Coronary heart disease (CHD) remains the major health threat in the U.S. The effectiveness of CHD prevention measures, such as dietary and lifestyle interventions vary greatly between individuals and may be ascribed partly to differences in gut microbiome composition and function. The proposed research aims to elucidate a critical metabolic pathway through which human microbiome may contribute to the CHD etiology and account for the between-person responses to diet and lifestyle choices. Specifically, we aim to evaluate, prospectively, the association of the estimated exposure to lipopolysaccharide (LPS), a major component of the Gram-negative bacteria membrane, on CHD risk. We will also examine dietary, lifestyle, and microbial predictors of LPS exposure and create an “empirical metabolic endotoxemia index”. Human LPS-infusion experiments clearly demonstrate that LPS exposure induces potent innate immune reactions, including inflammation and recruitment of leukocytes to endothelium, and predisposes humans to an elevated risk of developing atherosclerosis. Circulating LPS concentrations are challenging to measure in large-scale human population studies due to a short half-life and variability in assay effectiveness due to daily fluctuations and interference from proteins in blood samples. More reliable biomarkers, including LPS binding protein (LBP) and soluble cluster of differentiation 14 (sCD14), characterize LPS exposure, but have yet to be systematically examined in prospective epidemiological studies with documented incident CHD. Further knowledge gaps include a comprehensive evaluation of microbial composition and functional potential in relation to LPS exposure. To address these scientific gaps directly, we aim to 1) evaluate LBP and sCD14 in relation to CHD risk, 2) examine dietary and lifestyle predictors of LBP/sCD14 and derive an empirical metabolic endotoxemia index of these biomarkers, 3) establish temporal relationships of changes in the empirical index with changes in LBP/sCD14 levels, as well as CHD incidence, and 4) investigate the role of the microbiome in determining circulating levels of LBP/sCD14 and their impact on mediating diet/lifestyle associations. These aims will be realized among men and women participating in the Health Professionals Follow-up Study (HPFS) and the Nurses’ Health Study II (NHSII), who provided multiple blood and fecal samples. In addition to stored biospecimens, these studies also offer rich, existing data, including gut metagenome and metatranscriptome data, dietary assessments by 7-day diet records, and three decades of follow-up of diet, lifestyle, and CHD incidence, that allow us to examine the aims in an efficient and comprehensive manner. Data to be generated from this project will provide novel evidence that elucidates the complex inter-relationships between diet/lifestyle, microbial composition and functional potential, LPS exposure, and CHD incidence. The evidence will also inform the develo...