Endothelial dysfunction is a central mechanism in atherosclerotic cardiovascular disease (ASCVD) development. Traditional assessments of endothelial function have limitations in accuracy and reproducibility, motivating evaluation of blood-based indices. The Endothelial Activation and Stress Index (EASIX) has emerged as a candidate biomarker in prognostic research across several clinical contexts, but its relationship with ASCVD risk is underexplored. Genistein, a soy isoflavone with reported anti-inflammatory and endothelial effects, may modify relationships between endothelial stress and cardiovascular risk. This study aimed to assess the association between EASIX and estimated 10-year ASCVD risk classification and to test whether urinary genistein modifies that association.
This cross-sectional analysis used data from the National Health and Nutrition Examination Survey (NHANES) cycles 1999–2010. Participants aged 40–79 years were considered (initial n = 15,994). Exclusions included individuals with a history of cardiovascular disease (n = 2,246), those missing lactate dehydrogenase (LDH), creatinine (Cr) or platelet count (n = 92), and those without urinary genistein measurement (n = 9,178). The final analytic sample comprised 4,478 participants. The NHANES protocol received ethical approval from the NCHS Ethics Review Board, and participants provided informed consent.
Blood samples were collected in mobile examination centers and analyzed per NHANES laboratory procedures. Urinary genistein concentrations were normalized to urinary creatinine and expressed in μg/g. The upper quartile (75th percentile) of urinary genistein in the analytic sample—54.105 μg/g—served as the prespecified cutoff to define higher genistein exposure, consistent with prior epidemiologic convention.
The EASIX score was calculated using the formula LDH (U/L) × Cr (mg/dL) / platelet count (10^9/L). Analyses used the log2-transformed EASIX value. Urinary genistein was creatinine-normalized and dichotomized at 54.105 μg/g into lower (<54.105 μg/g) and higher (≥54.105 μg/g) exposure groups.
The outcome was estimated 10-year ASCVD risk classification derived from the 2013 ACC/AHA pooled cohort equations. The pooled cohort equation predicts the 10-year risk of stroke, nonfatal myocardial infarction, or coronary heart disease death in individuals aged 40–79 without prior ASCVD and with LDL-C <190 mg/dL. For this analysis, estimated 10-year risk was categorized as <7.5% versus ≥7.5%, aligning with prior studies and guideline-informed thresholds.
Covariates included demographic factors (age, sex, race, education, marital status, poverty-to-income ratio), lifestyle variables (smoking, drinking, physical activity, total energy intake), clinical conditions (diabetes mellitus, hypertension, dyslipidemia), medication use (antihypertensive agents, lipid-lowering drugs), anthropometrics (body mass index), and biomarkers (C-reactive protein, uric acid). Dietary data were derived from two 24-hour recalls when available and otherwise from a single recall.
Multivariable logistic regression models assessed the association between log2-EASIX and estimated 10-year ASCVD risk classification (≥7.5% vs <7.5%). Models were adjusted for the covariates listed above. Effect modification by urinary genistein was evaluated with multiplicative interaction terms and stratified analyses by genistein group (<54.105 vs ≥54.105 μg/g). Dose–response relationships were explored using restricted cubic spline (RCS) models, and nonlinearity was tested.
In the fully adjusted logistic regression, higher log2-transformed EASIX was independently associated with greater odds of estimated 10-year ASCVD risk ≥7.5% (OR per unit increase = 1.407; 95% CI 1.208–1.640; P < 0.001). This indicates that, after adjusting for multiple confounders, elevated endothelial activation/stress as reflected by EASIX correlates with higher estimated 10-year cardiovascular risk.
A statistically significant multiplicative interaction between log2-EASIX and urinary genistein strata was observed (interaction OR = 1.66; 95% CI 1.17–2.35; P = 0.005), indicating that the strength or shape of the association between EASIX and estimated ASCVD risk differs by genistein exposure.
Stratified analyses demonstrated the association persisted in both genistein groups but was slightly attenuated in the higher-genistein group. In the lower-genistein group, the OR per unit increase in log2-EASIX was 1.397 (95% CI 1.202–1.624; P < 0.001). In the higher-genistein group, the OR was 1.360 (95% CI 1.066–1.736; P = 0.015).
Restricted cubic spline analyses revealed differing dose–response patterns across genistein strata. In the lower-genistein group, the relationship between log2-EASIX and estimated ASCVD risk exhibited a non-linear S-shaped pattern (P nonlinear = 0.020). In contrast, the higher-genistein group showed a predominantly linear association (P nonlinear = 0.108), suggesting genistein exposure may influence the functional form of the EASIX–ASCVD risk relationship.
These results indicate that higher EASIX—a composite biomarker of endothelial activation and stress—associates with higher estimated 10-year ASCVD risk in a US population sample aged 40–79. Urinary genistein appears to modify this association, with slightly attenuated effect estimates and a more linear dose–response in the higher-genistein group. The authors note genistein’s reported endothelial and anti-inflammatory actions as biologic plausibility for effect modification, but emphasize findings are hypothesis-generating.
As a cross-sectional analysis of NHANES data, causal inference is limited. The authors excluded substantial numbers due to missing urinary genistein measures, and residual confounding is possible despite multivariable adjustment. The study used estimated ASCVD risk rather than observed cardiovascular outcomes. The authors provided the minimal dataset via Figshare to support reproducibility (DOI in the original report).
In this NHANES-based study of adults aged 40–79, higher EASIX was associated with increased estimated 10-year ASCVD risk, and urinary genistein modified that association. The findings warrant further investigation in prospective studies to clarify causal relationships and potential clinical implications of EASIX and isoflavone exposure for cardiovascular risk assessment.