprandially. Examination of 24-hour urine specimens following the phosphatidylcholine challenge also showed the presence of trimethylamine-N- oxide and d9-trimethylamine-N-oxide (Supplementary Fig. 4-2, left panels). A strong correlation was observed between plasma and both absolute urine trimethylamine-N-oxide concentrations (Spearman’s R=0.58, P<0.001) and urinary trimethylamine-N-oxide-to-creatinine ratio (Spearman’s R=0.91,

P<0.001). Time dependent increases in the plasma levels of both the natural isotopes and d9-tracer forms of choline and betaine also increased following ingestion the phosphatidylcholine challenge (Supplementary Fig. 4-1c,d, left panels).

Suppression of intestinal microflora by the administration of oral broad- spectrum antibiotics for one week (in six of the participants) resulted in near- complete suppression of detectable trimethylamine-N-oxide in fasting plasma

(during Visit 2), as well as both trimethylamine-N-oxide and d9-trimethylamine-N- oxide following phosphatidylcholine challenge in both plasma (Fig.4-1, center panels) and urine (Supplementary Fig. 4-2, center panels). In parallel analyses, post-prandial elevations in plasma trimethlyamine and d9-trimethylamine were

142

observed following phosphatidylcholine challenge, but were completely

suppressed to non-detectable levels following antibiotics (data not shown). In

contrast, the time courses for post-prandial changes in free choline or betaine

(naturally-occurring and d9-isotopologues) were not altered by suppression of

intestinal microflora (Supplementary Fig. 4-1, center panels).

Following cessation of antibiotics and reacquisition of intestinal microflora

over the ensuing one month or longer, phosphatidylcholine challenge (at Visit 3)

again resulted in readily detectable and time-dependent changes in

trimethylamine-N-oxide and d9-trimethylamine-N-oxide in plasma (Fig. 4-1, right panels) and urine (Supplementary Fig. 4-1, right panels). Consistent with recent reports observing variable recovery of intestinal microbiota composition after antibiotic cessation48,158, the extent to which trimethylamine-N-oxide levels in plasma at Visit 3 returned to pre-antibiotic levels was variable.

Correlation of plasma levels of trimethylamine-N-oxide with major adverse cardiovascular events

The baseline characteristics of the 4,007 participants in the clinical outcomes

study are shown in Table 4-1. The mean age of the participants was 63 years, and two-thirds were male; the prevalence of cardiovascular risk factors was high

and most had at least single-vessel coronary disease. Participants with incident

major adverse cardiovascular events during three years of follow-up had higher

risk profiles than those without events, including greater age, higher rates of

143

diabetes, hypertension, and prior myocardial infarction, and higher fasting

glucose levels.

As noted in Table 1, participants with major adverse cardiovascular events

at three years of follow-up also had higher baseline levels of trimethylamine-N-

oxide (median(interquartile range) 5.0(3.0-8.8) µM versus 3.5(2.4-5.9) µM,

P<0.001). Compared to participants in the lowest quartile level of trimethylamine-

N-oxide, the highest quartile had a 2.5-fold increased risk of an event (HR 2.5,

95% CI 2.0-3.2; P<0.001, Table 4-2 and Supplementary Table 4-1). After

adjusting for traditional risk factors and other baseline covariates, elevated

plasma levels of trimethylamine-N-oxide remained a significant predictor of risk of

major adverse cardiovascular events (Table 4-2). We observed a graded

increase in the risk of major adverse cardiovascular events associated with

increasing levels of trimethylamine-N-oxide, as illustrated in the Kaplan-Meier

analysis shown in Figure 4-2. A similar graded increase in risk was observed when levels of trimethylamine-N-oxide were analyzed as a continuous variable in increments of one standard deviation (unadjusted HR 1.4, 95% CI 1.3-1.5;

P<0.01; adjusted HR 1.3, 95% CI 1.2-1.4, P<0.01).

When components of the composite primary outcome (major adverse cardiovascular events) were analyzed separately, higher levels of trimethylamine-N-oxide remained significantly associated with higher risk of death (HR 3.2, 95%CI 2.1-4.8; P<0.001) and non-fatal myocardial infarction or stroke (HR 2.3, 95%CI 1.5-3.6; P<0.001). Inclusion of trimethylamine-N-oxide as a covariate resulted in a significant improvement in risk estimation over traditional

144

risk factors (net reclassification improvement 8.6%, P<0.001; integrated discrimination improvement 9.2%, P<0.001; C-statistic 68.3% vs. 66.4%,

P=0.01). In a separate analysis, we excluded all participants who underwent revascularization within the 30 days following enrollment in the study. In this sub- cohort (n = 3,475), trimethylamine-N-oxide remained significantly associated with risk of major adverse cardiovascular events [highest quartile versus lowest quartile, unadjusted HR (95% CI), 2.47 (1.87-3.27); adjusted HR (95% CI) 1.79

(1.34-2.4); both P<0.001].

Correlation of trimethylamine-N-oxide levels with risk in low-risk subgroups

The prognostic value of elevated plasma levels of trimethylamine-N-oxide remained significant in various subgroups associated with reduced overall cardiac risks (Supplementary Fig. 4-3). Subgroups examined included those who were younger, females, those without known history of coronary artery disease or coronary disease risk equivalents, those with lower-risk lipid and apolipoprotein levels, those with normal blood pressure, non-smokers, and those with lower levels of other known risk markers such as C-reactive protein, myeloperoxidase, or white blood cell count.

Discussion

Recent animal model studies with germ-free mice suggest a role for the intestinal microbial community in the pathogenesis of atherosclerosis in the setting of a diet

145

rich in phosphatidylcholine via formation of the metabolite trimethylamine and

conversion to trimethylamine-N-oxide (Fig. 4-3) 69,70. Herein we demonstrate the generation of the pro-atherogenic metabolite trimethylamine-N-oxide from dietary phosphatidylcholine in humans through use of stable isotope tracer feeding studies. We further demonstrate a role for the intestinal microbiota in production of trimethylamine-N-oxide in humans via both its suppression with oral broad- spectrum antibiotics, and then reacquisition of trimethylamine and trimethylamine-N-oxide production from dietary phosphatidylcholine following

cessation of antibiotics and intestinal recolonization. Finally, we demonstrate the potential clinical prognostic significance of this intestinal microbiota-dependent metabolite by showing that fasting plasma trimethylamine-N-oxide levels predict

development of incident major adverse cardiovascular events independent of

traditional cardiovascular risk factors, presence or extent of coronary artery

disease, and within multiple lower risk subgroups, including both primary

prevention subjects and subjects with lower-risk lipid and apolipoprotein levels.

The present findings suggest that intestinal microbial organism-dependent

pathways may contribute to the pathophysiology of atherosclerotic coronary

artery disease in humans, and suggest new potential therapeutic targets.

The intestinal microflora have previously been implicated in complex metabolic diseases like obesity42,151,152,159-161. However, involvement of

microflora in the inception of atherosclerosis in humans has only recently been

suggested69,162. The ability of oral broad-spectrum antibiotics to temporarily

suppress the production of trimethylamine-N-oxide is a direct demonstration that

146

intestinal micro-organisms play an obligatory role in trimethylamine-N-oxide

production from phosphatidylcholine in humans. Intestinal microbiota convert the choline moiety of dietary phosphatidylcholine into trimethylamine, which is subsequently converted into trimethylamine-N-oxide by hepatic flavin-containing mono-oxygenases (Fig. 4-3)71,163. The requirement for trimethylamine to be converted into trimethylamine-N-oxide by hepatic flavin-containing mono- oxygenases164 may help to explain the observed delay in the detection of plasma

d9-trimethylamine-N-oxide levels following oral ingestion of d9-

phosphatidylcholine, since separate analyses monitoring trimethylamine and d9-

trimethylamine production show a time course consistent with a precursor-to-

product relationship (not shown). Interestingly, trimethylamine-N-oxide has been

identified in fish as an important osmolite,165 and fish ingestion raises urinary

trimethylamine-N-oxide levels. Nevertheless, the high correlation between urine and plasma levels argues for effective urinary clearance of trimethylamine-N- oxide. Hence, an efficient excretion mechanism may be protective in preventing the accumulation of trimethylamine-N-oxide and does not undermine the mechanistic link between trimethylamine-N-oxide and cardiovascular risk.

While an association between infectious organisms and atherosclerosis has previously been postulated, studies looking at the role of antimicrobial therapy in preventing disease progression have been disappointing166,167. It is important to recognize that the choice of antimicrobial therapy in prior intervention trials was largely based on targeting postulated organisms rather than modulating intestinal microflora composition or their metabolites. Further,

147

even if an antibiotic initially suppressed trimethylamine-N-oxide levels, the

durability of that effect with chronic intervention remains unknown. Indeed, in

unpublished studies we observed that chronic use (e.g. half year) of a single

antibiotic (ciprofloxacin) that initially fully suppressed plasma TMAO levels in a

rodent model completely lost its suppressive effect, consistent with expansion of

antibiotic resistant intestinal microflora (Z. Wang and S.L. Hazen, unpublished).

Thus, instead of suggesting that intestinal microbes should be eradicated with

chronic antibiotics, the present findings imply that plasma trimethylamine-N-oxide

levels may potentially identify a pathway within intestinal microflora amenable to therapeutic modulation. For example, our data suggest that excessive

consumption of dietary phosphatidylcholine and choline should be avoided; a

vegetarian or high-fiber diet can reduce total choline intake159. It also should be

noted that choline is a semi-essential nutrient and should not be completely eliminated from the diet, as this can result in a deficiency state. However, standard dietary recommendations, if adopted, will limit phosphatidylcholine- and choline-rich foods since these are also typically high in fat and cholesterol content148. An alternative potential therapeutic intervention is targeting intestinal

microbial organism composition or biochemical pathways, either with a

“functional food” such as a probiotic160, or even a pharmacologic intervention.

This latter intervention hypothetically could take the form of either an inhibitor to

block specific microbial metabolic pathways, or even a short course of non-

systemic antibiotics to reduce the “burden” of trimethylamine-N-oxide-producing microbes, as seen in the treatment of irritable bowel syndrome168. Further

148

studies are warranted to establish whether antimicrobial targeted therapies can

significantly reduce cardiovascular risk.

In summary, we demonstrated that intestinal microbes participate in

phosphatidylcholine metabolism to form circulating and urinary trimethylamine-N-

oxide in humans. We also established a correlation between high plasma levels of trimethylamine-N-oxide and higher risk of incident major adverse cardiovascular events independent of traditional risk factors, even in lower-risk

cohorts.

Acknowledgements

We thank Linda Kerchenski and Cindy Stevenson for assistance in subject

recruitment, and Amber Gist and Naomi Bongorno for assistance in the

preparation of figures and the manuscript. Mass spectrometry instrumentation

used was housed within the Cleveland Clinic Mass Spectrometry Facility with

partial support through a Center of Innovation by AB SCIEX.

Methods

Study patients and design

We designed and performed two prospective clinical studies, which were funded

by the National Institutes of Health and approved by the Cleveland Clinic

Institutional Review Board. All participants gave written informed consent. The

first study (the phosphatidylcholine challenge study) enrolled 40 healthy volunteers 18 years of age or above, who were without chronic illness (including

149

known history of heart, renal, pulmonary, or hematologic disease), without active

infection and not currently (or within preceding month) taking antibiotics or

probiotics. Participants underwent a dietary phosphatidylcholine challenge (see

below) during Visit 1. Among these study participants, six were then given

metronidazole 500 mg twice daily plus ciprofloxacin 500 mg once daily for one week, and a repeat phosphatidylcholine challenge was performed after antibiotics (Visit 2). A third and final phosphatidylcholine challenge was performed one month or longer following cessation of antibiotics and re- acquisition of gut flora (Visit 3). After each challenge, choline metabolites were measured in plasma and urine as described below.

The second study (the clinical outcomes study) enrolled 4,007 stable adults 18 years of age or older, who were undergoing elective diagnostic cardiac catheterization with cardiac troponin I less than 0.03 ug/L and no evidence of acute coronary syndrome. History of cardiovascular disease was defined as a documented history of coronary artery disease, peripheral artery disease, coronary or peripheral revascularization, 50% or greater stenosis of one or more

vessels during coronary angiography, or remote history of either myocardial infarction or stroke. Fasting blood samples were obtained at the time of cardiac catheterization on all participants. Routine laboratory tests were measured on the Abbott Architect platform (Abbott Laboratories, Abbott Park IL) except for myeloperoxidase, which was determined using the CardioMPO test (Cleveland

Heart Labs, Inc., Cleveland, OH). Creatinine clearance was estimated by the

Cockcroft-Gault equation. Trimethylamine-N-oxide was measured in plasma as

150

described below. Major adverse cardiovascular events (defined as all-cause

mortality, non-fatal myocardial infarction, and non-fatal stroke) were ascertained

and adjudicated for all participants over the ensuing three years following

enrollment.

Dietary phosphatidylcholine challenge

A simple dietary phosphatidylcholine-choline challenge test was administered to

all participants in the first study. For each participant, baseline blood and spot urine samples were obtained following an overnight (12 hours or longer) fast. At baseline, participants were provided two large hard-boiled eggs including yolk

(containing approximately 250 mg of total choline each) to be eaten within a 10-

minute period together with 250 mg of deuterium-labeled phosphatidylcholine

[(d9-trimethyl)-dipalmitoylphosphatidylcholine, d9-phosphatidylcholine] contained

in a gelatin capsule as a tracer (administered under an Investigational New Drug

exemption). Serial venous blood sampling was performed at 1, 2, 3, 4, 6 and 8

hours post-baseline, along with a 24-hour urine collection.

The high-purity d9-(trimethyl)-phosphatidylcholine (greater than 98%

isotope enrichment) provided was synthesized from 1-palmitoyl,2-palmitoyl,sn- glycero-3-phosphoethanolamine following exhaustive methylation with d3- methyliodide (Cambridge Isotopes Laboratories Inc, Andover MA). The d9- phosphatidylcholine was isolated by sequential preparative thin layer chromatography and high performance liquid chromatography, and crystallized

151

and dried under vacuum. Its purity (greater than 99%) was confirmed by both multinuclear nuclear magnetic resonance spectroscopy and mass spectrometry.

Measurements of choline metabolites

Plasma aliquots were isolated from whole blood collected into ethylenediaminetetraacetic acid tubes, maintained at 0 to 4°C until processing within four hours, and stored at -80°C. An aliquot from each 24-hour urine collection was spun to precipitate any potential cellular debris, and supernatants were stored at -80°C until analysis. Trimethylamine-N-oxide, trimethylamine, choline, betaine and their d9-isotopologues were quantified using stable isotope dilution high-performance liquid chromatography (HPLC) with on-line electrospray ionization tandem mass spectrometry on an AB SCIEX QTRAP

5500 mass spectrometer, using d4(1,1,2,2)-choline, d3(methyl)-trimethylamine-

N-oxide, and d3(methyl)-trimethylamine as internal standards. For measurement of trimethylamine in plasma, a sample aliquot was acidified (60 mM HCl final) prior to storage at -80ºC. Concentrations of trimethylamine-N-oxide in urine were adjusted for urinary dilution by analysis of urine creatinine concentration.

Statistical analysis for the clinical outcomes study

Student’s t-test, the Wilcoxon rank-sum test for continuous variables, and the chi- square test for categorical variables were used to examine the differences between participants in the clinical outcomes study who had major adverse cardiovascular events during follow-up and those who did not. For most

152

analyses of outcomes, plasma trimethylamine-N-oxide levels were divided into

quartiles. Where indicated, trimethylamine-N-oxide was also analyzed as a continuous variable with hazard ratio (HR) determined per standard deviation change in trimethylamine-N-oxide level. Kaplan–Meier analysis with Cox proportional hazards regression was used for time-to-event analysis to determine

HR and 95% confidence intervals (95% CI) for major adverse cardiovascular events. Logistic regression analyses were performed adjusting for traditional cardiac risk factors (age, gender, systolic blood pressure, history of diabetes mellitus, low-density and high-density lipoprotein cholesterol, triglycerides, and smoking history) with log-transformed high-sensitivity C-reactive protein, both alone and with myeloperoxidase, log-transformed estimated glomerular filtration

rate (GFR), total leukocyte count, body mass index (BMI), medications, and

angiographic extent of coronary artery disease. For subgroup analyses, logistic

regression analyses were performed by adjusting for traditional cardiac risk

factors and log-transformed high-sensitivity C-reactive protein. Improvement in

model performance introduced by the inclusion of trimethylamine-N-oxide was

evaluated using net reclassification improvement. The C-statistic was calculated

using the area under the receiver-operating-characteristic (ROC) curve. Three-

year predicted probabilities of a major adverse cardiovascular event were

estimated from the Cox model. All analyses were performed using R version

2.8.0 (Vienna, Austria). P values <0.05 (two-sided) were considered statistically

significant.

153

Variable Whole cohort (n=4,007) Without Events With P value (n=3,494) Events Age (years) 63±11 62±11 68±10 <0.001

Male Gender (%) 64 65 62 0.161

Body mass index 28.7(25.6-32.5) 28.7(25.7-32.5) 28.1 (24.8-32.4) 0.033

Diabetes mellitus (%) 32 30 43 <0.001

Hypertension (%) 72 71 79 <0.001

History of MI (%) 42 40 53 <0.001

Number of CAD vessels* Smoking (%) 65 65 69 0.053

LDL-c (mg/dL) 96 (78-117) 96 (78-117) 96 (75-116) 0.337

HDL-c (mg/dL) 34(28-41) 34(28-41) 33(28-40) 0.034

Triglycerides (mg/dL) 118 (85-170) 118 (85-169) 124 (86-173) 0.521

ApoB (mg/dL) 82 (69-96) 82 (69-96) 82 (68-96) 0.862

ApoA1 (mg/dL) 116 (103-133) 117 (103-133) 114 (100-129) 0.002

Fasting glucose 102 (93-119) 102 (92-117) 106 (94-135) <0.001 hsCRP (ng/L) 2.4 (1-5.9) 2.3(1-5.5) 3.9(1.8-9.8) <0.001

MPO (pM) 115.2 (76.4-245.7) 113.2 (75.4-238.3) 136.3 (84.7-329.3) <0.001

GFR(ml/min/1.73m2) 82 (69-95) 83 (71-96) 75 (56-89) <0.001

Total leukocyte count 6.1 (5.1-7.5) 6.1 (5-7.5) 6.4 (5.3-8.1) 0.001 (WBC, x109) Baseline drugs (%):

Aspirin 74 74 70 0.038

ACE inhibitor/ARB 50 49 58 <0.001

Statin 60 61 56 0.057

Beta blockers 63 63 65 0.414

TMAO (µM) 3.7 (2.4-6.2) 3.5 (2.4-5.9) 5.0 (3.0-8.8) <0.001

Values expressed in mean ± standard deviation or median (interquartile range). Abbreviations: ACE, angiotensin converting enzyme; ARB, angiotensin receptor blocker; ApoA1, apolipoprotein A1; ApoB, apolipoprotein B; CAD, coronary artery disease; GFR, estimated glomerular filtration rate; HDL-c, high-density lipoprotein cholesterol; hsCRP, high sensitivity C-reactive protein; LDL-c, low-density lipoprotein cholesterol; MI, myocardial infarction; MPO, myeloperoxidase; TMAO, trimethylamine N-oxide; WBC, white blood cell *Defined as a coronary stenosis of 50% or greater.

