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From the First Cardiology Clinic, School of Medicine, University of Athens, Athens, Greece (CP, CC, ME, and CS), and the Department of Nutrition and Dietetics, Harokopio University, Athens, Greece (DBP, NT, and AZ)
2 The ATTICA study is supported by research grants from the Hellenic Cardiological Society (HCS2002). 3 Reprints not available. Address correspondence to DB Panagiotakos, 46 Paleon Polemiston Street, 166 74, Attica, Greece. E-mail: d.b.panagiotakos{at}usa.net.
ABSTRACT
Background: Greater adherence to the Mediterranean diet has been associated with a lower incidence of cardiovascular disease and cancer.
Objective: We studied the effect of the Mediterranean diet on total antioxidant capacity (TAC) in 3042 participants who had no clinical evidence of cardiovascular disease.
Design: During 2001–2002, a random sample of 1514 men and 1528 women aged 18–89 y from the Attica area of Greece was selected. TAC was measured with an immune-diagnostic assay. Food consumption was evaluated with a validated food-frequency questionnaire, and adherence to the Mediterranean diet was assessed on the basis of a diet score that incorporated the inherent characteristics of this diet.
Results: TAC was positively correlated with diet score. The participants in the highest tertile of the diet score had, on average, 11% higher TAC levels than did the participants in the lowest tertile, even after adjustment for relevant confounders (P < 0.01). On the other hand, the participants in the highest tertile of the diet score had, on average, 19% lower oxidized LDL-cholesterol concentrations than did the participants in the lowest tertile (P < 0.01). An additional analysis showed that TAC was positively correlated with the consumption of olive oil ( = 0.54, P = 0.002) and of fruit and vegetables ( = 0.34 and = 0.31, respectively; P < 0.001 for both), whereas it was inversely associated with the consumption of red meat ( = –0.35, P = 0.02).
Conclusion: Greater adherence to the Mediterranean diet is associated with elevated TAC levels and low oxidized LDL-cholesterol concentrations, which may explain the beneficial role of this diet on the cardiovascular system.
Key Words: Mediterranean diet • antioxidant capacity • oxidized LDL cholesterol • cardiovascular disease
INTRODUCTION
Several observational studies and large-scale clinical trials have provided scientific evidence that diets rich in fruit, vegetables, legumes, whole grains, fish, and low-fat dairy products are associated with a lowered incidence of various chronic diseases (1, 2). The dietary pattern that was found in the olive growing areas of the Mediterranean region (such as Greece, Spain, Italy, and France) in the late 1950s and early 1960s encompasses these dietary characteristics and has been associated with a lowered incidence of cardiovascular diseases, metabolic disorders, and several types of cancer (3–10). Many investigators have already underlined the beneficial role of this dietary pattern on lipid metabolism, blood pressure levels (1, 6, 7), and body mass index (6, 8), as well as on inflammation and coagulation processes (9).
The determination of antioxidative capacity is now considered a tool in the medical diagnosis and treatment of several diseases, including cardiovascular disease, cancer, diabetes mellitus, and aging (11). Total antioxidant capacity (TAC) considers the cumulative action of all antioxidants that are present in plasma and body fluids and provides an integrated measurement rather than the simple sum of measurable antioxidants. A wide range of evidence indicates the importance of TAC in plasma and tissues, of its modification during the development of oxidative stress, and of its feasibility as a tool for investigating the association between diet and oxidative stress (12). In addition, the oxidative conversion of LDL cholesterol to oxidized LDL cholesterol is now considered to be a key event in the initiation and acceleration of the development of the early atherosclerotic lesion, the fatty streak. Diet seems to play a fundamental role in LDL-cholesterol oxidation (13–15). In particular, high dietary intakes of ß-carotene and of vitamins C and E and intakes of phenolic compounds in red wine (16) have been associated in some studies with low concentrations of oxidized LDL cholesterol. However, in most of these studies the approach has been to assess single nutrients or food items instead of dietary patterns. Recently, Martinez-Gonzalez and Estruch (17) underlined the need for randomized trials that use a whole-diet approach and not a simple antioxidant supplement to evaluate the role of the Mediterranean dietary pattern in human health. Moreover, in a recent review article, Martinez-Gonzalez and Sanchez-Villegas (5) underlined that not all components of the Mediterranean diet are protective, or at least they may not provide equal levels of protection. Thus, because food items and nutrients could have a synergistic and antagonistic effect on health outcome, the study of overall dietary patterns and not single nutrients has been suggested. In a recent editorial, Trichopoulos and Lagiou (3) suggested that the evaluation of whole dietary patterns, and consequently the use of diet scores, seemed attractive because whole dietary patterns capture the extremes of dietary habits, preempt nutritional confounding, capture possible effect modifications between nutritional variables with the use of diet scores, and tend not to be biased.
