Publications by authors named "Xinmin S Li"

21 Publications

  • Page 1 of 1

Plasma trimethylamine N-oxide (TMAO) levels predict future risk of coronary artery disease in apparently healthy individuals in the EPIC-Norfolk prospective population study.

Am Heart J 2021 Feb 21. Epub 2021 Feb 21.

Center for Microbiome and Human Health, Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland, OH; Department of Cardiovascular Medicine, Heart, Vascular and Thoracic Institute, Cleveland, OH.

Background: Recent studies show a mechanistic link between gut microbiota-dependent formation of the atherosclerosis- and thrombosis-promoting metabolite trimethylamine N-oxide (TMAO) and cardiovascular disease (CVD). The clinical utility of TMAO in apparently healthy subjects for predicting incident CVD risks is unclear.

Methods And Results: In the EPIC-Norfolk community-based study, we examined baseline fasting levels of TMAO and two of its nutrient precursors, choline and betaine, in a case:control design study comparing apparently European healthy middle-aged participants who subsequently develop CVD (Cases, n = 908) vs those who did not (Controls, n = 1,273) over an ensuing average follow-up period of 8 years. In participants who developed CVD vs controls, higher plasma TMAO (3.70 [IQR 2.50-6.41]μM vs 3.25 [IQR 2.19-52,1.15]μM; P < .001) and choline levels (9.09 [IQR 7.87-10.53]μM vs 8.89 [IQR 7.66-10.13]μM; P = .001) were observed. Following adjustments for traditional risk factors, elevated TMAO (adjusted odds ratio (OR) 1.58 [95% confidence interval (CI) 1.21-2.06], P < .001) and choline levels (adjusted OR 1.31 [95%CI 1.00-1.72], P < .05) remained predictive of incident CVD development. The clinical prognostic utility of TMAO remained significant and essentially unchanged regardless of the level of cutoff chosen between 1.5 uM (10%ile) to 10.5 uM (90%ile).

Conclusion: In apparently healthy participants of the community-based middle-aged EPIC-Norfolk population, elevated plasma levels of the gut microbe-dependent metabolite TMAO, and its nutrient precursor choline, predict incident risk for CVD development independent of traditional risk factors.
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http://dx.doi.org/10.1016/j.ahj.2021.01.020DOI Listing
February 2021

A Novel Cell-Free Fluorescent Assay for HDL Function: Low Apolipoprotein A1 Exchange Rate Associated with Increased Incident Cardiovascular Events.

J Appl Lab Med 2020 05;5(3):544-557

Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, OH.

Background: Cholesterol efflux capacity is a tissue culture assay for HDL function that is not amenable for high-throughput monitoring of risk assessment.

Methods: We devised a cell-free HDL function assay to measure the exchange rate of exogenous apoA1 into serum HDL using NBD/Alexa647 double-labeled apoA1, whose NBD/Alexa647 emission ratio increased upon exchange into HDL. ApoA1 exchange rate (AER) was assayed by incubating labeled apoA1 with human serum, and the rate of the increase of the NBD/Alexa647 ratio over time was calculated as AER.

Results: Fast protein liquid chromatography analysis of serum confirmed that the labeled apoA1 selectively exchanged into the HDL lipoprotein fraction. Characterization studies demonstrated that the AER assay had excellent intra- and inter-day reproducibility, was stable over 3 freeze-thaw cycles, and yielded similar results with serum or plasma. We quantified AER in serum from randomly selected stable subjects undergoing elective diagnostic coronary angiography (n = 997). AER was correlated with HDL-cholesterol (r = 0.58, P < 0.0001) and apoA1 levels (r = 0.56, P < 0.0001). Kaplan-Meier survival plot showed subjects in the lowest quartile of AER experienced a significantly higher rate of incident major adverse cardiovascular events (MACE = myocardial infarction, stroke, or death) (P < 0.0069 log rank). Moreover, compared to subjects in the lowest AER quartile, the remaining subjects showed significantly lower incident (3 year) risk for MACE, even after adjustment for traditional risk factors and apoA1 (HR 0.58; 95% CI 0.40-0.85; P = 0.005).

Conclusions: In a prospective cohort of stable subjects undergoing elective diagnostic cardiac evaluations, low AER was associated with increased incident risk of MACE.
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http://dx.doi.org/10.1093/jalm/jfaa002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192547PMC
May 2020

Gut Microbiota-Dependent Trimethylamine N-oxide and Cardiovascular Outcomes in Patients With Prior Myocardial Infarction: A Nested Case Control Study From the PEGASUS-TIMI 54 Trial.

J Am Heart Assoc 2020 05 5;9(10):e015331. Epub 2020 May 5.

TIMI Study Group Division of Cardiovascular Medicine Brigham and Women's Hospital Harvard Medical School Boston MA.