Table 4-1. Baseline characteristics147

154

TMAO (range)

Quartile 1 Quartile 2 Quartile 3 Quartile 4

Range <2.43 2.43-3.66 3.67 -6.18 ≥ 6.18

Major adverse cardiac events (Death, myocardial infarction, stroke)

Unadjusted HR 1 1.2 (0.9-1.6) 1.5 (1.2-2.0)** 2.5 (2.0-3.2)**

Adjusted HR

Model 1 1 1.1 (0.8-1.5) 1.3 (0.97-1.7) 1.9 (1.4-2.4)**

Model 2 1 1.1 (0.8-1.4) 1.2 (0.8-1.6) 1.6 (1.1-2.1)**

Model 3 1 1.1 (0.8-1.5) 1.1 (0.8-1.5) 1.4 (1.1-1.9)*

** p<0.01; HR, * p<0.05. Cox Proportional Hazards analyses variables were adjusted to +1 standard deviation increment for continuous variables. Model 1: Adjusted for traditional risk factors (age, gender, smoking, systolic blood pressure, low density lipoprotein cholesterol [LDL], high-density lipoprotein cholesterol [HDL], and diabetes mellitus), plus log-transformed hsCRP Model 2: Adjusted for traditional risk factors, plus log-transformed hsCRP, myeloperoxidase, log-transformed estimated GFR, total leukocyte count, body mass index, aspirin, statins, ACE inhibitor/ARB, and beta blockers Model 3: Adjusted for traditional risk factors, plus log-transformed hsCRP, myeloperoxidase, log-transformed estimated GFR, total leukocyte count, body mass index, aspirin, statins, ACE inhibitor/ARB, beta blockers, and angiographic extent of disease.147

Table 4-2. Unadjusted and adjusted hazard ratio for risks of MACE at 3-years stratified by quartile levels of TMAO147

155

Figure 4-1. Human plasma levels of phosphatidylcholine Metabolites (TMAO, choline, betaine) after oral ingestion of two hard-boiled eggs and d9-Phosphatidylcholine before and after antibiotics. At the top of the figure, the visit sequence is shown. All 40 study participants (healthy volunteers) participated in the first dietary phosphatidylcholine challenge (Visit 1). Six participants were then administered broad-spectrum antibiotics for one week, followed by a second phosphatidylcholine challenge (Visit 2). These same participants returned again at least one month after discontinuing antibiotics for a third challenge (Visit 3). The panels in Rows A and B show the results of assays for trimethylamine-N-oxide (Panel a) and d9-trimethylamine-N-oxide (Panel b) after the phosphatidylcholine challenge, using stable isotope dilution high-performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry. Panels c and d show the time course of plasma concentrations of betaine, choline and trimethylamine-N-oxide (Panel c) and of their d9 isotopologues (Panel d). Note that, in Panel d, the concentrations of d9-trimethylamine-N-oxide are multiplied by 4; in Panel c, the concentrations of d9-trimethylamine-N-oxide are multiplied by 12, and those of choline are multiplied by 4. All left panels show data from Visit 1; center panels, from Visit 2; and right panels, from Visit 3.147

156

Figure 4-2. Kaplan-Meier estimates of long-term major adverse cardiac events, according to TMAO Quartiles.147

157

Figure 4-3. Pathways linking dietary phosphatidylcholine, intestinal microflora (gut flora), and incident adverse cardiovascular events. Ingested phosphatidylcholine (lecithin), the major dietary source of total choline, is acted on by intestinal lipases to form a variety of metabolic products including the choline-containing nutrients glycerophosphocholine, phosphocholine, and choline. Choline-containing nutrients that reach the cecum and large bowel may serve as fuel for intestinal microbiota (gut flora), producing trimethylamine (TMA). TMA is rapidly further oxidized to trimethylamine-N-oxide (TMAO) by hepatic flavin-containing monooxygenases (FMOs). TMAO enhances macrophage cholesterol accumulation, foam cell accumulation in the artery wall and atherosclerosis69, and incident risk of heart attack, stroke, and death. Choline can also be oxidized to betaine in both liver and kidney169. Dietary betaine can also serve as a substrate for bacteria to form TMA117 and presumably TMAO.147

158

Supplementary Tables and Figures

Characteristic Quartile 1 Quartile 2 (n=998) Quartile 3 (n=1003) Quartile 4 P-value (n=1001) (n=1005) (2.4-3.6) (3.7-6.2) (TMAO, µM) (<2.4) (>6.2)

Age (years) 59±11 62±11 65±10 66±10 <0.001

Male Gender (%) 67 67 63 61 0.008

Body mass index 28.4 28.7 28.7 28.4 0.138 (25.4-32) (25.6-32.8) (25.9-32.6) (25.7-33.1) Diabetes mellitus (%) 24 28 31 42 <0.001

Hypertension (%) 68 69 70 79 <0.001

History of MI (%) 41 39 42 43 0.317

# of diseased vessels

0 30 26 27 21 <0.001

1 22 22 18 18 0.017

2 21 21 19 18 0.233

3 27 30 36 42 <0.001

Smoking (%) 63 65 67 65 0.314

LDL-c (mg/dL) 97 98 95 92 <0.001 (79-117) (81-120) (78-116) (74-114) HDL-c (mg/dL) 34 35 34 33 <0.001 (29-42) (29-42) (29-41) (27-40) Triglycerides (mg/dL) 115 115 121 123 0.089 (84-166) (84-163) (86-178) (86-180) ApoB (mg/dL) 82 83 82 80 0.05 (70-97) (69-98) (69-95) (68-94) ApoA1 (mg/dL) 116 117 117 115 0.121 (103-134) (104-132) (104-133) (101-132) Fasting glucose 100 101 102 107 <0.001 (91-114) (92-116) (93-119) (95-134) hsCRP (ng/L) 2.3 2.2 2.3 3.1 <0.001 (0.9-6.3) (1-5.6) (1.1-5) (1.2-6.8) MPO (pM) 127.3 115.3 112.9 110.8 0.092 (78.7-264.6) (76.9-242.4) (75.8-235.2) (74.2-226.8) eGFR(ml/min/1.73m2) 92 86 79 69 <0.001 (81-103) (75-96) (67-91) (52-83) Total leukocyte count (WBC, x109) 6.2 6.1 6.1 6.1 0.524 (5.1-7.6) (5.1-7.5) (5-7.5) (5-7.5) Baseline drugs (%):

Aspirin 76 76 73 70 0.007

ACE inhibitor/ARB 44 47 53 57 <0.001

Statin 64 61 58 57 0.005

Beta blockers 62 64 63 64 0.823 Supplementary Table 4-1. Baseline characteristics of cohort according to TMAO quartiles values expressed in mean ± standard deviation or median (interquartile range). Abbreviations: MI, myocardial infarction; LDL-c, low-density lipoprotein cholesterol; HDL-c, high-density lipoprotein cholesterol; ApoB, apolipoprotein B; ApoA1, apolipoprotein A1; hsCRP, high sensitivity C-reactive protein; MPO, myeloperoxidase; WBC, white blood cell; ACE, angiotensin converting enzyme; ARB, angiotensin receptor blocker; TMAO, trimethylamine N-oxide.147

159

Supplementary Figure 4-1: Human plasma levels of phosphatidylcholine metabolites (TMAO, choline, betaine) after oral ingestion of two hard-boiled eEggs and d9-phosphatidylcholine before and after antibiotics. At the top of the figure, the visit sequence is shown. All 40 study participants (healthy volunteers) participated in the first dietary phosphatidylcholine challenge (Visit 1). Six participants were then administered broad-spectrum antibiotics for one week, followed by a second phosphatidylcholine challenge (Visit 2). These same participants returned again at least one month after discontinuing antibiotics for a third challenge (Visit 3). Panels a and b show the time course of plasma concentrations of betaine, choline and trimethylamine- N-oxide (Panel a) and of their d9 isotopologues (Panel b). Note that, in Panel a, the concentrations of choline are multiplied by 4, and the concentration of trimethylamine-N- oxide are multiplied by 12; in Panel b, the concentrations of d9-trimethylamine-N-oxide are multiplied by 4. All left panels show data from Visit 1; center panels, from Visit 2; and right panels, from Visit 3.147

160

Supplementary Figure 4-2. Human 24-hour urine levels of TMAO after oral ingestion of two hard-boiled eggs and d9-phosphatidylcholine before and after antibiotics. Numbers in label represent the mass-to-charge ratios for the precursor → product ion transitions monitored for TMAO and d9-TMAO.147

161

Supplementary Figure 4-3: Risks of major adverse cardiac events (MACE) among patient subgroups, according to baseline TMAO levels. Hazard ratios compare top to bottom quartiles. Significant interactions were observed between plasma trimethylamine-N-oxide and cigarette smoking (P=0.027) as well as plasma trimethylamine-N-oxide and plasma myeloperoxidase (P=0.012).147

162

CHAPTER 5: 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

Authors: Robert A. Koeth, Bruce S. Levison, Zeneng Wang, Jennifer A. Buffa,

Elin Org, Miranda Culley, Yuping Wu, Lin Li, Jonathan D. Smith, Joseph A.

DiDonato, W. H. Wilson Tang, Aldons J. Lusis, and Stanley L. Hazen

Abstract

Carnitine, an abundant nonessential nutrient found in red meat, was recently shown to promote atherosclerosis via generation of a gut microbiota dependent compound trimethylamine-N-oxide (TMAO)73. These studies did not explore the possibility of an intermediate compound formed in the gut microbiota metabolism of carnitine to TMAO. Further analysis of plasma from mice fed a carnitine supplemented diet demonstrates the production of a second gut microbiota dependent trimethylamine metabolite, γ-butyrobetaine (γBB) that is both a quantitatively dominant product of gut microbiome dependent carnitine metabolism and an inducible trait. γBB is endogenously produced as the terminal precursor in the synthesis of carnitine, but serves no other known physiological function81,82. Atherosclerotic prone mice supplemented with a γBB diet develop significantly more aortic root lesion area compared to mice on a normal chow diet and gut flora suppressed animals supplemented with γBB. Importantly, the increase in atherosclerotic disease burden cannot be attributed to abnormalities

163

in plasma or liver lipid metabolism. These data suggest the terminal gut flora

product TMA/TMAO is promoting the atherosclerotic disease process and that

γBB is a gut flora intermediate in carnitine metabolism to TMA/TMAO.

Remarkably, microbiome analysis of mice fed carnitine diet and γBB diets

demonstrate differing bacterial compositions suggesting cooperation of two

distinct microbiota populations in the sequential 2 step gut microbiota dependent

metabolism of carnitine to TMAO (e.g. carnitine to γBB to TMAO). γBB further

extends our knowledge on gut flora metabolism of carnitine by defining a second,

dominant pathway for TMAO formation and revealing further complexity of the

proatherogenic microbiome.

Introduction

The gut microbiome participates in the pathogenesis of complex disease

phenotypes such as diabetes, obesity, and more recently

atherosclerosis36,43,67,69. The gut microbiome promotes atherosclerosis by the

gut microbiota metabolism of phosphatidylcholine, the major dietary source of choline, and resulting production of the proatherogenic metabolite TMAO (Gut

Pathway 1 )69. TMAO increases scavenger receptor expression (CD36 and SRA)

resulting in enhanced foam cell formation and causes gut microbiota dependent

dysfunction in the reverse cholesterol transport pathway by disruption of bile acid

synthesis73. Moreover, plasma levels of TMAO associate with cardiovascular

disease and independently predict prospective near and long-term Major

Adverse Cardiovascular Events (MACE)147. Together these data suggest a like

between dietary trimethylamines, the gut microbiota, and atherosclerosis. 164

Microbiota FMOs Gut Pathway 1: Choline TMA TMAO

Recently, we demonstrated that L-carnitine, a dietary trimethylamine found

principally in red meat, can also be metabolized by the gut microbiota to produce

TMA/TMAO73. Animals fed a L-carnitine supplemented diet developed gut

microbiota dependent accelerated atherosclerosis that associates with

TMA/TMAO production. Further analyses reveal that TMAO significantly

associates with carnivorous eating habits and together with microbiome

composition can distinguish dietary patterns in humans and mice. This suggests

gut microbiome metabolism of carnitine can partly explain the commonly

observed association between a carnivorous diet and atherosclerosis73 (Gut

Pathway 2).

Microbiota FMOs Gut Pathway 2: L-carnitine TMA TMAO

Although the metabolism of L-carnitine to TMA/TMAO is clear, the pathways and

enzymes bacteria use to metabolize are not. Our previous studies did not

consider the possibility that carnitine may produce other gut microbiota

metabolites. Studies in a rat model using a radioactive isotope of L-carnitine

suggested that another trimethylamine, γ-butyrobetaine (γBB), is produced from

L-carnitine (Gut Pathway 3)94.

Microbiota Gut Pathway 3: L-carnitine γ-BB

165

γ-Butyrobetaine (γBB) is a trimethylamine containing compound that is used as a dietary supplement and endogenously produced as the terminal precursor in the production of endogenous carnitine (Endogenous Pathway)81,82. Little is known

regarding the relationship between γBB and the gut microbiota.

Endogenous Pathway: Lysine TML HTML TMABA γBB L-carnitine

The gut microbiota metabolism of carnitine to γBB raised the possibility that

direct formation of TMA/TMAO from L-carnitine is principally mediated an intermediate metabolite γBB formation (Hypothesized Gut Pathway).

Microbiota ? Hypothesized Gut Pathway: L-carnitine γ-BB TMA FMOs TMAO

Herein, we demonstrate that the gut microbiota metabolite, γBB, is the dominant metabolite of gut microbiota metabolism of carnitine. γBB produces TMA/TMAO and promotes atherosclerosis in a gut microbiota dependent fashion and production of γBB from L-carnitine is an inducible trait. Moreover, gut microbiome

characterization studies reveal the production of γBB from L-carnitine and the

concordant production of TMA/TMAO from γBB associate with entirely separate

bacterial taxa suggesting a more complex microbiome than previously

anticipated.

166

Results

Gut microbiota metabolism of L-carnitine produces γBB

Survey of the literature revealed that the production of γBB by the gut microbiota

may be a gut microbiota product of L-carnitine94. This observation raised the

possibility that γBB could be another gut microbiota metabolite contributing to the atherosclerotic disease process directly or by further metabolism into

TMA/TMAO. Quantification of γBB in plasma by LC/MS/MS from mice on a L- carnitine supplemented diet demonstrated an almost 100 fold increase in plasma concentration compared to chow fed control or antibiotic controls (Figure 5-1).

Remarkably, plasma concentrations of γBB exceeded the concentration of plasma TMA or TMAO in L-carnitine supplemented mice by approximately 2-fold suggesting its production was also the major gut microbiota metabolite produced from L-carnitine (Figure 5-1). The gut microbiota dependent production of γBB from L-carnitine was confirmed by performing a L-carnitine challenge (d3-L- carnitine direct gastric challenge) in female Germ Free Swiss Webster mice.

Post challenge measurements of d3-γBB in Germ Free mice demonstrate absolutely no production of d3-γBB. However, following acquisition of the gut microbiota, mice rechallanged demonstrated the ability to produce d3-γBB

(Figure 5-2).

γBB produces TMA/TMAO in a gut microbiota dependent manner

The production of γBB from L-carnitine raised the possibility that γBB could contribute to TMA/TMAO formation by serving as an intermediate in the gut

167

microbiota metabolism of L-carnitine. C57BL/6J 12 week old female mice were

challenged with d9-γBB chloride and followed with serial venous bleeds for 12

hours. Quantification of d9-containing trimethylamines in plasma by LC/MS/MS

revealed that both d9-TMA and d9-TMAO were produced (Figure 5-3, first panel). Interestingly, d9-L-carnitine was also produced. Gut microbiota

suppression with broad spectrum antibiotics and rechallenge demonstrated the

complete absence of d9-TMA/TMAO production confirming gut microbiota

dependence (Figure 5-3, second panel). In contrast, d9-carnitine was produced

in a similar concentration compared to the initial conventional challenge

establishing that L-carnitine is not a gut microbiota product of γBB. Instead, d9-

γBB appears to be absorbed and shuttled through the endogenous L-carnitine

synthetic pathway. This is supported by the early peak of d9-γBB in plasma

followed by the gradual increase in d9-L-carnitine concentration in plasma over

the 12 hour period. Conventionalization of the experimental mice demonstrated

reacquisition of the capacity of the gut microbiota to produce d9TMA/TMAO while

d9-L-carnitine levels remained similar to concentrations in the two previous

challenges (Figure 5-3, last panel). The gut microbiota dependent formation of

d9TMA/TMAO from d9-γBB was confirmed by challenging Germ free female

Swiss Webster mice immediately upon receipt (Figure 5-4). As observed in

antibiotic suppressed C57BL/6J female mice, both d9-TMA/TMAO was absent

and d9 –L-carnitine was produced. Conventionalization of germ free mice with in-

house mice, demonstrate the acquisition of the capacity for the gut microbiota to

168

produce d9-TMA/TMAO while the amount of d9-carnitine production remained

the same(Figure 5-4).

TMA formation occurs in the cecum and γBB is the dominant gut

microbiota product of L-carnitine gut microbiota metabolism

Incubation of equal molar amounts of d3-L-carnitine or d9-γBB with segments of

mouse intestines demonstrate the production of TMA mainly in the bacterial rich

cecum of mice for both d3-L-carnitine and d9-γBB suggesting the cecum is the major site of TMA generation from dietary trimethylamines (Figure 5-5). In

contrast, the production of γBB was more uniformly distributed along the distal

intestinal tract (Figure 5-5). d9-γBB was also more readily metabolized to TMA

than d3-L-carnitine by approximately 2-fold (Figure 5-5). Moreover, quantitative

comparison of the production d3-γBB and d3-TMA from in vitro cecal studies

show a remarkable 1000 fold increase in d3-γBB production over d3-TMA

demonstrating that d3-γBB is the dominant gut microbiota metabolite produced

from the intestinal gut microbiota (Figure 5-5; lower panel).

Metabolism of γBB by the gut microbiota to TMA/TMAO promotes

atherosclerosis

The gut microbiota dependent production of TMA/TMAO from γBB and the

dominant production of γBB from L-carnitine raised the possibility that γBB was

contributing to the development of atherosclerosis either indirectly by serving as

an intermediate between the terminal metabolism of L-carnitine to TMA or as a

169

direct gut microbiota L-carnitine metabolite. C57BL/6J, Apoe-/- female mice were placed on a chow diet or γBB (1.3%) diet with respective gut microbiota suppression controls (+ABS) at weaning for 15 weeks before necroscopy (Figure

5-6). Quantification of atherosclerotic plaque at the aortic root revealed an approximately 1.5 fold increase in total area of plaque in γBB animals compared to controls (Figure 5-6). Importantly, there was no increase in total plaque area in mice on a γBB diet with gut microbiota suppression (+ABS) suggesting that

TMA and TMAO was the gut microbiota product promoting atherosclerotic disease and not γBB. These data were confirmed by quantification of

TMA/TMAO in terminal mouse plasma samples by LC/MS/MS. Both TMA/TMAO were produced in a quantitatively dominant amount in mice fed a γBB diet compared to the respective chow or gut microbiota suppressed controls (Figure

5-7). Additionally, +ABS, γBB supplemented mice failed to demonstrate an increase in atherosclerotic aortic root plaque yet had the highest plasma γBB concentrations among mice in the study (Figure 5-7).

