The effect of polyphenols in olive oil on heart disease risk factors: a randomized trial.

Context Olive oil, the main fat in the Mediterranean diet, contains polyphenols, which have antioxidant properties and may affect serum lipid levels.  The authors studied virgin olive oil (high in polyphenols), refined olive oil (low in polyphenols), and a mixture of the 2 oils in equal parts. Two hundred healthy young men consumed 25 mL of an olive oil daily for 3 weeks followed by the other olive oils in a randomly assigned sequence. Olive oils with greater polyphenol content increased high-density lipoprotein (HDL) cholesterol levels and decreased serum markers of oxidation.  The increase in HDL cholesterol level was small.  Virgin olive oil might have greater health benefits than refined olive oil. The Editors Polyphenol intake has been associated with low cancer and coronary heart disease (CHD) mortality rates (1). Antioxidant and anti-inflammatory properties and improvements in endothelial dysfunction and the lipid profile have been reported for dietary polyphenols (2). Studies have recently suggested that Mediterranean health benefits may be due to a synergistic combination of phytochemicals and fatty acids (3). Olive oil, rich in oleic acid (a monounsaturated fatty acid), is the main fat of the Mediterranean diet (4). To date, most of the protective effect of olive oil within the Mediterranean diet has been attributed to its high monounsaturated fatty acid content (5). However, if the effect of olive oil can be attributed solely to its monounsaturated fatty acid content, any type of olive oil, rapeseed or canola oil, or monounsaturated fatty acidenriched fat would provide similar health benefits. Whether the beneficial effects of olive oil on the cardiovascular system are exclusively due to oleic acid remains to be elucidated. The minor components, particularly the phenolic compounds, in olive oil may contribute to the health benefits derived from the Mediterranean diet. Among olive oils usually present on the market, virgin olive oils produced by direct-press or centrifugation methods have higher phenolic content (150 to 350 mg/kg of olive oil) (6). In experimental studies, phenolic compounds in olive oil showed strong antioxidant properties (7, 8). Oxidized low-density lipoprotein (LDL) is currently thought to be more damaging to the arterial wall than native LDL cholesterol (9). Results of randomized, crossover, controlled clinical trials on the antioxidant effect of polyphenols from real-life daily doses of olive oil in humans are, however, conflicting (10). Growing evidence suggests that dietary phenols (1115) and plant-based diets (16) can modulate lipid and lipoprotein metabolism. The Effect of Olive Oil on Oxidative Damage in European Populations (EUROLIVE) Study is a multicenter, randomized, crossover, clinical intervention trial that aims to assess the effect of sustained daily doses of olive oil, as a function of its phenolic content, on the oxidative damage to lipid and LDL cholesterol levels and the lipid profile as cardiovascular risk factors.  We recruited healthy men, 20 to 60 years of age, from 6 European cities through newspaper and university advertisements. Of the 344 persons who agreed to be screened, 200 persons were eligible (32 men from Barcelona, Spain; 33 men from Copenhagen, Denmark; 30 men from Kuopio, Finland; 31 men from Bologna, Italy; 40 men from Postdam, Germany; and 34 men from Berlin, Germany) and were enrolled from September 2002 through June 2003 . Participants were eligible for study inclusion if they provided written informed consent, were willing to adhere to the protocol, and were in good health. We preselected volunteers when clinical record, physical examination, and blood pressure were strictly normal and the candidate was a nonsmoker. Next, we performed a complete blood count, biochemical laboratory analyses, and urinary dipstick tests to measure levels of serum glucose, total cholesterol, creatinine, alanine aminotransferase, and triglycerides. We included candidates with values within the reference range. Exclusion criteria were smoking; use of antioxidant supplements, aspirin, or drugs with established antioxidant properties; hyperlipidemia; obesity; diabetes; hypertension; intestinal disease; or any other disease or condition that would impair adherence. We excluded women to avoid the possible interference of estrogens, which are considered to be potential antioxidants (17). All participants provided written informed consent, and the local institutional ethics committees approved the protocol. Figure 1. Study flow diagram. Sequence of olive oil administration: 1) high-, medium-, and low-polyphenol olive oil; 2) medium-, low-, and high-polyphenol olive oil; and 3) low-, high-, and medium-polyphenol olive oil.  