Background Trimethylamine N-oxide (TMAO) may have prothrombotic properties. We examined the association of TMAO quartiles with major adverse cardiovascular events (MACE) and the effect of TMAO on the efficacy of ticagrelor. Methods and Results PEGASUS-TIMI 54 (Prevention of Cardiovascular Events in Patients With Prior Heart Attack Using Ticagrelor Compared to Placebo on a Background of Aspirin - Thrombolysis in Myocardial Infarction 54) randomized patients with prior myocardial infarction to ticagrelor or placebo (median follow-up 33 months). Baseline plasma concentrations of TMAO were measured in a nested case-control study of 597 cases with cardiovascular death, myocardial infarction, or stroke (MACE) and 1206 controls matched for age, sex, and estimated glomerular filtration rate [eGFR]. Odds ratios (OR) were used for the association between TMAO quartiles and MACE, adjusting for baseline clinical characteristics (age, sex, eGFR, region, body mass index, hypertension, hypercholesterolemia, diabetes mellitus, smoking, peripheral artery disease, index event, aspirin dosage and treatment arm), and cardiovascular biomarkers (hs-TnT [high-sensitivity troponin T], hs-CRP [high-sensitivity C-reactive protein], NT-proBNP [N-terminal-pro-B-type natriuretic peptide]). Higher TMAO quartiles were associated with risk of MACE (OR for quartile 4 versus quartile 1, 1.43, 95% CI, 1.06-1.93, trend=0.015). The association was driven by cardiovascular death (OR 2.25, 95% CI, 1.28-3.96, trend=0.003) and stroke (OR 2.68, 95% CI, 1.39-5.17, trend<0.001). After adjustment for clinical factors, the association persisted for cardiovascular death (OR 1.89, 95% CI, 1.03-3.45, trend=0.027) and stroke (OR 2.01, 95% CI, 1.01-4.01, trend=0.022), but was slightly attenuated after adjustment for cardiovascular biomarkers (cardiovascular death: OR 1.74, 95% CI, 0.88-3.45, trend=0.079; and stroke: OR 1.82, 95% CI, 0.88-3.78, trend=0.056). The reduction in MACE with ticagrelor was consistent across TMAO quartiles ( interaction=0.92). Conclusions Among patients with prior myocardial infarction, higher TMAO levels were associated with cardiovascular death and stroke but not with recurrent myocardial infarction. The efficacy of ticagrelor was consistent regardless of TMAO levels. Registration URL: https://www.clini​caltr​ials.gov; Unique identifiers: PEGASUS-TIMI 54, NCT01225562.
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http://dx.doi.org/10.1161/JAHA.119.015331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7660879PMC
May 2020

Site-specific 5-hydroxytryptophan incorporation into apolipoprotein A-I impairs cholesterol efflux activity and high-density lipoprotein biogenesis.

J Biol Chem 2020 04 25;295(15):4836-4848. Epub 2020 Feb 25.

Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195

Apolipoprotein A-I (apoA-I) is the major protein constituent of high-density lipoprotein (HDL) and a target of myeloperoxidase-dependent oxidation in the artery wall. In atherosclerotic lesions, apoA-I exhibits marked oxidative modifications at multiple sites, including Trp Site-specific mutagenesis studies have suggested, but have not conclusively shown, that oxidative modification of Trp of apoA-I impairs many atheroprotective properties of this lipoprotein. Herein, we used genetic code expansion technology with an engineered tryptophanyl tRNA-synthetase (Trp-RS):suppressor tRNA pair to insert the noncanonical amino acid 5-hydroxytryptophan (5-OHTrp) at position 72 in recombinant human apoA-I and confirmed site-specific incorporation utilizing MS. In functional characterization studies, 5-OHTrp apoA-I (compared with WT apoA-I) exhibited reduced ABC subfamily A member 1 (ABCA1)-dependent cholesterol acceptor activity (41.73 ± 6.57% inhibition; < 0.01). Additionally, 5-OHTrp apoA-I displayed increased activation and stabilization of paraoxonase 1 (PON1) activity (μmol/min/mg) when compared with WT apoA-I and comparable PON1 activation/stabilization compared with reconstituted HDL (WT apoA-I, 1.92 ± 0.04; 5-OHTrp apoA-I, 2.35 ± 0.0; and HDL, 2.33 ± 0.1; < 0.001, < 0.001, and < 0.001, respectively). Following injection into apoA-I-deficient mice, 5-OHTrp apoA-I reached plasma levels comparable with those of native apoA-I yet exhibited significantly reduced (48%; < 0.01) lipidation and evidence of HDL biogenesis. Collectively, these findings unequivocally reveal that site-specific oxidative modification of apoA-I via 5-OHTrp at Trp impairs cholesterol efflux and the rate-limiting step of HDL biogenesis both and .
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http://dx.doi.org/10.1074/jbc.RA119.012092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152772PMC
April 2020

Non-Linear Relationship between Anti-Apolipoprotein A-1 IgGs and Cardiovascular Outcomes in Patients with Acute Coronary Syndromes.

J Clin Med 2019 Jul 9;8(7). Epub 2019 Jul 9.

Division of Cardiology, Cardiology Center, Geneva University Hospital, 1205 Geneva Switzerland.

Autoantibodies against apolipoprotein A-I (anti-apoA-I IgGs) are prevalent in atherosclerosis-related conditions. It remains elusive whether they improve the prognostic accuracy of the Global Registry of Acute Coronary Events (GRACE) score 2.0 (GS) in acute coronary syndromes (ACS). In this prospective multicenter registry, 1713 ACS patients were included and followed for 1 year. The primary endpoint (major adverse cardiovascular events (MACE)) was defined as the composite of myocardial infarction, stroke (including transient ischemic attack), or cardiovascular (CV) death with individual events independently adjudicated. Plasma levels of anti-apoA-I IgGs upon study inclusion were assessed using ELISA. The association between anti-apoA-I IgGs and incident MACE was assessed using Cox models with splines and C-statistics. One-year MACE incidence was 8.4% (144/1713). Anti-apoA-I IgG levels were associated with MACE with a non-linear relationship ( = 0.01), which remained unchanged after adjusting for the GS ( = 0.04). The hazard increased progressively across the two first anti-apoA-I IgG quartiles before decreasing thereafter. Anti-apoA-I IgGs marginally improved the prognostic accuracy of the GS (c-statistics increased from 0.68 to 0.70). In this multicenter study, anti-apoA-I IgGs were predictive of incident MACE in ACS independently of the GS but in a nonlinear manner. The practical implications of these findings remain to be defined.
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http://dx.doi.org/10.3390/jcm8071002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679072PMC
July 2019

Trimethyllysine, a trimethylamine N-oxide precursor, provides near- and long-term prognostic value in patients presenting with acute coronary syndromes.

