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 Lcarnitine 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 LCarnitine .......................................................................................................... 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
Supp. Table 2-2
107
Subject characteristics, demographics, and laboratory
values in the whole cohort (n = 2595), and across
quartiles of plasma carnitine
108
Plasma levels of triglycerides, cholesterol, glucose, and
insulin from mice on normal chow vs. carnitine
supplemented diet
109
Liver levels of triglycerides and total cholesterol in mice
on normal chow versus carnitine supplemented diet
110
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 2-3
Supp. Table 2-4
Supp. Table 2-5
Supp. Table 4-1
Baseline characteristics of cohort according to TMAO
quartiles values expressed in mean ± standard deviation
159
or median (interquartile range)
Table 5-1
Plasma and liver lipid levels in C57BL/6J, Apoe-/female mice used in γBB atherosclerosis study
186
Baseline clinical characteristics of n = 1445 Genebank
subjects used in analyses with γBB
217
Quantification of carnitine and γBB in beef, lamb,
chicken, and perch samples
218
Baseline clinical characteristics of n = 836 Genebank
subjects used in analyses with TC
238
Plasma levels of triglycerides, cholesterol, and glucose
from mice on normal chow vs. transcrotonobetaine
supplemented diet
239
Table 6-1
Table 6-2
Table 7-1
Table 7-2
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
TMAO production from carnitine is a microbiota
dependent process in humans
101
The formation of TMAO from ingested L-carnitine is
negligible in vegans, and fecal microbiota composition
associates with plasma TMAO concentrations
102
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
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
Figure 2-1
Figure 2-2
Figure 2-3
Supp. Figure 2-2
10
Supp. Figure 2-3
Supp. Figure 2-4
Standard curves for LC/MS/MS quantification of
carnitine and d3-(methyl)-carnitine in plasma matrix
114
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
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
Plasma levels of d3-carnitine following d3-carnitine
challenge (no steak) in omnivorous (n = 5) versus
vegan subjects (n = 5)
118
Human fecal microbiota taxa associate with plasma
TMAO
119
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 2-7
Supp. Figure 2-8
Supp. Figure 2-9
116
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
Figure 4-1
Figure 4-2
Figure 4-3
Supp. Figure 4-1
Carnitine supplementation can induce the gut
microbiota
138
Human plasma levels of phosphatidylcholine
Metabolites (TMAO, choline, betaine) after oral
ingestion of two hard-boiled eggs and d9Phosphatidylcholine before and after antibiotics
156
Kaplan-Meier estimates of long-term major adverse
cardiac events, according to TMAO Quartiles
157
Pathways linking dietary phosphatidylcholine, intestinal
microflora (gut flora), and incident adverse
cardiovascular events
158
Human plasma levels of phosphatidylcholine
metabolites (TMAO, choline, betaine) after oral
ingestion of two hard-boiled eEggs and d912
phosphatidylcholine before and after antibiotics
160
Human 24-hour urine levels of TMAO after oral
ingestion of two hard-boiled eggs and d9phosphatidylcholine before and after antibiotics
161
Risks of major adverse cardiac events (MACE) among
patient subgroups, according to baseline TMAO levels
162
γBB is produced as a major gut microbiota metabolite
of L-carnitine
187
γBB is produced from L-carnitine in a gut microbiota
dependent manner
188
TMA/TMAO is a gut a microbiota dependent product of
γBB metabolism
189
Confirmatory studies that TMA/TMAO is a gut a
microbiota dependent product of γBB metabolism
190
γBB is the dominant gut microbiota metabolite of Lcarnitine and is metabolized to TMA at a great
equamolar capacity than L-carnitine
191
γBB promotes atherosclerosis in a gut microbiota
dependent manner
192
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
γBB production from L-carnitine and microbiome
composition associate with dietary status
196
TMA/TMAO production from γBB associates with
microbiome composition
197
TMAO production from γBB and microbiome
composition associate with dietary status
198
Liver Expression of Bile acid enzymes
199
Supp. Figure 4-2
Supp. Figure 4-3
Figure 5-1
Figure 5-2
Figure 5-3
Figure 5-4
Figure 5-5
Figure 5-6
Figure 5-7
Figure 5-10
Figure 5-11
Figure 5-12
Figure 5-13
13
Figure 5-14
Scheme of endogenous and exogenous γBB
production
200
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
TMAO is the major gut microbiota metabolite in human
carnitine catabolism
223
The formation of γBB from ingested L-carnitine is
similar in vegans and vegetarians compared to
omnivores
224
Figure 6-1
Figure 6-5
Figure 6-6
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 Lcarnitine
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
Plasma TC is associated with MACE over a 3-year
period
244
Plasma TC is not associated with MACE over a 3-year
period after adjustment with other CVD risk factors in
n = 836 subjects.
