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Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. The gut microbiome has been implicated in outcomes for HCC, and gut microbe-derived products may serve as potential non-invasive indices for early HCC detection. This study evaluated differences in plasma concentrations of gut microbiota-derived metabolites. Methods: Forty-one patients with HCC and 96 healthy controls were enrolled from surgical clinics at the Cleveland Clinic from 2016 to 2020. Gut microbiota-derived circulating metabolites detectable in plasma were compared between patients with HCC and healthy controls. Hierarchical clustering was performed for generating heatmaps based on circulating metabolite concentrations using ClustVis, with Euclidean and Ward settings and significant differences between metabolite concentrations were tested using a binary logistic regression model. Results: In patients with HCC, 25 (61%) had histologically confirmed cirrhosis. Trimethylamine (TMA)-related metabolites were found at higher concentrations in those with HCC, including choline (p < 0.001), betaine (p < 0.001), carnitine (p = 0.007), TMA (p < 0.001) and trimethylamine N-oxide (TMAO, p < 0.001). Notably, concentrations of P-cresol glucuronide (p < 0.001), indole-lactic acid (p = 0.038), 5-hydroxyindoleacetic acid (p < 0.0001) and 4-hydroxyphenyllactic acid (p < 0.001) were also increased in those with HCC compared to healthy controls. Hierarchical clustering of the metabolite panel separated patients based on the presence of HCC (p < 0.001), but was not able to distinguish between patients with HCC based on the presence of cirrhosis (p = 0.42). Conclusions: Gut microbiota-derived metabolites were differentially abundant in patients with HCC versus healthy controls. The observed perturbations of the TMAO pathway in HCC seem particularly promising as a target of future research and may have both diagnostic and therapeutic implications.

Plant growth is coordinated with the availability of nutrients that ensure its development. Nitrate is a major source of nitrogen (N), an essential macronutrient for plant growth. It also acts as a signaling molecule to modulate gene expression, metabolism, and a variety of physiological processes. Recently, it has become evident that the calcium signal appears to be part of the nitrate signaling pathway. New key players have been discovered and described in Arabidopsis thaliana (Arabidopsis). In addition, knowledge of the molecular mechanisms of how N signaling affects growth and development, such as the nitrate control of the flowering process, is increasing rapidly. Here, we review recent advances in the identification of new components involved in nitrate signal transduction, summarize newly identified mechanisms of nitrate signaling-modulated flowering time in Arabidopsis, and suggest emerging concepts and existing open questions that will hopefully be informative for further discoveries.

ABSTRACT Trimethylamine-N-oxide (TMAO) is a major pro-atherogenic and pro-thrombotic metaorganismal molecule produced through the initial conversion of the dietary L-carnitine and other precursors into trimethylamine (TMA). We recently identified a dual-microbe anaerobic pathway for the metabolism of L-carnitine into TMA, in which the widely distributed cai operon in Enterobacteriaceae converts L-carnitine into gamma-butyrobetaine (γBB), followed by the degradation of γBB into TMA by the relatively rare gamma-butyrobetaine utilization (gbu) gene cluster present in Emergencia timonensis and few other related microbes. Studies of this pathway in animal models, however, have been limited by the lack of single microbes harboring the whole L-carnitine→γBB→TMA transformation pathway. Such recombinant microbes would both serve as a tool to further prove the contribution of this pathway to gut microbial TMA production and for future in vivo studies investigating the diet linkage to cardiovascular disease and the involvement of the TMAO pathway in this linkage. Here, we recapitulate the whole pathway in a single microbe by cloning the E. timonensis gbu gene cluster into Escherichia fergusonii, which naturally harbors the cai operon. We then show that the native E. timonensis GroES/GroEL-like proteins are needed for the proper functioning of the gbu cluster at 37°C. Finally, we demonstrate that inoculating germ-free mice with this recombinant E. fergusonii strain is sufficient to raise serum TMAO to pathophysiological levels upon dietary L-carnitine supplementation. The recombinant E. fergusonii strain developed will be a useful tool to facilitate future studies on the role of anaerobic gut microbial L-carnitine metabolism in cardiovascular and metabolic diseases. IMPORTANCE The key atherosclerotic TMAO originates from the initial gut microbial conversion of L-carnitine and other dietary compounds into TMA. Developing therapeutic strategies to block gut microbial TMA production needs a detailed understanding of the different production mechanisms and their relative contributions. Recently, we identified a two-step anaerobic pathway for TMA production from L-carnitine through initial conversion by some microbes into the intermediate γBB which is then metabolized by other microbes into TMA. Investigational studies of this pathway, however, are limited by the lack of single microbes harboring the whole pathway. Here, we engineered E. fergusonii strain to harbor the whole two-step pathway and optimized the expression through cloning a specific chaperone from the original host. Inoculating germ-free mice with this recombinant E. fergusonii is enough to raise serum TMAO to pathophysiological levels upon L-carnitine feeding. This engineered microbe will facilitate future studies investigating the contribution of this pathway to cardiovascular disease.

