Background Trimethylamine‐N‐oxide (TMAO) is a small molecule derived from the metabolism of dietary nutrients by gut microbes and contributes to cardiovascular disease. Plasma TMAO increases following consumption of red meat. This metabolic change is thought to be partly because of the expansion of gut microbes able to use nutrients abundant in red meat. Methods and Results We used data from a randomized crossover study to estimate the degree to which TMAO can be estimated from fecal microbial composition. Healthy participants received a series of 3 diets that differed in protein source (red meat, white meat, and non‐meat), and fecal, plasma, and urine samples were collected following 4 weeks of exposure to each diet. TMAO was quantitated in plasma and urine, while shotgun metagenomic sequencing was performed on fecal DNA. While the cai gene cluster was weakly correlated with plasma TMAO (rho=0.17, P=0.0007), elastic net models of TMAO were not improved by abundances of bacterial genes known to contribute to TMAO synthesis. A global analysis of all taxonomic groups, genes, and gene families found no meaningful predictors of TMAO. We postulated that abundances of known genes related to TMAO production do not predict bacterial metabolism, and we measured choline‐ and carnitine‐trimethylamine lyase activity during fecal culture. Trimethylamine lyase genes were only weakly correlated with the activity of the enzymes they encode. Conclusions Fecal microbiome composition does not predict systemic TMAO because, in this case, gene copy number does not predict bacterial metabolic activity. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT01427855.

L-alpha glycerylphosphorylcholine (GPC), a nutritional supplement, has been demonstrated to improve neurological function. However, a new study suggests that GPC supplementation increases incident stroke risk thus its potential adverse effects warrant further investigation. Here we show that GPC promotes atherosclerosis in hyperlipidemic Apoe−/− mice. GPC can be metabolized to trimethylamine N-oxide, a pro-atherogenic agent, suggesting a potential molecular mechanism underlying the observed atherosclerosis progression. GPC supplementation shifted the gut microbial community structure, characterized by increased abundance of Parabacteroides, Ruminococcus, and Bacteroides and decreased abundance of Akkermansia, Lactobacillus, and Roseburia, as determined by 16S rRNA gene sequencing. These data are consistent with a reduction in fecal and cecal short chain fatty acids in GPC-fed mice. Additionally, we found that GPC supplementation led to an increased relative abundance of choline trimethylamine lyase (cutC)-encoding bacteria via qPCR. Interrogation of host inflammatory signaling showed that GPC supplementation increased expression of the proinflammatory effectors CXCL13 and TIMP-1 and activated NF-κB and MAPK signaling pathways in human coronary artery endothelial cells. Finally, targeted and untargeted metabolomic analysis of murine plasma revealed additional metabolites associated with GPC supplementation and atherosclerosis. In summary, our results show GPC promotes atherosclerosis through multiple mechanisms and that caution should be applied when using GPC as a nutritional supplement.

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(72). Site-specific mutagenesis studies have suggested, but have not conclusively shown, that oxidative modification of Trp(72) of apoA-I impairs many atheroprotective properties of this lipoprotein. Herein, we used genetic code expansion technology with an engineered Saccharomyces cerevisiae 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(72) apoA-I (compared with WT apoA-I) exhibited reduced ABC subfamily A member 1 (ABCA1)-dependent cholesterol acceptor activity in vitro (41.73 ? 6.57% inhibition; p < 0.01). Additionally, 5-OHTrp(72) apoA-I displayed increased activation and stabilization of paraoxonase 1 (PON1) activity (?mol/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(72) apoA-I, 2.35 ? 0.0; and HDL, 2.33 ? 0.1; p < 0.001, p < 0.001, and p < 0.001, respectively). Following injection into apoA-I?deficient mice, 5-OHTrp(72) apoA-I reached plasma levels comparable with those of native apoA-I yet exhibited significantly reduced (48%; p < 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(72) impairs cholesterol efflux and the rate-limiting step of HDL biogenesis both in vitro and in vivo.