Metabolism of γBB from L-carnitine is an inducible trait

We previously reported that metabolism of TMA/TMAO from L-carnitine by the gut microbiota is an inducible trait. This suggested that the production of γBB from L-carnitine may also be inducible. To test this possibility, a L-carnitine challenge (d3-L-carnitine) was performed on mice on a L-carnitine supplemented diet and control chow fed mice respectively over a 12 hour period. LC/MS/MS

170

analysis of plasma from serial venous draws from mice reveal that production of

plasma d3-γBB is an inducible trait (Figure 5-8).

γBB associates with a microbiome composition that differs from

TMA/TMAO formation

We previously demonstrated that production of TMA/TMAO associated with

microbiome genera of mice on a L-carnitine diet73. The gut microbiota dependent

production of γBB from L-carnitine naturally raised the possibility that production

of γBB may also associate with the gut microbiome. Compositional analysis of

the microbiota of mice on a carnitine diet with plasma γBB demonstrated

significant associations even after adjustment for multiple testing (Fig. 5-9,5-10).

Remarkably, analysis of the gut microbiome composition revealed distinctly

different microbiome associations than previously reported with TMA/TMAO73.

γBB associates with the bacterial genera of Parasutterella, Prevotella, and

Bacteroides and we previously had demonstrated that TMA/TMAO production

from L-carnitine associates with Prevotella, Anaeroplasma, and Mucispirillium73.

Although these associations overlapped with the genus Prevotella, TMA/TMAO production generally had a negative association with bacteria from the

Bacteroides and Proteobacteria phyla suggesting that distinct microbiota participate in the metabolism of L-carnitine. Combined analysis of plasma γBB (x- axis) and bacterial operational taxonomic units (OTUs; y Axis) further demonstrate mice can be distinguished by dietary status.

171

TMAO production from γBB associates with microbiome composition

The dietary contribution of γBB and the production of γBB from L-carnitine data

suggested that γBB can also be involved in shaping the gut microbiota.

Compositional analysis of mice on a γBB diet reveal that gut microbiota

metabolite TMAO associates with gut microbiome composition of microbiota from

the phyla Verrucombria. This appears to be mostly driven by an association with

the genus Akkermansia (Figure 5-11,5-12). Remarkably, plasma γBB

metabolized from L-carnitine and microbiota composition studies demonstrate

virtually no association with this genus. Together these data suggest cooperation

between bacterial microbiota species in the sequential production of TMA from L-

carnitine.

Mice on a γBB diet have significant decreased liver expression of Cyp7a1,

but not Cyp27a1

We previously demonstrated that mice on trimethylamine supplemented diets

(e.g. carnitine or choline) have a significant gut microbiota dependent reduction

in total reverse cholesterol transport that may be attributed to a decrease in total

bile acid pool size and related bile acid synthetic enzyme73. Thus, in a final set of studies liver expression of two key bile acid producing enzymes (Cyp7a1 and

Cyp27a1) were examined (Figure 5-13). Consistent with previous studies the expression of liver Cyp7a1, the rate-limiting enzyme in the classic pathway of bile acid synthesis from cholesterol, was significantly decreased in γBB liver compared to chow fed control liver. In contrast to previous studies, expression of

172

Cyp27a1, an important enzyme in the classic pathway and the alternative acidic

pathways of bile acid synthesis from cholesterol, was not significantly different170

(Figure 5-13).

Discussion

Early studies demonstrate that mammals lack the capacity to catabolize L- carnitine81,82,119. We previously demonstrated that catabolism of L-carnitine

directly to TMA/TMAO is a gut microbiota dependent pathway. The present

studies unambiguously show that gut microbiota metabolism to γBB, mainly

thought to be involved only in the endogenous L-carnitine synthesis, is the

dominant metabolite in L-carnitine degradation by the gut microbiota. As our data shows, even in the proximal small bowel where the bacterial load is relatively small compared to more distal parts of the GI tract L-carnitine catabolism to γBB

begins. This pathway (e.g. L-carnitine catabolism to γBB) is kinetically favored

over a 1,000 fold compared to direct metabolism of L-carnitine to TMA/TMAO

(Figure 5-14).

The importance of γBB in mammalian physiology has traditionally centered on its role in L-carnitine synthesis. γBB serves as the terminal substrate in endogenous

L-carnitine production that begins with metabolism of lysine and methionine.

Indeed, the majority of studies on γBB in mice and humans have been in the context of understanding endogenous L-carnitine production and its effect on L- carnitine levels171-173. Here, we demonstrate an important role for γBB in gut

173

microbiota metabolism of L-carnitine by demonstrating for the first time the

microbiota mediated metabolism of γBB to TMA. Not only does γBB serve as the

dominant metabolite of gut microbiota dependent L-carnitine metabolism, but

more TMA is produced from γBB than L-carnitine on an equamolar basis.

As with dietary supplementation with choline and carnitine, supplementation of

γBB also increased atherosclerotic plaque area in a gut microbiota dependent manner. Importantly, these data also clarify that the gut microbiota metabolism of

γBB to TMA/TMAO and not γBB directly promote atherosclerosis. This

conclusion is supported by the following evidence. First, mice supplemented with

γBB and cosuppression of the microbiota with antibiotics did not have an

increase in total plaque area at the aortic root. Secondly, mice with the highest plasma concentrations of γBB (e.g. γBB, +ABS) did have increased atherosclerotic plaque compared to the chow control. Finally, mice with the highest plasma concentrations of TMA/TMAO had the most plaque at the aortic root compared to chow and antibiotic controls. Together these data further suggest the terminal microbiota production of TMA/TMAO is responsible.

Interestingly, there was a significant decrease in plasma L-carnitine concentration in mice fed γBB diet compared to chow controls suggesting that high plasma γBB concentrations have a suppressive effect on endogenous L- carnitine production. The difference in plasma γBB in mice fed γBB to the respective gut microbiota suppressed control would suggest that γBB is being

174

metabolized by the gut microbiota into TMA and not being readily absorbed. This may also explain why there is also a significant decrease in plasma L-carnitine when comparing the γBB and γBB gut microbiota suppressed control (e.g. the more plasma γBB there is the less plasma L-carnitine is observed).

The metabolism of γBB from L-carnitine appears to be more evenly distributed

between the cecum and colon. However, the site of proatherogenic TMA

production from both L-carnitine and γBB appears to be primarily localized in the

cecum. The observation that both the production of γBB from carnitine and

TMA/TMAO production from γBB is inducible indicates a more complex microbiome than previously anticipated.

Microbiome analysis of cecums from L-carnitine supplemented mice demonstrate

a gut microbiome that associates with plasma γBB. Remarkably, this analysis

revealed that γBB associated with a different microbiome composition than

TMA/TMAO from L-carnitine supplemented mice. Close examination between the

trends of γBB and TMA/TMAO association with the gut microbiota reveal that

only Prevotella commonly associated taxa between both studies (γBB and

TMA/TMAO). Moreover, the microbiota taxa that associated with γBB production tended to have no association or inversely associate with TMA/TMAO production.

175

To further understand the role of the gut microbiota in L-carnitine metabolism, we performed compositional microbiota studies of animals on a γBB diet and

analyzed it with corresponding mouse plasma TMA /TMAO concentrations.

Interestingly, correlation heat maps between the gut microbiota composition of

TMAO from mice on a γBB diet and TMAO from mice on a L-carnitine diet are

different suggesting more complexity in the gut microbiome metabolism of

carnitine than previously anticipated. Together, these data suggest the

participation of two distinct microbiomes in the 2 step metabolism of L-carnitine to

TMA. It also suggests cooperation between microorganisms metabolizing L-

carnitine to γBB and γBB to TMA/TMAO.

These observations suggest that there are multiple potential clinical therapeutic

targets. Disruption of the enzymes involved in the metabolism of L-carnitine to

γBB and/or inhibition of γBB to TMA , for example, may be more important

potential therapeutic targets than enzymes involved in the direct production of

TMA from L-carnitine. One would anticipate that a probiotic could be used that

would allow one to consume steak without metabolizing carnitine to TMAO.

However, further studies in human subjects to confirm the dominance of gut

microbiota metabolism of carnitine to γBB are needed.

The exact microorganisms responsible for metabolizing carnitine to γBB remain

unclear. There are reports demonstrating that microorganisms found in the GI

tract have the capacity to metabolize L-carnitine to γBB81. Initially, the recognition

176

that γBB was a gut metabolite of L-carnitine was demonstrated from Escherichia

coli, purified from rat intestine174. Subsequent studies using other gut

microorganisms in the Enterobacteriaceae family also have been found to

metabolize L-carnitine to γBB (e.g. Escherichia coli, Proteus vulgaris, and

Salmonella typhimurium). Interestingly, these microorganisms are contained with

the Proteobacteria phyla. In the present studies many of the taxa positively

associating with γBB production are also found in this same phyla. Although

none of the associated taxa contained these bacterial genera, it does raise the

possibility that this phyla of bacteria have the capacity to L-carnitine to γBB (e.g..

Proteobacteria). In contrast, there are no reports of microorganisms metabolizing TMA from γBB.

Previously, we have demonstrated that the mechanisms accounting for the contribution of the terminal gut microbiota end products TMA/TMAO in the

pathogenesis of cardiovascular disease are multifactorial. Mice fed a

trimethylamine diet can promote atherogenesis by increasing foam cell formation

by upregulation of scavenger receptors CD36 and SRA69. Follow-up work

demonstrated that dietary trimethylamines also promote atherogenesis by

reducing reverse cholesterol transport by decreasing the bile acid pool size. This

dysfunction was apparently being mediated by the downregulation of enzymes

involved in cholesterol metabolism into bile acids and bile acid transporters73.

Here we demonstrate that like mice fed a TMAO supplemented diet, animals on

a γBB diet also have dysfunctional bile synthesis evidenced by the parallel

177

decrease in Cyp7a1, the rate limiting enzyme in bile acid synthesis. The exact

molecular explanation for the role of TMA/TMAO in dysfunctional bile acid

metabolism and upregulation of SRA and CD36 remains unclear, but could be

mediated by a TMA/TMAO interaction with a ‘TMAO specific’ receptor.

In summary, we have discovered that γBB, a trimethylamine, serves as a gut

microbiota intermediate compound in the metabolism of L-carnitine to

TMA/TMAO and provides an important mechanistic link in understanding the L- carnitine gut microbiota dependent promotion of atherosclerosis.

Methods

Materials and general procedures

Mice and/or breeders were obtained from Jackson Laboratories. All animal studies were performed under approval of the Animal Research Committee of the

Cleveland Clinic. Mouse plasma total cholesterol, triglycerides, and glucose were

measured using the Abbott ARCHITECT platform model ci8200 (Abbott

Diagnostics, Abbott Park, IL). HDL cholesterol concentration in mice used for the

γBB atherosclerosis study was enzymatically determined (Stan bio, Houston, TX)

from plasma HDL isolated using density ultracentrifugation as previously described73. Liver triglyceride content was measured using the GPO reagent

(Pointe Scientific, Canton, MI) and normalized to liver mass (g) grams as

previously described (millard). Liver cholesterol was quantified in liver

homoginates with added coprostanol (Steraloids, Inc, Newport, RI) internal

178

standard. Liver were lipids extracted by the Folch method (chloroform:methanol

(2:1, v/v)), and then cholesterol quantified as its trimethylsilane (TMS) derivative

(Sylon HTP, Sigma-Aldrich, Sigma St. Louis, MO) by GC/MS (Agilent 5973N

model, Santa Clara CA) as previously described126. Gut microbiota suppression

studies were performed by dissolving antibiotics in mouse drinking and included

0.1% Ampicillin sodium salt (Fisher Scientific), 0.1% Metronidazole, 0.05%

Vancomycin (Chem Impex Intl.), and 0.1% Neomycin sulfate (Gibco) as

previously described35,69.

Mouse challenge and atherosclerosis studies

An oral γBB or L-carnitine challenge in mice consisted of a gastric gavage of d9-

γBB (prepared as described below) or d3-L-carnitine (Cambridge Isotope

Laboratories; Andover, MA) dissolved in water respectively. 10-week-old female

Taconic Swiss Webster germ-free mice were γBB challenged immediately upon

arrival in a microisolater. The mice were then conventionalized by being housed

in cages with non-sterile C57BL/6J female mice for approximately 1 month

before the γBB challenge was perform again. The γBB challenge was also performed on 12-week old C57BL/6J female mice in the native state, after gut microbiota suppression with broad spectrum antibiotics for 1 month, and finally, after being housed with native mice for an approximately 3 month conventionalization period69. Gut microbiota inducibility studies were completed

by performing L-carnitine or the γBB challenge on 12 week old C57BL/6J, Apoe-

/- mice on a chow diet or an L-carnitine supplemented diet for at least a 10 week

179

period. For the atherosclerosis study, C57BL/6J, Apoe-/- were placed on a standard chow control diet (Teklad 2018) or γBB supplemented diet (mouse drinking water with 1.3% γBB; BOC scientific) with and without antibiotics at time of weaning for a 15 week duration. The antibiotic regimen used was provided to the mouse in the drinking water as described above. Mouse aortic root plaque was prepared and quantified as previously described69. Quantification of natural abundance and isotope labeled forms of carnitine, γBB, TMA and TMAO in mouse plasma was performed using stable isotope dilution LC/MS/MS as described below.

Mouse microbiome studies

Microbial community composition was assessed by pyrosequencing 16S rRNA genes derived from the mice cecal samples of normal chow diet (n=16), transcrotonobetaine (n=11) and γ-butrobetaine (n=12). DNA was isolated using the MoBio PowerSoil DNA Isolation Kit according to the manufacturer’s instructions. The V4 region of the 16S rRNA gene was amplified using bar-coded fusion primers (F515/R806) with the 454 a Titanium sequencing adapter. The barcoded primers were achieved following the protocol described by Hamady et133. Sample preparation was performed similarly to that described by Costello et al.134. Each sample was amplified in triplicate, combined in equal amounts and cleaned using the PCR clean-up kit (Mo Bio). Cleaned amplicons were quantified using Picogreen dsDNA reagent (Invitrogen) before sequencing using 454 GS

FLX titanium chemistry at the GenoSeq Facility at the University of California,

180

Los Angeles. The raw data from the 454 pyrosequencing machine were first

processed through a quality filter that removed sequence reads that did not meet

the quality criteria. Sequences were removed if they were shorter than 200

nucleotides, longer than 1,000 nucleotides, contained primer mismatches,

ambiguous bases, uncorrectable barcodes, or homopolymer runs in excess of six

bases. The remaining sequences were analyzed using the open source software

package Quantitative Insights Into Microbial Ecology (QIIME)135,136. A total of

49,458 quality filtered reads were obtained from 39 samples (three samples were

removed due to low sequence count). Individual reads that passed filtering were

distributed to each sample based on bar-code sequences. Demultiplexed

sequences were assigned to operational taxonomic units (OTUs) using UCLUST

with a threshold of 97% pair-wise identity. Representative sequences were

selected and BLASTed against a reference Greengenes reference database. For

each resulting OTU, a representative sequences were selected by choosing the most abundant sequence from the original post-quality filtered sequence collection. The taxonomic composition was assigned to the representative sequence of each OTU using Ribosomal Database Project (RDP) Classifier

2.0.1137. The relative abundances of bacteria at each taxonomic level (e.g.,

phylum, class, order, family and genus) were computed for each mouse.

Correlations between relative abundance of gut microbiota and TMA and TMAO

levels and association testing were performed in R. False discovery rates (FDR)

of the multiple comparisons were estimated for each taxon based on the P-

values resulted from correlation estimates.

181

d9-γ-Butyrobetaine chloride preparation

(3-Carboxypropyl)trimethyl(d9)ammonium Chloride (d9-γ-butyrobetaine Chloride, d9-γBB Cl) was prepared from γ-aminobutyric (GABA) acid (Sigma #A2129) in methanol with potassium hydrogen carbonate and d3-methyl iodide by the method of Cain Morano, Xin Zhang, and Lloyd D. Fricker 175. After 72 hours, the

entire reaction mixture was quantitatively transferred onto a short silica gel

column (grade 60, 230-400 mesh) equilibrated in methanol in a coarse fritted

Buchner funnel. Non-polar material was removed by elution of the column with

the 1.25 column volumes of methanol. The product d9-γBB was eluted in 2.5

column volumes of 30%v/v water in methanol. Rotary evaporation of this second

eluate gave the crude product as an oily semisolid which was dissolved in water

and titrated to pH 7.2 with dilute hydrochloric acid. The water was removed by

rotary evaporation and final traces of moisture were removed azeotropically by

distillation of absolute ethanol from the residue. The white to off-white solid was

dissolved in absolute ethanol and filtered to remove residual inorganic salts. The

material was concentrated to dryness and dissolved in excess diluted

hydrochloric acid (3M). The resulting straw colored solution was concentration to

dryness by rotary evaporation was followed by re-dissolution of the semi-

crystalline light amber colored salt in a minimal amount of methanol. This

methanolic solution was treated with 5 volumes of acetone; the resulting almost

clear solution was allowed to sit at room temperature for several hours. The

resulting plate-like crystals were isolated by suction filtration, transferred to a

clean container, and dried under vacuum at 60oC. This material darkened slightly

182

to an off white, slightly amber free flowing powder which was stored refrigerated over desiccant. Concentrations of stock solutions of this material were determined relative to a standard curve of authentic γBB Cl, by LC/MS/MS as described below. ESI positive ion mode mass spectrum for d9-γ-butyrobetaine chloride (5µM in 50% v/v Methanol in water plus 0.1% v/v formic acid) shows a

base peak at m/z 155.2 [M]+, a peak at m/z 178.2 corresponding to [M+Na]+ and a peak at m/z 194.2 corresponding to [M+K]+ , MS2 positive ion mode for m/z

155.2 (collision energy 20) shows a base peak at m/z 87.2 corresponding to [M-

+ + N(CD3)3] and a peak at 69.3 corresponding to [HN(CD3)3] a peak at m/z 45.1

+ + corresponding to [CO2H] , and a peak at m/z 43.2 corresponding to [C2OH3]

Quantification of TMAO, TMA, a γBB, and L-carnitine

Stable isotope dilution LC/MS/MS was used to quantify trimethylamine

compounds from mouse plasma samples in positive MRM mode using the

supernatant from methanolic plasma precipitation. Precursor → product ion

transitions at m/z 76 to 58 (TMAO), m/z 60 to 44 (TMA), m/z 146 to 60 (γBB), m/z

162 to 60 (carnitine) and were used. d9(trimethyl)TMAO (d9-TMAO),

d9(trimethyl)TMA (d9-TMA), d9 (trimethyl) γBB, and d9(trimethyl)carnitine (d9-

carnitine), were added to mouse plasma to quantify native compound

concentrations. d4-Choline was used to quantify d9-γBB and d9 gut microbiota

mouse products (d9-TMA, d9-TMAO) from d9-γBB-challenge studies. Increasing

concentrations of the trimethylamines with a fixed amount of internal standard

183

were added to control plasma to generate calibration curves for determining plasma concentrations of each respective analyte as previously described69.

