Milk products supplemented with Phytosterols or omega-3 oils and biomarkers of cardiovascular disease

INTERVENTION: All subjects were submitted to two 28‐day treatment sequences, separated by a 4 weeks washout period. Before the initiation of the intervention, individuals were submitted to a 2‐week run‐in period. During the run‐in and wash‐out periods, participants received 250 mL/day commercially available plain low‐fat milk (without PhyS or ?‐3), with the same composition to that used for preparing the PhyS‐enriched‐ and ?‐3 enriched milks. At the end of the run‐in period, subjects were randomly allocated to receive one of the two treatment sequences: Study arm 1: Phytosterol (PhyS)‐enriched milk in the first intervention period and ?‐3 enriched milk in the second intervention period Study arm 2: Omega‐3 (?3)‐enriched milk in the first intervention period and PhyS‐enriched milk in the second intervention period Participants were instructed to consume a pack of milk (250 mL) per day, distributed in one or more portions according to their normal habits, replacing their habitual milk‐products consumption. Subjects were requested to maintain their habitual diet at levels consistent with maintenance of a stable body weight and to continue their normal pattern of physical activity throughout the study period. Lipid content varied from 0.4 g per 100 ml milk in the control low‐fat milk to 0.8 g in the ?3‐enriched milk and 0.9 g in the PhyS‐enriched milk. The 20.6% of the lipid content in the ?3‐enriched milk were omega‐3 fatty acids, mainly consisting in EPA and DHA (150 mg EPA+DHA / 100 mL milk) that represented the 90.8% of the total omega‐3 fatty acid content. In addition to lipid content, every 100 mL PhyS‐enriched milk contained 0.64 g plant sterols (expressed as free PhyS). According to the provider’s information, the supplemented vegetable oil‐based sterols (VEGAPURE® 95E) were mainly found as sterol esters (>94%) with 68% poly‐unsaturated FA (C 18:2), 19% monounsaturated FA (18:1), and 13% saturated FA (16:0, 18:0). PhyS mix mainly consisted of sitosterol, campesterol, and CONDITION: Arteriosclerosis ; Circulatory System ; Atherosclerosis SECONDARY OUTCOME: 1. Alcohol intake, smoking and dietary habits, taken from participants' medical records ; 2. Dietary habits, assessed using a questionnaire at baseline; 3. Inflammatory variables related to atherosclerosis; 4. Gene expression measured by RT‐PCR and arrays either in the plasma or the blood cellular fraction after the intake of supplemented milk; 5. Circulating microparticles assessed using blood samples or the cells of the vascular wall; 6. Potential gene polymorphisms associated with the response to the interventions at protemic and lipidomic levels; Measured at baseline and after each intervention period INCLUSION CRITERIA: 1. Healthy adult males and females between 25 and 70 years old 2. Overweight or grade 1 obese (body mass index greater than 25 kg/m^2) 3. Signed informed consent PRIMARY OUTCOME: 1. Triglyceride and cholesterol levels, measured using biochemical analysis; 2. Oxidative profile: measure of the lipidic peroxidation in plasma and LDL, the plasma antioxidant behavior and LDL resistance to oxidation; 3. Fatty acids (FA) and lipidomic pattern measured in plasma and LDL samples respectively and analyzed by LC/MS/MS. The analysis of FA is performed after the derivatization of the samples to convert FAs into thrimethylaminoethyl esther iodide derivates. LDL lipidomic profile is analyzed in mixture with an internal lipid standard mixture; 4. Proteomic profile:; 4.1. Analysis of the differential proteomic profile of the lipid and lipid‐free fraction of serum and plasma samples measured by 2DE electrophoresis, followed by protein identification by mass spectrometry identification (MALDI‐TOF); 4.2. Validation of the results by western blot analysis and enzyme‐linked immunosorbent assay (ELISA) kits; Measured at baseline and after each intervention period