L -Carnitine, a nutrient in red meat, accelerates atherosclerosis in a gut microbiota-dependent pathway by the formation of trimethylamine- N -oxide (TMAO). We now show that a novel compound, crotonobetaine (crotono), identified in a metabolomics study, is a product of L -carnitine gut microbial

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, gamma-butyrobetaine (gamma BB). The contribution of gamma BB in gut microbiota-dependent L-carnitine metabolism in humans is unknown. METHODS. Omnivores and vegans/vegetarians ingested deuterium-labeled L-carnitine (d(3)-L-carnitine) or gamma BB (d(9)-gamma BB), 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 ->gamma BB -> TMA transformation were identified. RESULTS. Studies with oral d(3)-L-carnitine or d(9)-gamma BB before versus after antibiotic exposure revealed gut microbiota contribution to the initial 2 steps in a metaorganismal L-carnitine ->gamma BB -> TMA -> TMAO pathway in subjects. Moreover, a striking increase in d(3)-TMAO generation was observed in omnivores over vegans/vegetarians (>20-fold; P = 0.001) following oral d(3)-L-carnitine ingestion, whereas fasting endogenous plasma L-carnitine and gamma BB 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 gamma BB, whereas the second gut microbial transformation, gamma BB -> 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 gamma BB, and only 1 Clostridiales bacterium, Emergencia timonensis, that converted gamma BB 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 gamma BB, followed by (b) transformation into TMA via low-abundance microbiota in omnivores, and to a markedly lower extent, in vegans/vegetarians. Gut microbiota gamma BB -> TMA/TMAO transformation is induced by omnivorous dietary patterns and chronic L-carnitine exposure.

Microcystins are potent hepatotoxins that have become a global health concern in recent years. Their actions in at-risk populations with pre-existing liver disease is unknown. We tested the hypothesis that the No Observed Adverse Effect Level (NOAEL) of Microcystin-LR (MC-LR) established in healthy mice would cause exacerbation of hepatic injury in a murine model (Leprdb/J) of Non-alcoholic Fatty Liver Disease (NAFLD). Ten-week-old male Leprdb/J mice were gavaged with 50 μg/kg, 100 μg/kg MC-LR or vehicle every 48 h for 4 weeks (n = 15–17 mice/group). Early mortality was observed in both the 50 μg/kg (1/17, 6%), and 100 μg/kg (3/17, 18%) MC-LR exposed mice. MC-LR exposure resulted in significant increases in circulating alkaline phosphatase levels, and histopathological markers of hepatic injury as well as significant upregulation of genes associated with hepatotoxicity, necrosis, nongenotoxic hepatocarcinogenicity and oxidative stress response. In addition, we observed exposure dependent changes in protein phosphorylation sites in pathways involved in inflammation, immune function, and response to oxidative stress. These results demonstrate that exposure to MC-LR at levels that are below the NOAEL established in healthy animals results in significant exacerbation of hepatic injury that is accompanied by genetic and phosphoproteomic dysregulation in key signaling pathways in the livers of NAFLD mice.

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.

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, gamma-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.

Trimethylamine N-oxide (TMAO) is a gut microbiota-derived metabolite that enhances both platelet responsiveness and in vivo thrombosis potential in animal models, and TMAO plasma levels predict incident atherothrombotic event risks in human clinical studies. TMAO is formed by gut microbe-dependent metabolism of trimethylamine (TMA) moiety-containing nutrients, which are abundant in a Western diet. Here, using a mechanism-based inhibitor approach targeting a major microbial TMA-generating enzyme pair, CutC and CutD (CutC/D), we developed inhibitors that are potent, time-dependent, and irreversible and that do not affect commensal viability. In animal models, a single oral dose of a CutC/D inhibitor significantly reduced plasma TMAO levels for up to 3 d and rescued diet-induced enhanced platelet responsiveness and thrombus formation, without observable toxicity or increased bleeding risk. The inhibitor selectively accumulated within intestinal microbes to millimolar levels, a concentration over 1-million-fold higher than needed for a therapeutic effect. These studies reveal that mechanism-based inhibition of gut microbial TMA and TMAO production reduces thrombosis potential, a critical adverse complication in heart disease. They also offer a generalizable approach for the selective nonlethal targeting of gut microbial enzymes linked to host disease limiting systemic exposure of the inhibitor in the host.