In vitro mouse cecum study

C57BL/6J female mouse (n=3) cecums were harvested, sectioned longitudinally into 2 halves, and placed into 10mM Hepes PH 7.4 containing either a 150 µM d9-γBB or d3-L-carnitine respectively. Samples were placed into a sealed falcon tubes under anaerobic (in the presence of Argon) and acidic conditions (in the presence of 0.1% formic acid) for a 16 hour incubation at 37oC. Reactions were

halted by the mixing of the reaction mixture and 0.1% formic acid. A methanolic

precipitation was performed and the supernatant of samples were analyzed by

LC/MS/MS using d4-choline as internal standard as described above.

RNA preparation and real time PCR analysis

RNA was first purified from liver using the animal tissue protocol from the Qiagen

rneasy mini kit. Purified total RNA and random primers were used to synthesize

first strand cDNA using the High Capacity cDNA Reverse Transcription Kit

(Applied Biosystems, Foster City, CA) reverse transcription protocol. Quantitative

real-time PCR was performed using Taqman qRT-PCR probes (Applied

Biosystems, Foster City, CA) and normalized to tissue β-Actin by the ∆∆CT

method using StepOne Software v2.1 (Applied Biosystems, Foster City, CA).

184

General Statistics

The Wilcoxon Rank-Sum test was used for two-group comparison and Spearman associations were performed for correlation studies. A robust Hotelling T2 test was used to assess differences between dietary groups (chow or 1.3% γBB) by utilizing the proportion of specific bacterial genera and mouse plasma TMA or

TMAO concentrations132. All data was analyzed using R software version 2.15,

JMP (SAS Inc, Cary NC), and Prism (Graphpad Software, San Diego, CA).

185

Table 5-1. Plasma and liver lipid levels in C57BL/6J, Apoe-/- female mice used in γBB atherosclerosis study. Data is expressed as means +SD.

Plasma Lipids Chow γ-butyrobetaine (1.3%) P (n = 20) (n = 17) Triglyceride (mg/dL) 113+26 139+25 <0.01 Total Cholesterol 304+53 300+56 0.84 (mg/dL) HDL (mg/dL) 32+10 33+22 0.82 Total Glucose (mg/dL) 217+45 194+35 0.10

Lipids Chow,+ABS γ-butyrobetaine (1.3%), +ABS P (n = 7) (n = 18) Triglyceride (mg/dL) 75+13 90+23 0.10 Total Cholesterol 368+52 419+53 0.06 (mg/dL) HDL (mg/dL) 29+9 27+7 0.80 Total Glucose (mg/dL) 203+44 188+43 0.43

Liver Lipids Chow γ-butyrobetaine (1.3%) P (n=20) (n=17) Triglyceride (mg/g 46+21 35+11 0.09 liver) Cholesterol (mg/g liver) 1.6+0.32 1.1+0.25 <0.01

Lipids Chow,+ABS γ-butyrobetaine (1.3%), +ABS P (n=7) (n=18) Triglyceride (mg/g 38+15 28+9.4 0.10 liver) Cholesterol (mg/g liver) 1.7+0.22 1.6+0.21 0.61

Table 5-1. Plasma and liver lipid levels in C57BL/6J, Apoe-/- female mice used in γBB atherosclerosis study. Data is expressed as means +SD.

186

Figure 5-1. γBB is produced as a major gut microbiota metabolite of L-carnitine. Stable isotope dilution of LC/MS/MS of plasma γBB, carnitine, TMA, and TMAO in female terminal plasma of C57BL/6J, Apoe-/- mice on respective diets. Data is expressed as means + SE.

187

Figure 5-2. γBB is produced from L-carnitine in a gut microbiota dependent manner. Female Swiss Webster Germ Free mice challenged with d3-L-carnitine before and after conventionalization. Post challenge measurement of d3-L-carnitine and d3- γBB was performed in serial venous blood draws by stable isotope dilution LC/MS/MS.

188

Figure 5-3. TMA/TMAO is a gut a microbiota dependent product of γBB metabolism. C57BL/6J female mice (n=5) challenged with d9-γBB gastric gavage (left panels; upper (d9-carnitine and d9- γBB) and lower (d9TMA/TMAO)) followed with serial blood venous blood draws and quantification of plasma deuterated analytes by stable isotope dilution LC/MS/MS. Middle panels-Repeat gastric gavage with d9-γBB after 1 month gut suppression with a cocktail of broad spectrum antibiotics as described in Methods. Right Panels- A final d9-γBB gastric challenge and sequential measurement of deuterated plasma compounds was performed after a month long reconventionalization period.

189

Figure 5-4. Confirmatory studies that TMA/TMAO is a gut a microbiota dependent product of γBB metabolism. Female Swiss Webster Germ Free mice (n=5) were challenged with d9- γBB before and after conventionalization. Post challenge measurement of d9-carnitine, d9-γBB (upper panels), d9-TMA, and d9-TMAO (lower panels) was performed in serial venous blood draws by stable isotope dilution LC/MS/MS.

190

Figure 5-5. γBB is the dominant gut microbiota metabolite of L-carnitine and is metabolized to TMA at a great equamolar capacity than L-carnitine. C57BL/6J Female mouse intestine (n=3) was sectioned into two complementary pieces for incubation with equamolar amounts of d3-L-carnitine or d9-γBB under anaerobic conditions at 37oC for 12 hours. Deuterated trimethylamine analytes were quantified by stable isotope dilution LC/MS/MS as detailed in Methods. d9/d3-TMA production by the gut microbiota from d9-γBB and d3-L-carnitine respectively occurs primarily in the cecum (top and middle panels) whereas d3-γBB production from d3-L-carnitine is more evenly distributed in the cecum and colon. d9-γBB produced more d9-TMA on an equamolar basis than d3-L-carnitine produced d3- TMA (top and middle panels). d3-γBB production from d3-L-carnitine is approximately 1000 fold higher (bottom panel) than d3-TMA production (middle panel).

191

Figure 5-6. γBB promotes atherosclerosis in a gut microbiota dependent manner. (A) Oil-red-O stained and hematoxylin counterstained representative aortic roots slides of 19 week old C57BL/6J, Apoe-/- female mice on the respective diets in the presence versus absence of gut microbiota suppression (+ antibiotics (ABS)) as described under Experimental Procedures. (B) Quantification of mouse aortic root plaque lesion area of 19 week-old C57BL/6J, Apoe-/- female mice. Mice were started on the indicated diets at the time of weaning (4 weeks of age). Lesion area was quantified as described under Methods.

192

Figure 5-7. Plasma trimethylamine concentrations of C57BL/6J, Apoe-/- female mice used in γBB atherosclerosis study. Carnitine, TMA, γBB, and TMAO were determined using stable isotope dilution LC/MS/MS analysis of terminal plasma recovered from γBB atherosclerotic mice. Data is expressed as means + SE.

193

Figure 5-8. γBB production from L-carnitine is an inducible trait. d3-L-carnitine challenge of mice on a L-carnitine supplemented diet (1.3%) at 10 weeks and age or age-matched normal chow controls. Plasma d3- γBB was measured in sequential venous blood draws at the indicated times post d3-L- carnitine oral gavage.

194

Figure 5-9. γBB production from L-carnitine associates with microbiome composition. A correlation heat map demonstrating the association between the indicated gut microbiota taxonomic taxa and γBB plasma levels of mice grouped by dietary status (chow, n=10 and L-carnitine, n=13). Red denotes a positive association, blue a negative association, and white no association. The single asterisk indicates a significant FDR adjusted association of P ≤ 0.1

195

Figure 5-10. γBB production from L-carnitine and microbiome composition associate with dietary status. Plasma γBB concentrations were determined by stable isotope dilution LC/MS/MS (plotted on x axes) and the proportion of taxonomic operational units (OTUs, plotted on Y axes) were determined as described in Experimental Procedures. The P value shown is for comparisons between dietary groups using a robust Hotelling T2 test.

196

Figure 5-11. TMA/TMAO production from γBB associates with microbiome composition. A correlation heat map demonstrating the association between the indicated gut microbiota taxonomic taxa and TMA/TMAO plasma levels of mice grouped by dietary status (chow, n=15 and γBB, n=11 ). Red denotes a positive association, blue a negative association, and white no association. The single asterisk indicates a significant FDR adjusted association of P ≤ 0.1.

197

Figure 5-12. TMAO production from γBB and microbiome composition associate with dietary status. Plasma TMAO concentrations were determined by stable isotope dilution LC/MS/MS (plotted on x axes) and the proportion of taxonomic operational units (OTUs, plotted on Y axes) were determined as described in Experimental Procedures. The P value shown is for comparisons between dietary groups using a robust Hotelling T2 test.

198

Figure 5-13. Liver Expression of Bile acid enzymes. Relative mRNA levels (to β-actin) of mouse bile acid synthetic enzymes liver Cyp7a1 and Cyp27a1.

199

Figure 5-14. Scheme of endogenous and exogenous γBB production. γBB is endogenously produced as part of the L-carnitine synthetic pathway, but can also be produced by the metabolism of sources of TMA production by the gut microbiota.

200

CHAPTER 6: γ-Butyrobetaine is a Gut Microbiota Dependent Product of L-

Carnitine

Authors: Robert A. Koeth, Bruce S. Levison, Zeneng Wang, Yuping Wu, Lin Li,

W. H. Wilson Tang, and Stanley L. Hazen

Introduction

The gut microbiota, the consumption of dietary trimethylamines, and

cardiovascular disease (CVD) has recently been linked together by gut

microbiota dependent formation of proatherogenic TMAO from dietary

phosphatidylcholine (the major source of dietary choline)69. Human plasma

TMAO is an independent prognostic indicator of MACE over a 3-year period and

dietary supplementation of TMAO promotes gut microbiota dependent

atherogenesis in mice69,147. Together these data suggest a mechanistic link

between the dietary trimethylamines, CVD, and the gut microbiota.

Microbiota Gut Pathway 1: Choline TMA FMOs TMAO

Carnitine, a nutrient found primarily found in red meat and a frequent additive to

energy drinks, is also metabolized by the gut microbiota to proatherogenic TMAO

in humans and mice. We recently demonstrated that carnitine dependent TMAO

formation associates with omnivorous eating habits and an omnivorous gut

microbiota has a greater capacity to metabolize TMAO from carnitine compared

to vegans/vegetarians73. Moreover, tandem analysis of plasma TMAO and gut

201

microbiota composition are sufficient to distinguish dietary patterns in humans73.

These studies provide a mechanistic link between the commonly observed

association between high red meat consumption and atherosclerosis.

Microbiota FMOs Gut Pathway 2: L-carnitine TMA TMAO

Despite these intriguing observations, the exact mechanism(s) for gut microbiota

dependent formation of TMAO from carnitine were not known and secondary

pathways in the formation of TMAO from carnitine had not been considered.

Recent studies have demonstrated the existence of an alternative, dominant gut microbiota mediated pathway for TMAO generation form carnitine in mice. L- carnitine can be metabolized by the gut microbiota to γBB and then further

metabolized by the gut microbiota to TMA/TMAO. γBB supplementation

promotes atherosclerosis by formation of TMAO and is associated with

dysfunctional bile acid synthesis from cholesterol (Chapter 5). Surprisingly, gut microbiota characterization of mice on γBB or carnitine diets respectively demonstrated distinct dominant populations suggesting more complexity in gut

microbiota dependent TMAO formation from carnitine.

Microbiota ? Hypothesized Gut Pathway: L-carnitine γ-BB TMA FMOs TMAO

These data raised the possibility that γBB may also be a carnitine gut microbiota product in humans and may serve as an intermediate in the gut microbiota production of TMAO. Herein, we demonstrate that γBB is a gut microbiota

202

metabolite of carnitine in humans, but is quantitatively produced at a lower level

than TMAO. Moreover, it does not associate with an omnivorous diet. Finally,

increasing plasma γBB concentrations independently associates with major

adverse cardiovascular events (MACE = death, MI, stroke) from subjects with

near and long term outcomes collected, but only in the context of concurrent high

plasma TMAO levels.

Results

γBB associates with CVD prevalence

Previous studies in mice suggested that γBB is the major gut microbiota

metabolite from carnitine and serves as an intermediate in the production of

TMAO. As such, we first investigated the relationship between fasting plasma

levels of γBB and CVD disease prevalence in an independent large cohort of

stable subjects undergoing elective cardiac cauterization for cardiovascular

disease (n = 1,445). Subject demographics, laboratory values, and clinical

characteristics are provided in Table 6-1. Subjects were first stratified by tertiles

of increasing plasma γBB and then an analysis between CVD and γBB was

performed. A significant dose – dependent association between γBB levels and

risk of overall CVD was noted even after adjustment for traditional cardiovascular

risk factors (Age, sex, gender, systolic blood pressure, low-density lipoprotein

cholesterol, high-density lipoprotein cholesterol, smoking and diabetes)(Fig. 6-1).

Prevalent coronary artery disease (CAD) and peripheral artery disease (PAD) also had significant associations with fasting plasma γBB levels (Fig. 6-1).

203

Interestingly, whereas this association remained significant following adjustments for traditional CVD risk factors (noted above) with CAD, the association between

PAD and γBB did not (P < 0.05) (Fig. 6-1).

γBB is associated with MACE, but not after TMAO adjustment

We next investigated the relationship between fasting plasma levels of γBB and prospective risk for major adverse cardiac events (MACE = composite of death,

MI, stroke) over a 3-year period. Kaplan-Meier analysis of tertiles of γBB and incident MACE revealed a significant association (Fig. 6-2). To understand the relationship between tertiles and MACE in the context of traditional CVD risk,

Cox regression analyses were performed. Consistent with the Kaplan-Meier analysis of γBB tertiles demonstrated a dose–dependent association with MACE that remained significant after adjustment for traditional cardiovascular risk factors. The observation that γBB serves as an intermediate in carnitine metabolism in mice (e.g. carnitine to γBB to TMAO) suggested that it may also serve as an intermediate in humans. Moreover, it also suggested the association observed between CVD and plasma concentrations of γBB may be attributed to

TMAO. Consistent with our supposition, after further adjustment for TMAO the significant relationship between γBB and MACE risk was completely abolished suggesting TMAO is accounting for the significant association with MACE (Fig.

6-2, Model 2, open squares).

204

To further define the relationship between γBB and TMAO, subjects were

stratified into groups based on binary classification of γBB and TMAO plasma

levels (e.g. high and low levels of γBB and TMAO respectively). Analysis of these

groups with MACE revealed a significant association between γBB and incident

cardiovascular event risks, but only in subject with high plasma TMAO levels.

This association remained significant even after multivariate adjustment (Fig. 6-

3). These data are consistent with the observation that TMAO, and not γBB, associates with cardiovascular risks and suggests γBB may be metabolized by a gut microbiota mechanism to TMAO in humans.

γBB is produced from carnitine in a gut microbiota dependent manner in humans

We have recently demonstrated the participation of gut microbiota in γBB production from dietary L-carnitine in mice, but γBB production from carnitine in humans has not yet been demonstrated. In two omnivorous subjects, an "L- carnitine challenge test" was performed that has been previously described73.

The challenge included a simultaneous challenge of an 8 ounce sirloin steak

(contains an estimated mass of 180 mg L-carnitine) 83,84,85 a major dietary

source of L-carnitine and a capsule of a heavy stable isotope labeled L-carnitine

(250 mg synthetic d3-(methyl)-L-carnitine). At the initial baseline (Visit 1) L-

carnitine challenge, post-prandial increases in d3-γBB and d3-L-carnitine in

plasma were observed. However, there was virtually no change in the plasma

concentration of native carnitine or γBB in either subject (Fig. 6-4). The absent

205

change in plasma carnitine may be explained by the large endogenous pool of native carnitine present. However, γBB is typically found at 1-2 µM in human plasma and there was relatively little or no change observed upon consumption of a large oral load of carnitine or with suppression of the gut microbiota with broad spectrum antibiotics. These data are consistent with endogenous γBB production being the major source of plasma γBB in humans.

We next examined the contribution of gut microbiota to γBB formation from dietary L-carnitine. After the initial baseline challenge subjects were placed on oral poorly absorbed broad spectrum antibiotics to suppress intestinal microbiota for a week, and the L-carnitine challenge was repeated (Visit 2). There was almost complete suppression of d3-γBB in plasma. However, there was no change in plasma γBB. This is consistent with an endogenous source of γBB being the major contributor to plasma. d3-L-carnitine and unlabeled L-carnitine were also detected following carnitine challenge, and showed little or no change compared to Visit 1 in both subjects. After discontinuation of the oral antibiotics and a reconventionalization period (>4 weeks), subjects were re-challenged.

Both subjects studied demonstrated production of d3-γBB post carnitine challenge consistent with recolonization of the gut microbiota. However, the production of d3-γBB was notably lower after the reconventionalization period suggesting residual effects of the antibiotic suppression on the gut microbiota even several weeks post discontinuation of antibiotics. Together, these data

206

show that γBB production from dietary L-carnitine in humans is gut intestinal microbiota dependent.

TMAO is the major gut microbiota metabolite of L-carnitine in humans

Although these data demonstrate that γBB can be produced in a gut microbiota

dependent manner in humans, the concentrations found in human plasma were extremely low suggesting γBB may be a minor metabolite in human plasma.

Comparison of 24 hour urine collections from individuals participating in the carnitine challenge either with concomitant challenge of an 8 ounce steak and a capsule of a heavy stable isotope labeled L-carnitine (250 mg d3-L-carnitine) or a capsule of d3-L-carnitine (250 mg) alone (n=12) demonstrate an almost 10,000 fold increase in d3-TMAO production compared to d3-γBB (Fig. 6-5). These data suggest that TMAO is the major gut microbiota metabolite in humans.

γBB does not associate with a omnivorous diet

We previously demonstrated that the gut microbiota metabolite of carnitine,

TMAO was higher in baseline plasma levels in omnivores compared to vegans and vegetarians73. Additionally, omnivores had a greater capacity to generate

TMAO from carnitine than vegans and vegetarians. This raised the possibility

that γBB may also associate with an omnivorous diet. To test this hypothesis,

fasting baseline concentrations of γBB were quantified by LC/MS/MS in (n = 27)

omnivores and (n = 30) vegans and vegetarians. Surprisingly, plasma

concentrations of γBB were similar between the two groups (Fig. 6-6). We

207

reasoned that the gut microbiota contribution to the total plasma pool of γBB may be small given relatively low plasma concentrations generated from d3-carnitine.

Thus, we sought to examine the specific gut microbiota capacity of omnivores vs. vegans/vegetarians to metabolize carnitine to γBB by performing a carnitine challenge test (250 mg d3-L-carnitine in a capsules only). Remarkably, post plasma quantification of d3-γBB in n=5 omnivores and n=5 vegans or vegetarians demonstrated no observable difference between the capacity of the gut microbiota from omnivores and vegan/vegetarians to generate γBB from carnitine (Fig. 6-6).

Red meat is an exogenous source of γBB, but is found at lower concentrations compared to carnitine

γBB serves as the terminal precursor in endogenous carnitine synthesis and can be found in plasma (presumably to serve as a storage depot for carnitine synthesis) indicate that meat may serve as an exogenous source of γBB.

Despite its synthetic association with carnitine, a survey of the literature revealed no reports on the amount of γBB in meat or other possible exogenous sources of

γBB. Thus, samples of beef, poultry, lamb, and fish were analyzed for tandem

γBB and carnitine concentrations. γBB is quantitatively at similar amounts among meats sampled, but is found at significantly lower concentration than carnitine

(Table 6-2). Although quantifiably at a lower concentration than carnitine, the presence of γBB adds yet another dietary trimethylamine to the possible pool of substrates to TMA/TMAO formation and promotion of atherosclerosis.

