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nm.3145

nm.3145

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    N  a   t  u  r  e   A  m  e  r   i  c  a ,   I  n  c .   A   l   l  r   i  g   h   t  s  r  e  s  e  r  v  e   d .
articles
nature medicine
 
advance online publication
The high eve of meat consumption in the deveoped word is inked toCVD risk, presumaby owing to the arge content of saturated fats andchoestero in meat
. However, a recent meta-anaysis of prospectivecohort studies showed no association between dietary saturated fat intakeand CVD, prompting the suggestion that other environmenta exposuresinked to increased meat consumption are responsibe
3
. In fact, the sus-picion that the choestero and saturated fat content of red meat may not be sufficienty high enough to account for the observed associationbetween CVD and meat consumption has stimuated investigation of aternative disease-promoting exposures that accompany dietary meatingestion, such as high sat content or heterocycic compounds gener-ated during cooking
. To our knowedge, no studies have yet exporedthe participation of commensa intestina microbiota in modifying thediet-host interaction with reference to red meat consumption.The microbiota of humans has been inked to intestina heath,immune function, bioactivation of nutrients and vitamins, and, morerecenty, compex disease phenotypes such as obesity and insuin resist-ance
. We recenty reported a pathway in both humans and mice ink-ing microbiota metaboism of dietary choine and phosphatidychoineto CVD pathogenesis
9
. Choine, a trimethyamine-containing com-pound and part of the head group of phosphatidychoine, is metabo-ized by gut microbiota to produce an intermediate compound knownas TMA (
). TMA is rapidy further oxidized by hepatic favinmonooxygenases to form TMAO, which is proatherogenic and associ-ated with cardiovascuar risks. These findings raise the possibiity thatother dietary nutrients possessing a trimethyamine structure may aso generate TMAO from gut microbiota and promote acceeratedatheroscerosis. TMAO has been proposed to induce upreguation of macrophage scavenger receptors and thereby potentiay contributeto enhanced “forward choestero transport.”
. Whether TMAO isinked to the deveopment of acceerated atheroscerosis through addi-tiona mechanisms, and which specific microbia species contributeto TMAO formation, have not been fuy carified.l-carnitine is an abundant nutrient in red meat and containsa trimethyamine structure simiar to that of choine (
).Athough dietary ingestion is a major source of l-carnitine in omni- vores, it is aso endogenousy produced in mammas from ysineand serves an essentia function in transporting fatty acids into the
1
Department o Cellular & Molecular Medicine, Cleveland Clinic, Cleveland, Ohio, USA.
2
Center or Cardiovascular Diagnostics & Prevention, Cleveland Clinic,Cleveland, Ohio, USA.
3
Department o Medicine, Division o Cardiology, David Geen School o Medicine, University o Caliornia–Los Angeles, Los Angeles,Caliornia, USA.
4
Department o Mathematics, Cleveland State University, Cleveland, Ohio, USA.
5
Department o Cardiovascular Medicine, Cleveland Clinic,Cleveland, Ohio, USA.
6
Department o Microbiology, Center or Clinical Epidemiology and Biostatistics, Perelman School o Medicine at the University o Pennsylvania,Philadelphia, Pennsylvania, USA.
7
Division o Gastroenterology, Perelman School o Medicine at the University o Pennsylvania, Philadelphia, Pennsylvania, USA.
8
Department o Medicine, Perelman School o Medicine at the University o Pennsylvania, Philadelphia, Pennsylvania, USA.
9
Department o Pathology,Section on Lipid Sciences, Wake Forest School o Medicine, Winston-Salem, North Carolina, USA.
