Abstract 23: Gut Microbiota Related Plasma Metabolites and Risk of Cardiovascular Disease in the PREDIMED Study
Authors: Guasch-Ferre Marta , Hu Frank B , Ruiz-Canela Miguel , Bullo Monica , Yu Edward , Zheng Yan , Toledo Estefania , Wang Dong D , Hruby Adela , Corella Dolores , Gomez-Gracia Enrique , Fiol Miquel , Estruch Ramon , Lapetra José , Fito Montserrat , Aros Fernando , Serra-Majem Lluis , Ros Emilio , Liang Liming , Clish Clary , Martinez-Gonzalez Miguel A , Salas-Salvado Jordi 
Published: March 2022

Background:
Metabolites associated with betaine and choline metabolism and the gut-microbiota-dependent metabolite trimethylamine N-oxide (TMAO) have been linked to the risk of cardiovascular disease (CVD). However, the relationship between plasma concentrations of other gut microbiota-related metabolites and major CVD endpoints remains unclear.

Objectives:
To evaluate the association between gut microbiota-related metabolites and risk of incident CVD and the potential modifying effect of Mediterranean diet (MedDiet) interventions.

Methods:
We designed a case-cohort study nested within the PREDIMED trial. We used liquid chromatography–tandem mass spectrometry to measure plasma gut microbiota-related metabolites. A score including the sum of quartile values of 8 metabolites was constructed (TMAO, betaine, choline, phosphocholine, alphaglycerophosphocholine, proline, hydroxyproline, allantoin). The primary outcome was a composite of myocardial infarction, stroke, and cardiovascular death. Blood samples from a randomly selected PREDIMED sub-cohort (n=751) and all available incident CVD cases (n=229) after 4.8-y of follow-up were included in the analysis. We used weighted Cox regression models to estimate multivariable-adjusted hazard ratios (HR) and their 95% confidence intervals (CI). Models were adjusted for age, sex, BMI, family history of premature heart disease, and smoking, physical activity (metabolic equivalent tasks in min/d), hypertension, dyslipidemia, diabetes and was stratified by intervention group.

Results:
Baseline plasma concentrations of choline and hydroxyproline were associated with higher CVD risk independent of traditional risk factors, while no significant association between plasma concentrations of TMAO and CVD was found. The HRs comparing extreme quartiles (lowest quartile as the reference) were 1.72 (95% CI: 1.05, 2.81; P for trend=0.01) and 1.65 (95% CI: 1.03, 2.65; P for trend =0.04), respectively. The microbiota metabolite score was associated with a 2.13-fold higher risk of CVD across extreme quartiles (95% CI: 1.32, 3.43; P for trend <0.001) and a 1.99-fold higher risk of stroke (95% CI: 1.08, 3.65; P for trend=0.02). Baseline betaine/choline ratio was inversely associated with CVD. Compared to participants with a score below the median and randomized to the Mediterranean diet, the HR of developing CVD was 2.56 (95% CI: 1.59, 4.11) for participants with a gut microbiota score above the median and randomized to the control group.

Conclusions:
Plasma gut microbiota-related metabolites were associated with an increased risk of CVD in a Mediterranean population at high cardiovascular risk, independent of traditional CVD risk factors.