Eur Heart J 2019 08;40(32):2700-2709

Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA.

Aims: Trimethyllysine (TML) serves as a nutrient precursor of the gut microbiota-derived metabolite trimethylamine N-oxide (TMAO) and is associated with incident cardiovascular (CV) events in stable subjects. We examined the relationship between plasma TML levels and incident CV events in patients presenting with acute coronary syndromes (ACS).

Methods And Results: Plasma levels of TML were quantified in two independent cohorts using mass spectrometry, and its relationship with CV events was investigated. In a Cleveland Cohort (N = 530), comprised of patients presenting to the emergency department with chest pain and suspected ACS, TML was associated with major adverse cardiac events (MACE, myocardial infarction, stroke, need for revascularization, or all-cause mortality) over both 30 days [3rd tertile (T3), adjusted odds ratio (OR) 1.77, 95% confidence interval (CI) 1.04-3.01; P < 0.05] and 6 months (T3, adjusted OR 1.95, 95% CI 1.15-3.32; P < 0.05) of follow-up independent of traditional CV risk factors and indices of renal function. Elevated TML levels were also associated with incident long-term (7-year) all-cause mortality [T3, adjusted hazard ratio (HR) 2.52, 95% CI 1.50-4.24; P < 0.001], and MACE even amongst patients persistently negative for cardiac Troponin T at presentation (e.g. 30-day MACE, T3, adjusted OR 4.49, 95% CI 2.06-9.79; P < 0.001). Trimethyllysine in combination with TMAO showed additive significance for near- and long-term CV events, including patients with 'negative' high-sensitivity Troponin T levels. In a multicentre Swiss Cohort (N = 1683) comprised of ACS patients, similar associations between TML and incident 1-year adverse cardiac risks were observed (e.g. mortality, adjusted T3 HR 2.74, 95% CI 1.28-5.85; P < 0.05; and MACE, adjusted T3 HR 1.55, 95% CI 1.04-2.31; P < 0.05).

Conclusion: Plasma TML levels, alone and together with TMAO, are associated with both near- and long-term CV events in patients with chest pain and ACS.
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http://dx.doi.org/10.1093/eurheartj/ehz259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7963132PMC
August 2019

Impact of chronic dietary red meat, white meat, or non-meat protein on trimethylamine N-oxide metabolism and renal excretion in healthy men and women.

Eur Heart J 2019 02;40(7):583-594

Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Euclid Ave, Cleveland, OH, USA.

Aims: Carnitine and choline are major nutrient precursors for gut microbiota-dependent generation of the atherogenic metabolite, trimethylamine N-oxide (TMAO). We performed randomized-controlled dietary intervention studies to explore the impact of chronic dietary patterns on TMAO levels, metabolism and renal excretion.

Methods And Results: Volunteers (N = 113) were enrolled in a randomized 2-arm (high- or low-saturated fat) crossover design study. Within each arm, three 4-week isocaloric diets (with washout period between each) were evaluated (all meals prepared in metabolic kitchen with 25% calories from protein) to examine the effects of red meat, white meat, or non-meat protein on TMAO metabolism. Trimethylamine N-oxide and other trimethylamine (TMA) related metabolites were quantified at the end of each diet period. A random subset (N = 13) of subjects also participated in heavy isotope tracer studies. Chronic red meat, but not white meat or non-meat ingestion, increased plasma and urine TMAO (each >two-fold; P < 0.0001). Red meat ingestion also significantly reduced fractional renal excretion of TMAO (P < 0.05), but conversely, increased fractional renal excretion of carnitine, and two alternative gut microbiota-generated metabolites of carnitine, γ-butyrobetaine, and crotonobetaine (P < 0.05). Oral isotope challenge revealed red meat or white meat (vs. non-meat) increased TMA and TMAO production from carnitine (P < 0.05 each) but not choline. Dietary-saturated fat failed to impact TMAO or its metabolites.

Conclusion: Chronic dietary red meat increases systemic TMAO levels through: (i) enhanced dietary precursors; (ii) increased microbial TMA/TMAO production from carnitine, but not choline; and (iii) reduced renal TMAO excretion. Discontinuation of dietary red meat reduces plasma TMAO within 4 weeks.
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http://dx.doi.org/10.1093/eurheartj/ehy799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374688PMC
February 2019

l-Carnitine in omnivorous diets induces an atherogenic gut microbial pathway in humans.

J Clin Invest 2019 01 10;129(1):373-387. Epub 2018 Dec 10.

Department of Cellular and Molecular Medicine, Lerner Research Institute.

Background: l-Carnitine, an abundant nutrient in red meat, accelerates atherosclerosis in mice via gut microbiota-dependent formation of trimethylamine (TMA) and trimethylamine N-oxide (TMAO) via a multistep pathway involving an atherogenic intermediate, γ-butyrobetaine (γBB). The contribution of γBB in gut microbiota-dependent l-carnitine metabolism in humans is unknown.

Methods: Omnivores and vegans/vegetarians ingested deuterium-labeled l-carnitine (d3-l-carnitine) or γBB (d9-γBB), and both plasma metabolites and fecal polymicrobial transformations were examined at baseline, following oral antibiotics, or following chronic (≥2 months) l-carnitine supplementation. Human fecal commensals capable of performing each step of the l-carnitine→γBB→TMA transformation were identified.

