Associations of serum trimethylamine N-oxide and its precursors with colorectal cancer risk in the Prostate, Lung, Colorectal, Ovarian Cancer Screening Trial Cohort.

BACKGROUND: Dietary intake influences gut microbiome composition, which in turn may be associated with colorectal cancer (CRC). Associations of the gut microbiome with colorectal carcinogenesis may be mediated through bacterially regulated, metabolically active metabolites, including trimethylamine N-oxide (TMAO) and its precursors, choline, L-carnitine, and betaine. METHODS: Prospective associations of circulating TMAO and its precursors with CRC risk were investigated. TMAO, choline, betaine, and L-carnitine were measured in baseline serum samples from 761 incident CRC cases and 1:1 individually matched controls in the prospective Prostate, Lung, Colorectal, Ovarian Cancer Screening Trial Cohort using targeted fully quantitative liquid chromatography tandem mass spectrometry panels. Prospective associations of the metabolites with CRC risk, using multivariable conditional logistic regression, were measured. Associations of a priori-selected dietary exposures with the four metabolites were also investigated. RESULTS: TMAO and its precursors were not associated with CRC risk overall, but TMAO and choline were positively associated with higher risk for distal CRC (continuous ORQ90 vs. Q10 [95% CI] = 1.90 [CI, 1.24-2.92; p = .003] and 1.26 [1.17-1.36; p < .0001], respectively). Conversely, choline was inversely associated with rectal cancer (ORQ90 vs. Q10 [95% CI] = 0.77 [0.76-0.79; p < .001]). Red meat, which was previously associated with CRC risk in the Prostate, Lung, Colorectal, Ovarian Cancer Screening Trial Cohort , was positively associated with TMAO (Spearman rho = 0.10; p = .0003). CONCLUSIONS: Serum TMAO and choline may be associated with higher risk of distal CRC, and red meat may be positively associated with serum TMAO. These findings provide insight into a potential microbially mediated mechanism underlying CRC etiology.

Tumor cell-derived spermidine promotes a pro-tumorigenic immune microenvironment in glioblastoma via CD8+ T cell inhibition.

The glioblastoma microenvironment is enriched in immunosuppressive factors that potently interfere with the function of cytotoxic T lymphocytes. Cancer cells can directly impact the immune system, but the mechanisms driving these interactions are not completely clear. Here we demonstrate that the polyamine metabolite spermidine is elevated in the glioblastoma tumor microenvironment. Exogenous administration of spermidine drives tumor aggressiveness in an immune-dependent manner in pre-clinical mouse models via reduction of CD8+ T cell frequency and phenotype. Knockdown of ornithine decarboxylase, the rate-limiting enzyme in spermidine synthesis, did not impact cancer cell growth in vitro but did result in extended survival. Furthermore, glioblastoma patients with a more favorable outcome had a significant reduction in spermidine compared to patients with a poor prognosis. Our results demonstrate that spermidine functions as a cancer cell-derived metabolite that drives tumor progression by reducing CD8+T cell number and function.

Trimethylamine N-oxide is associated with long-term mortality risk: the multi-ethnic study of atherosclerosis.

AIMS: Little is known about associations of trimethylamine N-oxide (TMAO), a novel gut microbiota-generated metabolite of dietary phosphatidylcholine and carnitine, and its changes over time with all-cause and cause-specific mortality in the general population or in different race/ethnicity groups. The study aimed to investigate associations of serially measured plasma TMAO levels and changes in TMAO over time with all-cause and cause-specific mortality in a multi-ethnic community-based cohort. METHODS AND RESULTS: The study included 6,785 adults from the Multi-Ethnic Study of Atherosclerosis. TMAO was measured at baseline and year 5 using mass spectrometry. Primary outcomes were adjudicated all-cause mortality and cardiovascular disease (CVD) mortality. Secondary outcomes were deaths due to kidney failure, cancer, or dementia obtained from death certificates. Cox proportional hazards models with time-varying TMAO and covariates assessed the associations with adjustment for sociodemographics, lifestyles, diet, metabolic factors, and comorbidities. During a median follow-up of 16.9 years, 1704 participants died and 411 from CVD. Higher TMAO levels associated with higher risk of all-cause mortality [hazard ratio (HR): 1.12, 95% confidence interval (CI): 1.08-1.17], CVD mortality (HR: 1.09, 95% CI: 1.00-1.09), and death due to kidney failure (HR: 1.44, 95% CI: 1.25-1.66) per inter-quintile range, but not deaths due to cancer or dementia. Annualized changes in TMAO levels associated with higher risk of all-cause mortality (HR: 1.10, 95% CI: 1.05-1.14) and death due to kidney failure (HR: 1.54, 95% CI: 1.26-1.89) but not other deaths. CONCLUSION: Plasma TMAO levels were positively associated with mortality, especially deaths due to cardiovascular and renal disease, in a multi-ethnic US cohort.

