Sex-Associated Metabolites and Incident Stroke, Incident Coronary Heart Disease, Hypertension, and Chronic Kidney Disease in the REGARDS Cohort.

BACKGROUND: Sex disparities exist in cardiometabolic diseases. Metabolomic profiling offers insight into disease mechanisms, as the metabolome is influenced by environmental and genetic factors. We identified metabolites associated with sex and determined if sex-associated metabolites are associated with incident stoke, incident coronary heart disease, prevalent hypertension, and prevalent chronic kidney disease. METHODS AND RESULTS: Targeted metabolomics was conducted for 357 metabolites in the REGARDS (Reasons for Geographic and Racial Differences in Stroke) case-cohort substudy for incident stroke. Weighted logistic regression models were used to identify metabolites associated with sex in REGARDS. Sex-associated metabolites were replicated in the HyperGEN (Hypertension Genetic Epidemiology Network) and using the literature. Weighted Cox proportional hazard models were used to evaluate associations between metabolites and incident stroke. Cox proportional hazard models were used to evaluate associations between metabolites and incident coronary heart disease. Weighted logistic regression models were used to evaluate associations between metabolites and hypertension and chronic kidney disease. Fifty-one replicated metabolites were associated with sex. Higher levels of 6 phosphatidylethanolamines were associated with incident stroke. No metabolites were associated with incident coronary heart disease. Higher levels of uric acid and leucine and lower levels of a lysophosphatidylcholine were associated with hypertension. Higher levels of indole-3-lactic acid, 7 phosphatidylethanolamines, and uric acid, and lower levels of betaine and bilirubin were associated with chronic kidney disease. CONCLUSIONS: These findings suggest that the sexual dimorphism of the metabolome may contribute to sex differences in stroke, hypertension, and chronic kidney disease.

Omega-3 Fatty Acids and Risk of Ischemic Stroke in REGARDS.

We examined associations between lipidomic profiles and incident ischemic stroke in the REasons for Geographic and Racial Differences in Stroke (REGARDS) cohort. Plasma lipids (n = 195) were measured from baseline blood samples, and lipids were consolidated into underlying factors using exploratory factor analysis. Cox proportional hazards models were used to test associations between lipid factors and incident stroke, linear regressions to determine associations between dietary intake and lipid factors, and the inverse odds ratio weighting (IORW) approach to test mediation. The study followed participants over a median (IQR) of 7 (3.4-11) years, and the case-cohort substudy included 1075 incident ischemic stroke and 968 non-stroke participants. One lipid factor, enriched for docosahexaenoic acid (DHA, an omega-3 fatty acid), was inversely associated with stroke risk in a base model (HR = 0.84; 95%CI 0.79-0.90; P = 8.33 × 10-8) and fully adjusted model (HR = 0.88; 95%CI 0.83-0.94; P = 2.79 × 10-4). This factor was associated with a healthy diet pattern (ß = 0.21; 95%CI 0.12-0.30; P = 2.06 × 10-6), specifically with fish intake (ß = 1.96; 95%CI 0.95-2.96; P = 1.36 × 10-4). DHA was a mediator between fish intake and incident ischemic stroke (30% P = 5.78 × 10-3). Taken together, DHA-containing plasma lipids were inversely associated with incident ischemic stroke and mediated the relationship between fish intake and stroke risk.

Plasma Metabolites and Life's Simple 7 in REGARDS.