Results: Studies with oral d3-l-carnitine or d9-γBB before versus after antibiotic exposure revealed gut microbiota contribution to the initial 2 steps in a metaorganismal l-carnitine→γBB→TMA→TMAO pathway in subjects. Moreover, a striking increase in d3-TMAO generation was observed in omnivores over vegans/vegetarians (>20-fold; P = 0.001) following oral d3-l-carnitine ingestion, whereas fasting endogenous plasma l-carnitine and γBB levels were similar in vegans/vegetarians (n = 32) versus omnivores (n = 40). Fecal metabolic transformation studies, and oral isotope tracer studies before versus after chronic l-carnitine supplementation, revealed that omnivores and vegans/vegetarians alike rapidly converted carnitine to γBB, whereas the second gut microbial transformation, γBB→TMA, was diet inducible (l-carnitine, omnivorous). Extensive anaerobic subculturing of human feces identified no single commensal capable of l-carnitine→TMA transformation, multiple community members that converted l-carnitine to γBB, and only 1 Clostridiales bacterium, Emergencia timonensis, that converted γBB to TMA. In coculture, E. timonensis promoted the complete l-carnitine→TMA transformation.

Conclusion: In humans, dietary l-carnitine is converted into the atherosclerosis- and thrombosis-promoting metabolite TMAO via 2 sequential gut microbiota-dependent transformations: (a) initial rapid generation of the atherogenic intermediate γBB, followed by (b) transformation into TMA via low-abundance microbiota in omnivores, and to a markedly lower extent, in vegans/vegetarians. Gut microbiota γBB→TMA/TMAO transformation is induced by omnivorous dietary patterns and chronic l-carnitine exposure.

Trial Registration: ClinicalTrials.gov NCT01731236.

Funding: NIH and Office of Dietary Supplements grants HL103866, HL126827, and DK106000, and the Leducq Foundation.
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http://dx.doi.org/10.1172/JCI94601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307959PMC
January 2019

Loss of HDAC6 alters gut microbiota and worsens obesity.

FASEB J 2019 01 13;33(1):1098-1109. Epub 2018 Aug 13.

Department of Medicine New York University School of Medicine (NYUSM), New York, New York, USA.

Alterations in gut microbiota are known to affect intestinal inflammation and obesity. Antibiotic treatment can affect weight gain by elimination of histone deacetylase (HDAC) inhibitor-producing microbes, which are anti-inflammatory by augmenting regulatory T (T) cells. We asked whether mice that lack HDAC6 and have potent suppressive T cells are protected from microbiota-induced accelerated weight gain. We crossed wild-type and HDAC6-deficient mice and subjected the offspring to perinatal penicillin, inducing weight gain via microbiota disturbance. We observed that male HDAC6-deficient mice were not protected and developed profoundly accelerated weight gain. The antibiotic-exposed HDAC6-deficient mice showed a mixed immune phenotype with increased CD4 and CD8 T-cell activation yet maintained the enhanced T cell-suppressive function phenotype characteristic of HDAC6-deficient mice. 16S rRNA sequencing of mouse fecal samples reveals that their microbiota diverged with time, with HDAC6 deletion altering microbiome composition. On a high-fat diet, HDAC6-deficient mice were depleted in representatives of the S24-7 family and Lactobacillus but enriched with Bacteroides and Parabacteroides; these changes are associated with obesity. Our findings further our understanding of the influence of HDACs on microbiome composition and are important for the development of HDAC6 inhibitors in the treatment of human diseases.-Lieber, A. D., Beier, U. H., Xiao, H., Wilkins, B. J., Jiao, J., Li, X. S., Schugar, R. C., Strauch, C. M., Wang, Z., Brown, J. M., Hazen, S. L., Bokulich, N. A., Ruggles, K. V., Akimova, T., Hancock, W. W., Blaser, M. J. Loss of HDAC6 alters gut microbiota and worsens obesity.
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http://dx.doi.org/10.1096/fj.201701586RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355060PMC
January 2019

Gut Microbiota-Dependent Trimethylamine N-Oxide Predicts Risk of Cardiovascular Events in Patients With Stroke and Is Related to Proinflammatory Monocytes.

Arterioscler Thromb Vasc Biol 2018 09;38(9):2225-2235

From the Department of Cardiology (A.H., D.S., F.Z., N.K., U.L.).

Objective- Gut microbiota-dependent metabolites, in particular trimethylamine N-oxide (TMAO), have recently been reported to promote atherosclerosis and thrombosis. Here, we examined for the first time the relation of TMAO and the risk of incident cardiovascular events in patients with recent first-ever ischemic stroke in 2 independent prospective cohorts. Moreover, the link between TMAO and proinflammatory monocytes as a potential contributing factor for cardiovascular risk in stroke patients was studied. Approach and Results- In a first study (n=78), higher TMAO plasma levels were linked with an increased risk of incident cardiovascular events including myocardial infarction, recurrent stroke, and cardiovascular death (fourth quartile versus first quartile; hazard ratio, 2.31; 95% CI, 1.25-4.23; P<0.01). In the second independent validation cohort (n=593), high TMAO levels again heralded marked increased risk of adverse cardiovascular events (fourth quartile versus first quartile; hazard ratio, 5.0; 95% CI, 1.7-14.8; P<0.01), and also after adjustments for cardiovascular risk factors including hypertension, diabetes mellitus, LDL (low-density lipoprotein) cholesterol, and estimated glomerular filtration rate (hazard ratio, 3.3; 95% CI, 1.2-10.9; P=0.04). A significant correlation was also found between TMAO levels and percentage of proinflammatory intermediate CD14CD16 monocytes ( r=0.70; P<0.01). Moreover, in mice fed a diet enriched with choline to increase TMAO synthesis, levels of proinflammatory murine Ly6C monocytes were higher than in the chow-fed control group (choline: 9.2±0.5×10 per mL versus control: 6.5±0.5×10 per mL; P<0.01). This increase was abolished in mice with depleted gut microbiota (choline+antibiotics: 5.4±0.7×10 per mL; P<0.001 versus choline). Conclusions- The present study demonstrates for the first time a graded relation between TMAO levels and the risk of subsequent cardiovascular events in patients with recent prior ischemic stroke. Our data support the notion that TMAO-related increase of proinflammatory monocytes may add to elevated cardiovascular risk of patients with increased TMAO levels.
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http://dx.doi.org/10.1161/ATVBAHA.118.311023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202215PMC
September 2018

Untargeted metabolomics identifies trimethyllysine, a TMAO-producing nutrient precursor, as a predictor of incident cardiovascular disease risk.