Background The association of circulating trimethylamine‐N‐oxide (TMAO) with stroke has received limited attention. To address this gap, we examined the associations of serial measures of plasma TMAO with incident ischemic stroke. Methods and Results We used a prospective cohort design with data pooled from 2 cohorts. The settings were the CHS (Cardiovascular Health Study), a cohort of older adults, and the MESA (Multi‐Ethnic Study of Atherosclerosis), both in the United States. We measured plasma concentrations of TMAO at baseline and again during the follow‐up using high‐performance liquid chromatography and mass spectrometry. We assessed the association of plasma TMAO with incident ischemic stroke using proportional hazards regression adjusted for risk factors. The combined cohorts included 11 785 participants without a history of stroke, on average 73 (CHS) and 62 (MESA) years old at baseline, including 60% (CHS) and 53% (MESA) women. We identified 1031 total incident ischemic strokes during a median 15‐year follow‐up in the combined cohorts. In multivariable analyses, TMAO was significantly associated with incident ischemic stroke risk (hazard ratios comparing a doubling of TMAO: 1.11 [1.03–1.18], P=0.004). The association was linear over the range of TMAO concentrations and appeared restricted to those without diagnosed coronary heart disease. An association with hemorrhagic stroke was not found. Conclusions Plasma TMAO levels are associated with incident ischemic stroke in a diverse population. Registration URL: https://www.clinicaltrials.gov. Unique identifier: NCT00005133.

Background: Hydrogen sulfide (H2S), a gaseous signaling molecule that impacts multiple physiological processes including aging, is produced via select mammalian enzymes and enteric sulfur-reducing bacteria. H2S research is limited by the lack of an accurate internal standard-containing assay for its quantitation in biological matrices. Methods: After synthesizing [34S]H2S and developing sample preparation protocols that avoid sulfide contamination with the addition of thiol-containing standards or reducing reagents, we developed a stable isotope-dilution high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS) method for the simultaneous quantification of Total H2S and other abundant thiols (cysteine, homocysteine, glutathione, glutamylcysteine, cysteinylglycine) in biological matrices, conducted a 20-day analytical validation/normal range study, and then both analyzed circulating Total H2S and thiols in plasma from 400 subjects, and within 20 volunteers before and after antibiotic-induced suppression of gut microbiota. Results: Using the new assay, all analytes showed minimal interference, no carryover, and excellent intra- and inter-day reproducibility (≤7.6%, and ≤12.7%, respectively), linearity (r2 > 0.997), recovery (90.9%–110%) and stability (90.0%–100.5%). Only circulating Total H2S levels showed significant age-associated reductions in both males and females (p

There is growing appreciation of the importance of the intestinal microbiota in Parkinson's disease (PD), and one potential mechanism by which the intestinal microbiota can communicate with the brain is via bacteria-derived metabolites. In this study, plasma levels of bacterial-derived metabolites including trimethylamine-N-oxide (TMAO), short chain fatty acids (SCFA), the branched chain fatty acid isovalerate, succinate, and lactate were evaluated in PD subjects (treatment naïve and treated) which were compared to (1) population controls, (2) spousal / household controls (similar lifestyle to PD subjects), and (3) subjects with multiple system atrophy (MSA). Analyses revealed an increase in the TMAO pathway in PD subjects which was independent of medication status, disease characteristics, and lifestyle. Lactic acid was decreased in treated PD subjects, succinic acid positively correlated with disease severity, and the ratio of pro-inflammatory TMAO to the putative anti-inflammatory metabolite butyric acid was significantly higher in PD subjects compared to controls indicating a pro-inflammatory shift in the metabolite profile in PD subjects. Finally, acetic and butyric acid were different between PD and MSA subjects indicating that metabolites may differentiate these synucleinopathies. In summary, (1) TMAO is elevated in PD subjects, a phenomenon independent of disease characteristics, treatment status, and lifestyle and (2) metabolites may differentiate PD and MSA subjects. Additional studies to understand the potential of TMAO and other bacterial metabolites to serve as a biomarker or therapeutic targets are warranted.

Abstract Patients with chronic kidney disease (CKD) have elevated circulating levels of trimethylamine N-oxide (TMAO), a metabolite derived from gut microbes and associated with cardiovascular diseases. High circulating levels of TMAO and its dietary precursor, choline, predict increased risk for development of CKD in apparently healthy subjects, and studies in mice fed TMAO or choline suggest that TMAO can contribute to kidney impairment and renal fibrosis. Here we examined the interactions between TMAO, kidney disease, and cardiovascular disease in mouse models. We observed that while female hyperlipidemic apoE KO mice fed a 0.2% adenine diet for 14 weeks developed CKD with elevated plasma levels of TMAO, provision of a non-lethal inhibitor of gut microbial trimethylamine (TMA) production, iodomethylcholine (IMC), significantly reduced multiple markers of renal injury (plasma creatinine, cystatin C, FGF23, and TMAO), reduced histopathologic evidence of fibrosis, and markedly attenuated development of microalbuminuria. In addition, while the adenine-induced CKD model significantly increased heart weight, a surrogate marker for myocardial hypertrophy, this was largely prevented by IMC supplementation. Surprisingly, adenine feeding did not increase atherosclerosis and significantly decreased the expression of inflammatory genes in the aorta compared to the control groups, effects unrelated to TMAO levels. Our data demonstrate that inhibition of TMAO production attenuated CKD development and cardiac hypertrophy in mice, suggesting that TMAO reduction may be a novel strategy in treating CKD and its cardiovascular disease complications.