A gut microbial metabolite of dietary polyphenols reverses obesity-driven hepatic steatosis.

The molecular mechanisms by which dietary fruits and vegetables confer cardiometabolic benefits remain poorly understood. Historically, these beneficial properties have been attributed to the antioxidant activity of flavonoids. Here, we reveal that the host metabolic benefits associated with flavonoid consumption hinge, in part, on gut microbial metabolism. Specifically, we show that a single gut microbial flavonoid catabolite, 4-hydroxyphenylacetic acid (4-HPAA), is sufficient to reduce diet-induced cardiometabolic disease (CMD) burden in mice. The addition of flavonoids to a high fat diet heightened the levels of 4-HPAA within the portal plasma and attenuated obesity, and continuous delivery of 4-HPAA was sufficient to reverse hepatic steatosis. The antisteatotic effect was shown to be associated with the activation of AMP-activated protein kinase α (AMPKα). In a large survey of healthy human gut metagenomes, just over one percent contained homologs of all four characterized bacterial genes required to catabolize flavonols into 4-HPAA. Our results demonstrate the gut microbial contribution to the metabolic benefits associated with flavonoid consumption and underscore the rarity of this process in human gut microbial communities.

Disruption of the Gut Microbiota Confers Cisplatin Resistance in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the leading cause of gynecologic cancer death. Despite initial responses to intervention, up to 80% of patient tumors recur and require additional treatment. Retrospective clinical analysis of patients with ovarian cancer indicates antibiotic use during chemotherapy treatment is associated with poor overall survival. Here, we assessed whether antibiotic (ABX) treatment would impact growth of EOC and sensitivity to cisplatin. Immunocompetent or immunocompromised mice were given untreated control or ABX-containing (metronidazole, ampicillin, vancomycin, and neomycin) water prior to intraperitoneal injection with EOC cells, and cisplatin therapy was administered biweekly until endpoint. Tumor-bearing ABX-treated mice exhibited accelerated tumor growth and resistance to cisplatin therapy compared with control treatment. ABX treatment led to reduced apoptosis, increased DNA damage repair, and enhanced angiogenesis in cisplatin-treated tumors, and tumors from ABX-treated mice contained a higher frequency of cisplatin-augmented cancer stem cells than control mice. Stool analysis indicated nonresistant gut microbial species were disrupted by ABX treatment. Cecal transplants of microbiota derived from control-treated mice was sufficient to ameliorate chemoresistance and prolong survival of ABX-treated mice, indicative of a gut-derived tumor suppressor. Metabolomics analyses identified circulating gut-derived metabolites that were altered by ABX treatment and restored by recolonization, providing candidate metabolites that mediate the cross-talk between the gut microbiome and ovarian cancer. Collectively, these findings indicate that an intact microbiome functions as a tumor suppressor in EOC, and perturbation of the gut microbiota with ABX treatment promotes tumor growth and suppresses cisplatin sensitivity. SIGNIFICANCE: Restoration of the gut microbiome, which is disrupted following antibiotic treatment, may help overcome platinum resistance in patients with epithelial ovarian cancer. See related commentary by Hawkins and Nephew, p. 4511.

Longitudinal Associations of Plasma TMAO and Related Metabolites with Cognitive Impairment and Dementia in Older Adults: The Cardiovascular Health Study.

BACKGROUND: Animal studies suggest that gut microbiome metabolites such as trimethylamine N-oxide (TMAO) may influence cognitive function and dementia risk. However potential health effects of TMAO and related metabolites remain unclear. OBJECTIVE: We examined prospective associations of TMAO, γ-butyrobetaine, crotonobetaine, carnitine, choline, and betaine with risk of cognitive impairment and dementia among older adults aged 65 years and older in the Cardiovascular Health Study (CHS). METHODS: TMAO and metabolites were measured in stored plasma specimens collected at baseline. Incident cognitive impairment was assessed using the 100-point Modified Mini-Mental State Examination administered serially up to 7 times. Clinical dementia was identified using neuropsychological tests adjudicated by CHS Cognition Study investigators, and by ICD-9 codes from linked Medicare data. Associations of each metabolite with cognitive outcomes were assessed using Cox proportional hazards models. RESULTS: Over a median of 13 years of follow-up, 529 cases of cognitive impairment, and 522 of dementia were identified. After multivariable adjustment for relevant risk factors, no associations were seen with TMAO, carnitine, choline, or betaine. In contrast, higher crotonobetaine was associated with 20-32% higher risk of cognitive impairment and dementia per interquintile range (IQR), while γ-butyrobetaine was associated with ∼25% lower risk of the same cognitive outcomes per IQR.∥Conclusion:These findings suggest that γ-butyrobetaine, crotonobetaine, two gut microbe and host metabolites, are associated with risk of cognitive impairment and dementia. Our results indicate a need for mechanistic studies evaluating potential effects of these metabolites, and their interconversion on brain health, especially later in life.