The trial was a randomized, crossover, controlled study. We randomly assigned participants consecutively to 1 of 3 sequences of olive oil administration. Participants received a daily dose of 25 mL (22 g) of 3 olive oils with high (366 mg/kg), medium (164 mg/kg), and low (2.7 mg/kg) polyphenol content  in replacement of other raw fats. Sequences were high-, medium-, and low-polyphenol olive oil (sequence 1); medium-, low-, and high-polyphenol olive oil (sequence 2); and low-, high-, and medium-polyphenol olive oil (sequence 3). In the coordinating center, we prepared random allocation to each sequence, taken from a Latin square, for each center by blocks of 42 participants (14 persons in each sequence), using specific software that was developed at the Municipal Institute for Medical Research, Barcelona, Spain (Aleator, Municipal Institute for Medical Research). The random allocation was faxed to the participating centers upon request for each individual included in the study. Treatment containers were assigned a code number that was concealed from participants and investigators, and the coordinating center disclosed the code number only after completion of statistical analyses. Olive oils were specially prepared for the trial. We selected a virgin olive oil with high natural phenolic content (366 mg/kg) and measured its fatty acid and vitamin E composition. We tested refined olive oil harvested from the same cultivar and soil to find an olive oil with similar quantities of fatty acid and a similar micronutrient profile. Vitamin E was adjusted to values similar to those of the selected virgin olive oil. Because phenolic compounds are lost in the refinement process, the refined olive oil had a low phenolic content (2.7 mg/kg). By mixing virgin and refined olive oil, we obtained an olive oil with an intermediate phenolic content (164 mg/kg). Olive oils did not differ in fat and micronutrient composition (that is, vitamin E, triterpenes, and sitosterols), with the exception of phenolic content. Three-week interventions were preceded by 2-week washout periods, in which we requested that participants avoid olive and olive oil consumption. We chose the 2-week washout period to reach the equilibrium in the plasma lipid profile because longer intervention periods with fat-rich diets did not modify the lipid concentrations (18). Daily doses of 25 mL of olive oil were blindly prepared in containers delivered to the participants at the beginning of each intervention period. We instructed participants to return the 21 containers at the end of each intervention period so that the daily amount of unconsumed olive oil could be registered. Dietary Adherence We measured tyrosol and hydroxytyrosol, the 2 major phenolic compounds in olive oil as simple forms or conjugates (7), by gas chromatography and mass spectrometry in 24-hour urine before and after each intervention period as biomarkers of adherence to the type of olive oil ingested. We asked participants to keep a 3-day dietary record at baseline and after each intervention period. We requested that participants in all centers avoid a high intake of foods that contain antioxidants (that is, vegetables, legumes, fruits, tea, coffee, chocolate, wine, and beer). A nutritionist also personally advised participants to replace all types of habitually consumed raw fats with the olive oils (for example, spread the assigned olive oil on bread instead of butter, put the assigned olive oil on boiled vegetables instead of margarine, and use the assigned olive oil on salads instead of other vegetable oils or standard salad dressings).  Main outcome measures were changes in biomarkers of oxidative damage to lipids. Secondary outcomes were changes in lipid levels and in biomarkers of the antioxidant status of the participants. We assessed outcome measures at the beginning of the study (baseline) and before (preintervention) and after (postintervention) each olive oil intervention period. We collected blood samples at fasting state together with 24-hour urine and recorded anthropometric variables. We measured blood pressure with a mercury sphygmomanometer after at least a 10-minute rest in the seated position. We recorded physical activity at baseline and at the end of the study and assessed it by using the Minnesota Leisure Time Physical Activity Questionnaire (19). We measured 1) glucose and lipid profile, including serum glucose, total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglyceride levels determined by enzymatic methods (2023) and LDL cholesterol levels calculated by the Friedewald formula; 2) oxidative damage to lipids, including plasma-circulating oxidized LDL measured by enzyme immunoassay, plasma total F2-isoprostanes determined by using high-performance liquid chromatography and stable isotope-dilution and mass spectrometry, plasma C18 hydroxy fatty acids measured by gas chromatography and mass spectrometry, and serum LDL cholesterol uninduced conjugated dienes measured by spectrophotometry and adjusted for the cholesterol concentration in LDL cholesterol levels; 3) antioxidant sta