BACKGROUND: The American Heart Association's Life's Simple 7 (LS7) is a health metric that captures important factors associated with cardiovascular and cerebrovascular health. Previous studies highlight the potential of plasma metabolites to serve as a marker for lifestyle and health behavior that could be a target for stroke prevention. The objectives of this study were to identify metabolites that were associated with LS7 and incident ischemic stroke and mediate the relationship between the two. METHODS: Targeted metabolomic profiling of 162 metabolites by liquid chromatography-tandem mass spectrometry was used to identify candidate metabolites in a stroke case-cohort nested within the REGARDS study (Reasons for Geographic and Racial Differences in Stroke). Weighted linear regression and weighted Cox proportional hazard models were used to identify metabolites that were associated with LS7 and incident ischemic stroke, respectively. Effect measures were based on a 1-SD change in metabolite level. Metabolite mediators were examined using inverse odds ratio weighting mediation analysis. RESULTS: The study comprised 1075 ischemic stroke cases and 968 participants in the random cohort sample. Three out of 162 metabolites were associated with the overall LS7 score including guanosine (ß, -0.46 [95% CI, -0.65 to -0.27]; P=2.87×10-6), cotinine (ß, -0.49 [95% CI, -0.70 to -0.28]; P=7.74×10-6), and acetylneuraminic acid (ß, -0.59 [95% CI, -0.77 to -0.42]; P=4.29×10-11). Guanosine (hazard ratio, 1.47 [95% CI, 1.31-1.65]; P=6.97×10-11), cotinine (hazard ratio, 1.30 [95% CI, 1.16-1.44]; P=2.09×10-6), and acetylneuraminic acid (hazard ratio, 1.29 [95% CI, 1.15-1.45]; P=9.24×10-6) were associated with incident ischemic stroke. The mediation analysis identified guanosine (27% mediation, indirect effect; P=0.002), cotinine (30% mediation, indirect effect; P=0.004), and acetylneurminic acid (22% mediation, indirect effect; P=0.041) partially mediated the relationship between LS7 and ischemic stroke. CONCLUSIONS: We identified guanosine, cotinine, and acetylneuraminic acid that were associated with LS7, incident ischemic stroke, and mediated the relationship between LS7 and ischemic stroke.

Trimethylamine N-Oxide and White Matter Hyperintensity Volume Among Patients With Acute Ischemic Stroke.

Importance: Although increasing evidence suggests that trimethylamine N-oxide (TMAO) is associated with atherosclerosis, little is known about whether TMAO and its related metabolites (ie, choline, betaine, and carnitine) are associated with small vessel disease. Objective: To evaluate the association between TMAO and its related metabolites with features of cerebral small vessel disease, including white matter hyperintensity volume (WMHV) and acute lacunar infarction. Design, Setting, and Participants: This cross-sectional study included patients enrolled in the Specialized Programs of Translational Research in Acute Stroke biorepository. The registry included 522 patients with acute ischemic stroke who were 18 years or older who presented at the Massachusetts General Hospital or Brigham and Women's Hospital within 9 hours after onset between January 2007 and April 2010. The analyses in this study were conducted between November 2022 and April 2023. Exposures: Plasma TMAO, choline, betaine, and carnitine were measured by liquid chromatography-tandem mass spectrometry. Main Outcomes and Measures: WMHV was quantified by a semiautomated approach using signal intensity threshold with subsequent manual editing. Ischemic stroke subtype was classified using the Causative Classification System. Results: Among 351 patients included in this study, the mean (SD) age was 69 (15) years; 209 patients (59.5%) were male and had a median (IQR) admission National Institute of Health Stroke Scale of 6 (3-13). The magnetic resonance imaging subgroup consisted of 291 patients with a mean (SD) age of 67 (15) years. Among these, the median (IQR) WMHV was 3.2 (1.31-8.4) cm3. TMAO was associated with WMHV after adjustment for age and sex (ß, 0.15; 95% CI, 0.01-0.29; P < .001). TMAO remained significant in a multivariate analysis adjusted for age, sex, hypertension, diabetes, and smoking (ß, 0.14; 95% CI, 0-0.29; P = .05). TMAO was associated with lacunar stroke but not other ischemic stroke subtypes in a model adjusted for age, sex, hypertension, diabetes, and smoking (OR, 1.67; 95% CI, 1.05-2.66; P = .03). Conclusions and Relevance: In this observational study, TMAO was associated with cerebral small vessel disease determined by WMHV and acute lacunar infarction. The association was independent of traditional vascular risk factors.

Acetylglutamine Differentially Associated with First-Time Versus Recurrent Stroke.

