Choline metabolites and incident cardiovascular disease in a prospective cohort of adults: Coronary Artery Risk Development in Young Adults (CARDIA) Study.

BACKGROUND: The potential role for choline metabolite trimethylamine N-oxide (TMAO) in cardiovascular disease (CVD) has garnered much attention, but there have been limited data from diverse population-based cohorts. Furthermore, few studies have included circulating choline and betaine, which can serve as precursors to TMAO and may independently influence CVD. OBJECTIVE: We quantified prospective associations between 3 choline metabolites and 19-y incident CVD in a population-based cohort and tested effect modification of metabolite-CVD associations by kidney function. METHODS: Data were from the Coronary Artery Risk Development in Young Adults (CARDIA) Study, a prospective cohort with recruitment from 4 US urban centers (year 0: 1985-1986, n = 5115, ages 18-30). The analytic sample included 3444 White and Black males and females, aged 33 to 45, who attended the year 15 follow-up exam and did not have prevalent CVD. TMAO, choline, and betaine were quantitated from stored plasma (-70°C) using liquid-chromatography mass-spectrometry. Nineteen-year incident CVD events (n = 221), including coronary heart disease and stroke, were identified through adjudicated hospitalization records and linkage with the National Death Register. RESULTS: Plasma choline was positively associated with CVD in Cox proportional hazards regression analysis adjusted for demographics, health behaviors, CVD risk factors, and metabolites (hazard ratio: 1.24; 95% CI: 1.09, 1.40 per standard deviation-unit choline). TMAO and betaine were not associated with CVD in an identically adjusted analysis. There was statistical evidence for effect modification by kidney function with CVD positively associated with TMAO and negatively associated with betaine at lower values of estimated glomerular filtration rate (interaction P values: 0.0046 and 0.020, respectively). CONCLUSIONS: Our findings are consistent with a positive association between plasma choline and incident CVD. Among participants with lower kidney function, TMAO was positively, and betaine negatively, associated with CVD. These results further our understanding of the potential role for choline metabolism on CVD risk.

Conjugated bile acids are nutritionally re-programmable antihypertensive metabolites.

BACKGROUND: Hypertension is the largest risk factor affecting global mortality. Despite available medications, uncontrolled hypertension is on the rise, whereby there is an urgent need to develop novel and sustainable therapeutics. Because gut microbiota is now recognized as an important entity in blood pressure regulation, one such new avenue is to target the gut-liver axis wherein metabolites are transacted via host-microbiota interactions. Knowledge on which metabolites within the gut-liver axis regulate blood pressure is largely unknown. METHOD: To address this, we analyzed bile acid profiles of human, hypertensive and germ-free rat models and report that conjugated bile acids are inversely correlated with blood pressure in humans and rats. RESULTS: Notably intervening with taurine or tauro-cholic acid rescued bile acid conjugation and reduced blood pressure in hypertensive rats. Subsequently, untargeted metabolomics uncovered altered energy metabolism following conjugation of bile acids as a mechanism alleviating high blood pressure. CONCLUSION: Together this work reveals conjugated bile acids as nutritionally re-programmable anti-hypertensive metabolites.

Physical activity and diet associations with the gut microbiota in the Coronary Artery Risk Development in Young Adults (CARDIA) study.

BACKGROUND: Gut microbiota may influence metabolic pathways related to chronic health conditions. Evidence for physical activity and diet influences on gut microbial composition exists, but data from diverse population-based cohort studies are limited. OBJECTIVES: We hypothesized that gut microbial diversity and genera are associated with physical activity and diet quality. METHODS: Data were from 537 participants in the Coronary Artery Risk Development in Young Adults (CARDIA) Study, a prospective cohort, who attended the year 30 follow-up examination (2015-2016; aged 47-61 y; 45% Black race/55% White race; 45% men/55% women). The 16S ribosomal RNA marker gene was sequenced from stool DNA, and genus-level taxonomy was assigned. Within-person microbial diversity (α-diversity) was assessed with Shannon diversity index and richness scores; between-person diversity (ß-diversity) measures were generated with principal coordinates analysis (PCoA). Current and long-term physical activity and diet quality measures were derived from data collected over 30 y of follow-up. Multivariable-adjusted regression analysis controlled for: sociodemographic variables (age, race, sex, education, and field center), other health behaviors (smoking, alcohol consumption, and medication use), and adjusted for multiple comparisons with the false discovery rate (<0.20). RESULTS: Based on PCoA ß-diversity, participants' microbial community compositions differed significantly (P < 0.001), with respect to both current and long-term physical activity and diet quality. α-Diversity was associated only with current physical activity (positively) in multivariable-adjusted analysis. Multiple genera (n = 45) were associated with physical activity and fewer with diet (n = 5), including positive associations with Lachnospiraceae UCG-001 and Ruminococcaceae IncertaeSedis with both behaviors. CONCLUSIONS: Physical activity and diet quality were associated with gut microbial composition among 537 participants in the CARDIA study. Multiple genera were associated with physical activity. Physical activity and diet quality were associated with genera consistent with pathways related to inflammation and short-chain fatty acid production.