208

Discussion

γBB serves as the terminal substrate for synthetic carnitine production and has

traditionally been studied in the context of this pathway. The role of γBB in the

context of carnitine metabolism by the gut microbiota has been vastly

understudied. We recently reported that γBB serves as the major product of gut

microbiota mediated carnitine metabolism and can also be directly metabolized

to TMAO by a gut microbiota dependent pathway in mice. Here we demonstrate for the first time that carnitine can also be metabolized to γBB in a gut microbiota dependent manner in humans suggesting more complexity in gut microbiota mediated carnitine metabolism than previously anticipated.

The fact, that a major potential source of dietary γBB, red meat, has concentrations several fold lower than carnitine would suggest that more exogenous γBB is likely to derive from the gut flora metabolism of carnitine to

γBB than direct dietary absorption. This would only not be the case in a small number of athletes who make take γBB as a dietary supplement. It is often sold as “pre-carnitine” and marketed to increase carnitine levels and subsequently increase energy expenditure despite the lack of convincing studies. The pills come in doses of a 750 mg and may in this case account for a larger proportion of exogenous γBB.

209

The exact microbes that metabolize carnitine to γBB are unclear in humans.

There are reports of isolated specific microbes, some of which are found in the

human microbiota, that are able to metabolize carnitine to γBB, but the role of

these microbes in the context of greater human microbiota remain unclear81.

Characterization of the microbiome and association analyses with plasma γBB concentrations will provide insight into the gut microbes involved in the gut microbiota dependent metabolism of γBB from carnitine.

Notably, there was also little change of native plasma γBB between the baseline

(Visit1) and after gut microbiota suppression with broad spectrum antibiotics

(Visit 2). These data suggest that the majority of fasting plasma γBB is derived from endogenous production. There was also little change of native plasma γBB post challenge of a large dietary source (steak) of carnitine and γBB. Moreover, plasma concentrations of d3-γBB post d3-L-carnitine challenge were also low.

These data are consistent with a previous study in humans of carnitine metabolism that suggested only a small percentage (<4%) of oral carnitine is metabolized to γBB93. Indeed, the present studies demonstrate that, in fact,

TMAO appears to be the major gut microbiota metabolite of carnitine by almost

10,000 fold (as measured in urine). This may be at least partially attributed to

γBB serving as a substrate for TMAO production.

We previously reported that TMA/TMAO can be produced from γBB in a gut

microbiota dependent manner in mice. Additionally, γBB is the preferred

210

substrate for TMAO production by mouse microbiota compared to carnitine by at least 2-3 fold. Confirmation of these hypotheses with human studies using a heavy stable isotope challenge of γBB will no doubt need to be performed.

Consistent with our hypothesis that γBB may serve as a gut microbiota substrate for TMA/TMAO production in humans, is the observation that TMAO primarily accounts for the associations of incident MACE with γBB tertiles. Cox regression analysis with tertiles γBB initially showed an independent association with MACE that was completely abolished by addition of plasma TMAO to the model. Further analysis of the relationship of the γBB and TMAO demonstrate that γBB only associates with MACE at high levels of plasma TMAO. These data show that terminal gut microbiota metabolite, TMAO, accounts for the association with CVD and that γBB may serve as a substrate for TMAO production in humans.

These studies provide important insights for the development of therapeutic strategies for dietary trimethylamine metabolism. In humans the metabolism of the gut microbiota to γBB appears to be a minor pathway (>1000fold less) compared to the direct production of TMAO from carnitine. Thus, the development of probiotic inhibitors for the metabolism of γBB from carnitine would apparently be less beneficial than directly inhibiting the production of

TMAO from carnitine.

211

The relatively small amount of γBB produced from carnitine may also help

explain the little association γBB has with omnivorous dietary habits. The low

concentration would imply that the generation of γBB from carnitine is a minor

pathway utilized by the gut microbiota and the major route of gut microbiota

metabolism of carnitine would be via TMAO. Thus, carnitine to TMAO production

may be more likely to modify host metabolism and be influenced by dietary

changes. Together these data demonstrate the existence of an alternative

pathway of gut microbiota carnitine metabolism in humans.

Methods

Research subjects

All research subjects gave written informed consent to participate in these

studies and all protocols were approved by the Cleveland Clinic Institutional

Review Board. Two cohorts of subjects were used in the present studies. The

first group of volunteers had extensive dietary questioning and a subset of

subjects underwent oral carnitine challenge testing. Subjects (n = 12; n = 7

omnivores and n = 5 vegans) performed the oral carnitine challenge test

consisting of 250 mg d3(methyl)carnitine within a capsule and an 8 ounce steak

(consumed within 10 minutes) or a 250 mg d3(methyl)carnitine capsule alone.

Dietary habits of the subjects were determined using a questionnaire similar to

that conducted by the Atherosclerotic Risk in Community (ARIC) study.140

Subjects participating in the carnitine challenge test were excluded if they were pregnant, had chronic illness (including a known history of heart failure, renal

212

failure, pulmonary disease, gastrointestinal disorders, or hematologic diseases),

an active infection, received antibiotics within 2 months of study enrollment, used

any over the counter or prescriptive probiotic or bowel cleansing preparation

within the past 2 months, ingested yogurt within the past 7 days, or had

undergone bariatric or other intestinal (e.g. gall bladder removal, bowel resection) surgery.

Studies assessing the relationship between plasma γBB levels and both prevalent and incident cardiovascular risks were performed using archival plasma from GeneBank, a research tissue repository (n = 1,495) comprised of sequential consenting stable subjects undergoing elective cardiac evaluation with connecting clinical data over a 3-year period69,128. Exclusion criteria included

patients with a recent myocardial infarction (< 4 weeks) or elevated troponin I (>

0.03 mg dl–1) at enrollment. CVD was clinically defined as having a previous

history of coronary artery disease (CAD), peripheral artery disease (PAD),

cerebral vascular disease (history of a transient ischemic attack or cereberovascular accident), history of revascularization (coronary artery bypass

graft, angioplasty, or stent) or significant angiographic evidence of CAD (≥50%

stenosis) in at least one major coronary artery. Subjects with CAD were defined

as patients with adjudicated diagnoses of stable or unstable angina, myocardial infarction, history of coronary revascularization, or angiographic evidence of

≥50% stenosis of at least one major coronary artery. PAD was defined as subjects having any clinical evidence of extra-coronary atherosclerosis.

213

Human L-carnitine challenge test

Consented adults (n =10) performed a 12 hour fast overnight before performing

the "L-carnitine challenge test", which involved a baseline blood sample and then oral consumption (T = 0 at time of initial ingestion) of capsules containing 250 mg d3-L-carnitne (under Investigational New Drug exemption) and, in some cases, simultaneous challenge of a natural source of L-carnitine (an 8 ounce sirloin steak cooked medium on a George Forman Grill; estimated carnitine content 180 mg) within a 10 minute period as previously described73. In a second group of

subjects (n=10) a d3-carnitine challenge was performed with250 mg d3-L-

carnitne alone. Following the baseline blood draw and ingestion of the steak and

capsule of d3-L-carnitine, sequential venous serial blood draws were performed

at noted time points and a 24 hour urine collection was performed. After

completion of the initial carnitine challenge (Visit 1) an ensuing weeklong

treatment of oral antibiotics (Metronidazole 500 mg bid, Ciprofloxacin 500 mg

bid) was given to suppress intestinal microbiota and the challenge was then

repeated (Visit 2). A finally carnitine challenge was completed after at least 3

weeks off of all antibiotics allowing reacquisition of intestinal microbiota. Dietary

habits were determined using a questionnaire assessment of dietary L-carnitine

intake, similar to that conducted by the Atherosclerotic Risk in Community (ARIC)

study140. d3-Carnitine was prepared by dissolving sodium L-norcarnitine in

methanol and reacting it with d3-methyl iodide (Cambridge Isotope) in the

presence of potassium hydrogen carbonate to give d3-L-carnitine as previously

214

described73. Plasma d3-γBB was quantified by stable isotope dilution LC/MS/MS

as described below.

Quantification of L-carnitine, γBB, and TMAO in plasma samples

Human plasma and urine concentrations of carnitine, γBB, creatinine, and

TMAO isotopologues (d3-carnitine, d3-TMAO, and d3-γBB) and native

compounds in mouse and human plasma samples were determined by stable

isotope dilution LC/MS/MS in positive MRM mode using respective deuterated

(d9) internal standards (d9-carnitine, d9-TMAO, d3-creatinine and d9-γBB) on an

AB Sciex API 5000 triple quadrupole mass spectrometer (Applied Biosystems) as

previously described73.

γ-Butyrobetaine quantification in meat samples

1 gram of meat (ground beef, steak, fish, and poultry) was weighed and added to a PBS pH 7.4 solution. Samples were homogenized using a polytron homogenizer to a homogenous mixture and sampled for mass spec analysis.

Samples were extracted using a methanolic precipitation and quantified using

LC/MS/MS as described above.

General statistics

A Wilcoxon non parametric test were used to compare group means as deemed appropriate. Subjects were stratified into tertiles by increasing concentrations of

γBB (Tertile 1(n = 477, <0.8 µM γBB); Tertile 2 (n = 477, 0.8-1.1 µM γBB); Tertile

215

3(n = 477, ≥ 1.1 µM γBB)). Odds ratios for the cardiac phenotypes CAD, PAD, and CVD and 95% confidence intervals were calculated using logistic regression.

Kaplan–Meier analysis with γBB tertiles were performed with the composite

outcome of MACE (death, MI, stroke)over a 3 year period. Cox proportional

hazards regression was used for time-to-event analysis to determine Hazard

ratio (HR) and 95% confidence intervals (95%CI) for MACE. Adjustments were

made for individual traditional cardiac risk factors (Model1 = traditional CVD risk factors including age, gender, diabetes mellitus, systolic blood pressure, former

or current cigarette smoking, low-density lipoprotein cholesterol, high-density

lipoprotein cholesterol; Model 2 = traditional CVD risk factors and log (plasma

TMAO)). All data was analyzed using R software version 2.15 and Prism

(Graphpad Software).

216

Whole cohort

(n=1445) Age (years) 61±11 Male (%) 73

Former/current smokers (%) 71 Diabetes mellitus (%) 20 Hypertension (%) 73

Hyperlipidemia (%) 88 Prior coronary artery disease (%) 78 CAD (%) 80 PAD (%) 21 CVD (%) 82 Framingham ATP III Risk Score 8(6-10) BMI (kg/m2) 28.5(25.4-32) LDL cholesterol (mg/dL) 92(75-111) HDL cholesterol (mg/dL) 33(27-39) Total cholesterol (mg/dl) 156(135-181) Triglycerides (mg/dL) 115(82-164) hsCRP (mg/L) 2.05(0.93-4.69) MPO (pmol/L) 109(70-239) Creatinine clearance

(ml/min/1.73m2) 105(80-131) Creatinine (mg/dl) 0.85(0.76-0.98) Γ-butyrobetaine (µM) 0.93(0.77-1.16)

Baseline medications (%)

ACE inhibitors 50 Beta-blockers 68

Statin 66 Aspirin 78 Table 6-1. Baseline clinical characteristics of n = 1445 Genebank subjects used in analyses with γBB. Values expressed in mean ± standard deviation or median (interquartile range). Abbreviations: cTnI = LDL = low-density lipoprotein; HDL = high-density lipoprotein; hsCRP = high-sensitivity C-reactive protein; ATP III = Adult Treatment Panel III guidelines

217

Meat Carnitine γBB

ug/g weight ug/g weight

Beef Steak (Chuck Eye) 452 + 32 36 + 0.9 Lean Ground Beef 500 + 6.3 30 + 0.6

Lamb (Leg Steak) 14865 + 28 15.8 + 0.4

Pork (Center Cut Chop) 87.0 + 2.9 29.8 + 0.1

Ground Chicken 41.3 + 2.9 10.2 + 0.1

Fish (Ocean Perch) 5.84 + 3.1 1.04 + 0.5

Table 6-2. Quantification of carnitine and γBB in beef, lamb, chicken, and perch samples. Data is expressed as means +SD.

218

Figure 6-1. Relationship between plasma γBB and CVD prevalence. Forrest plots of odds ratio of CAD, PAD, and CVD and tertiles of γBB before (closed circles) and after (open circles) logistic regression adjustments with traditional cardiovascular risk factors including age, sex, history of diabetes mellitus, smoking, systolic blood pressure, low density lipoprotein cholesterol, and high density lipoprotein cholesterol. Bars represent 95% confidence intervals.

219

Figure 6-2. Relationship between plasma γBB and CVD risks. (a) Kaplan Meier plot (graph) plot of increasing concentrations of plasma γBB represented by tertiles of γBB with MACE over a 3-period. (b) Forrest plot of hazard ratio of MACE (death, non fatal-MI, stroke, and revascularization) and tertiles of γBB unadjusted (closed circles), and after adjusting for traditional cardiovascular risk factors (open circles), or traditional cardiac risk factors and TMAO levels (open squares). Bars represent 95% confidence intervals.

220

Figure 6-3. Relationship between plasma γBB, plasma TMAO, and CVD risks. Kaplan Meier plot (graph) and hazard ratios with 95% confidence intervals for unadjusted model, or following adjustments for traditional risk factors as in panel. Levels of γBB (0.84 µM) and TMAO (4.6 µM) within the cohort were used to stratify subjects as ‘high’ (≥ median) or ‘low’ (< median) concentrations.

221

Figure 6-4. γBB production from carnitine is a gut microbiota dependent process in humans. (a) Scheme of human carnitine challenge test. The carnitine challenge test consisted of a subject receiving an 8 ounce steak (estimated 180 mg L-carnitine) with a gel capsule of d3-carnitine (250mg) after a fasting overnight (12 hours) whereupon serial plasma was obtained. After a weeklong regimen of oral broad spectrum antibiotics to suppress the intestinal microflora, the challenge was repeated (visit 2), and then again a final third time after a ≥ 3 week period to permit repopulation of intestinal microflora (visit 3). Data shown in panels (b and c) are from a representative omnivorous subjects, and data is organized to vertically correspond with the indicated visit schedule above (visit 1, 2 or 3). Stable isotope dilution LC/MS/MS time course measurements of native carnitine and TMAO (Upper panels of a and b) and heavy stable isotope, d3-γBB and d3-carnitine (lower panels of a and b), in plasma collected from sequential venous blood draws at noted times.

222

Figure 6-5. TMAO is the major gut microbiota metabolite in human carnitine catabolism. d3- γBB and d3-TMAO was quantified in 24 hr urine samples by LC/MS/MS and normalized to tandem measurement of creatinine (Cr) from subjects participating in the human carnitine challenge tests (n=12) (e.g. d3-L-carnitine 250mg).

223

Figure 6-6. The formation of γBB from ingested L-carnitine is similar in vegans and vegetarians compared to omnivores (a) Baseline fasting plasma concentrations of γBB in (n = 26) vegans and vegetarians and (n = 30) omnivores. Boxes represent the 25th, 50th, and 75th percentile and whiskers represent the 10th and 90th percentile. (b) Plasma d3-γBB levels in male and female (n = 5) vegan/ vegetarian versus (n = 5) omnivores participating in a d3-carnitine (250 mg) challenge.

224

CHAPTER 7: Transcrotonobetaine, a Gut Microbiota Metabolite of Carnitine

Metabolism, Promotes Atherosclerosis

Introduction

We previously demonstrated that carnitine may be metabolized to γBB as a second pathway in the formation of proatherogenic TMAO (Chapters 5, 6). We now report the discovery of a third, lower abundance, proatherogenic gut microbiota metabolite generated from carnitine, transcrotonobetaine (TC). TC is a trimethylamine containing compound structurally related to carnitine that is not believed to be endogenously produced by mammalian species81,82. While some

studies have hypothesized a gut microbiota origin of TC, a survey of the literature

reveals no study to date that has definitively proven that TC is a gut microbiota-

dependent product of carnitine80,81. We therefore sought to investigate the role of

TC in the gut microbiota dependent metabolism of carnitine and its relationship to

TMAO and atherogenesis. Gut Gut Microbiota Microbiota FMOs Hypothesized Gut Pathway: L-carnitine TC TMA TMAO

Results

TC is a gut microbiota dependent product of L-carnitine

We first sought to test the hypothesis that TC is produced by L-carnitine in a gut

microbiota dependent manner. Gavage of d3-L-carnitine in Swiss Webster germ-

free mice was followed by the time dependent appearance of only minimal levels

of d3-TC in mouse plasma (Fig. 7-1). After a conventionalization period of >1

225

month accomplished by caging germ-free mice with conventional mice, the

carnitine challenge was repeated (Fig. 7-1). Sequential venous plasma measurements demonstrated a significant increased production of d3-TC, confirming that TC is a gut-microbiota dependent product of d3-carnitine. The small amount of d3-TC detected in post-challenge plasma samples may be attributed to spontaneous decomposition by SN2 elimination of water from

carnitine. This may be further promoted by the acidic environment of the

mammalian stomach.

TC is an abundant gut microbiota metabolite of L-carnitine in mice

Incubation of equal molar amounts of d3-L-carnitine with segments of mouse intestines demonstrated the production of TMA and TC mainly occurs in the bacterial rich cecum of mice (Figure 7-2). As previously described in Chapter 5, the production of γBB from L-carnitine was more uniformly distributed along the distal intestinal tract and the quantitative dominant product of L-carnitine metabolism (Figure 7-2). Interestingly, TC is also produced more than d3-TMA, but to a lesser extent than γBB. These studies suggest TC is an abundant gut microbiota product of L-carnitine (Figure 7-2; upper panel). They also show that in contrast to TMA, but similar to γBB, TC is also produced in the distal ileum, suggesting that net TMA production from carnitine ingested will be formed by three potential pathways (Figure 7-3).

226

TC produces both γ-butyrobetaine and TMA/TMAO in a gut microbiota

dependent manner

In Chapter 5 we demonstrated in mice that a major pathway for TMA/TMAO

production from oral carnitine is via production of γ-butyrobetaine. We also

hypothesized that TC could produce TMA/TMAO in a gut microbiota dependent

manner. Challenge of conventional C57BL/6J with d9-TC demonstrated

production of d9-γbutyrobetaine, d9-TMA, d9-TMAO, and d9-carnitine (Fig. 7-4).

Of note, the concentration of d9- γBB was 10-fold more than TMA. After a month

long suppression of the gut microbiota with broad spectrum antibiotics (+ABS)

the challenge was repeated revealing near complete suppression of all plasma

levels of metabolites (d9-γ-butyrobetaine, d9-TMA, and d9-TMAO) except for a

small concentration of d9-carnitine. Of note, the residual amount of d9-carnitine

found in the plasma of mice with +ABS suppression may be attributed to a small

percentage of d9-carnitine impurity from the preparation of d9-TC (~2% d9-

carnitine by mass of d9-TC). After a conventionalization period of >1 month

accomplished by caging experimental mice with conventional mice, the d9-TC

challenge was repeated. Sequential venous plasma measurements of d9-γ- butyrobetaine, d9-TMA, d9-TMAO, and d9-carnitine demonstrated production of these metabolites. These data confirm gut microbiota dependent formation of d9-

γ-butyrobetaine, d9-TMA, and d9-TMAO from d9-TC. Since d9-γ-butyrobetaine

from gut microbes may be absorbed by the mouse and shuttled into the endogenous synthetic pathway of carnitine production, the appearance of d9- carnitine in plasma following oral d9-TC ingestion likely arises from the

227

endogenous pathway. Together these data support that TMA/TMAO is produced from transcrotonobetaine in a gut microbiota dependent manner.