BackgroundIntestinal microbiota have been found to be linked to cardiovascular disease via conversion of the dietary compounds choline and carnitine to the atherogenic metabolite TMAO (trimethylamine‐N‐oxide). Specifically, a vegan diet was associated with decreased plasma TMAO levels and nearly absent TMAO production on carnitine challenge. Methods and ResultsWe performed a double‐blind randomized controlled pilot study in which 20 male metabolic syndrome patients were randomized to single lean vegan‐donor or autologous fecal microbiota transplantation. At baseline and 2 weeks thereafter, we determined the ability to produce TMAO from d6‐choline and d3‐carnitine (eg, labeled and unlabeled TMAO in plasma and 24‐hour urine after oral ingestion of 250 mg of both isotope‐labeled precursor nutrients), and fecal samples were collected for analysis of microbiota composition. 18F‐fluorodeoxyglucose positron emission tomography/computed tomography scans of the abdominal aorta, as well as ex vivo peripheral blood mononuclear cell cytokine production assays, were performed. At baseline, fecal microbiota composition differed significantly between vegans and metabolic syndrome patients. With vegan‐donor fecal microbiota transplantation, intestinal microbiota composition in metabolic syndrome patients, as monitored by global fecal microbial community structure, changed toward a vegan profile in some of the patients; however, no functional effects from vegan‐donor fecal microbiota transplantation were seen on TMAO production, abdominal aortic 18F‐fluorodeoxyglucose uptake, or ex vivo cytokine production from peripheral blood mononuclear cells. ConclusionsSingle lean vegan‐donor fecal microbiota transplantation in metabolic syndrome patients resulted in detectable changes in intestinal microbiota composition but failed to elicit changes in TMAO production capacity or parameters related to vascular inflammation. Clinical Trial RegistrationURL: http://www.trialregister.nl. Unique identifier: NTR 4338.

The protocols for solid-phase extraction (SPE) of six microcystins (MCs; MC-LR, MC-RR, MC-LA, MC-LF, MC-LW, and MC-YR) from mouse urine, mouse plasma, and human serum are reported. The quantification of those MCs in biofluids was achieved using HPLC-orbitrap-MS in selected-ion monitoring (SIM) mode, and MCs in urine samples were also quantified by ultra-HPLC-triple quadrupole-tandem mass spectrometry (UHPLC-QqQ-MS/MS) in multiple reaction monitoring (MRM) mode. Under optimal conditions, the extraction recoveries of MCs from samples spiked at two different concentrations (1 μg/L and 10 μg/L) ranged from 90.4% to 104.3% with relative standard deviations (RSDs) ≤ 4.7% for mouse urine, 90.4-106.9% with RSDs ≤ 6.3% for mouse plasma, and 90.0-104.8% with RSDs ≤ 5.0% for human serum. Matrix-matched internal standard calibration curves were linear with R2 ≥ 0.9950 for MC-LR, MC-RR and MC-YR, and R2 ≥ 0.9883 for MC-LA, MC-LF, and MC-LW. The limits of quantification (LOQs) in spiked urine samples were ∼0.13 μg/L for MC-LR, MC-RR, and MC-YR, and ∼0.50 μg/L for MC-LA, MC-LF, and MC-LW, while the LOQs in spiked plasma and serum were ∼0.25 μg/L for MC-LR, MC-RR, and MC-YR, and ∼1.00 μg/L for MC-LA, MC-LF, and MC-LW. The developed methods were applied in a proof-of-concept study to quantify urinary and blood concentrations of MC-LR after oral administration to mice. The urine of mice administered 50 μg of MC-LR per kg bodyweight contained on average 1.30 μg/L of MC-LR (n = 8), while mice administered 100 μg of MC-LR per kg bodyweight had average MC-LR concentration of 2.82 μg/L (n = 8). MC-LR was also quantified in the plasma of the same mice. The results showed that increased MC-LR dosage led to larger urinary and plasma MC-LR concentrations and the developed methods were effective for the quantification of MCs in mouse biofluids.