Gut microbiome and stages of diabetes in middle-aged adults: CARDIA microbiome study.

BACKGROUND: Animal and human studies suggest the gut microbiome is linked to diabetes but additional data are needed on the associations of the gut microbiome to specific diabetes characteristics. The aim of this study was to examine the associations of gut microbiome composition to insulin resistance [Homeostatic Model Assessment of Insulin Resistance (HOMA-IR)], duration of diabetes, and 4 stages of diabetes [normoglycemia, pre-diabetes, and diabetes with (+) and without (-) medication for diabetes]. METHODS: Data are from a sub-sample (n = 605) of Black and White participants from the 30-year follow-up exam of the prospectively followed community-based Coronary Artery Risk Development in Young Adults cohort (2015-2016; aged 48-60 years). Stool samples were collected and sequenced using the 16S ribosomal RNA method. Microbial measures included: α diversity (within-person), ß diversity (between-person), and taxonomies. All analyses were adjusted for demographic, clinical, lifestyle factors, and use of relevant medications (full adjustment). Multivariate linear regression models were used to assess the association of diabetes characteristics with α diversity and genera abundance, while the association with ß diversity was analyzed using permutational multivariate analysis of variance. Statistical significance was set to p-value < 0.05 for α and ß diversity analyses and to q-value < 0.1 for genera abundance analyses. RESULTS: There were 16.7% of participants with pre-diabetes, and 14.4% with diabetes (9% diabetes+) with median (interquartile range) diabetes duration of 5 (5-10) years. In the fully adjusted models, compared to those with no diabetes, longer diabetes duration and the diabetes + group had a lower α diversity. There were significant differences in ß diversity across diabetes-related characteristics. A significantly reduced abundance of butyrate-producing genera was associated with higher HOMA-IR (ex., Anaerostipes and Lachnospiraceae_UCG.004), longer diabetes duration (ex., Agathobacter and Ruminococcus), and diabetes + (ex., Faecalibacterium and Romboutsia). CONCLUSIONS: Our results suggest that an adverse alteration of gut microbiome composition is related to higher insulin resistance, longer diabetes duration, and is present in those persons with diabetes using medications. These diabetes-related characteristics were also associated with lower levels of certain butyrate-producing bacteria that produce health-promoting short-chain fatty acids. Understanding the role of gut microbiota in glucose regulation may provide new strategies to reduce the burden of diabetes.

Batch effects removal for microbiome data via conditional quantile regression.

Batch effects in microbiome data arise from differential processing of specimens and can lead to spurious findings and obscure true signals. Strategies designed for genomic data to mitigate batch effects usually fail to address the zero-inflated and over-dispersed microbiome data. Most strategies tailored for microbiome data are restricted to association testing or specialized study designs, failing to allow other analytic goals or general designs. Here, we develop the Conditional Quantile Regression (ConQuR) approach to remove microbiome batch effects using a two-part quantile regression model. ConQuR is a comprehensive method that accommodates the complex distributions of microbial read counts by non-parametric modeling, and it generates batch-removed zero-inflated read counts that can be used in and benefit usual subsequent analyses. We apply ConQuR to simulated and real microbiome datasets and demonstrate its advantages in removing batch effects while preserving the signals of interest.

Association of the Gut Microbiota With Cognitive Function in Midlife.