There is mounting evidence that microbes residing in the human intestine contribute to diverse alcohol-associated liver diseases (ALD) including the most deadly form known as alcohol-associated hepatitis (AH). However, mechanisms by which gut microbes synergize with excessive alcohol intake to promote liver injury are poorly understood. Furthermore, whether drugs that selectively target gut microbial metabolism can improve ALD has never been tested. We used liquid chromatography tandem mass spectrometry to quantify the levels of microbe and host choline co-metabolites in healthy controls and AH patients, finding elevated levels of the microbial metabolite trimethylamine (TMA) in AH. In subsequent studies, we treated mice with non-lethal bacterial choline TMA lyase (CutC/D) inhibitors to blunt gut microbe-dependent production of TMA in the context of chronic ethanol administration. Indices of liver injury were quantified by complementary RNA sequencing, biochemical, and histological approaches. In addition, we examined the impact of ethanol consumption and TMA lyase inhibition on gut microbiome structure via 16S rRNA sequencing. We show the gut microbial choline metabolite TMA is elevated in AH patients and correlates with reduced hepatic expression of the TMA oxygenase flavin-containing monooxygenase 3 (FMO3). Provocatively, we find that small molecule inhibition of gut microbial CutC/D activity protects mice from ethanol-induced liver injury. CutC/D inhibitor-driven improvement in ethanol-induced liver injury is associated with distinct reorganization of the gut microbiome and host liver transcriptome. The microbial metabolite TMA is elevated in patients with AH, and inhibition of TMA production from gut microbes can protect mice from ethanol-induced liver injury.

Obesity has repeatedly been linked to reorganization of the gut microbiome, yet to this point obesity therapeutics have been targeted exclusively toward the human host. Here, we show that gut microbe-targeted inhibition of the trimethylamine N-oxide (TMAO) pathway protects mice against the metabolic disturbances associated with diet-induced obesity (DIO) or leptin deficiency (Lepob/ob). Small molecule inhibition of the gut microbial enzyme choline TMA-lyase (CutC) does not reduce food intake but is instead associated with alterations in the gut microbiome, improvement in glucose tolerance, and enhanced energy expenditure. We also show that gut microbial CutC inhibition is associated with reorganization of host circadian control of both phosphatidylcholine and energy metabolism. This study underscores the relationship between microbe and host metabolism and provides evidence that gut microbe-derived trimethylamine (TMA) is a key regulator of the host circadian clock. This work also demonstrates that gut microbe-targeted enzyme inhibitors have potential as anti-obesity therapeutics.

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.

Background Trimethylamine N‐oxide (TMAO) is a gut microbiota‐dependent metabolite of dietary choline, L‐carnitine, and phosphatidylcholine‐rich foods. On the basis of experimental studies and patients with prevalent disease, elevated plasma TMAO may increase risk of atherosclerotic cardiovascular disease (ASCVD). TMAO is also renally cleared and may interact with and causally contribute to renal dysfunction. Yet, how serial TMAO levels relate to incident and recurrent ASCVD in community‐based populations and the potential mediating or modifying role of renal function are not established. Methods and Results We investigated associations of serial measures of plasma TMAO, assessed at baseline and 7 years, with incident and recurrent ASCVD in a community‐based cohort of 4131 (incident) and 1449 (recurrent) older US adults. TMAO was measured using stable isotope dilution liquid chromatography–tandem mass spectrometry (laboratory coefficient of variation,

Gut microbe–derived metabolites influence human physiology and disease. However, establishing mechanistic links between gut microbial metabolites and disease pathogenesis in animal models remains challenging. The major route of absorption for microbe-derived small molecules is venous drainage via the portal vein to the liver. In the event of presystemic hepatic metabolism, the route of metabolite administration becomes critical. To our knowledge, we describe here a novel portal vein cannulation technique using a s.c. implanted osmotic pump to achieve continuous portal vein infusion in mice. We first administered the microbial metabolite trimethylamine (TMA) over 4 weeks, during which increased peripheral plasma levels of TMA and its host liver-derived cometabolite, trimethylamine-N-oxide, were observed when compared with a vehicle control. Next, 4-hydroxyphenylacetic acid (4-HPAA), a microbial metabolite that undergoes extensive presystemic hepatic metabolism, was administered intraportally to examine effects on hepatic gene expression. As expected, hepatic levels of 4-HPAA were elevated when compared with the control group while peripheral plasma 4-HPAA levels remained the same. Moreover, significant changes in the hepatic transcriptome were revealed by an unbiased RNA-Seq approach. Collectively, to our knowledge this work describes a novel method for administering gut microbe–derived metabolites via the portal vein, mimicking their physiologic delivery in vivo.

AimsThe trimethylamine N-oxide (TMAO) pathway is related to intestinal microbiota and has been associated to risk of cardiovascular disease (CVD). We investigated associations between four plasma metabolites in the TMAO pathway and risk of all-cause mortality, CVD and deterioration in renal function in individuals with type 2-diabetes (T2D) and albuminuria.Materials and methodsPlasma concentrations of TMAO, choline, carnitine, and betaine were measured by liquid chromatography-tandem mass spectrometry at baseline in 311 individuals with T2D and albuminuria. Information on all-cause mortality and fatal/non-fatal CVD during follow-up was obtained from registries. The association of each metabolite, and a weighted sum score of all four metabolites, with the endpoints were examined. Serum creatinine was measured at follow-up visits and the renal endpoint was defined as eGFR-decline of ≥30%. Associations were analysed using proportional hazards models adjusted for traditional risk factors.ResultsBaseline mean(SD) age was 57.2(8.2) years and 75% were males. Follow-up was up to 21.9 years (median (IQR) follow-up 6.8 (6.1-15.5) years for mortality and 6.5 (5.5-8.1) years for CVD events). The individual metabolites and the weighted sum score were not associated with all-cause mortality (n = 106) or CVD (n = 116) (adjusted p≥0.09). Higher choline, carnitine and the weighted sum score of the four metabolites were associated with higher risk of decline in eGFR (n = 106) (adjusted p = 0.001, p = 0.03 and pConclusionsIn individuals with T2D and albuminuria, higher choline, carnitine and a weighted sum of four metabolites from the TMAO pathway were risk markers for deterioration in renal function during long-term follow-up. Metabolites from the TMAO pathway were not independently related to risk of all-cause mortality or CVD.

