**1. Introduction**

Cardiovascular diseases (CVD) remain the major cause of death worldwide. Therefore, the assessment and monitoring of cardiovascular (CV) risk through algorithms has shown to be an accurate tool to predict outcomes, as well as to improve treatment indication when compared with the isolated use of risk factors [1–3]. The estimates use risk factors that are the major contributors to cardiovascular events (i.e., age, sex, glycemia, blood pressure, and blood lipids) [3–5]. The ten-years CV risk estimation is relevant especially in moderaterisk patients because the intuitive ten-year period is important in making practical and usually therapeutic, decisions. Cardiovascular risk assessment models have been built to guide the treatment of modified cardiovascular risk factors and, in the last decade to help therapeutic goals based on statins. Moreover, the estimates provide insight into the individual contribution of variables to the patient's risk, guiding the preventive care [1]. However, the application of these estimates requires previous validation for the target population. Many CV risk estimates were developed based on American or European white populations, and the estimation of multi-ethnic populations is often overestimated [6–9].

**Citation:** Gonçalinho, G.H.F.; Sampaio, G.R.; Soares-Freitas, R.A.M.; Damasceno, N.R.T. Omega-3 Fatty Acids in Erythrocyte Membranes as Predictors of Lower Cardiovascular Risk in Adults without Previous Cardiovascular Events. *Nutrients* **2021**, *13*, 1919. https://doi.org/ 10.3390/nu13061919

Academic Editors: Carlo Agostoni and Hayato Tada

Received: 22 April 2021 Accepted: 26 May 2021 Published: 3 June 2021

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Nevertheless, the Framingham Risk Score (FRS) is the most popular estimating tool and its use is currently recommended by many guidelines, including in Brazil [10].

Omega-3 polyunsaturated fatty acids (*n*-3 PUFA) are often highlighted due to several mechanisms that modify CV risk factors, slow down the atherosclerotic process and, possibly change cardiovascular events. The eicosapentaenoic (EPA; C20:5 *n*-3) and docosahexaenoic acids (DHA; C22:6 *n*-3) are the main components of this family, is often linked to antiarrhythmic effects, autonomic function improvement, decreased platelet aggregation, vasodilatory effects, blood pressure reduction, endothelial function improvement, atherosclerotic plaque stabilization, increased adiponectin synthesis, reduction of collagen deposition in the arteries, anti-inflammatory effects, and reduction of plasma triglycerides and cholesterol, consequently reducing CVD risk [11]. Despite that, reports of randomized trials have shown small or even null effects on cardiovascular risk factors and outcomes [12].

Most of the studies show methodological differences and do not assess *n*-3 PUFA biomarkers. Circulating or tissue *n*-3 PUFA have proven their superiority in estimating habitual intake compared to dietary assessment [13]. Based on that, previous studies have associated *n*-3 PUFA in erythrocyte membranes with reduced CV risk and mortality [13–16]. Because *n*-3 PUFA alter some components included in CV risk estimates, it is possible to state that *n*-3 PUFA influence the overall CV risk which is frequently used to guide preventive care. Thus, the nutritional status of *n*-3 PUFA may be useful in CVD prevention. However, as far as it is known, no previous study investigated the association of isolated and clustered FA biomarkers with different cardiovascular risk estimates.

Therefore, the main goal of this study was to investigate the association of erythrocyte membranes *n*-3 PUFA with different cardiovascular risk estimate classifications in Brazilian individuals. In addition, we also evaluated the association of modified CV risk factors used in estimates with isolated and clusters *n*-3 PUFA.

## **2. Materials and Methods**

#### *2.1. Study Design and Participants*

This was a cross-sectional study, using the baseline data from the CARDIONUTRI clinical trial (ReBEC: RBR-2vfhfv), which included individuals from the outpatient clinic at the University Hospital of the University of São Paulo. The study selection was made public by poster, newspaper, and digital media (sites, electronic mailing, and social networks). Inclusion criteria were individuals of both sexes, 30 to 74 years, with at least one cardiovascular risk factor, and no previous cardiovascular events. Exclusion criteria were individuals with acute or chronic severe diseases, infectious diseases, pregnant, and/or lactating women. Individuals interested in participating in the study were submitted to a short phone interview to assess inclusion and exclusion criteria. Additionally, individuals were submitted to electrocardiogram assessment by a trained physician, and those with alterations suggesting previous cardiovascular events were excluded. Three hundred and seventy-four individuals were recruited for the study from 2011–2012. Two individuals declined after clarification of the study design. Fourteen were excluded due to altered electrocardiogram and one due to recent HIV diagnosis. At the end of the recruitment, 356 individuals were included in the study.
