Circulating gut microbial metabolites and risk of coronary heart disease: a prospective, multi-stage study.

Background: Despite growing evidence linking gut microbiota and microbial metabolites to human cardiometabolic health, few studies have systematically examined circulating microbial metabolites with incident coronary heart disease (CHD). Methods: We conducted a multi-stage metabolomics study involving five prospective cohorts. Discovery involved an untargeted plasma metabolite profiling among 896 incident cases and 896 age-/sex-/race-matched controls (∼300 pairs per race: Black, White, Asian) from the Southern Community Cohort Study (SCCS) and Shanghai Women's Health Study and Shanghai Men's Health Study (SWHS/SMHS). In-silico validation was conducted in the Atherosclerosis Risk in Communities Study (ARIC; N=3539; 663 cases) and Multi-Ethnic Study of Atherosclerosis (MESA; N=3860; 446 cases). Last, a quantitative assay was developed and applied to a new set of 864 cases and 864 age-/sex-/race-matched controls (∼260-340 pairs per race) from SCCS and SWHS/SMHS. Conditional logistic regression estimated odds ratios (ORs) of incident CHD per standard deviation (SD) increase in metabolite levels for discovery and quantitative stages with a nested case-control design. Cox regression was used in ARIC and MESA with a cohort design. All stages were adjusted for similar covariates. Results: The discovery stage identified 73 circulating microbiota-related metabolites associated with incident CHD (FDR<0.10). Sixty-one metabolites were available for in-silico validation, with 24 showing a significant association (p<0.05) in the same direction as discovery. The targeted assay quantified eight of these 24 metabolites, with five significantly associated with incident CHD: imidazole propionate, 3-hydroxy-2-ethylpropionate, 4-hydroxyphenylacetate, trans-4-hydroxyproline, and 3-hydroxybutyrate; OR per SD ranged from 1.18 to 1.27 after adjusting for sociodemographic and lifestyle factors. The targeted assay measured eight other promising microbial metabolites, four of which were significant: trimethylamine N-oxide, phenylacetyl-L-glutamine, 4-hydroxyhippuric acid, and indolepropionate. Most associations were consistent across participant subgroups, although some (e.g., 4-hydroxyphenylacetate) were stronger among Black than White/Asian participants. Other effect modifications were found by age, obesity, and hypertension history. Conclusions and relevance: We identified and validated circulating gut microbial metabolites associated with incident CHD across diverse populations. Our findings offer novel epidemiological evidence on the importance of gut microbial metabolism in CHD development and highlight specific metabolites to prioritize for mechanistic investigation, biomarker validation, and therapeutic development.