Exposure to some environmental pollutants can have potent endocrine-disrupting effects, thereby promoting hormone imbalance and cardiometabolic diseases such as non-alcoholic fatty liver disease (NAFLD), diabetes, and cardiorenal diseases. Recent evidence also suggests that many environmental pollutants can reorganize the gut microbiome to potentially impact these diverse human diseases. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is among the most potent endocrine-disrupting dioxin pollutants, yet our understanding of how TCDD impacts the gut microbiome and systemic metabolism is incompletely understood. Here, we show that TCDD exposure in mice profoundly stimulates the hepatic expression of flavin-containing monooxygenase 3 (Fmo3), which is a hepatic xenobiotic metabolizing enzyme that is also responsible for the production of the gut microbiome-associated metabolite trimethylamine N-oxide (TMAO). Interestingly, an enzymatic product of FMO3 (TMAO) has been associated with the same cardiometabolic diseases that these environmental pollutants promote. Therefore, here, we examined TCDD-induced alterations in the gut microbiome, host liver transcriptome, and glucose tolerance in Fmo3+/+ and Fmo3−/− mice. Our results show that Fmo3 is a critical component of the transcriptional response to TCDD, impacting the gut microbiome, host liver transcriptome, and systemic glucose tolerance. Collectively, this work uncovers a previously underappreciated role for Fmo3 in integrating diet–pollutant–microbe–host interactions.

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, P trend=0.015). The association was driven by cardiovascular death (OR 2.25, 95% CI, 1.28–3.96, P trend=0.003) and stroke (OR 2.68, 95% CI, 1.39–5.17, P trend

Abstract Background Aspirin-exacerbated respiratory disease (AERD) is a distinct eosinophilic phenotype of severe asthma with accompanying chronic rhinosinusitis, nasal polyposis, and hypersensitivity to aspirin. Urinary 3-bromotyrosine (uBrTyr) is a noninvasive marker of eosinophil-catalyzed protein oxidation. The lack of in vitro diagnostic test makes the diagnosis of AERD difficult. We aimed to determine uBrTyr levels in patients with AERD (n = 240) and aspirin-tolerant asthma (ATA) (n = 226) and to assess whether its addition to urinary leukotriene E4 (uLTE4) levels and blood eosinophilia can improve the prediction of AERD diagnosis. Methods Clinical data, spirometry and blood eosinophilis were evaluated. UBrTyr and uLTE4 levels were measured in urine by HPLC and ELISA, respectively. Results Both groups of asthmatics (AERD, n = 240; ATA, n = 226) had significantly higher uBrTyr, uLTE4 levels, and blood eosinophils than healthy controls (HC) (n = 71) (p 0.101 ng/mg Cr), uLTE4 levels (> 800 pg/mg Cr) and blood eosinophils (> 300 cells/ul) were 7 times more likely to have AERD.. However, uBrTyr did not increase the benefit for predicting AERD when uLTE4 and blood eosinophils were already taken into account (p = 0.57). Conclusion UBrTyr levels are elevated both in AERD and ATA as compared to HC, but they could not differentiate between these asthma phenotypes suggesting a similar eosinophilic activation. The addition of uBrTyr to elevated uLTE4 levels and blood eosinophils did not statistically enhance the prediction of AERD diagnosis.

Abstract Background Rheumatoid arthritis (RA) patients are at high risk of developing cardiovascular disease (CVD). In RA, chronic inflammation may lead to endothelial dysfunction, an early indicator of CVD, owing to diminished nitric oxide (NO) production. Because l-arginine is the sole precursor of NO, we hypothesized that levels of l-arginine metabolic products reflecting NO metabolism are altered in patients with RA. Methods Plasma samples from patients with RA (n = 119) and age- and sex-matched control subjects (n = 238) were used for this study. Using LC-MS/MS, we measured plasma levels of free l-arginine, l-ornithine, l-citrulline, l-N G-monomethyl arginine (MMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA). We compared global arginine bioavailability ratio (GABR) (i.e., ratio of l-arginine to l-ornithine + l-citrulline) and arginine methylation index (ArgMI) (i.e., ADMA + SDMA/MMA) in patients with RA vs. control subjects. Plasma arginase activity was measured using a sensitive arginase assay kit. The relationship of l-arginine metabolites and arginase activity to CVD risk factors was evaluated using Pearson’s chi-square test. Results Compared with healthy control subjects, the RA cohort showed significantly lower levels of plasma l-arginine (46.11 ± 17.29 vs. 74.2 ± 22.53 μmol/L, p

ABSTRACT Trillions of microbes inhabit the human gut, not only providing nutrients and energy to the host from the ingested food, but also producing metabolic bioactive signaling molecules to maintain health and elicit disease, such as cardiovascular disease (CVD). CVD is the leading cause of mortality worldwide. In this review, we presented gut microbiota derived metabolites involved in cardiovascular health and disease, including trimethylamine-N-oxide (TMAO), uremic toxins, short chain fatty acids (SCFAs), phytoestrogens, anthocyanins, bile acids and lipopolysaccharide. These gut microbiota derived metabolites play critical roles in maintaining a healthy cardiovascular function, and if dysregulated, potentially causally linked to CVD. A better understanding of the function and dynamics of gut microbiota derived metabolites holds great promise toward mechanistic predicative CVD biomarker discoveries and precise interventions.