Approximately one-quarter of strokes occur in individuals with prior stroke. Despite the advancement in secondary stroke prevention, the long-term risk of recurrent stroke has remained unchanged. The objective of this study was to identify metabolite risk markers that are associated with recurrent stroke. We performed targeted metabolomic profiling of 162 metabolites by liquid chromatography-tandem mass spectrometry in baseline plasma in a stroke case-cohort study nested within the Reasons for Geographic and Racial Differences in Stroke (REGARDS) study, an observational cohort study of 30,239 individuals aged 45 and older enrolled in 2003-2007. Weighted Cox proportional hazard models were used to identify metabolites that had a differential effect on first-time versus recurrent stroke using an interaction term between metabolite and prior stroke at baseline (yes or no). The study included 1391 incident stroke cases identified during 7.1 ± 4.5 years of follow-up and 1050 participants in the random cohort sample. Among 162 metabolites, 13 candidates had a metabolite-by-prior stroke interaction at a p-value <0.05, with one metabolite, acetylglutamine, surpassing the Bonferroni adjusted p-value threshold (p for interaction = 5.78 × 10-5). In an adjusted model that included traditional stroke risk factors, acetylglutamine was associated with recurrent stroke (HR = 2.27 per SD increment, 95% CI = 1.60-3.20, p = 3.52 × 10-6) but not with first-time stroke (HR = 0.96 per SD increment, 95% CI = 0.87-1.06, p = 0.44). Acetylglutamine was associated with recurrent stroke but not first-time stroke, independent of traditional stroke risk factors. Future studies are warranted to elucidate the pathogenesis of acetylglutamine and recurrent stroke risk.

Hypertension severity, apparent treatment resistant hypertension and hyperuricemia in the REasons for Geographic And Racial Differences in Stroke (REGARDS) study.

BACKGROUND: Prior studies have identified an association between hypertension and hyperuricemia; however, there has been limited research on the association between hypertension severity and hyperuricemia. METHOD: We studied 997 Black and white adults with serum urate data from the reasons for geographic and racial differences in stroke (REGARDS) study. Hypertension was defined as SBP ≥ 140 mmHg or DBP ≥ 90 mmHg or self-reported use of antihypertensive medication. Apparent treatment-resistant hypertension (aTRH) was defined as a SBP ≥ 140 mmHg or DBP ≥ 90 mmHg with concurrent use of three classes of antihypertensive medications, or taking four or more classes of antihypertensive medication regardless of BP level. Controlled BP was defined as SBP <140 mmHg and DBP <90 mmHg. RESULTS: Overall 5.9% of participants had aTRH and 36.6% had hyperuricemia, defined as serum urate >7.0 mg/dl for men and >6.0 mg/dl for women. After full multivariable adjustment, the odds ratio (OR) for hyperuricemia associated with hypertension was 1.60 [95% confidence interval (95% CI): 1.06-2.40]. Compared to participants not taking antihypertensive medication, the ORs for hyperuricemia for participants taking one, two and three classes of antihypertensive medication without aTRH were 1.98 (95% CI: 1.23-3.20), 2.08 (95% CI: 1.25-3.43), 4.31 (95% CI: 2.07-8.97), respectively, and 3.96 (95% CI: 1.75-8.96) for aTRH. Compared to participants without hypertension, the odds ratios for hyperuricemia were 1.67 (95% CI: 1.08-2.58) and 1.46 (95% CI: 0.88-2.44) among those with hypertension with and without controlled BP, respectively. Diuretic use was associated with a higher odds of hyperuricemia. CONCLUSION: This study suggests that individuals taking more classes of antihypertensive medication may benefit from monitoring for hyperuricemia.

Association of Circulating Metabolites With Racial Disparities in Hypertension and Stroke in the REGARDS Study.