To the best of our knowledge, the influence of the Mediterranean dietary pattern on the TAC of the human body has rarely been investigated. Because the Mediterranean diet has been suggested to protect against the development and progression of cardiovascular disease, we sought to evaluate whether this relation could be explained, at least in part, by its effect on the TAC of apparently healthy men and women.
SUBJECTS AND METHODS
The ATTICA epidemiologic study (18) was carried out in the province of Attica (an area that is 78% urban and 22% rural) from May 2001 to December 2002. During this time, 4056 inhabitants from Attica were randomly selected to enroll in the study. However, only 3042 of them agreed to participate (75% participation rate). Trained personnel (cardiologists, general practitioners, dietitians, and nurses) used standard questionnaires to interview all participants and evaluate their lifestyle habits and various sociodemographic, clinical, and biological characteristics. Five percent of men and 3% of women were excluded from the study because they had a history of cardiovascular disease, other atherosclerotic disease, or chronic viral infections, which was ascertained from their medical records. Moreover, the participants who were included in the study did not have a cold or the flu, acute respiratory infections, or dental problems and had not undergone any type of surgery in the weeks before the beginning of the study.
A power analysis showed that the number of enrolled participants was adequate to evaluate 2-sided standardized differences of >0.5 between diet subgroups and the investigated biochemical variables and could achieve statistical power >0.90 at the 5% probability level (P value). The study was approved by the Medical Research Ethics Committee of our Institution and was carried out in accordance with the Declaration of Helsinki (1983) of the World Medical Association.
Dietary assessment
Usual dietary intake of the participants over the year preceding enrollment was assessed with the use of a validated, semiquantitative food-frequency questionnaire, which included 156 foods and beverages that are commonly consumed in Greece (19). First, we asked all participants to report the daily or weekly average intake of several food items that they consumed. Then, the frequency of consumption was approximately quantified in terms of the number of times per month this food was consumed. Thus, daily consumption was multiplied by 30 and weekly consumption was multiplied by 4; a value of 0 was assigned to food items that were rarely or never consumed. Consumption of various alcoholic beverages was measured with a unit of one drink equivalent to a 100-mL glass of wine with a 12-g ethanol concentration. A dietary pyramid was developed a few years ago to describe the Mediterranean dietary pattern (20). This pattern consists of the following: 1) daily consumption of nonrefined cereals and products (whole-grain bread, pasta, brown rice, etc), fruit (4-6 servings/d), vegetables (2–3 servings/d), olive oil (as the main added lipid), and nonfat or low-fat dairy products (1–2 servings/d); 2) weekly consumption of fish, poultry, potatoes, olives, and nuts (4–6 servings/wk for each) and of eggs and sweets (1–3 servings/wk); and 3) monthly consumption of red meat and meat products (4–5 servings/mo). The Mediterranean diet is also characterized by moderate consumption of wine (1–2 glasses of wine/d), moderate consumption of fat, and a high ratio (>2) of monounsaturated to saturated fat. On the basis of food-composition tables, we also calculated the total energy intake (in kcal/d) of the participants.