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.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide, yet the pathogenesis of NAFLD is only partially understood. Here, we investigated the role of the gut bacteria in NAFLD by stimulating the gut bacteria via feeding mice the fermentable dietary fiber, guar gum (GG), and suppressing the gut bacteria via chronic oral administration of antibiotics. GG feeding profoundly altered the gut microbiota composition, in parallel with reduced diet-induced obesity and improved glucose tolerance. Strikingly, despite reducing adipose tissue mass and inflammation, GG enhanced hepatic inflammation and fibrosis, concurrent with markedly elevated plasma and hepatic bile acid levels. Consistent with a role of elevated bile acids in the liver phenotype, treatment of mice with taurocholic acid stimulated hepatic inflammation and fibrosis. In contrast to GG, chronic oral administration of antibiotics effectively suppressed the gut bacteria, decreased portal secondary bile acid levels, and attenuated hepatic inflammation and fibrosis. Neither GG nor antibiotics influenced plasma lipopolysaccharide levels. In conclusion, our data indicate a causal link between changes in gut microbiota and hepatic inflammation and fibrosis in a mouse model of NAFLD, possibly via alterations in bile acids.

Summary: 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. : Microbes resident in the human intestine represent a key transmissible environmental factor contributing to obesity and related disorders. Schugar et al. now show that expression of the TMAO-producing enzyme FMO3 is linked to obesity and energy expenditure in both mice and humans. Keywords: microbiota, nutrition, obesity, diabetes, adipose, flavin-containing monooxygenase 3, FMO3

Abstract Background The gut microbiome is a complex and metabolically active community that directly influences host phenotypes. In this study, we profile gut microbiota using 16S rRNA gene sequencing in 531 well-phenotyped Finnish men from the Metabolic Syndrome In Men (METSIM) study. Results We investigate gut microbiota relationships with a variety of factors that have an impact on the development of metabolic and cardiovascular traits. We identify novel associations between gut microbiota and fasting serum levels of a number of metabolites, including fatty acids, amino acids, lipids, and glucose. In particular, we detect associations with fasting plasma trimethylamine N-oxide (TMAO) levels, a gut microbiota-dependent metabolite associated with coronary artery disease and stroke. We further investigate the gut microbiota composition and microbiota–metabolite relationships in subjects with different body mass index and individuals with normal or altered oral glucose tolerance. Finally, we perform microbiota co-occurrence network analysis, which shows that certain metabolites strongly correlate with microbial community structure and that some of these correlations are specific for the pre-diabetic state. Conclusions Our study identifies novel relationships between the composition of the gut microbiota and circulating metabolites and provides a resource for future studies to understand host–gut microbiota relationships.

Background Recent studies have revealed sexually dimorphic associations between the carbamoyl‐phosphate synthase 1 locus, intermediates of the metabolic pathway leading from choline to urea, and risk of coronary artery disease (CAD) in women. Based on evidence from the literature, the atheroprotective association with carbamoyl‐phosphate synthase 1 could be mediated by the strong genetic effect of this locus on increased circulating glycine levels. Methods and Results We sought to identify additional genetic determinants of circulating glycine levels by carrying out a meta‐analysis of genome‐wide association study data in up to 30 118 subjects of European ancestry. Mendelian randomization and other analytical approaches were used to determine whether glycine‐associated variants were associated with CAD and traditional risk factors. Twelve loci were significantly associated with circulating glycine levels, 7 of which were not previously known to be involved in glycine metabolism (ACADM, PHGDH, COX18‐ADAMTS3, PSPH, TRIB1, PTPRD, and ABO). Glycine‐raising alleles at several loci individually exhibited directionally consistent associations with decreased risk of CAD. However, these effects could not be attributed directly to glycine because of associations with other CAD‐related traits. By comparison, genetic models that only included the 2 variants directly involved in glycine degradation and for which there were no other pleiotropic associations were not associated with risk of CAD or blood pressure, lipid levels, and obesity‐related traits. Conclusions These results provide additional insight into the genetic architecture of glycine metabolism, but do not yield conclusive evidence for a causal relationship between circulating levels of this amino acid and risk of CAD in humans.

Dietary choline metabolites, such as trimethylamine N-oxide and betaine, have been associated with coronary artery disease (CAD). Here, Hartiala et al. identify two genetic loci for betaine levels on chromosomes 2q34 and 5q14.1 and find that the 2q34 locus was also associated with other pathway intermediates, and decreased risk of CAD in women.