Recent studies reveal 2-aminoadipic acid (2-AAA) is both elevated in subjects at risk for diabetes and mechanistically linked to glucose homeostasis. Prior studies also suggest enrichment of protein-bound 2-AAA as an oxidative post-translational modification of lysyl residues in tissues associated with degenerative diseases of aging. While in vitro studies suggest redox active transition metals or myeloperoxidase (MPO) generated hypochlorous acid (HOCl) may produce protein-bound 2-AAA, the mechanism(s) responsible for generation of 2-AAA during inflammatory diseases are unknown. In initial studies we observed that traditional acid- or base-catalyzed protein hydrolysis methods previously employed to measure tissue 2-AAA can artificially generate protein-bound 2-AAA from an alternative potential lysine oxidative product, lysine nitrile (LysCN). Using a validated protease-based digestion method coupled with stable isotope dilution LC/MS/MS, we now report protein bound 2-AAA and LysCN are both formed by hypochlorous acid (HOCl) and the MPO/H2O2/Cl- system of leukocytes. At low molar ratio of oxidant to target protein Nε-lysine moiety, 2-AAA is formed via an initial Nε-monochloramine intermediate, which ultimately produces the more stable 2-AAA end-product via sequential generation of transient imine and semialdehyde intermediates. At higher oxidant to target protein Nε-lysine amine ratios, protein-bound LysCN is formed via initial generation of a lysine Nε-dichloramine intermediate. In studies employing MPO knockout mice and an acute inflammation model, we show that both free and protein-bound 2-AAA, and in lower yield, protein-bound LysCN, are formed by MPO in vivo during inflammation. Finally, both 2-AAA and to lesser extent LysCN are shown to be enriched in human aortic atherosclerotic plaque, a tissue known to harbor multiple MPO-catalyzed protein oxidation products. Collectively, these results show that MPO-mediated oxidation of protein lysyl residues serves as a mechanism for producing 2-AAA and LysCN in vivo. These studies further support involvement of MPO-catalyzed oxidative processes in both the development of atherosclerosis and diabetes risk.

Purpose of review: We have established that many metabolic biomes exist within the complex mammalian gut. Substantial metabolism occurs within these biomes and is called co-metabolism of the host and resident microorganisms. This gut-brain-endocrine metabolic interaction emphasizes how bacteria can affect the brain and the hormonal axes in the process of co-metabolism. This review highlights new findings in this regard.Recent findings: In this review, we explore how the gut microbiota affect the development and regulation of the hypothalamic-pituitary-adrenal axis and neurochemistry from mental health and behavioral health to memory, depression, mood, anxiety, obesity, and the development of the blood-brain barrier.Summary: This review describes the implications of the findings for clinical practice or research. Interaction of small molecules within these biomes is now described collectively as a "metabolic interactome". Metabolites of the gut-brain-endocrine axis and our overall gut health constantly shape the host phenotype in ways previously unimagined, and this niche represents potential targets for treatment and drug design, since the interaction or biochemical interplay results in net metabolite production and/or end products to exercise either positive or negative effects on human health.

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 mu M (interquartile range [IQR]: 3.4 to 9.8 mu M), 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. (C) 2016 by the American College of Cardiology Foundation.

Paraoxonase 1 (PON1) is a high density lipoprotein (HDL)-associated protein with atherosclerosis-protective and systemic anti-oxidant functions. We recently showed that PON1, myeloperoxidase, and HDL bind to one another in vivo forming a functional ternary complex (Huang, Y., Wu, Z., Riwanto, M., Gao, S., Levison, B. S., Gu, X., Fu, X., Wagner, M. A., Besler, C., Gerstenecker, G., Zhang, R., Li, X. M., Didonato, A. J., Gogonea, V., Tang, W. H., et al. (2013) J. Clin. Invest. 123, 3815–3828). However, specific residues on PON1 involved in the HDL-PON1 interaction remain unclear. Unambiguous identification of protein residues involved in docking interactions to lipid surfaces poses considerable methodological challenges. Here we describe a new strategy that uses a novel synthetic photoactivatable and click chemistry-taggable phospholipid probe, which, when incorporated into HDL, was used to identify amino acid residues on PON1 that directly interact with the lipoprotein phospholipid surface. Several specific PON1 residues (Leu-9, Tyr-185, and Tyr-293) were identified through covalent cross-links with the lipid probes using affinity isolation coupled to liquid chromatography with on-line tandem mass spectrometry. Based upon the crystal structure for PON1, the identified residues are all localized in relatively close proximity on the surface of PON1, defining a domain that binds to the HDL lipid surface. Site-specific mutagenesis of the identified PON1 residues (Leu-9, Tyr-185, and Tyr-293), coupled with functional studies, reveals their importance in PON1 binding to HDL and both PON1 catalytic activity and stability. Specifically, the residues identified on PON1 provide important structural insights into the PON1-HDL interaction. More generally, the new photoactivatable and affinity-tagged lipid probe developed herein should prove to be a valuable tool for identifying contact sites supporting protein interactions with lipid interfaces such as found on cell membranes or lipoproteins.

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.