The diet score
We calculated a special diet score for each participant according to the previous dietary pattern and the reported monthly frequency consumption of these food groups that assessed adherence to the Mediterranean diet. In particular, for the consumption of items presumed to be close to this pattern (ie, those that are suggested on a daily basis or >4 servings/wk: nonrefined cereals, fruit, vegetables, potatoes, legumes, olive oil, and fish), we assigned a score of 0 when a participant reported no consumption, a score of 1 when a participant reported consumption of 1–4 times/mo, a score of 2 for 5–8 times/mo, a score of 3 for 9–12 times/mo, a score of 4 for 13–18 times/mo, and a score of 5 for >18 times/mo. On the other hand, for the consumption of foods presumed to not be part of this dietary pattern (ie, meat and meat products, poultry, and high-fat dairy products), we assigned the opposite scores (ie, a score of 0 when a participant reported almost daily consumption of the food to a score of 5 for rare or no consumption). We did not use a monotonic function for alcohol consumption, but we assigned a score of 5 for consumption of <3 glasses of wine/d, a score of 0 for consumption of >7 glasses of wine/d, and scores of 1, 2, 3, and 4 for consumption of 3, 4–5, 6, and 7 glasses of wine/d, respectively. Thus, 11 components were used and a total score was then calculated (score range: 0-55). Higher values of this diet score indicate a greater adherence to the Mediterranean diet, whereas lower values indicate adherence to a westernized diet. We also calculated the tertiles of this score.
Sociodemographic and lifestyle variables
We recorded both the mean annual income during the past 3 y and the educational level of the participants (in years of school completed) as proxies of social status. Current smokers were defined as those who smoked 1 cigarette/d, former smokers were defined as those participants who had stopped smoking for 1 y, and the rest of the participants were defined as nonsmokers. Occasional smokers (<7 cigarettes/wk) were recorded and combined with current smokers because of their small sample size. For a more accurate evaluation of smoking habits we calculated the pack-years of smoking (cigarette packs/d x years of smoking). To take into account various types of cigarettes consumed (ie, light or heavy), we used a unit of 1 cigarette with a 0.8-mg nicotine content for measurements. For the ascertainment of physical activity status, we developed an index of weekly energy expenditure using frequency (times/wk), duration (in min/time), and intensity of sports or other habits related to physical activity. Intensity was gradated in qualitative terms, such as light (expended calories: <4 kcal/min; ie, walking slowly, stationary cycling, light stretching, etc), moderate (expended calories: 4–7 kcal/min; ie, walking briskly, cycling outdoors, swimming with moderate effort, etc), and high (expended calories: >7 kcal/min; ie, walking briskly uphill, long distance running, cycling fast or racing, swimming at a fast crawl, etc). The participants who did not report any physical activities were defined as sedentary. For the rest of the participants, we calculated a combined score by multiplying the weekly frequency, duration, and intensity of physical activity. Additional details about the evaluation of physical activity have been presented elsewhere (18). Standing heights and weights of the participants were recorded, and their body mass index was calculated [weight (in kg)/height2 (in m)].
Clinical and biochemical characteristics
The participants' resting arterial blood pressure was measured 3 times in the right arm at the end of the physical examination with the participant in a sitting position. The participants whose average blood pressure levels were 140/90 mm Hg or who were receiving antihypertensive medication were classified as hypertensive.