BACKGROUND AND OBJECTIVES: In the United States, the risk of stroke is greater among Black compared with that among White individuals. However, the reasons for the difference in stroke incidence are not fully elucidated. We aimed to identify metabolites that account for higher prevalent hypertension and incident ischemic stroke among Black adults. METHODS: We used a stroke case cohort nested within the REasons for Geographic and Racial Differences in Stroke (REGARDS) study. Targeted metabolomic profiling of 162 plasma metabolites was performed by liquid chromatography-tandem mass spectrometry. We identified metabolites that were associated with prevalent hypertension and incident ischemic stroke and mediated the relationship between hypertension and ischemic stroke by weighted logistic regression, Cox proportional hazard model, and inverse odds ratio weighting mediation analysis. RESULTS: Incident ischemic stroke cases adjudicated through April 1, 2019 (n = 1,075) were included in the study. The random cohort sample was derived from the full cohort using stratified sampling (n = 968). Among 162 metabolites, gluconic acid was associated with prevalent hypertension in Black adults (odds ratio [OR] 1.86, 95% CI 1.39-2.47, p = 2.58 × 10-5) but not in White adults (OR 1.00, 95% CI 0.80-1.24, p = 0.97; p for interaction = 4.57 × 10-4). Gluconic acid also demonstrated an association with incident ischemic stroke among Black participants (hazard ratio [HR] 1.53, 95% CI 1.28-1.81, p = 1.76 × 10-6) but not White participants (HR 1.16, 95% CI 1.00-1.34, p = 0.057; p for interaction = 0.019). In mediation analysis, gluconic acid mediated 25.4% (95% CI 4.1%-46.8%, p = 0.02) of the association between prevalent hypertension and incident ischemic stroke among Black individuals. Specific socioeconomic factors were linked to elevated gluconic acid level among Black adults in multivariable analysis, including a Southern dietary pattern (ß = 0.18, 95% CI 0.08-0.28, p < 0.001), lower educational attainment (ß = 0.45, 95% CI 0.19-0.72, p = 0.001), and a lack of exercise (ß = 0.26, 95% CI 0.01-0.51, p = 0.045). DISCUSSION: Gluconic acid is associated with prevalent hypertension and incident ischemic stroke and mediates the relationship between hypertension and ischemic stroke in Black but not White adults. Gluconic acid is a biomarker that is associated with social determinants of health including a Southern diet, low educational attainment, and low physical activity.

Gut microbiota-associated metabolites and risk of ischemic stroke in REGARDS.

Several metabolite markers are independently associated with incident ischemic stroke. However, prior studies have not accounted for intercorrelated metabolite networks. We used exploratory factor analysis (EFA) to determine if metabolite factors were associated with incident ischemic stroke. Metabolites (n = 162) were measured in a case-control cohort nested in the REasons for Geographic and Racial Differences in Stroke (REGARDS) study, which included 1,075 ischemic stroke cases and 968 random cohort participants. Cox models were adjusted for age, gender, race, and age-race interaction (base model) and further adjusted for the Framingham stroke risk factors (fully adjusted model). EFA identified fifteen metabolite factors, each representing a well-defined metabolic pathway. Of these, factor 3, a gut microbiome metabolism factor, was associated with an increased risk of stroke in the base (hazard ratio per one-unit standard deviation, HR = 1.23; 95%CI = 1.15-1.31; P = 1.98 × 10-10) and fully adjusted models (HR = 1.13; 95%CI = 1.06-1.21; P = 4.49 × 10-4). The highest tertile had a 45% increased risk relative to the lowest (HR = 1.45; 95%CI = 1.25-1.70; P = 2.24 × 10-6). Factor 3 was also associated with the Southern diet pattern, a dietary pattern previously linked to increased stroke risk in REGARDS (ß = 0.11; 95%CI = 0.03-0.18; P = 8.75 × 10-3). These findings highlight the role of diet and gut microbial metabolism in relation to incident ischemic stroke.

Plasma Metabolites Link Dietary Patterns to Stroke Risk.

