This study was an 8-week randomized, controlled, parallel, double-blinded clinical trial. It was registered at the Brazilian website of clinical trials (www.ensaiosclinicos.gov.br) as RBR-2vfhfv. Individuals were recruited from the University Hospital of the University of Sao Paulo. A total of 188 individuals responded to advertisements to participate in the study, and 168 were assigned to treatments. Eligible subjects to this study were 30-74 years old with at least one cardiovascular risk factor (dyslipidemia, high blood pressure, diabetes, or smoking). During a prescreening assessment, participants completed a battery of questionnaires to obtain demographic and clinical profiles addressing sex, age, clinical information, family history of chronic diseases (father and mother), smoking status, blood pressure, and regular medication use. Exclusion criteria included cardiovascular events (assessed by clinical history and electrocardiogram:ECG), malnourishment, pregnant or lactating individuals, participants in other intervention protocols, patients with acute or chronic severe and uncontrolled diseases, users of illicit drugs, alcoholics, people allergic or intolerant to any component of the intervention and individuals with uncontrolled psychiatric disorders.
All participants were randomly allocated into either the omega-6 group (omega-6) or the omega-3 group (omega-3). Individuals received three capsules for daily consumption containing 1 g of fish oil each (370 mg of EPA and 230 mg of DHA - total of omega-3/per day = 3 g; 1,110 mg of EPA and 690 mg of DHA) administered to omega-3 group or 1 g of sunflower oil each (760 mg of linoleic acid) for omega-6 group. Capsules were purchased from the Health Industry (Indaiatuba, Sao Paulo, Brazil) that blinded both interventions in color, shape, size, packing, and smell. High performance liquid chromatography (HPLC) analysis by an independent laboratory confirmed the contents of fatty acids in the capsules. Adherence to interventions was monitored by the number of capsules returned at final follow-up time (T8). Patients were requested to undertake their regular physical activity and not to take any extra nutritional supplements during the 8-week trial. All patients completed a 2-d food record and 1 physical activity record at the baseline of the study and at the end of the intervention. Both, 24 h recalls and physical activity questionnaire were applied by trained researchers and direct interviews. Energy and nutrient intakes were calculated by The Food Processor software (version 10.11.0) and intrapersonal variability was adjusted by The Multiple Source Method version 1.0.1 (DIfE, Nuthetal, German). After that, all nutrients were adjusted by energy. Anthropometric measures (weight, height, and waist circumference) and body composition (fat mass) were also evaluated by tetrapolar bioimpedance (Biodynamics 450 , TBW, Brazil). All procedures were obtained by direct evaluation and interview, except for 2nd food record, which was applied by phone by trained health professionals.
Participants were seen in the early morning after a 12-h fast. At enrollment and 8-week, blood samples were collected in vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) (1.0 mug/mL). For serum, blood samples were collected in dry tubes, and all samples were maintained at 4  C. Plasma and serum were separated by centrifugation at 1,500 g for 10 min at 4 C. Protease inhibitors containing aprotinin (10.0 mug/mL), benzamidine (10.0 mumol/L), and phenylmethylsulfonyl fluoride (PMSF) (5.0 mumol/L) and the antioxidant butylated hydroxytoluene (BHT) (100.0 mumol/L) were added. Plasma triacylglycerol (TAG), total cholesterol (TC), and HDL-C levels were measured using standard commercial kits (Labtest, Minas Gerais, Brazil). Low-density lipoprotein cholesterol (LDL-C) level was calculated using the Friedewald equation (1972). Apolipoproteins AI, B, CII, and CIII were determined by standard methods, using the autokit Apo AI, Apo B, Apo CII and Apo CII , respectively (Randox Laboratories Ltd., UK). Plasma and HDL non-esterified fatty acid (NEFAs) concentrations were analyzed by the NEFA-HR(2)  kit (Wako Chemicals GmbH, DE). Glucose and glycated hemoglobin (HbA1c) levels were measured by an enzymatic and colorimetric kit (Glucose PAP Liquiform  and HbA1c Turbiquest, Labtest, Minas Gerais, Brazil). ELISA was used to measure serum insulin (Insulin Human Direct ELISA Kit , Life Technologies, Grand Island, NY). Insulin resistance was calculated with the homeostasis model assessment-insulin resistance (HOMA-IR) as follows: fasting insulin concentration (muU/mL) x fasting glucose (mmol/L) / 22.5. The PON1 activity in HDL was analyzed as proposed by Mackness et al. and paraoxon-ethyl (Sigma, USA) as used as the substrate.
HDL subfractions were determined by the Lipoprint  system (Quantimetrix Inc., Redondo Beach, California). This method is based on the separation and quantification of lipoprotein subfractions using non-denaturing polyacrylamide gel. The system uses a lipophilic dye, which binds to cholesterol in the lipoprotein particles. Ten subfractions were identified and then classified as HDLLARGE (HDL1 to HDL3), HDLINTERMEDIATE (HDL4 to HDL7), or HDLSMALL (HDL8 to HDL10).