JCI Insight 2018 03 22;3(6). Epub 2018 Mar 22.

Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.

Using an untargeted metabolomics approach in initial (N = 99 subjects) and replication cohorts (N = 1,162), we discovered and structurally identified a plasma metabolite associated with cardiovascular disease (CVD) risks, N6,N6,N6-trimethyl-L-lysine (trimethyllysine, TML). Stable-isotope-dilution tandem mass spectrometry analyses of an independent validation cohort (N = 2,140) confirmed TML levels are independently associated with incident (3-year) major adverse cardiovascular event risks (hazards ratio [HR], 2.4; 95% CI, 1.7-3.4) and incident (5-year) mortality risk (HR, 2.9; 95% CI, 2.0-4.2). Genome-wide association studies identified several suggestive loci for TML levels, but none reached genome-wide significance; and d9(trimethyl)-TML isotope tracer studies confirmed TML can serve as a nutrient precursor for gut microbiota-dependent generation of trimethylamine (TMA) and the atherogenic metabolite trimethylamine N-oxide (TMAO). Although TML was shown to be abundant in both plant- and animal-derived foods, mouse and human fecal cultures (omnivores and vegans) showed slow conversion of TML to TMA. Furthermore, unlike chronic dietary choline, TML supplementation in mice failed to elevate plasma TMAO or heighten thrombosis potential in vivo. Thus, TML is identified as a strong predictor of incident CVD risks in subjects and to serve as a dietary precursor for gut microbiota-dependent generation of TMAO; however, TML does not appear to be a major microbial source for TMAO generation in vivo.
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http://dx.doi.org/10.1172/jci.insight.99096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5926943PMC
March 2018

The TMAO-Producing Enzyme Flavin-Containing Monooxygenase 3 Regulates Obesity and the Beiging of White Adipose Tissue.

Cell Rep 2017 06;19(12):2451-2461

Department of Cellular and Molecular Medicine, Cleveland Clinic, Cleveland, OH 44195, USA; Center for Microbiome and Human Health, Cleveland Clinic, Cleveland, OH 44195, USA. Electronic address:

Emerging evidence suggests that microbes resident in the human intestine represent a key environmental factor contributing to obesity-associated disorders. Here, we demonstrate that the gut microbiota-initiated trimethylamine N-oxide (TMAO)-generating pathway is linked to obesity and energy metabolism. In multiple clinical cohorts, systemic levels of TMAO were observed to strongly associate with type 2 diabetes. In addition, circulating TMAO levels were associated with obesity traits in the different inbred strains represented in the Hybrid Mouse Diversity Panel. Further, antisense oligonucleotide-mediated knockdown or genetic deletion of the TMAO-producing enzyme flavin-containing monooxygenase 3 (FMO3) conferred protection against obesity in mice. Complimentary mouse and human studies indicate a negative regulatory role for FMO3 in the beiging of white adipose tissue. Collectively, our studies reveal a link between the TMAO-producing enzyme FMO3 and obesity and the beiging of white adipose tissue.
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http://dx.doi.org/10.1016/j.celrep.2017.05.077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5672822PMC
June 2017

NMR quantification of trimethylamine-N-oxide in human serum and plasma in the clinical laboratory setting.

Clin Biochem 2017 Nov 15;50(16-17):947-955. Epub 2017 Jun 15.

LipoScience, Laboratory Corporation of America® Holdings, Raleigh, NC, United States. Electronic address:

Background And Objectives: Trimethylamine-N-oxide (TMAO) produced by gut microbiota metabolism of dietary choline and carnitine has been shown to be associated with increased risk of cardiovascular disease (CVD) and to provide incremental clinical prognostic utility beyond traditional risk factors for assessing a patient's CVD risk. The aim of this study was to develop an automated nuclear magnetic resonance (NMR) spectroscopy assay for quantification of TMAO concentration in serum and plasma using a high-throughput NMR clinical analyzer.

Methods: Key steps in assay development included: (i) shifting the TMAO analyte peak to a less crowded region of the spectrum with a pH buffer/reagent, (ii) attenuating the broad protein background signal in the spectrum and (iii) using a non-negative least squares algorithm for peak deconvolution. Assay performance was evaluated according to Clinical and Laboratory Standards Institute guidelines. A method comparison study was performed to compare TMAO concentrations quantified by NMR and mass spectrometry (MS).

Results: The within-run and within-lab imprecision ranged from 4.3 to 14.5%. Under the acquisition method employed, the NMR assay had a limit of blank, detection and quantitation of 1.6, 3.0 and 3.3μM, respectively. Linearity was demonstrated within the reportable range of 3.3 to 3000μM. TMAO measurements using the NMR assay, which involves minimal sample preparation, compared well with values obtained with the MS-based assay (R=0.98).

Conclusions: The NMR based assay provides a simple and accurate measurement of circulating TMAO levels amenable to the high-throughput demands of the clinical chemistry laboratory. Moreover, assay performance enables the levels of TMAO to be quantified in serum or plasma at clinically actionable concentrations for the assessment of cardiovascular disease risks and individualized dietary monitoring.
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http://dx.doi.org/10.1016/j.clinbiochem.2017.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5632584PMC
November 2017

Gut microbiota-dependent trimethylamine N-oxide in acute coronary syndromes: a prognostic marker for incident cardiovascular events beyond traditional risk factors.