Blood samples were collected from fasting participants from 0800 to 1000. The biochemical evaluation was carried out in the same laboratory and followed the criteria of the World Health Organization Reference Laboratories. TAC was measured with a colorimetric test on serum samples taken from the participants (ImAnOx; Immunodiagnostik AG, Bensheim, Germany). In brief, the measurement of antioxidant capacity was performed by the reaction of antioxidants in the serum sample with a defined amount of exogenously provided hydrogen peroxide. According to the manufacturer, a value <280 µmol/L indicates a low antioxidant capacity, whereas a value of 320 indicates a high antioxidant capacity. Oxidized LDL-cholesterol concentrations (in U/L) were measured in plasma samples with the use of an enzyme-linked immunosorbent assay kit (Mercodia AB, Uppsala, Sweden). The expected range of oxidized LDL cholesterol is considered between 30 and 120 U/L. The intra- and interassay CVs of TAC and oxidized LDL cholesterol did not exceed 3% and 7%, respectively. Additional blood lipid examinations, ie, serum total cholesterol, HDL-cholesterol, triacylglycerol, and glucose concentrations were measured with the use of a chromatographic enzymic method in an automatic analyzer (RA-1000; Mecon Ltd, Athens, Greece). The intra- and interassay CVs of all cholesterol and triacylglycerol concentrations did not exceed 4%. Patients were defined as having hypercholesterolemia if their total serum cholesterol concentrations were >200 mg/dL or if they were receiving lipid-lowering agents. Patients were defined as having diabetes mellitus if their fasting blood glucose concentrations were >125 mg/dL or if they were receiving antidiabetic medications. Finally, any family history of premature coronary heart disease was recorded for all participants.
Statistical analysis
Continuous variables are presented as means ± SDs. Categorical variables are presented as absolute and relative frequencies. Associations between categorical variables were tested with a chi-square test, whereas differences between categorical and several biochemical, clinical, and nutritional variables were tested with a Student's t test and a Mann-Whitney test (for the normally distributed and the skewed variables, respectively). Comparisons between TAC and tertiles of the diet score were performed with a one-factor analysis of variance after adjustment for sex. However, because of multiple comparisons, we used the Bonferroni correction to account for the increase in type I error. A multiple linear regression model was applied to test the association between the diet score and the investigated biomarkers, after controlling for several potential confounders. Colinearity between independent variables was evaluated through the condition index, whereas the model's goodness-of-fit was graphically evaluated (standardized residuals against fitted values). All reported P values are based on 2-sided tests and compared with a significance level of 5%. SPSS version 11.0.5 (SPSS Inc, Chicago, IL) software was used for all the statistical calculations.
RESULTS
The distribution of various characteristics of the participants according to Mediterranean diet score is shown in Table 1. TAC was positively correlated with diet score ( = 0.24, P < 0.001), which indicates that greater adherence to the Mediterranean diet was associated with increased TAC levels. In particular, the participants in the highest tertile of diet score had, on average, 11% higher TAC levels than did the participants in the lowest tertile. On the other hand, an inverse relation was observed between oxidized LDL-cholesterol concentrations and diet score ( = –0.10, P < 0.001). The participants who reported a greater adherence to the Mediterranean diet (ie, those in the 3rd tertile of the score) had, on average, 19% lower oxidized LDL-cholesterol concentrations than did the participants who reported a more westernized dietary pattern (ie, those in the lowest tertile of the score). Furthermore, both male and female participants in the highest tertile of the diet score were older, were more educated, had lower systolic blood pressure and triacylglycerol concentrations, and had higher HDL-cholesterol concentrations than did the men and women in the lowest tertile of the diet score. No associations were found between diet score and the other blood lipids measured, glucose concentrations, diastolic blood pressure levels, current smoking status (Table 1), and financial status (P = 0.12).
View this table:
TABLE 1. Lifestyle, clinical, and biochemical characteristics of the participants, according to Mediterranean diet score1
The weekly frequency consumption of major food groups, olive oil, and alcoholic beverages is shown in Table 2. Greater adherence to the Mediterranean diet was associated with less consumption of red meat and alcohol as well as increased consumption of fruit, potatoes, legumes, vegetables, and olive oil.
