The Mediterranean Diet, Cardiometabolic Biomarkers, and Risk of All-Cause Mortality: A 25-Year Follow-Up Study of the Women’s Health Study

Background Higher consumption of Mediterranean diet (MED) intake has been associated with reduced risk of all-cause mortality but limited data are available examining long-term outcomes in women or the underlying molecular mechanisms of this inverse association in human populations. We aimed to investigate the association of MED intake with long-term risk of all-cause mortality in women and to better characterize the relative contribution of traditional and novel cardiometabolic factors to the MED-related risk reduction in morality. Methods In a prospective cohort study of 25,315 initially healthy women from the Women’s Health Study, we assessed dietary MED intake using a validated semiquantitative food frequency questionnaire according to the usual 9-category measure of MED adherence. Baseline levels of more than thirty cardiometabolic biomarkers were measured using standard assays and targeted nuclear magnetic resonance spectroscopy, including lipids, lipoproteins, apolipoproteins, inflammation, glucose metabolism and insulin resistance, branched-chain amino acids, small metabolites, and clinical factors. Mortality and cause of death was ascertained prospectively through medical and death records. Results During a mean follow-up of 25 years, 3,879 deaths were ascertained. Compared to the reference group of low MED intake (0-3, approximately the bottom tertile), and adjusting for age, treatment, and energy intake, risk reductions were observed for the middle and upper MED groups with respective HRs of 0.84 (95% CI 0.78-0.90) and 0.77 (95% CI 0.70-0.84), p for trend <0.0001. Further adjusting for smoking, physical activity, alcohol intake and menopausal factors attenuated the risk reductions which remained significant with respective HRs of 0.92 (95% CI 0.85-0.99) and 0.89 (95% CI 0.82-0.98), p for trend 0.0011. Risk reductions were generally similar for CVD and non-CVD mortality. Small molecule metabolites (e.g., alanine and homocysteine) and inflammation made the largest contributions to lower mortality risk (accounting for 14.8% and 13.0% of the benefit of the MED-mortality association, respectively), followed by triglyceride-rich lipoproteins (10.2%), adiposity (10.2%) and insulin resistance (7.4%), with lesser contributions (<3%) from other pathways including branched-chain amino acids, high-density lipoproteins, low-density lipoproteins, glycemic measures, and hypertension. Conclusions In the large-scale prospective Women’s Health Study of 25,315 initially healthy US women followed for 25 years, higher MED intake was associated with approximately one fifth relative risk reduction in mortality. The inverse association was only partially explained by known novel and traditional cardiometabolic factors. ### Competing Interest Statement Dr. Mora is listed as co-inventor on a patent regarding the use of an NMR glycosylation biomarker (GlycA) in relation to colorectal cancer risk. All other coauthors have no conflict of interests. ### Funding Statement The Women's Health Study is supported by the National Institutes of Health (grants CA047988, HL043851, HL080467, HL099355, and UM1 CA182913). Dr Ahmad was supported through a career starting research grants from Swedish Research Council (2022-01460) and FORMAS (2020-00989) and also research grant from the EpiHealth, Sweden. Dr Demler was supported by a K award from the National Heart, Lung, and Blood Institute of the National Institutes of Health under award number K01HL135342-02. Dr Mora was supported by the research grants from the National Institute of Diabetes and Digestive and Kidney Diseases (grant DK112940); National Heart, Lung, and Blood Institute (grants R01HL134811, R01HL117861 and K24 HL136852); American Heart Association (grant 0670007N); and the Molino Family Trust. In addition, LabCorp provided the LipoProfile IV results to the study at no additional cost. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All study participants provided written consent and the study was approved by the institutional review board of Brigham and Women's Hospital, Boston, Massachusetts. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. 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