Eur Heart J 2017 Mar;38(11):814-824

Department of Cardiology, University Heart Center, University Hospital Zurich, Switzerland.

Aims: Systemic levels of trimethylamine N-oxide (TMAO), a pro-atherogenic and pro-thrombotic metabolite produced from gut microbiota metabolism of dietary trimethylamine (TMA)-containing nutrients such as choline or carnitine, predict incident cardiovascular event risks in stable primary and secondary prevention subjects. However, the prognostic value of TMAO in the setting of acute coronary syndromes (ACS) remains unknown.

Methods And Results: We investigated the relationship of TMAO levels with incident cardiovascular risks among sequential patients presenting with ACS in two independent cohorts. In the Cleveland Cohort, comprised of sequential subjects (n = 530) presenting to the Emergency Department (ED) with chest pain of suspected cardiac origin, an elevated plasma TMAO level at presentation was independently associated with risk of major adverse cardiac events (MACE, including myocardial infarction, stroke, need for revascularization, or death) over the ensuing 30-day (4th quartile (Q4) adjusted odds ratio (OR) 6.30, 95% confidence interval (CI), 1.89-21.0, P < 0.01) and 6-month (Q4 adjusted OR 5.65, 95%CI, 1.91-16.7; P < 0.01) intervals. TMAO levels were also a significant predictor of the long term (7-year) mortality (Q4 adjusted HR 1.81, 95%CI, 1.04-3.15; P < 0.05). Interestingly, TMAO level at initial presentation predicted risk of incident MACE over the near-term (30 days and 6 months) even among subjects who were initially negative for troponin T (< 0.1 ng/mL) (30 days, Q4 adjusted OR 5.83, 95%CI, 1.79-19.03; P < 0.01). The prognostic value of TMAO was also assessed in an independent multicentre Swiss Cohort of ACS patients (n = 1683) who underwent coronary angiography. Trimethylamine N-oxide again predicted enhanced MACE risk (1-year) (adjusted Q4 hazard ratios: 1.57, 95% CI, 1.03-2.41; P <0.05).

Conclusion: Plasma TMAO levels among patients presenting with chest pain predict both near- and long-term risks of incident cardiovascular events, and may thus provide clinical utility in risk stratification among subjects presenting with suspected ACS.
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http://dx.doi.org/10.1093/eurheartj/ehw582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837488PMC
March 2017

Serum Trimethylamine N-oxide, Carnitine, Choline, and Betaine in Relation to Colorectal Cancer Risk in the Alpha Tocopherol, Beta Carotene Cancer Prevention Study.

Cancer Epidemiol Biomarkers Prev 2017 06 11;26(6):945-952. Epub 2017 Jan 11.

Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, Maryland.

Trimethylamine N-oxide (TMAO), a choline-derived metabolite produced by gut microbiota, and its biomarker precursors have not been adequately evaluated in relation to colorectal cancer risk. We investigated the relationship between serum concentrations of TMAO and its biomarker precursors (choline, carnitine, and betaine) and incident colorectal cancer risk in a nested case-control study of male smokers in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study. We measured biomarker concentrations in baseline fasting serum samples from 644 incident colorectal cancer cases and 644 controls using LC/MS-MS. Logistic regression models estimated the ORs and 95% confidence interval (CI) for colorectal cancer by quartile (Q) of serum TMAO, choline, carnitine, and betaine concentrations. Men with higher serum choline at ATBC baseline had approximately 3-fold greater risk of developing colorectal cancer over the ensuing (median ± IQR) 14 ± 10 years (in fully adjusted models, Q4 vs. Q1, OR, 3.22; 95% CI, 2.24-4.61; < 0.0001). The prognostic value of serum choline for prediction of incident colorectal cancer was similarly robust for proximal, distal, and rectal colon cancers (all < 0.0001). The association between serum TMAO, carnitine, or betaine and colorectal cancer risk was not statistically significant ( = 0.25, 0.71, and 0.61, respectively). Higher serum choline concentration (but not TMAO, carnitine, or betaine) was associated with increased risk of colorectal cancer. Serum choline levels showed strong prognostic value for prediction of incident colorectal cancer risk across all anatomical subsites, suggesting a role of altered choline metabolism in colorectal cancer pathogenesis. .
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http://dx.doi.org/10.1158/1055-9965.EPI-16-0948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608021PMC
June 2017

Increased Trimethylamine N-Oxide Portends High Mortality Risk Independent of Glycemic Control in Patients with Type 2 Diabetes Mellitus.

Clin Chem 2017 Jan 18;63(1):297-306. Epub 2016 Nov 18.

Center for Cardiovascular Diagnostics & Prevention, Department of Cellular & Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH;

Background: Recent studies show a mechanistic link between intestinal microbial metabolism of dietary phosphatidylcholine and coronary artery disease pathogenesis. Concentrations of a proatherogenic gut microbe-generated metabolite, trimethylamine N-oxide (TMAO), predict increased incident cardiovascular disease risks in multiple cohorts. TMAO concentrations are increased in patients with type 2 diabetes mellitus (T2DM), but their prognostic value and relation to glycemic control are unclear.

Methods: We examined the relationship between fasting TMAO and 2 of its nutrient precursors, choline and betaine, vs 3-year major adverse cardiac events and 5-year mortality in 1216 stable patients with T2DM who underwent elective diagnostic coronary angiography.