245
Dietary TC gut microbiota metabolism accelerates
atherosclerosis
246
Plasma analytes from TC atherosclerosis study
247
Figure 7-5
Figure 7-6
Figure 7-7
Figure 7-8
14
Figure 8-1
Relationship of dietary trimethylamines,
atherosclerosis, and homocysteine formation
252
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, γbutyrobetaine (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 (d9PC) 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
Red Meat, Promotes Atherosclerosis73
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 Lcarnitine 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 wellestablished 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 Lcarnitine 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. 21, 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 d3TMAO (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. 2-1be; 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 Lcarnitine
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 postchallenge
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 Lcarnitine 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-carnitinedependent 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 proatherogenic 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 proatherosclerotic 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 microbiotagenerated 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]cholesterolloaded 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 Lcarnitine, 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 nonsignificant) 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 Lcarnitine 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 Lcarnitine 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 Lcarnitine 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 P40RR018603-06-08.
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, SigmaAldrich, 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, highdensity 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 Lcarnitine 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 Lcarnitine. 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 Lcarnitine 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,
d9(trimethyl)TMAO
and
(d9-TMAO),
TMA
respectively.
As
d9(trimethyl)carnitine
internal
standards,
(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,
(metagenomic).
COMBO:
SRX020587,
SRX020773,
SRX020379,
SRX020770).
Overall
SRX020378
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
with a higher Rf (> 0.1) than L-carnitine. 600MHz 1H-NMR (10 mg ml–1 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 Lnorcarnitine 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 Lcarnitine 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
Rf as L-carnitine. 600MHz 1H-NMR (10 mg ml–1 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 Lcarnitine under the same conditions and concentration except for the singlet peak
at 3.1 ppm corresponding to 9 protons on the trimethylammonium group on Lcarnitine 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
placement in vacuum dessicator.
13
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 nordeoxycholate.
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 cholesterol2,2,3,4,4,6-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 Lcarnitine), 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 nonparametric 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 d3TMAO 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 KruskalWallis 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
Patient Characteristics
Whole cohort
(n = 2595)
Carnitine
Quartiles
Q1
Q2
Q3
Q4
< 31.7 µM
31.7 - 37.8
37.9 - 45.2 µM
> 45.2 µM
(n = 649)
µM
(n = 649)
(n = 650)
P
(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
80
71
78
82
88
< 0.01
CVD (%)
2
BMI (kg/m )
29 (25-33)
28 (24-31)
29 (2-32)
29 (25-32)
29 (26-34)
< 0.01
–1
96 (78-117)
94 (74-111)
100 (80-122)
97 (80-117)
96 (77-117)
< 0.01
–1
34( 28-41)
35 (30-43)
34 (28-41)
32 (27-39)
32 (27-38)
< 0.01
160 (139-188)
159 (138-184)
160 (139-188)
161 (139-188)
< 0.01
117 (85-167)
103 (76-148)
164 (140194)
110 (84-159)
124 (88-170)
129 (96-192)
< 0.01
hsCRP (mg l )
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
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
LDL cholesterol (mg dl )
HDL cholesterol (mg dl )
–1
Total cholesterol (mg dl )
–1
Triglycerides (mg dl )
–1
Baseline medications (%)
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 d9carnitine.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 d9isotopologues. 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 d3carnitine (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 d3carnitine 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 d3carntine 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 d3carnitine 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 d3carnitine) 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 d3carnitine 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, 3hydroxy-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 nonparametric 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 d3carnitine 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 broadspectrum 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-Noxide, 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. 41c) 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). Timedependent increases in both the natural isotopes (Fig. 4-1c, left panel) and d9tracer 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-Noxide 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 broadspectrum antibiotics for one week (in six of the participants) resulted in nearcomplete suppression of detectable trimethylamine-N-oxide in fasting plasma
(during Visit 2), as well as both trimethylamine-N-oxide and d9-trimethylamine-Noxide 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-Noxide (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 trimethylamineN-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 subcohort (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 broadspectrum
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 monooxygenases164 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 d9trimethylamine production show a time course consistent with a precursor-toproduct 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-Noxide. 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 nonsystemic 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-Noxide 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 reacquisition 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 10minute 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,snglycero-3-phosphoethanolamine following exhaustive methylation with d3methyliodide (Cambridge Isotopes Laboratories Inc, Andover MA).