10
Children’s Hospital Oakland Research Institute, Oakland,Caliornia, USA. Correspondence should be addressed to S.L.H. (hazens@cc.org).Received 7 December 2012; accepted 27 February 2013; published online 7 April 2013;doi:10.1038/nm.3145
Intestina microbiota metaboism of l-carnitine,a nutrient in red meat, promotes atheroscerosis
Robert A Koeth
1,2
, Zeeg Wag
1,2
, Bruce S Leviso
1,2
, Jeifer A Buffa
1,2
, Eli Org
3
, Breda T Sheehy 
1
,Earl B Britt
1,2
, Xiaomig Fu
1,2
, Yupig Wu
4
, Li Li
1,2
, Joatha D Smith
1,2,5
, Joseph A DiDoato
1,2
, Ju Che
6
,Hogzhe Li
6
, Gary D Wu
7
, James D Lewis
6,8
, Maya Warrier
9
, J Mark Brow
9
, Roald M Krauss
10
,W H Wilso Tag
1,2,5
, Frederic D Bushma
5
, Aldos J Lusis
3
& Staley L Haze
1,2,5
Intestinal microbiota metabolism of choline and phosphatidylcholine produces trimethylamine (TMA), which is furthermetabolized to a proatherogenic species, trimethylamine-
-oxide (TMAO). We demonstrate here that metabolism by intestinalmicrobiota of dietary
L
-carnitine, a trimethylamine abundant in red meat, also produces TMAO and accelerates atherosclerosisin mice. Omnivorous human subjects produced more TMAO than did vegans or vegetarians following ingestion of
L
-carnitinethrough a microbiota-dependent mechanism. The presence of specific bacterial taxa in human feces was associated withboth plasma TMAO concentration and dietary status. Plasma
L
-carnitine levels in subjects undergoing cardiac evaluation(
= 2,595) predicted increased risks for both prevalent cardiovascular disease (CVD) and incident major adverse cardiac events(myocardial infarction, stroke or death), but only among subjects with concurrently high TMAO levels. Chronic dietary
L
-carnitinesupplementation in mice altered cecal microbial composition, markedly enhanced synthesis of TMA and TMAO, and increasedatherosclerosis, but this did not occur if intestinal microbiota was concurrently suppressed. In mice with an intact intestinalmicrobiota, dietary supplementation with TMAO or either carnitine or choline reduced
in vivo 
reverse cholesterol transport.Intestinal microbiota may thus contribute to the well-established link between high levels of red meat consumption and CVD risk.
 
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    N  a   t  u  r  e   A  m  e  r   i  c  a ,   I  n  c .   A   l   l  r   i  g   h   t  s  r  e  s  e  r  v  e   d .
articles
advance online publication
nature medicine
 mitochondria compartment
. l-Carnitine ingestion and sup-pementation in industriaized societies have markedy increased
.Whether there are potentia heath risks associated with the rapidy growing practice of consuming l-carnitine suppements has notbeen evauated.Herein we examine the gut microbiota–dependent metaboism of l-carnitine to produce TMAO in both rodents and humans (omnivoresand vegans or vegetarians). Using isotope tracer studies in humans,cinica studies to examine the effects on cardiovascuar disease risk,and anima modes incuding germ-free mice, we demonstrate a roefor gut microbiota metaboism of l-carnitine in atheroscerosis patho-genesis. We show that TMAO, and its dietary precursors choine andcarnitine, suppress reverse choestero transport (RCT) through gutmicrobiota–dependent mechanisms
in vivo
. Finay, we define micro-bia taxa in feces of humans whose proportions are associated withboth dietary carnitine ingestion and pasma TMAO concentrations.We aso show microbia compositiona changes in mice associatedwith chronic carnitine ingestion and a consequent marked enhance-ment in TMAO synthetic capacity 
in vivo
.
RESULTSMetabolomic studies link
L
-carnitine with CVD
Given the simiarity in structure between l-carnitine and choine(
), we hypothesized that dietary l-carnitine in humans, ikechoine and phosphatidychoine, might be metaboized to pro-duce TMA and TMAO in a gut microbiota–dependent fashionand be associated with atheroscerosis risk. To test this hypothesis,we initiay examined data from our recenty pubished unbiasedsma-moecue metaboomics anayses of pasma anaytes andCVD risks
9
.An anayte with identica moecuar weight and retention time tol-carnitine was not in the top tier of anaytes that met the stringent
vaue cutoff for association with CVD. However, a hypothesis-drivenexamination of the data using ess stringent criteria (no adjustment formutipe testing) reveaed an anayte with the appropriate moecuarweight and retention time for l-carnitine that was associated with car-diovascuar event risk (
= 0.04) (
Suppementary Tabe 1
). In furtherstudies we were abe to confirm the identity of the pasma anayte asl-carnitine and deveop a quantitative stabe-isotope-diution iquidchromatography tandem mass spectrometry (LC-MS/MS) method formeasuring endogenous l-carnitine concentrations in a subsequentinvestigations (
Suppementary Figs. 1
3
).