Stable isotope dilution mass spectrometry quantification of hydrogen sulfide and thiols in biological matrices.

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 < 0.001), and a marked reduction following gut microbiota suppression (mean 33.8 ±â€¯17.7%, p < 0.001), with large variations in gut microbiota contribution among subjects (range 6.0-66.7% reduction with antibiotics). CONCLUSIONS: A stable-isotope-dilution LC-MS/MS method is presented for the simultaneous quantification of Total H2S and multiple thiols in biological matrices. We then use this assay panel to show a striking age-related decline and gut microbiota contribution to circulating Total H2S levels in humans.

The pattern of apolipoprotein A-I lysine carbamylation reflects its lipidation state and the chemical environment within human atherosclerotic aorta.

Protein lysine carbamylation is an irreversible post-translational modification resulting in generation of homocitrulline (N-ε-carbamyllysine), which no longer possesses a charged ε-amino moiety. Two distinct pathways can promote protein carbamylation. One results from urea decomposition, forming an equilibrium mixture of cyanate (CNO-) and the reactive electrophile isocyanate. The second pathway involves myeloperoxidase (MPO)-catalyzed oxidation of thiocyanate (SCN-), yielding CNO- and isocyanate. Apolipoprotein A-I (apoA-I), the major protein constituent of high-density lipoprotein (HDL), is a known target for MPO-catalyzed modification in vivo, converting the cardioprotective lipoprotein into a proatherogenic and proapoptotic one. We hypothesized that monitoring site-specific carbamylation patterns of apoA-I recovered from human atherosclerotic aorta could provide insights into the chemical environment within the artery wall. To test this, we first mapped carbamyllysine obtained from in vitro carbamylation of apoA-I by both the urea-driven (nonenzymatic) and inflammatory-driven (enzymatic) pathways in lipid-poor and lipidated apoA-I (reconstituted HDL). Our results suggest that lysine residues within proximity of the known MPO-binding sites on HDL are preferentially targeted by the enzymatic (MPO) carbamylation pathway, whereas the nonenzymatic pathway leads to nearly uniform distribution of carbamylated lysine residues along the apoA-I polypeptide chain. Quantitative proteomic analyses of apoA-I from human aortic atheroma identified 16 of the 21 lysine residues as carbamylated and suggested that the majority of apoA-I carbamylation in vivo occurs on "lipid-poor" apoA-I forms via the nonenzymatic CNO- pathway. Monitoring patterns of apoA-I carbamylation recovered from arterial tissues can provide insights into both apoA-I structure and the chemical environment within human atheroma.

Gut microbiota-dependent metabolite trimethylamine N-oxide (TMAO) and cardiovascular risk in patients with suspected functionally relevant coronary artery disease (fCAD).

BACKGROUND: Trimethylamine N-oxide (TMAO) has been associated with cardiovascular outcomes. However, the diagnostic value of TMAO and its precursors have not been assessed for functionally relevant coronary artery disease (fCAD) and its prognostic potential in this setting needs to be evaluated. METHODS: Among 1726 patients with suspected fCAD serum TMAO, and its precursors betaine, choline and carnitine, were quantified using liquid chromatography tandem mass spectrometry. Diagnosis of fCAD was performed by myocardial perfusion single photon emission tomography (MPI-SPECT) and coronary angiography blinded to marker concentrations. Incident all-cause death, cardiovascular death (CVD) and myocardial infarction (MI) were assessed during 5-years follow-up. RESULTS: Concentrations of TMAO, betaine, choline and carnitine were significantly higher in patients with fCAD versus those without (TMAO 5.33 µM vs 4.66 µM, p < 0.001); however, diagnostic accuracy was low (TMAO area under the receiver operating curve [AUC]: 0.56, 95% CI [0.53-0.59], p < 0.001). In prognostic analyses, TMAO, choline and carnitine above the median were associated with significantly (p < 0.001 for all) higher cumulative events for death and CVD during 5-years follow-up. TMAO remained a significant predictor for death and CVD even in full models adjusted for renal function (HR = 1.58 (1.16, 2.14), p = 0.003; HR = 1.66 [1.07, 2.59], p = 0.025). Prognostic discriminative accuracy for TMAO was good and robust for death and CVD (2-years AUC for CVD 0.73, 95% CI [0.65-0.80]). CONCLUSION: TMAO and its precursors, betaine, choline and carnitine were significantly associated with fCAD, but with limited diagnostic value. TMAO was a strong predictor for incident death and CVD in patients with suspected fCAD. CLINICAL TRIAL REGISTRATION: NCT01838148.