Importance: Animal experiments and small clinical studies support a role for the gut microbiota in cognitive functioning. Few studies have investigated gut microbiota and cognition in large community samples. Objective: To examine associations of gut microbial composition with measures of cognition in an established population-based study of middle-aged adults. Design, Setting, and Participants: This cross-sectional study analyzed data from the prospective Coronary Artery Risk Development in Young Adults (CARDIA) cohort in 4 US metropolitan centers between 2015 and 2016. Data were analyzed in 2019 and 2020. Exposures: Stool DNA were sequenced, and the following gut microbial measures were gathered: (1) ß-diversity (between-person) derived with multivariate principal coordinates analysis; (2) α-diversity (within-person), defined as richness (genera count) and the Shannon index (integrative measure of genera richness and evenness); and (3) taxonomy (107 genera, after filtering). Main Outcomes and Measures: Cognitive status was assessed using 6 clinic-administered cognitive tests: Montreal Cognitive Assessment (MoCA), Digit Symbol Substitution Test (DSST), Rey-Auditory Verbal Learning Test (RAVLT), Stroop, category fluency, and letter fluency. A global score measure derived using principal components analysis was also assessed; the first principal component explained 56% of variability. Results: Microbiome data were available on 597 CARDIA participants; mean (SD) age was 55.2 (3.5) years, 268 participants (44.7%) were men, and 270 (45.2%) were Black. In multivariable-adjusted principal coordinates analysis, permutational multivariate analysis of variance tests for ß-diversity were statistically significant for all cognition measures (principal component analysis, P = .001; MoCA, P = .001; DSST, P = .001; RAVLT, P = .001; Stroop, P = .007; category fluency, P = .001) with the exception of letter fluency (P = .07). After adjusting for sociodemographic variables (age, race, sex, education), health behaviors (physical activity, diet, smoking, medication use), and clinical covariates (body mass index, diabetes, hypertension), Barnesiella was positively associated with the first principal component (ß, 0.16; 95% CI, 0.08-0.24), DSST (ß, 1.18; 95% CI, 0.35-2.00), and category fluency (ß, 0.59; 95% CI, 0.31-0.87); Lachnospiraceae FCS020 group was positively associated with DSST (ß, 2.67; 95% CI, 1.10-4.23), and Sutterella was negatively associated with MoCA (ß, -0.27; 95% CI, -0.44 to -0.11). Conclusions and Relevance: In this cross-sectional study, microbial community composition, based on ß-diversity, was associated with all cognitive measures in multivariable-adjusted analysis. These data contribute to a growing body of literature suggesting that the gut microbiota may be associated with cognitive aging, but must be replicated in larger samples and further researched to identify relevant pathways.

Gut Microbiota Composition and Blood Pressure.

Animal models support a role for the gut microbiota in the development of hypertension. There has been a lack of epidemiological cohort studies to confirm these findings in human populations. We examined cross-sectional associations between measures of gut microbial diversity and taxonomic composition and blood pressure (BP) in 529 participants of the biracial (black and white) CARDIA study (Coronary Artery Risk Development in Young Adults). We sequenced V3-V4 regions of the 16S ribosomal RNA marker gene using DNA extracted from stool samples collected at CARDIA's Year 30 follow-up examination (2015-2016; aged 48-60 years). We quantified associations between BP (hypertension [defined as systolic BP ≥140 mm Hg or diastolic BP ≥90 mm Hg or antihypertension medication use] and systolic BP) and within and between-person diversity measures. We conducted genera-specific multivariable-adjusted regression analysis, accounting for multiple comparisons using the false discovery rate. Hypertension and systolic BP were inversely associated with measures of α-diversity, including richness and the Shannon Diversity Index, and were distinguished with respect to principal coordinates based on a similarity matrix of genera abundance. Several specific genera were significantly associated with hypertension and systolic BP, though results were attenuated with adjustment for body mass index. Our findings support associations between within-person and between-person gut microbial community diversity and taxonomic composition and BP in a diverse population-based cohort of middle-aged adults. Future study is needed to define functional pathways that underlie observed associations and identify specific microbial targets for intervention.

Use of the mouse ear vesicant model to evaluate the effectiveness of ebselen as a countermeasure to the nitrogen mustard mechlorethamine.

Previous studies in this and other laboratories have demonstrated that ebselen (EB-1), an organoselenium compound, spares cells from mechlorethamine (HN2) toxicity in vitro. In the present study, the hypothesis that EB-1 will reduce dermal toxicity of HN2 in vivo is put forward and found to have merit. Using the mouse ear vesicant model (MEVM), HN2, applied topically, showed a dose-dependent effect upon ear swelling and thickness 24 h after treatment; whereas tissue injury consistent with vesication was observed at the higher test doses of HN2 (≥ 0.250 µmol per ear). To examine HN2 countermeasure activity using the MEVM, either hydrocortisone (HC), as a positive control, or EB-1, the test countermeasure, was administered as three topical treatments 15 min, 4 and 8 h after HN2 exposure. Using this approach, both HC and EB-1 were found to reduce tissue swelling associated with HN2 toxicity 24 h after exposure to the vesicant. Taken together, these data demonstrate for the first time the effectiveness of EB-1 as a vesicant countermeasure in a relevant in vivo model.

Ebselen reduces the toxicity of mechlorethamine in A-431 cells via inhibition of apoptosis.

A series of test compounds were evaluated for an ability to reduce the toxicity of the nitrogen mustard mechlorethamine (HN2) in vitro. The test compounds included resveratrol, pterostilbene, vitamin C, ebselen, ebselen diselenide, and ebselen-sulfur. Among them, ebselen demonstrated the highest degree of protection against HN2 toxicity. To this end, pretreatment of the cells with ebselen offered protection against the toxicant whereas no protection was observed when cells were first incubated with HN2 and then treated with ebselen. Significant increases in caspase 3 and caspase 9 activities were observed in response to HN2, and ebselen was found to reduce these effects. Taken together, the data presented here indicate that ebselen is an effective countermeasure to nitrogen mustard in vitro, which is worthy of future investigation in vivo.