Human gut microbiota is required to form TMAO from
L
-carnitine
The participation of gut microbiota in TMAO production fromdietary l-carnitine in humans has not previousy been shown. Ininitia subjects (omnivores), we deveoped an l-carnitine chaengetest in which the subjects were fed a arge amount of l-carnitine(an 8-ounce siroin steak, corresponding to an estimated 180 mgof l-carnitine)
, together with a capsue containing 250 mgof a heavy isotope–abeed l-carnitine (synthetic d3-(methy)-l-carnitine). At visit 1 post-prandia increases in pasma d3-TMAOand d3- l-carnitine concentrations were readiy detected, and 24-hurine coections aso reveaed the presence of d3-TMAO (
e
 and
Suppementary Figs. 4
and
5
).
and
Suppementary Figure 4
show tracings from a representative omnivorous subject, of five studied with sequentia seria bood draws after carnitine cha-enge. In most subjects examined, despite cear increases in pasmad3-carnitine and d3-TMAO concentrations over time (
), post-prandia changes in endogenous (unabeed) carnitine and TMAOconcentrations were modest (
Suppementary Fig. 5
), consistent withtota body poos of carnitine and TMAO that are reativey very argein reation to the amounts of carnitine ingested and TMAO producedfrom the carnitine chaenge.To examine the potentia contribution of gut microbiota toTMAO formation from dietary l-carnitine, we paced the five vounteers studied above on orabroad-spectrum antibioticsto suppress intestina microbiota for a week and then performeda second l-carnitine chaenge (visit 2). We noted near competesuppression of detectabe endogenous TMAO in both pasmaand urine after a week-ong treatment with the antibiotics (visit 2)
a
AtherosclerosisCarnitineGut floraCholineTMAOTMAFMO
b
Visit 1Steak+d3-carnitineVisit 2Steak+d3-carnitineVisit 3Steak+d3-carnitine
e
   P   l  a  s  m  a   (
     µ
   M   )
d3-TMAOd3-carnitine2.501.2500 12 24Time (h)d3-TMAOd3-carnitine2.501.2500 12 24Time (h)d3-TMAOd3-carnitine12.002.501.2500 12 24Time (h)
c
   I  n   t  e  n  s   i   t  y   (   %   )
1005000 5 10TMAO
 m
 / 
 z
=76
58
1005000510TMAO
 m
 / 
 z
=76
581005000510TMAO
 m
 / 
 z
=76
58
d
   I  n   t  e  n  s   i   t  y   (   %   )
1005000510d3-TMAO
 m
 / 
 z
=79
61Time (min)1005000510d3-TMAO
 m
 / 
 z
=79
61Time (min)1005000510d3-TMAO
 m
 / 
 z
=79
61Time (min)Gut florasuppressionof gut floraReacquisition
Figure 1
TMAO production rom
l
-carnitine is a microbiota-dependentprocess in humans. (
a
) Structure o carnitine and scheme o carnitineand choline metabolism to TMAO.