Olfactory receptor 2 in vascular macrophages drives atherosclerosis by NLRP3-dependent IL-1 production.

Atherosclerosis is an inflammatory disease of the artery walls and involves immune cells such as macrophages. Olfactory receptors (OLFRs) are G protein­coupled chemoreceptors that have a central role in detecting odorants and the sense of smell. We found that mouse vascular macrophages express the olfactory receptor Olfr2 and all associated trafficking and signaling molecules. Olfr2 detects the compound octanal, which activates the NLR family pyrin domain containing 3 (NLRP3) inflammasome and induces interleukin-1ß secretion in human and mouse macrophages. We found that human and mouse blood plasma contains octanal, a product of lipid peroxidation, at concentrations sufficient to activate Olfr2 and the human ortholog olfactory receptor 6A2 (OR6A2). Boosting octanal levels exacerbated atherosclerosis, whereas genetic targeting of Olfr2 in mice significantly reduced atherosclerotic plaques. Our findings suggest that inhibiting OR6A2 may provide a promising strategy to prevent and treat atherosclerosis.

Gut Microbiome-Dependent Metabolic Pathways and Risk of Lethal Prostate Cancer: Prospective Analysis of a PLCO Cancer Screening Trial Cohort.

BACKGROUND: Diet and the gut microbiome have a complex interaction that generates metabolites with an unclear effect on lethal prostate cancer risk. Identification of modifiable risk factors for lethal prostate cancer is challenging given the long natural history of this disease and difficulty of prospectively identifying lethal cancers. METHODS: Mass spectrometry was performed on baseline serum samples collected from 173 lethal prostate cancer cases and 519 controls enrolled in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening trial. Baseline serum levels of choline, carnitine, betaine, γ-butyrobetaine, crotonobetaine, phenylacetylglutamine, hippuric acid, and p-cresol sulfate were quantified and analyzed by quartile. Conditional multivariable logistic regression analysis associated analyte levels with lethal prostate cancer incidence after adjusting for body mass index and PSA. The Cochran-Armitage test evaluated analyte level trends across quartiles. RESULTS: Relative to those in the first quartile, cases with the highest baseline levels of choline (Q4 OR: 2.19; 95% CI, 1.23-3.90; P-trend: 0.005) and betaine (Q4 OR: 1.86; 95% CI, 1.05-3.30; P-trend: 0.11) exhibited increased odds of developing lethal prostate cancer. Higher baseline serum levels of phenylacetylglutamine (Q4 OR: 2.55; 95% CI, 1.40-4.64; P-trend: 0.003), a gut microbiome metabolite of phenylalanine with adrenergic activity, were also associated with lethal prostate cancer. CONCLUSIONS: Baseline serum levels of one-carbon methyl donors and adrenergic compounds resulting from human and gut microbiota-mediated metabolism are associated with increased lethal prostate cancer risk. IMPACT: Dietary composition, circulating metabolite levels, and downstream signaling pathways may represent modifiable risk factors associated with incident lethal prostate cancer. Beta-adrenergic blockade represents an additional target for oncologic risk reduction.

Longitudinal Plasma Measures of Trimethylamine N-Oxide and Risk of Atherosclerotic Cardiovascular Disease Events in Community-Based Older Adults.