phosphatidylcholine
was
isolated
by
sequential
preparative
The d9thin
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)-trimethylamineN-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 chisquare 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. Threeyear 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)
With
Events
68±10
P value
63±11
Without Events
(n=3,494)
62±11
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
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
2.4 (1-5.9)
2.3(1-5.5)
3.9(1.8-9.8)
<0.001
115.2 (76.4-245.7)
113.2 (75.4-238.3)
136.3 (84.7-329.3)
<0.001
GFR(ml/min/1.73m )
82 (69-95)
83 (71-96)
75 (56-89)
<0.001
Total leukocyte count
9
(WBC, x10 )
Baseline drugs (%):
6.1 (5.1-7.5)
6.1 (5-7.5)
6.4 (5.3-8.1)
0.001
Aspirin
74
74
70
0.038
ACE inhibitor/ARB
50
49
58
<0.001
Statin
60
61
56
0.057
63
63
65
0.414
3.7 (2.4-6.2)
3.5 (2.4-5.9)
5.0 (3.0-8.8)
<0.001
Age (years)
Triglycerides (mg/dL)
hsCRP (ng/L)
MPO (pM)
2
Beta blockers
TMAO (µM)
<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
<2.43
2.43-3.66
3.67 -6.18
≥ 6.18
Range
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)**
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)*
Adjusted HR
** 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
147
disease.
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
(n=1001)
Quartile 2 (n=998)
Quartile 3 (n=1003)
(2.4-3.6)
(3.7-6.2)
Quartile 4
(n=1005)
P-value
(TMAO, µM)
(<2.4)
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
(25.4-32)
24
28.7
(25.6-32.8)
28
28.7
(25.9-32.6)
31
28.4
(25.7-33.1)
42
0.138
<0.001
Hypertension (%)
68
69
70
79
<0.001
History of MI (%)
41
39
42
43
0.317
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
63
65
67
65
0.314
97
(79-117)
34
(29-42)
115
(84-166)
82
(70-97)
116
(103-134)
100
(91-114)
2.3
(0.9-6.3)
127.3
(78.7-264.6)
92
(81-103)
6.2
(5.1-7.6)
98
(81-120)
35
(29-42)
115
(84-163)
83
(69-98)
117
(104-132)
101
(92-116)
2.2
(1-5.6)
115.3
(76.9-242.4)
86
(75-96)
6.1
(5.1-7.5)
95
(78-116)
34
(29-41)
121
(86-178)
82
(69-95)
117
(104-133)
102
(93-119)
2.3
(1.1-5)
112.9
(75.8-235.2)
79
(67-91)
6.1
(5-7.5)
92
(74-114)
33
(27-40)
123
(86-180)
80
(68-94)
115
(101-132)
107
(95-134)
3.1
(1.2-6.8)
110.8
(74.2-226.8)
69
(52-83)
6.1
(5-7.5)
<0.001
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
Diabetes mellitus (%)
(>6.2)
# of diseased vessels
Smoking (%)
LDL-c (mg/dL)
HDL-c (mg/dL)
Triglycerides (mg/dL)
ApoB (mg/dL)
ApoA1 (mg/dL)
Fasting glucose
hsCRP (ng/L)
MPO (pM)
2
eGFR(ml/min/1.73m )
9
Total leukocyte count (WBC, x10 )
Baseline drugs (%):
<0.001
0.089
0.05
0.121
<0.001
<0.001
0.092
<0.001
0.524
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 trimethylamineN-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-Noxide 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
69
Pathway 1 ) . 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
Gut Pathway 1: Choline
Microbiota
FMOs
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
Gut Pathway 2: L-carnitine
TMA
FMOs
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.
Gut Pathway 3: L-carnitine
Microbiota
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).
Hypothesized Gut Pathway: L-carnitine
Microbiota
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 Lcarnitine 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-Lcarnitine 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, d9γBB 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 inhouse 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 (xaxis) 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 Lcarnitine.