OBJECTIVE: While dietary intake is linked to stroke risk, surrogate markers that could inform personalized dietary interventions are lacking. We identified metabolites associated with diet patterns and incident stroke in a nested cohort from the REasons for Geographic and Racial Differences in Stroke (REGARDS) study. METHODS: Levels of 162 metabolites were measured in baseline plasma from stroke cases (n = 1,198) and random controls (n = 904). We examined associations between metabolites and a plant-based diet pattern previously linked to reduced stroke risk in REGARDS. Secondary analyses included 3 additional stroke-associated diet patterns: a Mediterranean, Dietary Approaches to Stop Hypertension (DASH), and Southern diet. Metabolites were tested using Cox proportional hazards models with incident stroke as the outcome. Replication was performed in the Jackson Heart Study (JHS). Inverse odds ratio-weighted mediation was used to determine whether metabolites mediated the association between a plant-based diet and stroke risk. RESULTS: Metabolites associated with a plant-based diet included the gut metabolite indole-3-propionic acid (ß = 0.23, 95% confidence interval [CI] [0.14, 0.33], p = 1.14 × 10-6 ), guanosine (ß = -0.13, 95% CI [-0.19, -0.07], p = 6.48 × 10-5 ), gluconic acid (ß = -0.11, 95% CI [-0.18, -0.04], p = 2.06 × 10-3 ), and C7 carnitine (ß = -0.16, 95% CI [-0.24, -0.09], p = 4.14 × 10-5 ). All of these metabolites were associated with both additional diet patterns and altered stroke risk. Mediation analyses identified guanosine (32.6% mediation, p = 1.51 × 10-3 ), gluconic acid (35.7%, p = 2.28 × 10-3 ), and C7 carnitine (26.2%, p = 1.88 × 10-2 ) as mediators linking a plant-based diet to reduced stroke risk. INTERPRETATION: A subset of diet-related metabolites are associated with risk of stroke. These metabolites could serve as surrogate markers that inform dietary interventions. ANN NEUROL 2023;93:500-510.

Nucleosides Associated With Incident Ischemic Stroke in the REGARDS and JHS Cohorts.

BACKGROUND AND OBJECTIVES: Both genetic and environmental factors contribute to stroke risk. We sought to identify novel metabolites associated with incident stroke in the Reasons for Geographic and Racial Differences in Stroke (REGARDS) cohort and determine whether they reflected genetic or environmental variation. METHODS: This was a stroke case-cohort observational study nested in REGARDS. Cases were defined as incident stroke and metabolomic profiles were compared to a randomly selected control cohort. In baseline plasma samples, 162 metabolites were measured using liquid chromatography-tandem mass spectrometry. Cox proportional hazards models were adjusted for age, sex, race, and age by race in the base model. Fully adjusted models included traditional stroke risk factors. Mediation analyses conducted for these stroke risk factors used the metabolite as mediator. Genome-wide associations with the leading candidate metabolites were calculated using array data. Replication analyses in the Jackson Heart Study (JHS) were conducted using random effects meta-analysis. RESULTS: There were 2,043 participants who were followed over an average period of 7.1 years, including 1,075 stroke cases and 968 random controls. Nine metabolites were associated with stroke in the base model, 8 of which were measured and remained significant in meta-analysis with JHS. In the fully adjusted model in REGARDS, guanosine (hazard ratio [HR] 1.34, 95% CI 1.18-1.53; p = 7.26 × 10-6) and pseudouridine (HR 1.28, 95% CI 1.13-1.45; p = 1.03 × 10-4) were associated with incident ischemic stroke following Bonferroni adjustment. Guanosine also partially mediated the relationship between hypertension and stroke (17.6%) and pseudouridine did not mediate any risk factor. Genome-wide association analysis identified loci rs34631560 and rs34631560 associated with pseudouridine, but these did not explain the association of pseudouridine with stroke. DISCUSSION: Guanosine and pseudouridine are nucleosides associated with incident ischemic stroke independently of other risk factors. Genetic and mediation analyses suggest that environmental exposures rather than genetic variation link nucleoside levels to stroke risk. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that guanosine and pseudouridine are associated with incident stroke.

Genetic correlations between traits associated with hyperuricemia, gout, and comorbidities.