We evaluated associations of smoking heaviness markers and the effects of smoking cessation on the intestinal microbiota and cardiovascular disease risk factors in current smokers undertaking a quit attempt. Participants were current smokers enrolled in a prospective randomized clinical trial of smoking cessation therapies with visits at baseline, 2, and 12 weeks. Genomic DNA was extracted from fecal samples followed by 16S rRNA gene sequencing and analysis using the QIIME2 software workflow. Relative abundances of bacterial taxa and alpha- and beta-diversity measures were used for comparisons. The 36 smokers were (mean (standard deviation)) 51.5 (11.1) years old (42% male) and smoked 15.1 (6.4) cigarettes per day for 22.7 (11.9) pack-years. Relative abundances of the phylum Actinobacteria correlated with pack-years (rho = −0.44, p = 0.008) and Cyanobacteria correlated with CO levels (rho = 0.39, p = 0.021). After 12 weeks, relative abundances of the phylum Bacteroidetes increased (pANCOVA = 0.048) and Firmicutes decreased (pANCOVA = 0.036) among abstainers compared to continuing smokers. Increases in alpha-diversity were associated with heart rates (rho = −0.59, p = 0.037), systolic blood pressures (rho = −0.58, p = 0.043), and C-reactive protein (rho = −0.60, p = 0.034). Smoking cessation led to minor changes in the intestinal microbiota. It is unclear if the proven health benefits of smoking cessation lead to salutary changes in the intestinal microbiota.

Trimethylamine (TMA) is a gut microbial metabolite—rendered by the enzymatic cleavage of nutrients containing a TMA moiety in their chemical structure. TMA can be oxidized as trimethylamine N-oxide (TMAO) catalyzed by hepatic flavin monooxygenases. Circulating TMAO has been demonstrated to portend a pro-inflammatory state, contributing to chronic diseases such as cardiovascular disease and chronic kidney disease. Consequently, TMAO serves as an excellent candidate biomarker for a variety of chronic inflammatory disorders. The highly positive correlation between plasma TMAO and urine TMAO suggests that urine TMAO has the potential to serve as a less invasive biomarker for chronic disease compared to plasma TMAO. In this study, we validated a method to simultaneously measure urine TMA and TMAO concentrations by liquid chromatography–mass spectrometry (LC/MS). Urine TMA and TMAO can be extracted by hexane/butanol under alkaline pH and transferred to the aqueous phase following acidification for LC/MS quantitation. Importantly, during sample processing, none of the nutrients with a chemical structure containing a TMA moiety were spontaneously cleaved to yield TMA. Moreover, we demonstrated that the acidification of urine prevents an increase of TMA after prolonged storage as was observed in non-acidified urine. Finally, here we demonstrated that TMAO can spontaneously degrade to TMA at a very slow rate.

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.

We performed silencing and overexpression studies of flavin containing monooxygenase (FMO) 3 in hyperlipidemic mouse models to examine its effects on trimethylamine N-oxide (TMAO) levels and atherosclerosis. Knockdown of hepatic FMO3 in LDL receptor knockout mice using an antisense oligonucleotide resulted in decreased circulating TMAO levels and atherosclerosis. Surprisingly, we also observed significant decreases in hepatic lipids and in levels of plasma lipids, ketone bodies, glucose, and insulin. FMO3 overexpression in transgenic mice, on the other hand, increased hepatic and plasma lipids. Global gene expression analyses suggested that these effects of FMO3 on lipogenesis and gluconeogenesis may be mediated through the PPARα and Kruppel-like factor 15 pathways. In vivo and in vitro results were consistent with the concept that the effects were mediated directly by FMO3 rather than trimethylamine/TMAO; in particular, overexpression of FMO3 in the human hepatoma cell line, Hep3B, resulted in significantly increased glucose secretion and lipogenesis. Our results indicate a major role for FMO3 in modulating glucose and lipid homeostasis in vivo, and they suggest that pharmacologic inhibition of FMO3 to reduce TMAO levels would be confounded by metabolic interactions.

Circulating levels of the gut microbe-derived metabolite trimethylamine-N-oxide (TMAO) have recently been linked to cardiovascular disease (CVD) risk. Here, we performed transcriptional profiling in mouse models of altered reverse cholesterol transport (RCT) and serendipitously identified the TMAO-generating enzyme flavin monooxygenase 3 (FMO3) as a powerful modifier of cholesterol metabolism and RCT. Knockdown of FMO3 in cholesterol-fed mice alters biliary lipid secretion, blunts intestinal cholesterol absorption, and limits the production of hepatic oxysterols and cholesteryl esters. Furthermore, FMO3 knockdown stimulates basal and liver X receptor (LXR)-stimulated macrophage RCT, thereby improving cholesterol balance. Conversely, FMO3 knockdown exacerbates hepatic endoplasmic reticulum (ER) stress and inflammation in part by decreasing hepatic oxysterol levels and subsequent LXR activation. FMO3 is thus identified as a central integrator of hepatic cholesterol and triacylglycerol metabolism, inflammation, and ER stress. These studies suggest that the gut microbiota-driven TMA/FMO3/TMAO pathway is a key regulator of lipid metabolism and inflammation.

Sphingomyelin (SM) levels in the circulation correlate positively with atherosclerosis burden. SM is a ubiquitous component of human diets, but it is unclear if dietary SM increases circulating SM levels. Dietary choline increases atherosclerosis by raising circulating trimethylamine N-oxide (TMAO) levels in mice and humans. As SM has a choline head group, we ask in this study if dietary SM accelerates atherosclerotic lesion development by increasing circulating SM and TMAO levels. Three studies were performed: (Study 1) C57BL/6 mice were maintained on a high fat diet with or without SM supplementation for 4 weeks prior to quantification of serum TMAO and SM levels; (Study 2) atherosclerosis was studied in apoE-/- mice after 16 weeks of a high fat diet without or with SM supplementation and (Study 3) apoE-/- mice were maintained on a chow diet for 19 weeks without or with SM supplementation and antibiotic treatment prior to quantification of atherosclerotic lesions and serum TMAO and SM levels. SM consumption did not increase circulating SM levels or atherosclerosis in high fat-fed apoE-/- mice. Serum TMAO levels in C57BL/6 mice were low and had no effect atherosclerosis lesion development. Dietary SM supplementation significantly reduced atherosclerotic lesion area in the aortic arch of chow-fed apoE-/- mice. This study establishes that dietary SM does not affect circulating SM levels or increase atherosclerosis in high fat-fed apoE-/- mice, but it is anti-atherogenic in chow-fed apoE-/- mice.