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 Lcarnitine81,82,119. 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 Lcarnitine 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 Lcarnitine 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 Lcarnitine 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 Lcarnitine 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 d9γBB (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 Pvalues 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 semicrystalline 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 [MN(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 (d9carnitine), 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
Triglyceride (mg/dL)
Total Cholesterol
(mg/dL)
HDL (mg/dL)
Total Glucose (mg/dL)
Lipids
Triglyceride (mg/dL)
Total Cholesterol
(mg/dL)
HDL (mg/dL)
Total Glucose (mg/dL)
Chow
(n = 20)
113+26
304+53
γ-butyrobetaine (1.3%)
(n = 17)
139+25
300+56
P
<0.01
0.84
32+10
217+45
33+22
194+35
0.82
0.10
Chow,+ABS
(n = 7)
75+13
368+52
γ-butyrobetaine (1.3%), +ABS
(n = 18)
90+23
419+53
P
0.10
0.06
29+9
203+44
27+7
188+43
0.80
0.43
Chow
(n=20)
46+21
γ-butyrobetaine (1.3%)
(n=17)
35+11
P
0.09
1.6+0.32
1.1+0.25
<0.01
Chow,+ABS
(n=7)
38+15
γ-butyrobetaine (1.3%), +ABS
(n=18)
28+9.4
P
0.10
1.7+0.22
1.6+0.21
0.61
Liver
Lipids
Triglyceride (mg/g
liver)
Cholesterol (mg/g liver)
Lipids
Triglyceride (mg/g
liver)
Cholesterol (mg/g liver)
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 d9γBB) 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 d3TMA (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-Lcarnitine 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 LCarnitine
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
phosphatidylcholine (the major source of dietary choline)69.
from
dietary
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.
Gut Pathway 1: Choline
Microbiota
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.
Gut Pathway 2: L-carnitine
Microbiota
FMOs
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. Lcarnitine 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.
Hypothesized Gut Pathway: L-carnitine
Microbiota
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. 63). 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 "Lcarnitine 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) Lcarnitine 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-Lcarnitne 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)
Male (%)
Former/current smokers (%)
Diabetes mellitus (%)
Hypertension (%)
Hyperlipidemia (%)
Prior coronary artery disease (%)
CAD (%)
PAD (%)
CVD (%)
Framingham ATP III Risk Score
BMI (kg/m2)
LDL cholesterol (mg/dL)
HDL cholesterol (mg/dL)
Total cholesterol (mg/dl)
Triglycerides (mg/dL)
hsCRP (mg/L)
MPO (pmol/L)
61±11
73
71
20
73
88
78
80
21
82
8(6-10)
28.5(25.4-32)
92(75-111)
33(27-39)
156(135-181)
115(82-164)
2.05(0.93-4.69)
109(70-239)
Creatinine clearance
(ml/min/1.73m2)
Creatinine (mg/dl)
Γ-butyrobetaine (µM)
105(80-131)
0.85(0.76-0.98)
0.93(0.77-1.16)
Baseline medications (%)
ACE inhibitors
Beta-blockers
Statin
Aspirin
50
68
66
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 microbiotadependent 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.
Hypothesized Gut Pathway: L-carnitine
Gut
Microbiota
TC
Gut
Microbiota
TMA
FMOs
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 germfree 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% d9carnitine 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-γbutyrobetaine, d9-TMA, d9-TMAO, and d9-carnitine demonstrated production of
these metabolites. These data confirm gut microbiota dependent formation of d9γ-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 d9carnitine 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, highdensity 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 72). 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 Lcarnitine 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. 73).
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 downstream 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 10week 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, d9γBB,
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)
Male (%)
61±11
78
Former/current smokers (%)
Diabetes mellitus (%)
Hypertension (%)
Hyperlipidemia (%)
Prior coronary artery disease (%)
CAD (%)
76
12
72
89
80
83
PAD (%)
20
CVD (%)
83
Framingham ATP III Risk Score
7(5-10)
2
BMI (kg/m )
LDL cholesterol (mg/dL)
HDL cholesterol (mg/dL)
Total cholesterol (mg/dl)
Triglycerides (mg/dL)
hsCRP (mg/L)
MPO (pmol/L)
28.3(25.3-31.6)
88(72-109)
33(28-39)
151(131-178)
106(75-154)
1.89(0.86-4.3)
110(70-259)
Creatinine clearance
(ml/min/1.73m2)
Creatinine (mg/dl)
Transcrotonobetaine (µM)
104(82-128)
0.87(0.77-0.98)
0.09(0.07-0.11)
Baseline medications (%)
ACE inhibitors
Beta-blockers
Statin
Aspirin
49
70
70
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 d3TMA 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 fatalMI, 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
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