Trimethylamine (TMA) N-oxide (TMAO), a gut-microbiota-dependent metabolite, both enhances atherosclerosis in animal models and is associated with cardiovascular risks in clinical studies. Here, we investigate the impact of targeted inhibition of the first step in TMAO generation, commensal microbial TMA production, on diet-induced atherosclerosis. A structural analog of choline, 3,3-dimethyl-1-butanol (DMB), is shown to non-lethally inhibit TMA formation from cultured microbes, to inhibit distinct microbial TMA lyases, and to both inhibit TMA production from physiologic polymicrobial cultures (e.g., intestinal contents, human feces) and reduce TMAO levels in mice fed a high-choline or L-carnitine diet. DMB inhibited choline diet-enhanced endogenous macrophage foam cell formation and atherosclerotic lesion development in apolipoprotein e(-/-) mice without alterations in circulating cholesterol levels. The present studies suggest that targeting gut microbial production of TMA specifically and non-lethal microbial inhibitors in general may serve as a potential therapeutic approach for the treatment of cardiometabolic diseases.

Introduction: Cardiotonic steroids (CTS) such as marinobufagenin and telocinobufagin (TCB) are significantly elevated in chronic kidney disease (CKD) and heart failure and can directly lead to inflammation and cardiac fibrosis upon binding and signaling through the Na+/K+-ATPase. The 17β-lactone ring of CTS is critical for Na+/K+-ATPase binding. Paraoxonases are a family of hydrolytic enzymes which exhibit lactonase activity, although their physiologic substrate(s) are unclear. Decreased serum PON activity and increased levels of CTS are associated with increased oxidant stress and adverse clinical outcomes in the setting of HF and CKD, yet whether decreased PON activity is mechanistically linked to increased CTS levels is unclear. Hypothesis: We tested the hypothesis that PONs are capable of CTS hydrolysis and that hydrolysis of CTS to their open-ring form renders them inactive. Methods/Results: First we measured the ability of recombinant PONs to hydrolyze the lactone ring of active TCB to form open-ring TCB (ORT). Recombinant PON-1 (58.0±2.0 U/mg), PON-2 (0.49±0.01 U/mg) and PON-3 (10.8±0.6 U/mg) had specific lactonase activity for TCB as measured with a continuous pH sensitive colormetric assay. Further, plasma from PON-1 KO mice had a markedly diminished ability to open the 17β-lactone ring of TCB to form resulting ORT aldehyde as measured by a Purpald reagent assay, whereas plasma from PON1-overexpressing mice had increased production of ORT compared to wild type controls. Data from 86-Rubidium uptake experiments in LLC-PK1 cells demonstrated ORT had significantly reduced inhibition of Na+/K+-ATPase compared to TCB (0.25nM of TCB vs. 5000 nM of ORT, p<0.01). In both HK2 and human cardiac fibroblast cells, TCB but not ORT increased collagen-1 mRNA production in a dose dependent manner (1 to 100 nM, p<0.01). Finally, in plasma from CKD patients (n=22) both marinobufagenin (r2=0.58; p<0.01) and TCB (r2=0.31; p<0.01) levels were inversely proportional to lactonase activity, suggesting higher lactonase activity is associated with lower levels of CTS. Conclusion: These studies suggest CTS may be a novel physiologic substrate for PON and represent a previously unknown counter-regulatory mechanism of CTS in the setting of CKD and heart failure.

L-carnitine, a nutrient in red meat, was recently reported to accelerate atherosclerosis via a metaorganismal pathway involving gut microbial trimethylamine (TMA) formation and host hepatic conversion into trimethylamine-N-oxide (TMAO). Herein, we show that following L-carnitine ingestion, γ-butyrobetaine (γBB) is produced as an intermediary metabolite by gut microbes at a site anatomically proximal to and at a rate ∼1,000-fold higher than the formation of TMA. Moreover, we show that γBB is the major gut microbial metabolite formed from dietary L-carnitine in mice, is converted into TMA and TMAO in a gut microbiota-dependent manner (like dietary L-carnitine), and accelerates atherosclerosis. Gut microbial composition and functional metabolic studies reveal that distinct taxa are associated with the production of γBB or TMA/TMAO from dietary L-carnitine. Moreover, despite their close structural similarity, chronic dietary exposure to L-carnitine or γBB promotes development of functionally distinct microbial communities optimized for the metabolism of L-carnitine or γBB, respectively.