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 (p = 0.01), which remained unchanged after adjusting for the GS (p = 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.

Accumulating evidence linking trimethylamine N-oxide (TMAO) to cardiovascular disease (CVD) risk has prompted interest in developing therapeutic strategies to reduce its production. We compared two lifestyle intervention approaches: hypocaloric versus eucaloric diet, combined with exercise, on TMAO levels in relation to CVD risk factors. Sixteen obese adults (66.1 ± 4.4 years, BMI (body mass index): 35.9 ± 5.3 kg/m2, fasting glucose: 106 ± 16 mg/dL, 2-h PPG (postprandial glucose): 168 ± 37 mg/dL) were randomly assigned to 12 weeks of exercise (5 days/week, 80–85% HRmax (maximal heart rate)) plus either a hypocaloric (HYPO) (−500 kcal) or a eucaloric (EU) diet. Outcomes included plasma TMAO, glucose metabolism (oral glucose tolerance test (OGTT) and euglycemic-hyperinsulinemic clamps for glucose disposal rates (GDR)), exercise capacity (VO2max, maximal oxygen consumption), abdominal adiposity (computed tomography scans), cholesterol, and triglycerides. Results showed that body composition (body weight, subcutaneous adiposity), insulin sensitivity, VO2max, and cholesterol all improved (p < 0.05). HYPO decreased the percentage change in TMAO compared to an increase after EU (HYPO: −31 ± 0.4% vs. EU: 32 ± 0.6%, p = 0.04). Absolute TMAO levels were not impacted (HYPO: p = 0.09 or EU: p = 0.53 group). The change in TMAO after intervention was inversely correlated with baseline visceral adipose tissue (r = −0.63, p = 0.009) and GDR (r = 0.58, p = 0.002). A hypocaloric diet and exercise approach appears to be effective in reducing TMAO. Larger trials are needed to support this observation.

BackgroundProduction of the proatherogenic metabolite, trimethylamine N‐oxide (TMAO), from dietary nutrients by intestinal microbiota enhances atherosclerosis development in animal models and is associated with atherosclerotic coronary artery disease in humans. The utility of studying plasma levels of TMAO to risk stratify in patients with peripheral artery disease (PAD) has not been reported. Methods and ResultsWe examined the relationship between fasting plasma TMAO and all‐cause mortality (5‐year), stratified by subtypes of PAD and presence of coronary artery disease in 935 patients with PAD who underwent elective angiography for cardiac evaluation at a tertiary care hospital. Median plasma TMAO was 4.8 μmol/L (interquartile range, 2.9–8.0 μmol/L). Elevated TMAO levels were associated with 2.7‐fold increased mortality risk (fourth versus first quartiles, hazard ratio 2.86, 95% CI 1.82–3.97, P

BackgroundTrimethylamine‐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 ResultsWe 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

BackgroundThe choline‐derived metabolite trimethylamine N‐oxide (TMAO) has been demonstrated to contribute to atherosclerosis and is associated with coronary artery disease risk. Methods and ResultsWe explored the impact of TMAO on endothelial and smooth muscle cell function in vivo, focusing on disease‐relevant outcomes for atherogenesis. Initially, we observed that aortas of LDLR−/− mice fed a choline diet showed elevated inflammatory gene expression compared with controls. Acute TMAO injection at physiological levels was sufficient to induce the same inflammatory markers and activate the well‐known mitogen‐activated protein kinase, extracellular signal–related kinase, and nuclear factor‐κB signaling cascade. These observations were recapitulated in primary human aortic endothelial cells and vascular smooth muscle cells. We also found that TMAO promotes recruitment of activated leukocytes to endothelial cells. Through pharmacological inhibition, we further showed that activation of nuclear factor‐κB signaling was necessary for TMAO to induce inflammatory gene expression in both of these relevant cell types as well as endothelial cell adhesion of leukocytes. ConclusionsOur results suggest a likely contributory mechanism for TMAO‐dependent enhancement in atherosclerosis and cardiovascular risks.

Common forms of atherosclerosis involve multiple genetic and environmental factors. While human genome-wide association studies have identified numerous loci contributing to coronary artery disease and its risk factors, these studies are unable to control environmental factors or examine detailed molecular traits in relevant tissues. We now report a study of natural variations contributing to atherosclerosis and related traits in over 100 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP). The mice were made hyperlipidemic by transgenic expression of human apolipoprotein E-Leiden (APOE-Leiden) and human cholesteryl ester transfer protein (CETP). The mice were examined for lesion size and morphology as well as plasma lipid, insulin and glucose levels, and blood cell profiles. A subset of mice was studied for plasma levels of metabolites and cytokines. We also measured global transcript levels in aorta and liver. Finally, the uptake of acetylated LDL by macrophages from HMDP mice was quantitatively examined. Loci contributing to the traits were mapped using association analysis, and relationships among traits were examined using correlation and statistical modeling. A number of conclusions emerged. First, relationships among atherosclerosis and the risk factors in mice resemble those found in humans. Second, a number of trait-loci were identified, including some overlapping with previous human and mouse studies. Third, gene expression data enabled enrichment analysis of pathways contributing to atherosclerosis and prioritization of candidate genes at associated loci in both mice and humans. Fourth, the data provided a number of mechanistic inferences; for example, we detected no association between macrophage uptake of acetylated LDL and atherosclerosis. Fifth, broad sense heritability for atherosclerosis was much larger than narrow sense heritability, indicating an important role for gene-by-gene interactions. Sixth, stepwise linear regression showed that the combined variations in plasma metabolites, including LDL/VLDL-cholesterol, trimethylamine N-oxide (TMAO), arginine, glucose and insulin, account for approximately 30 to 40% of the variation in atherosclerotic lesion area. Overall, our data provide a rich resource for studies of complex interactions underlying atherosclerosis.