TC independently associates with cardiovascular disease, but not after multivariate model adjustment with TMAO

Our data shows the production of TC has an obligatory role of gut microbes.

This differentiates it from other trimethylamines like γ-butyrobetaine, carnitine, or choline that are endogenously produced by mammalians species. Moreover,

TMAO is the apparent terminal product of many gut microbiota metabolic pathways (including a pathway through TC). In contrast, TC is only known to be produced by the gut microbiota from carnitine suggesting it may be a specific marker of gut microbiota mediated carnitine metabolism. This raised the possibility that TC, a carnitine specific gut microbiota metabolite, would associate with CVD. To explore this relationship plasma samples from Genebank subjects(n = 836), a large tissue repository with connecting clinical data, were quantified by stable isotope dilution LC/MS/MS. Subject characteristics and laboratory values are recorded in Table 7-1. After stratification by tertiles of increasing concentrations of human plasma TC, subjects were analyzed by a time to event Kaplan Meier analysis with prospective MACE (composite MI, stroke, or death) over a 3-year period. Increasing tertiles of TC were significantly associated with MACE suggesting TC may be able to be used to risk stratify subjects for prospective CVD events. To further ascertain the relevance of human plasma TC in the context of other known CVD risk factors, Cox-

228

proportional hazard analysis was performed (Fig. 7-5). There was a step-wise

increase in the hazard ratio of tertiles of TC with MACE over three year period

that remained significant even after adjustment for traditional CVD risk factors

(age, gender, systolic blood pressure, low-density lipoprotein cholesterol, high-

density lipoprotein cholesterol, history of smoking, history of diabetes),

suggesting TC is an independent prognostic indicator of MACE (Fig. 7-6).

However, correlation analysis of human plasma TC with concurrent human

plasma TMAO revealed a significant association (Spearman ρ = 0.39, P<0.01),

suggesting TMAO may be accounting for the observed association with MACE.

Further, adjustment with TMAO to the model completely abolished the association of tertiles of TC and MACE (Fig. 7-6).

Dietary TC promotes atherosclerosis in a gut microbiota-dependent manner

To understand the direct role of transcrotonobetaine in atherosclerotic disease,

C57BL/6J, Apoe-/- female mice were placed on a 1.3% TC supplemented diet at

4 weeks of age (time of weaning) in the presence or absence of antibiotics. In parallel, mice on normal chow were examined. Consistent with previous studies with dietary trimethylamines, supplemented TC also promoted atherosclerosis in a gut microbiota dependent manner (Fig. 7-7, 7-8). There were no significant differences in plasma total cholesterol, HDL cholesterol, or glucose concentrations between study groups (chow vs. TC supplemented and chow/+ABS vs. TC/+ABS), but there was an isolated significant increase in

229

plasma triglycerides in the TC supplemented group compared to chow (Table 7-

2). Interestingly, terminal plasma concentrations of trimethylamines of mice in the atherosclerosis study demonstrated that TMAO was more abundant than TC in mice with an intact microbiota (Fig. 7-8). In contrast, with the suppression of the gut microbiota with antibiotics, TC was the most abundant analyte and TMA,

TMAO and γBB were all present at minimal levels, consistent with their gut microbe dependent pathways of formation. We also observed a significant decrease in atherosclerotic plaque at the aortic root of mice in the TC/+ABS arm of the study compared to the chow/+ABS supplemented mice (Fig. 7-7).

Examination of plasma lipid cholesterol, HDL, triglyceride, and glucose levels reveal no significant differences between the TC/+ABS and chow/+ABS arms of the atherosclerotic study compared to the chow/+ABS. This suggests that the decrease in atherosclerotic plaque is being mediated by mechanisms other than changes in lipid or glucose metabolism (Table 7-2).

Discussion

These studies together demonstrate unequivocally the existence of a third gut microbiota dependent carnitine metabolite and reveal more complexity in the gut microbiota dependent metabolism of carnitine. The production of TC from L- carnitine occurs in a gut microbiota manner and is the second most abundant gut microbiota product observed in mice. Interestingly, plasma levels of TC are uniformly lower than TMAO. This may be explained by the observation that the vast majority of gut microbiota dependent TC formation occurs in the distal gut

230

(e.g. in the cecum and beyond) and consequently past normal gut absorption

mechanisms. Production of gaseous TMA from carnitine in the large bowel, in

contrast, can diffuse through the gut epithelium into the portal circulation.

Quantitatively, tracer studies show that γBB is the major metabolite of gut

microbiota metabolism of TC. However, mice supplemented with a chronic TC

diet have higher terminal concentrations of plasma TMAO. This suggests that

TMAO is the dominant metabolite overall of TC gut microbiota metabolism and

γBB is serving as a proximal precursor for TMA/TMAO formation in mice (Fig. 7-

3).

Undoubtedly the generation of γBB from TC is contributing to the formation of

TMAO, but whether TC is directly metabolized to TMA/TMAO is unknown.

Additionally, the relationship between carnitine formation and TC is complex. The parallel formation of d9-γBB from d9-TC raises the possibility that d9-γBB may be absorbed in the mouse gut and shuttled into the endogenous carnitine synthesis pathway. Kinetically, the production of d9-carnitine parallels the formation of γBB and suggests a precursor (d9-γBB)-product (d9-carnitine) relationship. However, we cannot exclude the possibility that some carnitine may also be formed from

TC directly by the gut microbiota. Regardless of whether some d9-carnitine is formed directly, what remains clear is that the gut microbiota can influence the total body carnitine pool. Moreover, these studies also suggest that TC can

231

contribute to TMA/TMAO formation from carnitine metabolism by the gut

microbiota.

It is noteworthy that plasma levels of TC track with human CVD (e.g. higher

plasma concentrations of TC are positively associated with MACE), even after

adjustment with traditional CVD risk factors, but not after adjustment with TMAO.

Thus, these data are consistent with our mouse studies indicating it is not TC that

is accounting for the association between TC and MACE directly, but a down-

stream microbe-dependent metabolite (e.g. TMAO).

Consistent with previous studies, these data demonstrate that the gut microbiota

metabolism of another trimethylamine capable of formingTMA/TMAO (e.g. TC)

promotes atherosclerosis. We did not note any proatherogenic changes in

plasma HDL cholesterol , total cholesterol, triglycerides, or glucose.

Up to this point studies of carnitine or more broadly dietary trimethylamines have

shown that gut microbiota metabolism to TMA/TMAO promotes atherogenesis

and the native compound has no effect on atherogenesis (e.g. atherosclerosis

arms of dietary trimethylamines with gut microbiota suppression (choline, +ABS; carnitine, +ABS; γBB, +ABS) have no significant change in the amount of plaque

compared to controls arms). In the case of TC however, there was a significant

decrease in atherosclerotic plaque at the aortic root of mice fed a TC diet with coinciding gut microbiota suppression that cannot be easily explained by any

232

changes in glucose or lipid metabolism (Table 7-1). These studies suggest TC may have some alternative minor antiatherogenic biological activity. Further studies will be needed to confirm of these observations and the possible mechanisms contributing to this process.

Methods

Materials and general procedures

Mice and/or breeders were obtained from Jackson Laboratories. All animal studies were performed under approval of the Animal Research Committee of the

Cleveland Clinic. Mouse plasma total cholesterol, triglycerides, and glucose were measured using the Abbott ARCHITECT platform model ci8200 (Abbott

Diagnostics, Abbott Park, IL). HDL cholesterol concentration in mice used for the

TC atherosclerosis study was enzymatically determined (Stan bio, Houston, TX) from plasma HDL isolated using density ultracentrifugation as previously described73. Liver triglyceride content was measured using the GPO reagent

(Pointe Scientific, Canton, MI) and normalized to liver mass (g) grams as previously described125. Gut microbiota suppression studies were performed by dissolving antibiotics in mouse drinking and included 0.1% Ampicillin sodium salt

(Fisher Scientific), 0.1% Metronidazole, 0.05% Vancomycin (Chem Impex Intl.), and 0.1% Neomycin sulfate (Gibco) as previously described35,69.

233

Research subjects

All research subjects gave written informed consent to participate in these

studies and all protocols were approved by the Cleveland Clinic Institutional

Review Board. Studies assessing the relationship between plasma TC levels and

incident cardiovascular risks were performed using archival plasma from

GeneBank, a research tissue repository (n = 836) comprised of sequential

consenting stable subjects undergoing elective cardiac evaluation with

connecting clinical data over a 3-year period69,128. Exclusion criteria included

patients with a recent myocardial infarction (< 4 weeks) or elevated troponin I (>

0.03 mg dl–1) at enrollment.

Mouse challenge and atherosclerosis studies

An oral TC challenge in mice consisted of a gastric gavage of d9-TC (prepared as described below) dissolved in water. The TC challenge was performed on 10- week old C57BL/6J conventional female mice, after gut microbiota suppression with broad spectrum antibiotics for 1 month, and finally, after being housed with native mice for an approximately 1 month conventionalization period69.

Atherosclerosis studies with C57BL/6J, Apoe-/- on a standard chow control diet

(Teklad 2018) or TC supplemented diet (1.3% by weight synthesized TC as

described below) with and without antibiotics at time of weaning for a 14 week

duration. The antibiotic regimen used was provided to the mouse in the drinking

water as described above. Mouse aortic root plaque was prepared and quantified as previously described69. Quantification of natural abundance and isotope

234

labeled forms of TC, carnitine, γBB, TMA and TMAO in mouse plasma was

performed using stable isotope dilution LC/MS/MS as described below.

d9-TC and native TC preparation

TC and d9-TC was prepared by a modified method as previously described176.

Briefly, TC was prepared by gradually dissolving L-Carnitine (Chem Impex

International) or d9-L-carnitine (prepared as previously described) into

concentrated Sulfuric acid (Fisher) at 145oC. The reaction was allowed to cool to

80oC and then poured over crushed ice. Sodium Hydroxide (Fisher S318-3) was

then added to bring the pH of the reaction to 7.0 and chilled with an external ice methanol bath to less than 20 oC. The reaction was frozen overnight at -80oC and then lyophilized. TC was extracted in methanol, filtered with a Buchner funnel to remove impurities, and rotary evaporated to a beige crystalline semisolid.

Residual water was azeotroped away by rotary evaporating two additions of absolute Ethanol (Fisher Molecular) from the crystalline cake. The impure TC was dissolved in a minimal amount of absolute ethanol and filtered under house vacuum. A seed crystal of transcrotonobetaine and gradual additions of Ethyl

Acetate were added to the filtrate until transcrotonobetaine crystals were formed.

Crystals were isolated by using Coors Porcelain Buchner funnel with two washes of Ethyl Acetate. Residual Ethyl Acetate was removed in a Vacuum oven at 55oC for one hour and further dried under oil pump vacuum. TC was stored in

Polyethylene containers. Combination studies of NMR, mass spectrometry, and thin layer chromatography confirmed a purity of <98% TC.

235

Quantification of TC, TMAO, TMA, γBB, and L-carnitine

Stable isotope dilution LC/MS/MS was used to quantify trimethylamine compounds from in mouse and human plasma samples in positive MRM mode

using the supernatant from methanolic plasma precipitation. Precursor →

product ion transitions at m/z 144 to 59 (TC), m/z 76 to 58 (TMAO), m/z 60 to 44

(TMA), m/z 146 to 60 (γBB), m/z 162 to 60 (carnitine) and were used.

d9(trimethyl)TC, d9(trimethyl)TMAO (d9-TMAO), d9(trimethyl)TMA (d9-TMA), d9

(trimethyl) γBB, and d9(trimethyl)carnitine (d9-carnitine), were added to mouse

plasma to quantify native compound concentrations. d4-Choline was used to

quantify d9-TC and d9 gut microbiota mouse products (d9-TMA, d9-TMAO, d9-

γBB, d9-carnitine) from d9-TC-mouse challenge studies. Increasing

concentrations of the trimethylamines with a fixed amount of internal standard

were added to control plasma to generate calibration curves for determining

plasma concentrations of each respective analyte as previously described69,73 .

In vitro mouse cecum study

C57BL/6J female mouse (n=3) cecums were harvested, sectioned longitudinally into 2 halves, and placed into 10mM Hepes PH 7.4 containing either a 150 µM d9-γBB or d3-L-carnitine respectively. Samples were placed into a sealed falcon tubes under anaerobic (in the presence of Argon) and acidic conditions (in the presence of 0.1% formic acid) for a 16 hour incubation at 37oC. Reactions were

halted by the mixing of the reaction mixture and 0.1% formic acid. A methanolic

236

precipitation was performed and the supernatant of samples were analyzed by

LC/MS/MS using d4-choline as internal standard as described above.

General statistics

The Wilcoxon Rank-Sum test was used for two-group comparisons. Subjects were stratified into tertiles by increasing concentrations of TC (Tertile 1(n = 270,

<0.07 µM TC); Tertile 2 (n = 288, 0.07-0.1 µM TC); Tertile 3 (n = 278, ≥ 0.1 µM

TC)). Kaplan–Meier analysis with TC tertiles were performed with the composite outcome of MACE (death, MI, stroke)over a 3 year period. Cox proportional hazards regression was used for time-to-event analysis to determine Hazard ratio (HR) and 95% confidence intervals (95%CI) for MACE. Adjustments were made for individual traditional cardiac risk factors (Model1 = Traditional CVD risk factors: age, gender, diabetes mellitus, systolic blood pressure, former or current cigarette smoking, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol ) Model 2 = Traditional CVD risk factors + 1,2 or 3 vessel coronary disease, Coronary Artery Disease, History of MI, Left Ventricular Ejection

Fraction, TMAO). All data was analyzed using R software version 2.15, and

Prism (Graphpad Software, San Diego, CA).

237

Whole cohort

(n = 836)

Age (years) 61±11 Male (%) 78 Former/current smokers (%) 76 Diabetes mellitus (%) 12

Hypertension (%) 72 Hyperlipidemia (%) 89 Prior coronary artery disease (%) 80 CAD (%) 83

PAD (%) 20 CVD (%) 83 Framingham ATP III Risk Score 7(5-10) BMI (kg/m2) 28.3(25.3-31.6)

LDL cholesterol (mg/dL) 88(72-109) HDL cholesterol (mg/dL) 33(28-39) Total cholesterol (mg/dl) 151(131-178) Triglycerides (mg/dL) 106(75-154)

hsCRP (mg/L) 1.89(0.86-4.3) MPO (pmol/L) 110(70-259) Creatinine clearance

(ml/min/1.73m2) 104(82-128)

Creatinine (mg/dl) 0.87(0.77-0.98) Transcrotonobetaine (µM) 0.09(0.07-0.11) Baseline medications (%)

ACE inhibitors 49

Beta-blockers 70 Statin 70 Aspirin 79

Table 7-1. Baseline clinical characteristics of n = 836 Genebank subjects used in analyses with TC. Values expressed in mean ± standard deviation or median (interquartile range). Abbreviations: cTnI = LDL = low-density lipoprotein; HDL = high-density lipoprotein; hsCRP = high-sensitivity C-reactive protein; ATP III = Adult Treatment Panel III guidelines

238

Table 7-2. Plasma levels of triglycerides, cholesterol, and glucose from mice on normal chow vs. TC supplemented diet. C57BL/6J, Apoe–/– female mice at time of weaning were placed on the indicated diets until time of sacrifice for aortic root quantification of atherosclerosis (18 weeks of age). Parallel groups of animals were also provided an antibiotics cocktail in drinking water. Lipid profiles, glucose, and insulin levels shown were determined in plasma isolated at time of organ harvest at conclusion of study. Data shown are mean ± SD for each of the indicated feeding groups. Wilcoxon non-parametric comparisons are between chow and TC (1.3%) supplemented diets with the noted antibiotic (ABS) treatment status.

239

Figure 7-1: Demonstration of an obligatory role of the commensal gut microbiota of mice in the production of transcrotonobetaine from oral carnitine in germ-free and conventionalized mice. d3-Carnitine challenge (oral gavage of d3-carnitine) in germ-free female Swiss Webster mice before and after ensuing conventionalization (≥ 3 weeks in conventional cages with conventional mice). Each point represents mean ± SE of 4 independent replicates. Plasma levels of d3-transcrotonobetaine determined by stable isotope dilution LC/MS/MS.

240

Figure 7-2. TC is an abundant gut microbiota metabolite of L-carnitine. C57BL/6J Female mouse intestine (n=3) was sectioned into two complementary pieces for incubation with equamolar amounts of d3-L-carnitine under anaerobic conditions at 37oC for 12 hours. Deuterated trimethylamine analytes were quantified by stable isotope dilution LC/MS/MS as detailed in Methods. d3-TMA and d3-TC production by the gut microbiota from d3-L-carnitine occurs primarily in the cecum (top and middle panels) whereas d3-γBB production from d3-L-carnitine is more evenly distributed in the cecum and colon. d3-γBB production from d3-L-carnitine is approximately 1000 fold higher (bottom panel) than d3- TMA production (middle panel) and d3-TC production (top panel) is about 10 fold higher than d3-TMA (middle panel).

241

Figure 7-3. Proposed scheme of gut microbiota mediated carnitine metabolism and TC production. Carnitine can be metabolized by the gut microbiota via different pathways, but all leading to the terminal product TMA.* denotes a gut microbiota dependent pathway.

242

Figure 7-4. Demonstration of an obligatory role of commensal gut microbiota of mice in the production of TMA, TMAO, and γ-butyrobetaine from an oral d9-TC challenge. Left panels - C57BL/6Jfemale mice (n = 4) in conventional cages were given oral d9-transcrotonobetaine (d9-TC) via gavage at T = 0, and then serial blood draws were obtained at the indicated times. Plasma levels of d9-TMAO, d9-TMA, d9-γbutyrobetaine (d9-γBB), d9-carnitine, and d9-TC were determined by stable isotope dilution LC/MS/MS using d4-choline as an internal standard. Middle panel - Mice were then treated with a cocktail of oral broad spectrum antibiotics to suppress intestinal microbiota. Repeat gastric gavage with d9-TC was performed, and serial testing of plasma for quantification of respective analytes were determined. Right panel - Antibiotics were stopped and mice allowed to reacquire 1 month) their intestinal microbiota in conventional cages. Repeat gastric gavage with d9-TC was performed and its metabolites were then quantified by LC/MS/MS in serial plasma samples. Results shown are mean ± SE for 4 animals.

243

Figure 7-5. Plasma TC is associated with MACE over a 3-year period. Plasma levels (µM) of TC in sequential consenting subjects from Genebank (n = 836). Subjects were stratified into Tertiles by increasing concentrations of plasma TC. ((T1 (n = 270), < 0.07 µM; T2 (n = 288), 0.07 -0.1µM; T1 (n = 278), > 0.07 µM)

244

Figure 7-6. Plasma TC is not associated with MACE over a 3-year period after adjustment with other CVD risk factors in n = 836 subjects. Forrest plots of hazard ratios of MACE (death, non fatal- MI, stroke, and revascularization) and tertiles of TC unadjusted (closed circles), and after adjusting for traditional cardiovascular risk factors (open circles), or traditional cardiac risk factors, additional risk factors, and TMAO levels (open squares). Bars represent 95% confidence intervals.