The antioxidant capacity of HDL was based on the lag time test described by Esterbauer et al.. Briefly, an LDL standard sample was diluted in PBS without EDTA to a final concentration of 0.083 mg/mL and distributed in flat-bottom plates containing 96 wells. Subsequently, HDL samples (200 mg/mL) precipitated by tungsten acid (2:1; v/v) from each of the patients' serum were added (Labtest, Minas Gerais, Brazil). Peroxidation was induced by CuSO4 (30 mumol/L), and absorbance was measured at 234 nm for 5 h. The oxidation resistance phase (lag time), the propagation phase of the conjugated dienes (indicated by the increase in absorbance), and the decomposition phase of these compounds (plateau) were observed. From the results, the lag time phase was calculated, as well as the maximum rate of lipid peroxidation (Vmax), the maximum production of conjugated dienes (DOmax), the time for maximum production of conjugated dienes (Tmax) and the area under the curve generated (AUC).
For the HDL-fatty acid analysis, a solution formed by HDL (50 muL), methanol (1.75 mL), acetyl chloride (100 muL) and tridecane acid (50 muL) used as internal standard solution was mixed (30 s) in screw-capped glass tubes, then heated at 100  C for 60 min and after that, cooled to room temperature. Hexane (1.5 mL) was added, and tubes were vortexed for 1 min. The samples were centrifuged at 1,500 x g for 2 min at 4  C. The upper organic phase was collected, and this extraction procedure was repeated with hexane (0.75 mL) in order to optimize lipid extraction. The combined hexane solution was evaporated under nitrogen and the dry residue was then redissolved in hexane (100 muL). Samples were analyzed by gas chromatography with flame ionization detection (Trace 1310, Thermo Scientific) using a DB-FFAP column (15 m x 0.1 mm ID x 0.1 mum) (J and W Scientific from Agilent Technologies). Temperature program started at 150  C with a 0.25 min hold, which was increased at 35 C/min to 200 C, 8 C/min to 250 C, followed by 4 min of isothermal period. Run time for a single sample was 14 min. Fatty acids in HDL were identified by direct comparison with a FAME standard mix (Supelco 37 Component FAME Mix; Sigma-Aldrich). Each fatty acid peak was integrated and then normalized by internal standard. All analyses were conducted in duplicate, and coefficients of intra- and inter-assay variance were 1- 15%. Results are expressed in percentage of all peaks integrated after to exclude internal standard.
The sample size was calculated based on the method previously proposed for clinical trials (Browner, Newman and Hulley 2008), using Student's t test to compare means of continuous variables, with a standardized magnitude of effect and specific bilateral alpha values of 0.05 and beta of 0.10 (statistical power of 90%). For that, were considered biochemical parameters related to the outcomes: HDL-C, HDL size, HDL composition, and lag time. Based on this information, 60 individuals were the minimum required to be included in each intervention group.
All statistical analyses were performed by using SPSS , version 20 (IBM Corporation). Statistical significance was set at a 2-sided level of p < 0.05. The distribution of the data was checked by using normal plots and histograms and by performing the Kolmogorov-Smirnov test (p > 0.05). Time effect was performed for each group using paired Student's t or Wilcoxon tests. Differences between groups were assessed after calculating the magnitude of the intervention effect (Delta = T8-T0) and its percentage variation (Delta% = (Delta/T0)*100) and were compared using unpaired Student's t or Mann-Whitney tests. Categorical variables were evaluated using Pearson' s chi-square test. To identify the effect of fatty acids on HDL functionality, univariate logistic regression analysis was performed using the EPA, DHA, or omega-6/omega-3 ratio as an independent factor. Afterwards, variables that showed correlations with EPA, DHA, or omega-6/omega-3 values (p < 0.20) have been included in the final model. Multiple adjusts for confounding variables such as sex, smoking, previous diseases, and use of medications, were tested. The adjusted odds ratio and 95% confidence interval (CI) were determined. In addition, the Principal Component Analysis (PCA) was applied to the fatty acid (Delta = T8-T0) incorporated into HDL to analyze the covariance structure of the variables and reveal a restricted number of sample standards. The original data were self-scaled, dividing the mean by the standard deviation, resulting in variables with the same weight. The recalculated data were then submitted to the PCA, and the results were presented when loading. The Eigenvalue method were used to extract the Principal Components (PC) and the Kaiser criteria was used to consider the sample adequacy measure. The PCA projected 7 significant PC (Eigenvalue > 1), which were correlated with the HDL functionality parameters. Based in the PCA method a summarized number of groups were obtained according fatty acids profile more atherogenic (PCA 1, 2, 4, 5 and 6) and less atherogenic (PCA 3 and 5).