A precise and sensitive method for measuring cellular free and esterified cholesterol is required in order to perform studies of macrophage cholesterol loading, metabolism, storage, and efflux. Until now, the use of an enzymatic cholesterol assay, commonly used for aqueous phase plasma cholesterol assays, has not been optimized for use with solid phase samples such as cells, due to inefficient solubilization of total cholesterol in enzyme compatible solvents. We present an efficient solubilization protocol compatible with an enzymatic cholesterol assay that does not require chemical saponification or chromatographic separation. Another issue with enzyme compatible solvents is the presence of endogenous peroxides that interfere with the enzymatic cholesterol assay. We overcame this obstacle by pretreatment of the reaction solution with the enzyme catalase, which consumed endogenous peroxides resulting in reduced background and increased sensitivity in our method. Finally, we demonstrated that this method for cholesterol quantification in macrophages yields results that are comparable to those measured by stable isotope dilution gas chromatography with mass spectrometry detection. In conclusion, we describe a sensitive, simple, and high-throughput enzymatic method to quantify cholesterol in complex matrices such as cells.

BACKGROUND: Growing evidence indicates that trimethylamine N-oxide, a gut microbial metabolite of dietary choline and carnitine, promotes both cardiovascular disease and chronic kidney disease risk. It remains unclear how circulating concentrations of trimethylamine N-oxide and its related dietary and gut microbe-derived metabolites (choline, betaine, carnitine, γ-butyrobetaine, and crotonobetaine) affect incident heart failure (HF). METHODS: We evaluated 11 768 participants from the Cardiovascular Health Study and the Multi-Ethnic Study of Atherosclerosis with serial measures of metabolites. Cox proportional hazard models were used to examine the associations between metabolites and incident HF, adjusted for cardiovascular disease risk factors. RESULTS: In all, 2102 cases of HF occurred over a median follow-up of 15.9 years. After adjusting for traditional risk factors, higher concentrations of trimethylamine N-oxide (hazard ratio, 1.15 [95% CI, 1.09-1.20]; P<0.001), choline (hazard ratio, 1.44 [95% CI, 1.26-1.64]; P<0.001), and crotonobetaine (hazard ratio, 1.24 [95% CI, 1.16-1.32]; P<0.001) were associated with increased risk for incident HF. After further adjustment for renal function (potential confounder or mediator), these associations did not reach Bonferroni-corrected statistical significance (P=0.01, 0.049, and 0.006, respectively). Betaine and carnitine were nominally associated with a higher incidence of HF (P<0.05). In exploratory analyses, results were similar for subtypes of HF based on left ventricular ejection fraction, and associations appeared generally stronger among Black and Hispanic/Latino versus White adults, although there were no interactions for any metabolites with race. CONCLUSIONS: In this pooled analysis of 2 well-phenotyped, diverse, community-based cohorts, circulating concentrations of gut microbe-derived metabolites such as trimethylamine N-oxide, choline, and crotonobetaine were independently associated with a higher risk of developing HF. [ABSTRACT FROM AUTHOR]

Asthma is an inflammatory disease of the airways characterized by eosinophil recruitment, eosinophil peroxidase release, and protein oxidation through bromination, which following tissue remodeling results in excretion of 3-bromotyrosine. Predicting exacerbations and reducing their frequency is critical for the treatment of severe asthma. In this study, we aimed to investigate whether urinary total conjugated bromotyrosine can discriminate asthma severity and predict asthma exacerbations. We collected urine from participants with severe (n = 253) and nonsevere (n = 178) asthma, and the number of adjudicated exacerbations in 1-yr longitudinal follow-up was determined among subjects enrolled in the Severe Asthma Research Program, a largescale National Institutes of Health (NIH)-funded consortium. Urine glucuronidated bromotyrosine and total conjugated forms were quantified by hydrolysis with either glucuronidase or methanesulfonic acid, respectively, followed by liquid chromatography-tandem mass spectrometry analyses of free 3-bromotyrosine. Blood and sputum eosinophils were also counted. The majority of 3-bromotyrosine in urine was found to exist in conjugated forms, with glucuronidated bromotyrosine representing approximately a third, and free bromotyrosine less than 1% of total conjugated bromotyrosine. Total conjugated bromotyrosine was poorly correlated with blood (r² = 0.038) or sputum eosinophils (r² = 0.0069). Compared with participants with nonsevere asthma, participants with severe asthma had significantly higher urinary total conjugated bromotyrosine levels. Urinary total conjugated bromotyrosine was independently associated with asthma severity, correlated with the number of asthma exacerbations, and served as a predictor of asthma exacerbation risk over 1-yr of follow-up. [ABSTRACT FROM AUTHOR]