l
-Carnitine and choline are bothdietary trimethylamines that can be metabolized by microbiota to TMA.TMA is then urther oxidized to TMAO by lavin monooxygenases (FMOs).(
b
) Scheme o the human
l
-carnitine challenge test. Ater a 12-hovernight ast, subjects received a capsule o d3-(methyl)-carnitine(250 mg) alone, or in some cases (as in data or the subject shown)also an 8-ounce steak (estimated 180 mg
l
-carnitine), whereupon serialplasma and 24-h urine samples were obtained or TMA and TMAOanalyses (visit 1). Ater a weeklong regimen o oral broad-spectrumantibiotics to suppress the intestinal microbiota, the challenge wasrepeated (visit 2), and then again a inal third time ater a
3-week periodto permit repopulation o intestinal microbiota (visit 3). (
c
,
d
) LC-MS/MSchromatograms o plasma TMAO (
c
) and d3-TMAO (
d
) in an omnivoroussubject using speciic precursor
product ion transitions indicated at
= 8 h or each visit. (
e
) Stable-isotope-dilution LC-MS/MS time coursemeasurements o d3-labeled TMAO and carnitine in plasma collected romsequential venous blood draws at the indicated time points. Data shown in
c
e
are rom a representative emale omnivorous subject who underwentcarnitine challenge. Data are organized vertically to correspond with thevisit schedule indicated in
b
.
 
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    N  a   t  u  r  e   A  m  e  r   i  c  a ,   I  n  c .   A   l   l  r   i  g   h   t  s  r  e  s  e  r  v  e   d .
articles
nature medicine
 
advance online publication
(
e
and
Suppementary Fig. 5
). Moreover, we observed vir-tuay no detectabe formation of either native or d3-abeed TMAOin a post-prandia pasma sampes or 24-h urine sampes examinedafter carnitine chaenge, consistent with an obigatory roe for gutmicrobiota in TMAO formation from l-carnitine (
e
and
Suppementary Fig. 4
). In contrast, we detected both d3- l-carnitineand unabeed l-carnitine after the l-carnitine chaenge, and therewas itte change in the overa time course before (visit 1) versusafter (visit 2) antibiotic treatment (
and
Suppementary Fig. 5
). We rechaenged the same subjects severa weeks afterdiscontinuation of antibiotics (visit 3). Baseine and post-l-carnitinechaenge pasma and urine sampes again showed TMAO andd3-TMAO formation, consistent with intestina recoonization(
e
and
Suppementary Figs. 4
and
5
). Coectivey, thesedata show that TMAO production from dietary l-carnitine inhumans is dependent on intestina microbiota.
Vegans and vegetarians produce less TMAO from
L
-carnitine
The capacity to produce TMAO (native and d3-abeed) after l-carnitineingestion was variabe among individuas. A
 post hoc
nutritionasurvey that the vounteers competed suggested that antecedentdietary habits (red meat consumption) may infuence the capacity to generate TMAO from l-carnitine (data not shown). To test thisprospectivey, we examined TMAO and d3-TMAO production afterthe same l-carnitine chaenge, first in a ong-term (>5 years) veganwho consented to the carnitine chaenge (incuding both steak andd3-(methy)-carnitine consumption) (
). Aso shown forcomparison are data from a singe representative omnivore withsef-reported frequent (near daiy) dietary consumption of red meat(beef, venison, amb, mutton, duck or pork). Post-prandiay, theomnivore showed increases in TMAO and d3-TMAO concentra-tions in both sequentia pasma measurements (
) and in a24-h urine coection sampe (
). In contrast, the vegan showednomina pasma and urine TMAO eves at baseine, and virtuay nocapacity to generate TMAO or d3-TMAO in pasma after the carnitinechaenge (
,
b
). The vegan subject aso had ower fastingpasma eves of l-carnitine compared to the omnivorous subject(
Suppementary Fig. 6
).To confirm and extend these findings, we examined additiona vegans and vegetarians (
n
= 23) and omnivorous subjects (
n
= 51).