Results: TMAO [4.4 μmol/L (interquartile range 2.8-7.7 μmol/L) vs 3.6 (2.3-5.7 μmol/L); P < 0.001] and choline concentrations were higher in individuals with T2DM vs healthy controls. Within T2DM patients, higher plasma TMAO was associated with a significant 3.0-fold increased 3-year major adverse cardiac event risk (P < 0.001) and a 3.6-fold increased 5-year mortality risk (P < 0.001). Following adjustments for traditional risk factors and high-sensitivity C-reactive protein, glycohemoglobin, and estimated glomerular filtration rate, increased TMAO concentrations remained predictive of both major adverse cardiac events and mortality risks in T2DM patients [e.g., quartiles 4 vs 1, hazard ratio 2.05 (95% CI, 1.31-3.20), P < 0.001; and 2.07 (95% CI, 1.37-3.14), P < 0.001, respectively].

Conclusions: Fasting plasma concentrations of the proatherogenic gut microbe-generated metabolite TMAO are higher in diabetic patients and portend higher major adverse cardiac events and mortality risks independent of traditional risk factors, renal function, and relationship to glycemic control.
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http://dx.doi.org/10.1373/clinchem.2016.263640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5659115PMC
January 2017

Intestinal Microbiota-Generated Metabolite Trimethylamine-N-Oxide and 5-Year Mortality Risk in Stable Coronary Artery Disease: The Contributory Role of Intestinal Microbiota in a COURAGE-Like Patient Cohort.

J Am Heart Assoc 2016 06 10;5(6). Epub 2016 Jun 10.

Department of Cardiovascular Medicine, Heart and Vascular Institute, Cleveland Clinic, Cleveland, OH Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH

Background: Trimethylamine-N-oxide (TMAO), a metabolite derived from gut microbes and dietary phosphatidylcholine, is linked to both coronary artery disease pathogenesis and increased cardiovascular risks. The ability of plasma TMAO to predict 5-year mortality risk in patients with stable coronary artery disease has not been reported. This study examined the clinical prognostic value of TMAO in patients with stable coronary artery disease who met eligibility criteria for a patient cohort similar to that of the Clinical Outcomes Utilizing Revascularization and Aggressive Drug Evaluation (COURAGE) trial.

Methods And Results: We examined the relationship between fasting plasma TMAO and all-cause mortality over 5-year follow-up in sequential patients with stable coronary artery disease (n=2235) who underwent elective coronary angiography. We identified the COURAGE-like patient cohort as patients who had evidence of significant coronary artery stenosis and who were managed with optimal medical treatment. Higher plasma TMAO levels were associated with a 4-fold increased mortality risk. Following adjustments for traditional risk factors, high-sensitivity C-reactive protein, and estimated glomerular filtration rate, elevated TMAO levels remained predictive of 5-year all-cause mortality risk (quartile 4 versus 1, adjusted hazard ratio 1.95, 95% CI 1.33-2.86; P=0.003). TMAO remained predictive of incident mortality risk following cardiorenal and inflammatory biomarker adjustments to the model (adjusted hazard ratio 1.71, 95% CI 1.11-2.61; P=0.0138) and provided significant incremental prognostic value for all-cause mortality (net reclassification index 42.37%, P<0.001; improvement in area under receiver operator characteristic curve 70.6-73.76%, P<0.001).

Conclusions: Elevated plasma TMAO levels portended higher long-term mortality risk among patients with stable coronary artery disease managed with optimal medical treatment.
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http://dx.doi.org/10.1161/JAHA.115.002816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4937244PMC
June 2016

Plasma Trimethylamine N-Oxide, a Gut Microbe-Generated Phosphatidylcholine Metabolite, Is Associated With Atherosclerotic Burden.

J Am Coll Cardiol 2016 06;67(22):2620-8

Department of Cardiovascular Medicine, Heart and Vascular Institute, Cleveland Clinic, Cleveland, Ohio; Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio. Electronic address:

Background: Trimethylamine N-oxide (TMAO), a gut microbiota metabolite from dietary phosphatidylcholine, has mechanistic links to atherosclerotic coronary artery disease (CAD) pathogenesis and is associated with adverse outcomes.

Objectives: This study sought to examine the relationship between plasma TMAO levels and the complexity and burden of CAD and degree of subclinical myonecrosis.

Methods: We studied 353 consecutive stable patients with evidence of atherosclerotic CAD detected by elective coronary angiography between 2012 and 2014. Their high-sensitivity cardiac troponin T (hs-cTnT) levels were measured. SYNTAX (Synergy Between Percutaneous Coronary Intervention With Taxus and Cardiac Surgery) scores and lesion characteristics were used to quantify atherosclerotic burden. Fasting plasma TMAO was measured by mass spectrometry.

Results: In this prospective cohort study, the median TMAO level was 5.5 μM (interquartile range [IQR]: 3.4 to 9.8 μM), the median SYNTAX score was 11.0 (IQR: 4.0 to 18.5), and 289 (81.9%), 40 (11.3%), and 24 (6.8%) patients had low (0 to 22), intermediate (23 to 32), and high (≥33) SYNTAX scores, respectively. Plasma TMAO levels correlated (all p < 0.0001) with the SYNTAX score (r = 0.61), SYNTAX score II (r = 0.62), and hs-cTnT (r = 0.29). Adjusting for traditional risk factors, body mass index, medications, lesion characteristic, renal function, and high-sensitivity C-reactive protein, elevated TMAO levels remained independently associated with a higher SYNTAX score (odds ratio [OR]: 4.82; p < 0.0001), SYNTAX score II (OR: 1.88; p = 0.0001), but were not associated with subclinical myonecrosis (OR: 1.14; p = 0.3147). Elevated TMAO level was an independent predictor of the presence of diffuse lesions, even after adjustments for traditional risk factors and for hs-cTnT (OR: 2.05; 95% confidence interval: 1.45 to 2.90; p = 0.0001).