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TABLE 2. Frequency consumption of several food groups, according to Mediterranean diet score1
The results from a multiple linear regression analysis that evaluated the association between TAC, oxidized LDL-cholesterol concentrations, and diet score—after control for age, sex, daily energy intake, smoking habits, physical activity level, financial and education status, body mass index, presence of hypertension, diabetes, hypercholesterolemia, family history of coronary heart disease, and the use of lipid lowering agents, antihypertensive medication, and antidiabetic drugs—are shown in Table 3. For each 10 of 55 additional points in the diet score, we observed a 14 µmol/L increase in TAC levels and a 6.3 U/L decrease in oxidized LDL-cholesterol concentrations (Table 3). Moreover, the inclusion of diet scores in the models that evaluated TAC and oxidized LDL-cholesterol concentrations increased the explanatory ability (R2) of the regression models. We repeated these analyses after excluding patients with hypercholesterolemia, hypertension, or diabetes, and the previous relations between diet score, TAC, and oxidized LDL-cholesterol concentrations were still statistically significant.
View this table:
TABLE 3. Association between total antioxidant capacity (TAC) and oxidized LDL cholesterol (dependent variable) and adherence to Mediterranean diet (independent variable)1
An additional food-based analysis showed that TAC was positively correlated with the consumption of fruit ( = 0.34, P < 0.001), vegetables ( = 0.31, P < 0.001), and olive oil ( = 0.54, P = 0.002), whereas it was inversely associated with the consumption of red meat ( = –0.35, P = 0.02). No association was observed between TAC and either cereals ( = 0.05, P = 0.76) or whole grains ( = 0.06, P = 0.80). In addition, oxidized LDL-cholesterol concentrations were inversely associated with the consumption of fruit ( = –0.18, P = 0.03), vegetables ( = –0.19, P = 0.03), and olive oil ( = –0.48, P = 0.01).
However, even after adjustment for smoking habits, physical activity status, and body mass index, residual confounding may exist. Thus, we stratified the previous analyses by smoking status (ie, never or former smoker, <20 cigarettes/d, and 20 cigarettes/d), physical activity level (ie, sedentary or physically active), and obesity status. These analyses showed results similar to those observed in the regression models (data not shown).
DISCUSSION
We showed that a greater adherence to the Mediterranean diet is associated with increased TAC levels in apparently healthy men and women. Moreover, we found an inverse relation between oxidized LDL-cholesterol concentrations and diet score, which assessed adherence to this traditional diet. We also found that TAC was positively correlated with the consumption of fruit, vegetables, and olive oil, but it was inversely associated with the consumption of red meat. Additionally, oxidized LDL-cholesterol concentrations were inversely associated with the consumption of fruit, vegetables, and olive oil. Our findings may add to the current scientific knowledge concerning the benefits of this dietary pattern on cardiovascular disease because they provide another pathophysiologic mechanism, ie, the diet's ability to modulate the oxidation process.
Several observational studies have suggested that persons with high intakes of fruit, vegetables, and olive oil experience a low risk of coronary heart disease events (3–6, 10). However, the results from metabolic studies, in which antioxidant supplements were used to evaluate this theory, seem to be conflicting (21–23). Some investigators observed that -tocopherol treatment substantially reduces the rate of nonfatal myocardial infarction in patients with coronary heart disease, whereas other investigators reported that although vitamin E supplementation increased blood vitamin concentrations, it did not produce any significant reductions in the morbidity or mortality of any type of cardiovascular disease and cancer. Moreover, with a basis on a systematic review of the literature, Blomhoff (24) concluded that although observational studies have suggested that antioxidants may reduce oxidative stress, clinical trials do not support these benefits. Therefore, scientific research needs to clarify whether other plant antioxidants, the combination of plant antioxidants, or whole dietary patterns that induce the endogenous antioxidant defense can reduce the pathogenesis of cardiovascular disease.