Hypertension, obesity, chronic kidney disease and type 2 diabetes are comorbidities that have very high prevalence among persons with hyperuricemia (serum urate > 6.8 mg/dL) and gout. Here we use multivariate genetic models to test the hypothesis that the co-association of traits representing hyperuricemia and its comorbidities is genetically based. Using Bayesian whole-genome regression models, we estimated the genetic marker-based variance and the covariance between serum urate, serum creatinine, systolic blood pressure (SBP), blood glucose and body mass index (BMI) from two independent family-based studies: The Framingham Heart Study-FHS and the Hypertension Genetic Epidemiology Network study-HyperGEN. The main genetic findings that replicated in both FHS and HyperGEN, were (1) creatinine was genetically correlated only with urate and (2) BMI was genetically correlated with urate, SBP, and glucose. The environmental covariance among the traits was generally highest for trait pairs involving BMI. The genetic overlap of traits representing the comorbidities of hyperuricemia and gout appears to cluster in two separate axes of genetic covariance. Because creatinine is genetically correlated with urate but not with metabolic traits, this suggests there is one genetic module of shared loci associated with hyperuricemia and chronic kidney disease. Another module of shared loci may account for the association of hyperuricemia and metabolic syndrome. This study provides a clear quantitative genetic basis for the clustering of comorbidities with hyperuricemia.

Lipid changes due to fenofibrate treatment are not associated with changes in DNA methylation patterns in the GOLDN study.

Fenofibrate lowers triglycerides (TG) and raises high density lipoprotein cholesterol (HDLc) in dyslipidemic individuals. Several studies have shown genetic variability in lipid responses to fenofibrate treatment. It is, however, not known whether epigenetic patterns are also correlated with the changes in lipids due to fenofibrate treatment. The present study was therefore undertaken to examine the changes in DNA methylation among the participants of Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study. A total of 443 individuals were studied for epigenome-wide changes in DNA methylation, assessed using the Illumina Infinium HumanMethylation450 array, before and after a 3-week daily treatment with 160 mg of fenofibrate. The association between the change in DNA methylation and changes in TG, HDLc, and low-density lipoprotein cholesterol (LDLc) were assessed using linear mixed models adjusted for age, sex, baseline lipids, and study center as fixed effects and family as a random effect. Changes in DNA methylation were not significantly associated with changes in TG, HDLc, or LDLc after 3 weeks of fenofibrate for any CpG. CpG changes in genes known to be involved in fenofibrate response, e.g., PPAR-α, APOA1, LPL, APOA5, APOC3, CETP, and APOB, also did not show evidence of association. In conclusion, changes in lipids in response to 3-week treatment with fenofibrate were not associated with changes in DNA methylation. Studies of longer duration may be required to detect treatment-induced changes in methylation.

Epigenome-wide association study of fasting measures of glucose, insulin, and HOMA-IR in the Genetics of Lipid Lowering Drugs and Diet Network study.

Known genetic susceptibility loci for type 2 diabetes (T2D) explain only a small proportion of heritable T2D risk. We hypothesize that DNA methylation patterns may contribute to variation in diabetes-related risk factors, and this epigenetic variation across the genome can contribute to the missing heritability in T2D and related metabolic traits. We conducted an epigenome-wide association study for fasting glucose, insulin, and homeostasis model assessment of insulin resistance (HOMA-IR) among 837 nondiabetic participants in the Genetics of Lipid Lowering Drugs and Diet Network study, divided into discovery (N = 544) and replication (N = 293) stages. Cytosine guanine dinucleotide (CpG) methylation at ∼470,000 CpG sites was assayed in CD4(+) T cells using the Illumina Infinium HumanMethylation 450 Beadchip. We fit a mixed model with the methylation status of each CpG as the dependent variable, adjusting for age, sex, study site, and T-cell purity as fixed-effects and family structure as a random-effect. A Bonferroni corrected P value of 1.1 × 10(-7) was considered significant in the discovery stage. Significant associations were tested in the replication stage using identical models. Methylation of a CpG site in ABCG1 on chromosome 21 was significantly associated with insulin (P = 1.83 × 10(-7)) and HOMA-IR (P = 1.60 × 10(-9)). Another site in the same gene was significant for HOMA-IR and of borderline significance for insulin (P = 1.29 × 10(-7) and P = 3.36 × 10(-6), respectively). Associations with the top two signals replicated for insulin and HOMA-IR (P = 5.75 × 10(-3) and P = 3.35 × 10(-2), respectively). Our findings suggest that methylation of a CpG site within ABCG1 is associated with fasting insulin and merits further evaluation as a novel disease risk marker.