245

Figure 7-7. Dietary TC gut microbiota metabolism accelerates atherosclerosis. Quantification of mouse aortic root plaque lesion area of 18 week-old C57BL/6J, Apoe–/– female mice on respective diets. Transcrotonobetaine synthesized and supplemented in mouse chow with or without suppression of gut microbiota with oral broad spectrum antibiotics. P values shown are comparison of groups using a Wilcoxon non-parametric test.

246

Figure 7-8. Plasma analytes from TC atherosclerosis study. Carnitine, γBB, TC, TMA, and TMAO were determined using stable isotope dilution LC/MS/MS analysis of plasma recovered from mice at time of sacrifice.

247

CHAPTER 8: Summary, Conclusions, and Future Directions

The gut microbiome and host form a complex symbiotic relationship. The gut microbiota has important functions in the normal development and maintenance of mammalian physiological processes, but can also contribute to complex disease pathogenesis. Together, these data support a role for the gut microbiota

in the pathogenesis of atherosclerotic disease by the metabolism of dietary trimethylamines into TMAO. The relationship of CVD and dietary trimethylamines was first identified by the demonstration that the metabolism of dietary choline, a trimethylamine primarily found in meats and dairy, to TMAO promoted CVD69.

Subsequent studies show that another dietary trimethylamine, carnitine found

principally in red meat, can promote CVD by the direct, or indirect (through γBB

production) gut microbiota dependent metabolism to TMAO72,147. TMAO is an

independent prognostic indicator of MACE and preceding dietary habits shape

the gut microbiota composition and its capacity to metabolize dietary

trimethylamines to TMAO72. TMAO production promotes atherogenesis by

causing dysfunction in the balance of forward and reverse cholesterol transport.

These data provide a mechanistic link between foods commonly consumed in a

western diet, the gut microbiota, and atherosclerosis.

Clinical implications

There are several clinical implications for our studies. First, TMAO can serve as a functional plasma biomarker for CVD risk stratification. TMAO is an independent

248

prognostic indicator of MACE in > 4,000 subjects and may help identify

individuals at higher risk for CVD independent of traditional risk factors. Those at

risk may then benefit from direct intervention in metabolic pathways promoting

TMAO formation (e.g. the gut microbiota metabolism of carnitine and choline to

TMAO). Additionally, the development of human choline and carnitine challenges

represents a functional assay that can measure the capacity of the gut microbiota to produce TMAO from specific dietary trimethylamines. We have demonstrated

that chronic proceeding dietary habits can influence the capacity of the gut

microbiota to metabolize carnitine forming TMAO. However, we do not yet know if intervening to alter functional differences will translate into reduction in cardiovascular disease. Further, larger clinical studies are needed to determine whether these functional tests can be used in CVD diagnostics.

The identification of a metabolic pathway that promotes atherogenesis also provides therapeutic targets for CVD. The development of inhibitors of the gut

microbiota mediated pathways of carnitine and choline metabolism may be

reasonable targets for future therapeutic developments to prevent or treat

atherosclerosis. It is not known if a universal inhibitor could be developed to

block the metabolism of all dietary trimethylamines to TMAO or if multiple

individual inhibitors would be needed to target individual steps in the pathways.

For example, the metabolism of carnitine to TMAO by the gut microbiota clearly

involves multiple pathways. Each may represent a distinct target for inhibition.

Further characterization of the enzymes involved in these pathways may thus

249

help provide specific therapeutic targets. Another possible option is to develop probiotics and for fecal transplant regimens to change the gut microbiota from a high to low output TMAO producer.

A hypothetical role for the gut microbiota and TMAO in other disease states

High red meat consumption has been associated with the development of not just CVD but also other disease states like cancer177. Moreover, the consumption of choline and betaine is associated with adenoma formation, a premalignant mass in the gut178. The gut microbiota metabolism of dietary trimethylamines could thus provide a potential link for these observed associations.

It is interesting to note that choline metabolism is linked to one-carbon metabolism and homocysteine formation (Fig. 8-1). Plasma homocysteine, or more broadly one carbon methyl donor pathways, have been implicated in many physiologic and disease states such as cardiovascular disease, renal disease, bone disease, and cancer. It is interesting to note that both choline and betaine are involved in this metabolic pathway (Fig. 8-1). This may suggest a link between one carbon methylation cycles, dietary trimethylamines, and the gut microbiota (Fig. 8-1). Further studies will be needed explore these interactions.

Summary

These studies together demonstrate a clear and consistent relationship between dietary trimethylamines and CVD, and elucidate previously unrecognized

250

pathways that contribute to the pathogenesis of atherosclerotic disease.

Importantly, they thus suggest possible therapeutic targets for CVD. These data lay the foundation for testing various treatments.

251

+ L-carnitine

Figure 8-1. Relationship of dietary trimethylamines, atherosclerosis, and homocysteine formation. Dietary trimethylamines are metabolized to TMA and further oxidized by FMOs to TMAO thereby promoting atherosclerosis. Choline and betaine also participate in one-carbon metabolism and the generation of homocysteine.

252

REFERENCES

1. Libby, P. Inflammation in atherosclerosis. Nature 420, 868-874 (2002).

2. Lusis, A.J. Atherosclerosis. Nature 407, 233-241 (2000).

3. Soutar, A.K. & Naoumova, R.P. Mechanisms of disease: genetic causes

of familial hypercholesterolemia. Nature clinical practice. Cardiovascular

medicine 4, 214-225 (2007).

4. Brown, M.S. & Goldstein, J.L. Expression of the familial

hypercholesterolemia gene in heterozygotes: mechanism for a dominant

disorder in man. Science 185, 61-63 (1974).

5. Brown, M.S. & Goldstein, J.L. The SREBP pathway: regulation of

cholesterol metabolism by proteolysis of a membrane-bound transcription

factor. Cell 89, 331-340 (1997).

6. Tuzcu, E.M., et al. High prevalence of coronary atherosclerosis in

asymptomatic teenagers and young adults: evidence from intravascular

ultrasound. Circulation 103, 2705-2710 (2001).

7. Libby, P. & Theroux, P. Pathophysiology of coronary artery disease.

Circulation 111, 3481-3488 (2005).

8. Rader, D.J., Alexander, E.T., Weibel, G.L., Billheimer, J. & Rothblat, G.H.

The role of reverse cholesterol transport in animals and humans and

relationship to atherosclerosis. J Lipid Res 50 Suppl, S189-194 (2009).

9. Glomset, J.A. The plasma lecithins:cholesterol acyltransferase reaction. J

Lipid Res 9, 155-167 (1968).

253

10. Voight, B.F., et al. Plasma HDL cholesterol and risk of myocardial

infarction: a mendelian randomisation study. Lancet 380, 572-580 (2012).

11. Cuchel, M. & Rader, D.J. Macrophage reverse cholesterol transport: key

to the regression of atherosclerosis? Circulation 113, 2548-2555 (2006).

12. Zhang, Y., et al. Overexpression of apolipoprotein A-I promotes reverse

transport of cholesterol from macrophages to feces in vivo. Circulation

108, 661-663 (2003).

13. Moore, R.E., et al. Increased atherosclerosis in mice lacking

apolipoprotein A-I attributable to both impaired reverse cholesterol

transport and increased inflammation. Circ Res 97, 763-771 (2005).

14. Wang, X., et al. Macrophage ABCA1 and ABCG1, but not SR-BI, promote

macrophage reverse cholesterol transport in vivo. J Clin Invest 117, 2216-

2224 (2007).

15. McGillicuddy, F.C., et al. Inflammation impairs reverse cholesterol

transport in vivo. Circulation 119, 1135-1145 (2009).

16. Guarner, F. & Malagelada, J.R. Gut flora in health and disease. Lancet

361, 512-519 (2003).

17. Xu, J. & Gordon, J.I. Honor thy symbionts. Proc Natl Acad Sci U S A 100,

10452-10459 (2003).

18. Hawrelak, J.A. & Myers, S.P. The causes of intestinal dysbiosis: a review.

Alternative medicine review : a journal of clinical therapeutic 9, 180-197

(2004).

254

19. Control of infectious diseases. MMWR. Morbidity and mortality weekly

report 48, 621-629 (1999).

20. Sekirov, I., Russell, S.L., Antunes, L.C. & Finlay, B.B. Gut microbiota in

health and disease. Physiol Rev 90, 859-904 (2010).

21. O'Hara, A.M. & Shanahan, F. The gut flora as a forgotten organ. EMBO

reports 7, 688-693 (2006).

22. Harris, K., Kassis, A., Major, G. & Chou, C.J. Is the gut microbiota a new

factor contributing to obesity and its metabolic disorders? Journal of

obesity 2012, 879151 (2012).

23. Frank, D.N., et al. Molecular-phylogenetic characterization of microbial

community imbalances in human inflammatory bowel diseases. Proc Natl

Acad Sci U S A 104, 13780-13785 (2007).

24. Fanaro, S., Chierici, R., Guerrini, P. & Vigi, V. Intestinal microflora in early

infancy: composition and development. Acta Paediatr Suppl 91, 48-55

(2003).

25. Maynard, C.L., Elson, C.O., Hatton, R.D. & Weaver, C.T. Reciprocal

interactions of the intestinal microbiota and immune system. Nature 489,

231-241 (2012).

26. Mandar, R. & Mikelsaar, M. Transmission of mother's microflora to the

newborn at birth. Biology of the neonate 69, 30-35 (1996).

27. Yoshioka, H., Iseki, K. & Fujita, K. Development and differences of

intestinal flora in the neonatal period in breast-fed and bottle-fed infants.

Pediatrics 72, 317-321 (1983).

255

28. Round, J.L. & Mazmanian, S.K. The gut microbiota shapes intestinal

immune responses during health and disease. Nature reviews.

Immunology 9, 313-323 (2009).

29. Sprinz, H., et al. The response of the germfree guinea pig to oral bacterial

challenge with Escherichia coli and Shigella flexneri. The American journal

of pathology 39, 681-695 (1961).

30. Nardi, R.M., Silva, M.E., Vieira, E.C., Bambirra, E.A. & Nicoli, J.R.

Intragastric infection of germfree and conventional mice with Salmonella

typhimurium. Brazilian journal of medical and biological research = Revista

brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de

Biofisica ... [et al.] 22, 1389-1392 (1989).

31. Zachar, Z. & Savage, D.C. Microbial interference and colonization of the

murine gastrointestinal tract by Listeria monocytogenes. Infection and

immunity 23, 168-174 (1979).

32. Moreau, M.C. & Gaboriau-Routhiau, V. The absence of gut flora, the

doses of antigen ingested and aging affect the long-term peripheral

tolerance induced by ovalbumin feeding in mice. Research in immunology

147, 49-59 (1996).

33. Kagnoff, M.F. & Eckmann, L. Epithelial cells as sensors for microbial

infection. J Clin Invest 100, 6-10 (1997).

34. Cario, E., Gerken, G. & Podolsky, D.K. Toll-like receptor 2 controls

mucosal inflammation by regulating epithelial barrier function.

Gastroenterology 132, 1359-1374 (2007).

256

35. Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Edberg, S. &

Medzhitov, R. Recognition of commensal microflora by toll-like receptors

is required for intestinal homeostasis. Cell 118, 229-241 (2004).

36. Tremaroli, V. & Backhed, F. Functional interactions between the gut

microbiota and host metabolism. Nature 489, 242-249 (2012).

37. Simon, G.M., Cheng, J. & Gordon, J.I. Quantitative assessment of the

impact of the gut microbiota on lysine epsilon-acetylation of host proteins

using gnotobiotic mice. Proc Natl Acad Sci U S A 109, 11133-11138

(2012).

38. Wellen, K.E., et al. ATP-citrate lyase links cellular metabolism to histone

acetylation. Science 324, 1076-1080 (2009).

39. Hoverstad, T. & Midtvedt, T. Short-chain fatty acids in germfree mice and

rats. J Nutr 116, 1772-1776 (1986).

40. Wostmann, B.S., Larkin, C., Moriarty, A. & Bruckner-Kardoss, E. Dietary

intake, energy metabolism, and excretory losses of adult male germfree

Wistar rats. Laboratory animal science 33, 46-50 (1983).

41. Caesar, R., et al. Gut-derived lipopolysaccharide augments adipose

macrophage accumulation but is not essential for impaired glucose or

insulin tolerance in mice. Gut 61, 1701-1707 (2012).

42. Backhed, F., et al. The gut microbiota as an environmental factor that

regulates fat storage. Proc Natl Acad Sci U S A 101, 15718-15723 (2004).

43. Turnbaugh, P.J., et al. An obesity-associated gut microbiome with

increased capacity for energy harvest. Nature 444, 1027-1031 (2006).

257

44. Rabot, S., et al. Germ-free C57BL/6J mice are resistant to high-fat-diet-

induced insulin resistance and have altered cholesterol metabolism.

FASEB journal : official publication of the Federation of American

Societies for Experimental Biology 24, 4948-4959 (2010).

45. Ley, R.E., et al. Evolution of mammals and their gut microbes. Science

(New York, N.Y.) 320, 1647-1651 (2008).

46. Muegge, B.D., et al. Diet drives convergence in gut microbiome functions

across mammalian phylogeny and within humans. Science 332, 970-974

(2011).

47. Arumugam, M., et al. Enterotypes of the human gut microbiome. Nature

473, 174-180 (2011).

48. Jernberg, C., Lofmark, S., Edlund, C. & Jansson, J.K. Long-term impacts

of antibiotic exposure on the human intestinal microbiota. Microbiology

156, 3216-3223 (2010).

49. Wu, G.D., et al. Linking long-term dietary patterns with gut microbial

enterotypes. Science (New York, N.Y.) 334, 105-108 (2011).

50. Hildebrandt, M.A., et al. High-fat diet determines the composition of the

murine gut microbiome independently of obesity. Gastroenterology 137,

1716-1724 e1711-1712 (2009).

51. Manichanh, C., Borruel, N., Casellas, F. & Guarner, F. The gut microbiota

in IBD. Nature reviews. Gastroenterology & hepatology 9, 599-608 (2012).

52. Swidsinski, A., et al. Mucosal flora in inflammatory bowel disease.

Gastroenterology 122, 44-54 (2002).

258

53. Videla, S., et al. Role of intestinal microflora in chronic inflammation and

ulceration of the rat colon. Gut 35, 1090-1097 (1994).

54. Morrissey, P.J. & Charrier, K. Induction of wasting disease in SCID mice

by the transfer of normal CD4+/CD45RBhi T cells and the regulation of

this autoreactivity by CD4+/CD45RBlo T cells. Research in immunology

145, 357-362 (1994).

55. Casellas, F., et al. Antiinflammatory effects of enterically coated

amoxicillin-clavulanic acid in active ulcerative colitis. Inflammatory bowel

diseases 4, 1-5 (1998).

56. Macpherson, A., Khoo, U.Y., Forgacs, I., Philpott-Howard, J. & Bjarnason,

I. Mucosal antibodies in inflammatory bowel disease are directed against

intestinal bacteria. Gut 38, 365-375 (1996).

57. Brandtzaeg, P., et al. Immunobiology and immunopathology of human gut

mucosa: humoral immunity and intraepithelial lymphocytes.

Gastroenterology 97, 1562-1584 (1989).

58. Hampe, J., et al. Association between insertion mutation in NOD2 gene

and Crohn's disease in German and British populations. Lancet 357,

1925-1928 (2001).

59. Moore, W.E. & Moore, L.H. Intestinal floras of populations that have a high

risk of colon cancer. Applied and environmental microbiology 61, 3202-

3207 (1995).

259

60. Scanlan, P.D., et al. Culture-independent analysis of the gut microbiota in

colorectal cancer and polyposis. Environmental microbiology 10, 789-798

(2008).

61. Wu, S., et al. A human colonic commensal promotes colon tumorigenesis

via activation of T helper type 17 T cell responses. Nat Med 15, 1016-

1022 (2009).

62. Berg, R.D. Bacterial translocation from the gastrointestinal tract. Advances

in experimental medicine and biology 473, 11-30 (1999).

63. Klatt, N.R. & Brenchley, J.M. Th17 cell dynamics in HIV infection. Current

opinion in HIV and AIDS 5, 135-140 (2010).

64. Singh, N., Ranjan, A., Sur, S., Chandra, R. & Tandon, V. Inhibition of HIV-

1 Integrase gene expression by 10-23 DNAzyme. Journal of biosciences

37, 493-502 (2012).

65. Lichtman, S.M. Bacterial [correction of baterial] translocation in humans.

Journal of pediatric gastroenterology and nutrition 33, 1-10 (2001).

66. Turnbaugh, P.J., Backhed, F., Fulton, L. & Gordon, J.I. Diet-induced

obesity is linked to marked but reversible alterations in the mouse distal

gut microbiome. Cell host & microbe 3, 213-223 (2008).

67. Ley, R.E., Turnbaugh, P.J., Klein, S. & Gordon, J.I. Microbial ecology:

human gut microbes associated with obesity. Nature 444, 1022-1023

(2006).

68. Ley, R.E., et al. Obesity alters gut microbial ecology. Proc Natl Acad Sci U

S A 102, 11070-11075 (2005).

260

69. Wang, Z., et al. Gut flora metabolism of phosphatidylcholine promotes

cardiovascular disease. Nature 472, 57-63 (2011).

70. al-Waiz, M., Mikov, M., Mitchell, S.C. & Smith, R.L. The exogenous origin

of trimethylamine in the mouse. Metabolism: clinical and experimental 41,

135-136 (1992).

71. Lang, D.H., et al. Isoform specificity of trimethylamine N-oxygenation by

human flavin-containing monooxygenase (FMO) and P450 enzymes:

selective catalysis by FMO3. Biochem Pharmacol 56, 1005-1012 (1998).

72. Mitchell, S.C. & Smith, R.L. Trimethylaminuria: the fish malodor syndrome.

Drug metabolism and disposition: the biological fate of chemicals 29, 517-

521 (2001).

73. Koeth, R.A., et al. Intestinal microbiota metabolism of l-carnitine, a nutrient

in red meat, promotes atherosclerosis. Nat Med advance online

publication(2013).

74. Bernstein, A.M., et al. Major dietary protein sources and risk of coronary

heart disease in women. Circulation 122, 876-883 (2010).

75. Micha, R., Wallace, S.K. & Mozaffarian, D. Red and processed meat

consumption and risk of incident coronary heart disease, stroke, and

diabetes mellitus: a systematic review and meta-analysis. Circulation 121,

2271-2283 (2010).

76. Siri-Tarino, P.W., Sun, Q., Hu, F.B. & Krauss, R.M. Meta-analysis of

prospective cohort studies evaluating the association of saturated fat with

261

cardiovascular disease. The American Journal of Clinical Nutrition 91,

535-546 (2010).

77. Bibbins-Domingo, K., et al. Projected effect of dietary salt reductions on

future cardiovascular disease. The New England journal of medicine 362,

590-599 (2010).

78. Hansen, E.S. International Commission for Protection Against

Environmental Mutagens and Carcinogens. ICPEMC Working Paper

7/1/2. Shared risk factors for cancer and atherosclerosis--a review of the

epidemiological evidence. Mutation research 239, 163-179 (1990).