Conclusions: Fasting plasma TMAO levels are an independent predictor of a high atherosclerotic burden in patients with CAD.
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http://dx.doi.org/10.1016/j.jacc.2016.03.546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4893167PMC
June 2016

Elevated Plasma Marinobufagenin, An Endogenous Cardiotonic Steroid, Is Associated With Right Ventricular Dysfunction and Nitrative Stress in Heart Failure.

Circ Heart Fail 2015 Nov 14;8(6):1068-76. Epub 2015 Aug 14.

From the Department of Cellular and Molecular Medicine (D.J.K., K.S., B.S., X.S.L., A.G., Y.W., M.F., A.G., C.M.M., K.W., A.B., W.H.W.T.), Center for Cardiovascular Diagnostics and Prevention, Lerner Research Institute (W.H.W.T.), Department of Nephrology and Hypertension, Glickman Urological and Kidney Institute (D.J.K.), and Department of Cardiovascular Medicine, Heart and Vascular Institute (W.H.W.T.), Cleveland Clinic, Cleveland, OH; and Laboratory of Cardiovascular Science, Hypertension Unit, National Institute on Aging, National Institutes of Health, Baltimore, MD (O.F., A.Y.B.).

Background: Plasma levels of cardiotonic steroids are elevated in volume-expanded states, such as chronic kidney disease, but the role of these natriuretic hormones in subjects with heart failure (HF) is unclear. We sought to determine the prognostic role of the cardiotonic steroids marinobufagenin (MBG) in HF, particularly in relation to long-term outcomes.

Methods And Results: We first measured plasma MBG levels and performed comprehensive clinical, laboratory, and echocardiographic assessment in 245 patients with HF. All-cause mortality, cardiac transplantation, and HF hospitalization were tracked for 5 years. In our study cohort, median (interquartile range) MBG was 583 (383-812) pM. Higher MBG was associated with higher myeloperoxidase (r=0.42, P<0.0001), B-type natriuretic peptide (r=0.25, P=0.001), and asymmetrical dimethylarginine (r=0.32, P<0.001). Elevated levels of MBG were associated with measures of worse right ventricular function (RV s', r=-0.39, P<0.0001) and predicted increased risk of adverse clinical outcomes (MBG≥574 pmol/L: hazard ratio 1.58 [1.10-2.31], P=0.014) even after adjustment for age, sex, diabetes mellitus, and ischemic pathogenesis. In mice, a left anterior descending coronary artery ligation model of HF lead to increases in MBG, whereas infusion of MBG into mice for 4 weeks lead to significant increases in myeloperoxidase, asymmetrical dimethylarginine, and cardiac fibrosis.

Conclusions: In the setting of HF, elevated plasma levels of MBG are associated with right ventricular dysfunction and predict worse long-term clinical outcomes in multivariable models adjusting for established clinical and biochemical risk factors. Infusion of MBG seems to directly contribute to increased nitrative stress and cardiac fibrosis.
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http://dx.doi.org/10.1161/CIRCHEARTFAILURE.114.001976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4651837PMC
November 2015

Gut microbiota-dependent trimethylamine N-oxide (TMAO) pathway contributes to both development of renal insufficiency and mortality risk in chronic kidney disease.

Circ Res 2015 Jan 5;116(3):448-55. Epub 2014 Nov 5.

From the Department for Cellular and Molecular Medicine, Lerner Research Institute (W.H.W.T., Z.W., D.J.K., J.A.B., B.A.-B., X.S.L., B.S.L., S.L.H.); Department of Cardiovascular Medicine, Heart and Vascular Institute, Cleveland Clinic Foundation, OH (W.H.W.T., S.L.H.); and Department of Mathematics, Cleveland State University, OH (Y.W.).

Rationale: Trimethylamine-N-oxide (TMAO), a gut microbial-dependent metabolite of dietary choline, phosphatidylcholine (lecithin), and l-carnitine, is elevated in chronic kidney diseases (CKD) and associated with coronary artery disease pathogenesis.

Objective: To both investigate the clinical prognostic value of TMAO in subjects with versus without CKD, and test the hypothesis that TMAO plays a direct contributory role in the development and progression of renal dysfunction.

Methods And Results: We first examined the relationship between fasting plasma TMAO and all-cause mortality over 5-year follow-up in 521 stable subjects with CKD (estimated glomerular filtration rate, <60 mL/min per 1.73 m(2)). Median TMAO level among CKD subjects was 7.9 μmol/L (interquartile range, 5.2-12.4 μmol/L), which was markedly higher (P<0.001) than in non-CKD subjects (n=3166). Within CKD subjects, higher (fourth versus first quartile) plasma TMAO level was associated with a 2.8-fold increased mortality risk. After adjustments for traditional risk factors, high-sensitivity C-reactive protein, estimated glomerular filtration rate, elevated TMAO levels remained predictive of 5-year mortality risk (hazard ratio, 1.93; 95% confidence interval, 1.13-3.29; P<0.05). TMAO provided significant incremental prognostic value (net reclassification index, 17.26%; P<0.001 and differences in area under receiver operator characteristic curve, 63.26% versus 65.95%; P=0.036). Among non-CKD subjects, elevated TMAO levels portend poorer prognosis within cohorts of high and low cystatin C. In animal models, elevated dietary choline or TMAO directly led to progressive renal tubulointerstitial fibrosis and dysfunction.

Conclusions: Plasma TMAO levels are both elevated in patients with CKD and portend poorer long-term survival. Chronic dietary exposures that increase TMAO directly contributes to progressive renal fibrosis and dysfunction in animal models.
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http://dx.doi.org/10.1161/CIRCRESAHA.116.305360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4312512PMC
January 2015