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, <6%). Incident ASCVD (myocardial infarction, fatal coronary heart disease, stroke, sudden cardiac death, or other atherosclerotic death) was centrally adjudicated using medical records. Risk was assessed by multivariable Cox proportional hazards regression, including time-varying demographics, lifestyle factors, medical history, laboratory measures, and dietary habits. Potential mediating effects and interaction by estimated glomerular filtration rate (eGFR) were assessed. During prospective follow-up, 1766 incident and 897 recurrent ASCVD events occurred. After multivariable adjustment, higher levels of TMAO were associated with a higher risk of incident ASCVD, with extreme quintile hazard ratio (HR) compared with the lowest quintile=1.21 (95% CI, 1.02-1.42; P-trend=0.029). This relationship appeared mediated or confounded by eGFR (eGFR-adjusted HR, 1.07; 95% CI, 0.90-1.27), as well as modified by eGFR (P-interaction <0.001). High levels of TMAO were associated with higher incidence of ASCVD in the presence of impaired renal function (eGFR <60 mL/min per 1.73 m2: HR, 1.56 [95% CI, 1.13-2.14]; P-trend=0.007), but not normal or mildly reduced renal function (eGFR ≥60 mL/min per 1.73 m2: HR, 1.03 [95% CI, 0.85-1.25]; P-trend=0.668). Among individuals with prior ASCVD, TMAO associated with higher risk of recurrent ASCVD (HR, 1.25 [95% CI, 1.01-1.56]; P-trend=0.009), without significant modification by eGFR. Conclusions In this large community-based cohort of older US adults, serial measures of TMAO were associated with higher risk of incident ASCVD, with apparent modification by presence of impaired renal function and with higher risk of recurrent ASCVD.

A surgical method for continuous intraportal infusion of gut microbial metabolites in mice.

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.

Inhibition of microbiota-dependent TMAO production attenuates chronic kidney disease in mice.

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.

Bile acids profile, histopathological indices and genetic variants for non-alcoholic fatty liver disease progression.

OBJECTIVE: Metabolomic studies suggest plasma levels of bile acids (BAs) are elevated amongst subjects with non-alcoholic fatty liver disease (NAFLD) compared to healthy controls. However, it remains unclear whether or not specific BAs are associated with the clinically relevant transition from nonalcoholic fatty liver (i.e. simple steatosis) to non-alcoholic steatohepatitis (NASH), or enhanced progression of hepatic fibrosis, or genetic determinants of NAFLD/NASH. METHODS: Among sequential subjects (n=102) undergoing diagnostic liver biopsy, we examined the associations of a broad panel of BAs with distinct histopathological features of NAFLD, the presence of NASH, and their associations with genetic variants linked to NAFLD and NASH. RESULTS: Plasma BA alterations were observed through the entire spectrum of NAFLD, with several glycine conjugated forms of the BAs demonstrating significant associations with higher grades of inflammation and fibrosis. Plasma 7-Keto-DCA levels showed the strongest associations with advanced stages of hepatic fibrosis [odds ratio(95% confidence interval)], 4.2(1.2-16.4), NASH 24.5(4.1-473), and ballooning 18.7(4.8-91.9). Plasma 7-Keto-LCA levels were associated with NASH 9.4(1.5-185) and ballooning 5.9(1.4-28.8). Genetic variants at several NAFLD/NASH loci were nominally associated with increased levels of 7-Keto- and glycine-conjugated forms of BAs, and the NAFLD risk allele at the TRIB1 locus showed strong tendency toward increased plasma levels of GCA (p=0.02) and GUDCA (p=0.009). CONCLUSIONS: Circulating bile acid levels are associated with histopathological and genetic determinants of the transition from simple hepatic steatosis into NASH. Further studies exploring the potential involvement of bile acid metabolism in the development and/or progression of distinct histopathological features of NASH are warranted.

Effects of Smoking and Smoking Cessation on the Intestinal Microbiota.

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.

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.

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<0.001). After adjustment for clinical factors, the association persisted for cardiovascular death (ORadj 1.89, 95% CI, 1.03-3.45, P trend=0.027) and stroke (ORadj 2.01, 95% CI, 1.01-4.01, P trend=0.022), but was slightly attenuated after adjustment for cardiovascular biomarkers (cardiovascular death: ORadj 1.74, 95% CI, 0.88-3.45, P trend=0.079; and stroke: ORadj 1.82, 95% CI, 0.88-3.78, P trend=0.056). The reduction in MACE with ticagrelor was consistent across TMAO quartiles (P 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.

Effects of Lifestyle Intervention on Plasma Trimethylamine N-Oxide in Obese Adults.

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.

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

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.

The utility of biomarkers in diagnosis of aspirin exacerbated respiratory disease.

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 < 0.05). ULTE4 levels and blood eosinophils were significantly higher in AERD as compared to ATA (p = 0.004, p < 0.0001, respectively). whereas uBrTyr levels were not significantly different between both asthma phenotypes (p = 0.34). Asthmatics with high levels of uBrTyr (> 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.