79. Turnbaugh, P.J., et al. A core gut microbiome in obese and lean twins.

Nature 457, 480-484 (2009).

80. Goodman, A.L. & Gordon, J.I. Our unindicted coconspirators: human

metabolism from a microbial perspective. Cell metabolism 12, 111-116

(2010).

81. Bremer, J. Carnitine--metabolism and functions. Physiological Reviews

63, 1420-1480 (1983).

82. Rebouche, C.J. & Seim, H. Carnitine metabolism and its regulation in

microorganisms and mammals. Annual Review of Nutrition 18, 39-61

(1998).

83. Stanley, C.A. Carnitine deficiency disorders in children. Annals of the New

York Academy of Sciences 1033, 42-51 (2004).

262

84. Demarquoy, J., et al. Radioisotopic determination of L-carnitine content in

foods commonly eaten in western countries. Food Chemistry 86, 137-142

(2004).

85. Rigault, C., Mazue, F., Bernard, A., Demarquoy, J. & Le Borgne, F.

Changes in l-carnitine content of fish and meat during domestic cooking.

Meat Science 78, 331-335 (2008).

86. Zimmer, J., et al. A vegan or vegetarian diet substantially alters the human

colonic faecal microbiota. European journal of clinical nutrition 66, 53-60

(2012).

87. Febbraio, M., et al. Targeted disruption of the class B scavenger receptor

CD36 protects against atherosclerotic lesion development in mice. J Clin

Invest 105, 1049-1056 (2000).

88. Suzuki, H., et al. A role for macrophage scavenger receptors in

atherosclerosis and susceptibility to infection. Nature 386, 292-296 (1997).

89. Spann, N.J., et al. Regulated accumulation of desmosterol integrates

macrophage lipid metabolism and inflammatory responses. Cell 151, 138-

152 (2012).

90. Rader, D.J. Regulation of reverse cholesterol transport and clinical

implications. The American journal of cardiology 92, 42J-49J (2003).

91. Jia, L., Betters, J.L. & Yu, L. Niemann-pick C1-like 1 (NPC1L1) protein in

intestinal and hepatic cholesterol transport. Annual review of physiology

73, 239-259 (2011).

263

92. Gulewitsch, W. & Krimberg, R. Zur Kenntnis der Extrakivstoffe der

Muskein, II. Mitteilung. Uber das Carnitin. Hoppe-Seyler's Zeitschrift fur

Physiologische Chemie, 326-330 (1905).

93. Rebouche, C.J. & Chenard, C.A. Metabolic fate of dietary carnitine in

human adults: identification and quantification of urinary and fecal

metabolites. The Journal of nutrition 121, 539-546 (1991).

94. Rebouche, C.J., Mack, D.L. & Edmonson, P.F. L-Carnitine dissimilation in

the gastrointestinal tract of the rat. Biochemistry 23, 6422-6426 (1984).

95. Zhang, A.Q., Mitchell, S.C. & Smith, R.L. Dietary precursors of

trimethylamine in man: a pilot study. Food and chemical toxicology : an

international journal published for the British Industrial Biological Research

Association 37, 515-520 (1999).

96. Delany, J.P., snook, J.T., Vivian, V.M. & Cashmere, K. Metabolic effects of

a carnitine-free diet fed to college students. fed proc 45, 815 (1986).

97. Fraser, G.E. Vegetarian diets: what do we know of their effects on

common chronic diseases? Am J Clin Nutr 89, 1607S-1612S (2009).

98. Key, T.J., et al. Mortality in vegetarians and nonvegetarians: detailed

findings from a collaborative analysis of 5 prospective studies. Am J Clin

Nutr 70, 516S-524S (1999).

99. Snowdon, D.A., Phillips, R.L. & Fraser, G.E. Meat consumption and fatal

ischemic heart disease. Preventive medicine 13, 490-500 (1984).

264

100. Zeisel, S.H., Mar, M.H., Howe, J.C. & Holden, J.M. Concentrations of

choline-containing compounds and betaine in common foods. The Journal

of nutrition 133, 1302-1307 (2003).

101. Charach, G., Rabinovich, A., Argov, O., Weintraub, M. & Rabinovich, P.

The role of bile Acid excretion in atherosclerotic coronary artery disease.

International journal of vascular medicine 2012, 949672 (2012).

102. Charach, G., et al. Decreased fecal bile acid output in patients with

coronary atherosclerosis. Journal of medicine 29, 125-136 (1998).

103. Lu, Y., Feskens, E.J., Boer, J.M. & Muller, M. The potential influence of

genetic variants in genes along bile acid and bile metabolic pathway on

blood cholesterol levels in the population. Atherosclerosis 210, 14-27

(2010).

104. Miyake, J.H., et al. Transgenic expression of cholesterol-7-alpha-

hydroxylase prevents atherosclerosis in C57BL/6J mice. Arteriosclerosis,

thrombosis, and vascular biology 22, 121-126 (2002).

105. Post, S.M., de Crom, R., van Haperen, R., van Tol, A. & Princen, H.M.

Increased fecal bile acid excretion in transgenic mice with elevated

expression of human phospholipid transfer protein. Arteriosclerosis,

thrombosis, and vascular biology 23, 892-897 (2003).

106. Zong, C., et al. Chitosan oligosaccharides promote reverse cholesterol

transport and expression of scavenger receptor BI and CYP7A1 in mice.

Exp Biol Med (Maywood) 237, 194-200 (2012).

265

107. Altmann, S.W., et al. Niemann-Pick C1 Like 1 protein is critical for

intestinal cholesterol absorption. Science 303, 1201-1204 (2004).

108. Repa, J.J., et al. Disruption of the sterol 27-hydroxylase gene in mice

results in hepatomegaly and hypertriglyceridemia. Reversal by cholic acid

feeding. J Biol Chem 275, 39685-39692 (2000).

109. Schwarz, M., Russell, D.W., Dietschy, J.M. & Turley, S.D. Marked

reduction in bile acid synthesis in cholesterol 7alpha-hydroxylase-deficient

mice does not lead to diminished tissue cholesterol turnover or to

hypercholesterolemia. J Lipid Res 39, 1833-1843 (1998).

110. Bai, C., Biwersi, J., Verkman, A.S. & Matthay, M.A. A mouse model to test

the in vivo efficacy of chemical chaperones. Journal of pharmacological

and toxicological methods 40, 39-45 (1998).

111. Mello, C.C. & Barrick, D. Measuring the stability of partly folded proteins

using TMAO. Protein science : a publication of the Protein Society 12,

1522-1529 (2003).

112. Liberles, S.D. & Buck, L.B. A second class of chemosensory receptors in

the olfactory epithelium. Nature 442, 645-650 (2006).

113. Suska, A., Ibanez, A.B., Lundstrom, I. & Berghard, A. G protein-coupled

receptor mediated trimethylamine sensing. Biosensors & bioelectronics

25, 715 (2009).

114. Cordain, L., et al. Origins and evolution of the Western diet: health

implications for the 21st century. The American Journal of Clinical

Nutrition 81, 341-354 (2005).

266

115. Liszt, K., et al. Characterization of bacteria, clostridia and Bacteroides in

faeces of vegetarians using qPCR and PCR-DGGE fingerprinting. Annals

of nutrition & metabolism 54, 253-257 (2009).

116. Elssner, T., Preusser, A., Wagner, U. & Kleber, H.P. Metabolism of L(-)-

carnitine by Enterobacteriaceae under aerobic conditions. FEMS Microbiol

Lett 174, 295-301 (1999).

117. Moller, B., Hippe, H. & Gottschalk, G. Degradation of various amine

compounds by mesophilic clostridia. Archives of microbiology 145, 85-90

(1986).

118. Eckburg, P.B., et al. Diversity of the human intestinal microbial flora.

Science (New York, N.Y.) 308, 1635-1638 (2005).

119. Kleber, H.P. Bacterial carnitine metabolism. FEMS microbiology letters

147, 1-9 (1997).

120. Hedayati, S.S. Dialysis-related carnitine disorder. Seminars in dialysis 19,

323-328 (2006).

121. Mingorance, C., Rodriguez-Rodriguez, R., Justo, M.L., Alvarez de

Sotomayor, M. & Herrera, M.D. Critical update for the clinical use of L-

carnitine analogs in cardiometabolic disorders. Vascular health and risk

management 7, 169-176 (2011).

122. Sayed-Ahmed, M.M., Khattab, M.M., Gad, M.Z. & Mostafa, N. L-carnitine

prevents the progression of atherosclerotic lesions in

hypercholesterolaemic rabbits. Pharmacological research : the official

journal of the Italian Pharmacological Society 44, 235-242 (2001).

267

123. Dambrova, M., Liepinsh, E. & Kalvinsh, I. Mildronate: cardioprotective

action through carnitine-lowering effect. Trends in cardiovascular medicine

12, 275-279 (2002).

124. Vilskersts, R., et al. Mildronate, a regulator of energy metabolism, reduces

atherosclerosis in apoE/LDLR-/- mice. Pharmacology 83, 287-293 (2009).

125. Millward, C.A., et al. Genetic factors for resistance to diet-induced obesity

and associated metabolic traits on mouse chromosome 17. Mammalian

genome : official journal of the International Mammalian Genome Society

20, 71-82 (2009).

126. Folch, J., Lees, M. & Sloane Stanley, G.H. A simple method for the

isolation and purification of total lipides from animal tissues. The Journal of

biological chemistry 226, 497-509 (1957).

127. Robinet, P., Wang, Z., Hazen, S.L. & Smith, J.D. A simple and sensitive

enzymatic method for cholesterol quantification in macrophages and foam

cells. Journal of lipid research 51, 3364-3369 (2010).

128. Wang, Z., et al. Protein carbamylation links inflammation, smoking, uremia

and atherogenesis. Nature medicine 13, 1176-1184 (2007).

129. Chen, F.C. & Benoiton, L. A new method of quaternizing amines and its

use in amino acid and peptide chemistry. Can. J. Chem, 3310-3311

(1976).

130. Chen, J. Powerful statistical analysis for associating microbiomes to

environmental covariates using generalized UniFrac distances.

Bioinformatics 28, 2106-2113 (2012).

268

131. Wu, G.D., et al. Linking long-term dietary patterns with gut microbial

enterotypes. Science 334, 105-108 (2011).

132. Willems, G. A robust hotelling test. Metrika 55, 125-138 (2002).

133. Hamady, M., Walker, J.J., Harris, J.K., Gold, N.J. & Knight, R. Error-

correcting barcoded primers for pyrosequencing hundreds of samples in

multiplex. Nature methods 5, 235-237 (2008).

134. Costello, E.K., et al. Bacterial community variation in human body habitats

across space and time. Science 326, 1694-1697 (2009).

135. Caporaso, J.G., et al. QIIME allows analysis of high-throughput

community sequencing data. Nature methods 7, 335-336 (2010).

136. Kuczynski, J., et al. Using QIIME to analyze 16S rRNA gene sequences

from microbial communities. Current protocols in bioinformatics / editoral

board, Andreas D. Baxevanis ... [et al.] Chapter 10, Unit 10 17 (2011).

137. Wang, Q., Garrity, G.M., Tiedje, J.M. & Cole, J.R. Naive Bayesian

classifier for rapid assignment of rRNA sequences into the new bacterial

taxonomy. Applied and environmental microbiology 73, 5261-5267 (2007).

138. Caporaso, J.G., et al. PyNAST: a flexible tool for aligning sequences to a

template alignment. Bioinformatics 26, 266-267 (2010).

139. Lozupone, C.A. & Knight, R. Species divergence and the measurement of

microbial diversity. FEMS microbiology reviews 32, 557-578 (2008).

140. The Atherosclerosis Risk in Communities (ARIC) Study: design and

objectives. The ARIC investigators. American journal of epidemiology 129,

687-702 (1989).

269

141. Colucci, J.W., Turnbull, S.P. & Gandour, R.D. Preparation of crystalline

sodium norcarnitine: an easily handled precursor for the preparation of

carnitine analogs and radiolabeled carnitine. Anal.Biochem. 162, 459-462

(1986).

142. Sehayek, E. & Hazen, S.L. Cholesterol absorption from the intestine is a

major determinant of reverse cholesterol transport from peripheral tissue

macrophages. Arteriosclerosis, thrombosis, and vascular biology 28,

1296-1297 (2008).

143. Greenberg, M.E., Smith, J.D. & Sehayek, E. Moderately decreased

cholesterol absorption rates are associated with a large atheroprotective

effect. Arteriosclerosis, thrombosis, and vascular biology 29, 1745-1750

(2009).

144. Smith, J.D., et al. Cyclic AMP induces apolipoprotein E binding activity

and promotes cholesterol efflux from a macrophage cell line to

apolipoprotein acceptors. J Biol Chem 271, 30647-30655 (1996).

145. Goldstein, J.L., Ho, Y.K., Basu, S.K. & Brown, M.S. Binding site on

macrophages that mediates uptake and degradation of acetylated low

density lipoprotein, producing massive cholesterol deposition. Proc Natl

Acad Sci U S A 76, 333-337 (1979).

146. Sports drinks and energy drinks for children and adolescents: are they

appropriate? Pediatrics 127, 1182-1189 (2011).

147. Tang, W.H., et al. Intestinal microbial metabolism of phosphatidylcholine

and cardiovascular risk. N Engl J Med 368, 1575-1584 (2013).

270

148. Patterson, K.Y., Bhagwat, S.A., Williams, J.R., Howe, J.C. & Holden, J.M.

USDA Database for the Choline Content of Common Foods. Release

Two. Accessed on June 15, 2011 at

http://www.ars.usda.gov/SP2UserFiles/Place/12354500/Data/Choline/Chol

n02.pdf. (2008).

149. Zhang, A.Q., Mitchell, S.C. & Smith, R.L. Dietary precursors of

trimethylamine in man: a pilot study. Food Chem Toxicol 37, 515-520

(1999).

150. Zeisel, S.H. Choline: critical role during fetal development and dietary

requirements in adults. Annu Rev Nutr 26, 229-250 (2006).

151. Dumas, M.E., et al. Metabolic profiling reveals a contribution of gut

microbiota to fatty liver phenotype in insulin-resistant mice. Proc Natl Acad

Sci U S A 103, 12511-12516 (2006).

152. Wen, L., et al. Innate immunity and intestinal microbiota in the

development of Type 1 diabetes. Nature 455, 1109-1113 (2008).

153. de la Huerga, J. & Popper, H. Urinary excretion of choline metabolites

following choline administration in normals and patients with hepatobiliary

diseases. J Clin Invest 30, 463-470 (1951).

154. Simenhoff, M.L., Saukkonen, J.J., Burke, J.F., Wesson, L.G. & Schaedler,

R.W. Amine metabolism and the small bowel in uraemia. Lancet 2, 818-

821 (1976).

155. Ihle, B.U., Cox, R.W., Dunn, S.R. & Simenhoff, M.L. Determination of body

burden of uremic toxins. Clin Nephrol 22, 82-89 (1984).

271

156. Bain, M.A., Fornasini, G. & Evans, A.M. Trimethylamine: metabolic,

pharmacokinetic and safety aspects. Curr Drug Metab 6, 227-240 (2005).

157. Erdmann, C.C. On the Alleged Occurrence of Trimethylamine in the Urine.

J Biol Chem 8, 57-60 (1910).

158. Hooper, L.V. & Gordon, J.I. Commensal host-bacterial relationships in the

gut. Science 292, 1115-1118 (2001).

159. Stella, C., et al. Susceptibility of human metabolic phenotypes to dietary

modulation. J Proteome Res 5, 2780-2788 (2006).

160. Martin, F.P., et al. Probiotic modulation of symbiotic gut microbial-host

metabolic interactions in a humanized microbiome mouse model. Mol Syst

Biol 4, 157 (2008).

161. Gill, S.R., et al. Metagenomic analysis of the human distal gut microbiome.

Science 312, 1355-1359 (2006).

162. Loscalzo, J. Lipid metabolism by gut microbes and atherosclerosis. Circ

Res 109, 127-129 (2011).

163. Bennett, B.J., et al. Trimethylamine-N-oxide, a metabolite associated with

atherosclerosis, exhibits complex genetic and dietary regulation. Cell

Metab 17, 49-60 (2013).

164. Al-Waiz, M., Mitchell, S.C., Idle, J.R. & Smith, R.L. The relative importance

of N-oxidation and N-demethylation in the metabolism of trimethylamine in

man. Toxicology 43, 117-121 (1987).

165. Yancey, P.H., Rhea, M.D., Kemp, K.M. & Bailey, D.M. Trimethylamine

oxide, betaine and other osmolytes in deep-sea animals: depth trends and

272

effects on enzymes under hydrostatic pressure. Cell Mol Biol (Noisy-le-

grand) 50, 371-376 (2004).

166. Cannon, C.P., et al. Antibiotic treatment of Chlamydia pneumoniae after

acute coronary syndrome. N Engl J Med 352, 1646-1654 (2005).

167. Grayston, J.T., et al. Azithromycin for the secondary prevention of

coronary events. N Engl J Med 352, 1637-1645 (2005).

168. Pimentel, M., et al. Rifaximin therapy for patients with irritable bowel

syndrome without constipation. N Engl J Med 364, 22-32 (2011).

169. Craig, S.A. Betaine in human nutrition. Am J Clin Nutr 80, 539-549 (2004).

170. Cali, J.J. & Russell, D.W. Characterization of human sterol 27-

hydroxylase. A mitochondrial cytochrome P-450 that catalyzes multiple

oxidation reaction in bile acid biosynthesis. J Biol Chem 266, 7774-7778

(1991).

171. Fujita, M., et al. Hepatic uptake of gamma-butyrobetaine, a precursor of

carnitine biosynthesis, in rats. American journal of physiology.

Gastrointestinal and liver physiology 297, G681-686 (2009).

172. Shekhawat, P.S., Sonne, S., Carter, A.L., Matern, D. & Ganapathy, V.

Enzymes involved in l-carnitine biosynthesis are expressed by small

intestinal enterocytes in mice: Implications for gut health. Journal of

Crohn's & colitis (2012).

173. Liepinsh, E., et al. Mildronate treatment alters gamma-butyrobetaine and l-

carnitine concentrations in healthy volunteers. The Journal of pharmacy

and pharmacology 63, 1195-1201 (2011).

273

174. Seim, H., Kleber, H.P. & Strack, E. [Reduction of L-carnitine to gamma-

butyrobetaine by Escherichia coli]. Zeitschrift fur allgemeine Mikrobiologie

19, 753-758 (1979).

175. Morano, C., Zhang, X. & Fricker, L.D. Multiple isotopic labels for

quantitative mass spectrometry. Analytical chemistry 80, 9298-9309

(2008).

176. Heinz, L. & Hermann, S. SYNTHESIS OF [methyl-

14C1CROTONOBETAlNE FROM DL-[methyl-14CICARNITINE. Journal of

Labelled Compounds and Radiopharmaceuricals 38, 179-186 (1996).

177. Pan, A., et al. Red meat consumption and mortality: results from 2

prospective cohort studies. Archives of internal medicine 172, 555-563

(2012).

178. Cho, E., et al. Dietary choline and betaine and the risk of distal colorectal

adenoma in women. Journal of the National Cancer Institute 99, 1224-

1231 (2007).

274