Figure 2
The ormation o TMAO romingested
l
-carnitine is negligible in vegans,and ecal microbiota composition associateswith plasma TMAO concentrations. (
a
,
b
) Datarom a male vegan subject in the carnitinechallenge consisting o co-administrationo 250 mg d3-(methyl)-carnitine and an8-ounce sirloin steak and, or comparison,a representative emale omnivore whorequently consumes red meat. PlasmaTMAO and d3-TMAO were quantiied ater
l
-carnitine challenge (
a
) and in a 24-h urinecollection (
b
). Urine TMAO and d3-TMAOreported as ratio with urinary creatinine(Cr) to adjust or urinary dilution. Data areexpressed as means ± s.e.m. (
c
) Baselineasting plasma concentrations o TMAO andd3-TMAO rom male and emale vegans andvegetarians (
= 26) and omnivores (
= 51).Boxes represent the 25th, 50th, and 75thpercentiles and whiskers represent the 10thand 90th percentiles. (
d
) Plasma d3-TMAOconcentrations in male and emale vegansand vegetarians (
= 5) and omnivores(
= 5) participating in a d3-(methyl)-carnitine (250 mg) challenge withoutconcomitant steak consumption. The
valueshown is or the comparison o the area underthe curve (AUC) o groups using the Wilcoxonnonparametric test. Data points representmean
±
s.e.m. o
= 5 per group. (
e
) BaselineTMAO plasma concentrations associate withenterotype 2 (
Prevotella 
) in male and emalesubjects with a characterized gut microbiomeenterotype. Boxes represent the 25th,50th (middle lines) and 75th percentiles,and whiskers represent the 10th and 90thpercentiles. (
f
) Plasma TMAO concentrations(plotted on
axes) and the proportion otaxonomic operational units (OTUs, plottedon
 y 
axes), determined as described in
Supplementary Methods
. Subjects weregrouped by dietary status as either veganor vegetarian (
= 23) or omnivore (
= 30).
value shown is or comparisons between dietary groups using a robust Hotelling
2
test. Data areexpressed as means ± s.e.m. or both TMAO concentration (
axis) and the proportion o OTUs (
 y 
axis).
a
6400 12 24
   P   l  a  s  m  a   (
     µ
   M   )
TMAOOmnivoreVeganTime (h)0.2500.12500 12 24
   P   l  a  s  m  a   (
     µ
   M   )
d3-TMAOOmnivoreVeganTime (h)
b
50250Vegan OmnivoreUrine TMAO
   T   M   A   O   /   C  r   (  m  m  o   l   /  m  o   l   )
210Vegan OmnivoreUrine d3-TMAO
   d   3  -   T   M   A   O   /   C  r   (  m  m  o   l   /  m  o   l   )
c
8
40
   P   l  a  s  m  a   T   M   A   O    (
     µ
   M   )
Omnivore(
n
= 51)Vegan/ vegetarian(
n
= 26)
P
< 0.05
d
3015001224
   P   l  a  s  m  a   d   3  -   T   M   A   O    (
     µ
   M   )
P
< 0.05
 
  ) 
Vegan/vegetarian(
n
= 5)Time (h)
   P  r  o  p  o  r   t   i  o  n   O   T   U  s   (
     ×
   1   0
  –   4
   )
Clostridiales
 incertae sedis
XII4201.82.73.6TMAO (
µ
M)
P
= 0.13
Fusibacterium
4201.82.73.6TMAO (
µ
M)
P
= 0.13
Lachnospira
50
2501.82.73.6TMAO (
µ
M)
P
< 0.05
Sporobacter 
241201.82.73.6TMAO (
µ
M)
P
= 0.10
f
40200
   P  r  o  p  o  r   t   i  o  n   O   T   U  s   (
     ×
   1   0
  –   4
   )
Omnivore(
n
= 30)Peptostreptococcaceae
 incertae sedis
Vegan/ vegetarian(
n
= 23)1.82.73.6TMAO (
µ
M)
P
< 0.05Peptostreptococcaceae402001.82.73.6TMAO (
µ
M)
P
< 0.05
Clostridium
301501.82.73.6TMAO (
µ
M)
P
< 0.05301501.82.73.6TMAO (
µ
M)
P
< 0.05Clostridiaceae
e
840
   P   l  a  s  m  a   T   M   A   O    (
     µ
   M   )
P
< 0.05Enterotype 1
Bacteroides
(
n
= 49)Enterotype 2
Prevotella
(
n
= 4)

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