In the present study, we showed that a greater adherence to the Mediterranean diet is associated with high antioxidant capacity and low oxidized LDL-cholesterol concentrations. Particularly, we observed that a 20% increment in the diet score, which assessed greater adherence to the Mediterranean dietary pattern, was associated with an increase in TAC of 6% and a 10% reduction in oxidized LDL-cholesterol concentrations irrespective of various potential confounding factors. Moreover, the inclusion of the Mediterranean diet score increased the explanatory ability of the models that evaluated TAC and oxidized LDL-cholesterol concentrations (Table 3). The antioxidative effects of the various food groups that are included in the traditional Mediterranean diet, such as fruit, vegetables, red wine, and olive oil, have already been reported (25–28). In agreement with the previous studies, we found that TAC was positively correlated with the consumption of fruit, vegetables, and olive oil and inversely associated with red meat consumption, whereas oxidized LDL-cholesterol concentrations were inversely associated with the consumption of fruit, vegetables, and olive oil. The high content of vegetables, fresh fruit, cereals, and olive oil in the diet, as well as the moderate intake of wine, guarantees a high intake of ß-carotene; vitamins B-6, B-12, C, and E; folic acid, polyphenols, and various minerals, all of which are known for their antioxidant effects. However, the influence of the Mediterranean dietary pattern on the antioxidant capacity of the human body has rarely been investigated. To the best of our knowledge, only Leighton et al (29), on the basis of an intervention study, reported that total antioxidant reactivity increased by 28% above basal levels in the Mediterranean diet group compared with the high-fat diet group. In the present study, we have expanded the findings from previous studies because we studied a free-eating population. Thus, our findings could be applicable to the general population because the doses of this dietary pattern were not excessive, even in the high-consumption group. Moreover, our findings may explain, at least in part, the conflicting results regarding the effect of various food groups and nutrient supplements on the oxidation process that were observed in metabolic studies. We conclude from the present study that it is the balance of food consumption, and not the consumption of specific foods or nutrients, that matters for the protection of the cardiovascular system.
Limitations
Because of the cross-sectional design of the study, we cannot establish causal relations but only generate hypotheses for the association of a dietary pattern and the TAC of the human body. Another limitation is the misreporting of food items consumed, especially alcohol consumption, because of either a recall bias or the social class of the participants. Moreover, the food-frequency questionnaire has been validated only in a sample of schoolteachers (19), whereas we applied it to the general population. This may hide any over- or underestimation of the various nutrients examined. However, in the present study we did not use nutrient intake that may be influenced by the validation of the questionnaire.
Conclusions
Until now, traditional analyses in the field of nutritional epidemiology had typically examined health status with a single or a few nutrients or foods. However, people do not eat isolated nutrients; rather, they eat meals consisting of a variety of foods with complex combinations of nutrients (30). To overcome the limitations of a single nutrient approach, ie, interactions and intercorrelation between nutrients, several scientists have proposed to study overall dietary patterns by considering how foods and nutrients are consumed in combination (2, 5, 16, 31, 32). Under this concept, we evaluated a whole dietary pattern, ie, the traditional Mediterranean diet, on TAC, and we observed that the Mediterranean diet enhances antioxidant defenses, whereas a diet high in saturated fats induces oxidative stress. On the basis of these findings, we underline the need for action from public health care professionals to prevent the development and progression of atherosclerotic diseases by encouraging patients to adopt diets low in animal fats, such as the Mediterranean diet.
ACKNOWLEDGMENTS
We thank the field investigators of the ATTICA study: John Skoumas (principal field investigator); Natasa Katinioti, Akis Zeimbekis, Spiros Vellas, Efi Tsetsekou, Dina Massoura, and Lambros Papadimitriou for the physical examinations; and Efi Tsetsekou for the psychological evaluations. We also thank the technical team: Marina Toutouza (principal investigator) for the biochemical analyses, Carmen Vasiliadou for the genetic analyses, Manolis Kambaxis and Konstadina Paliou for the nutritional evaluations, Konstadina Tselika and Sia Poulopoulou for the biochemical evaluations, and Maria Toutouza for the database management.
DBP designed the study, performed the data analyses, interpreted the results, and wrote the manuscript. CP and CC wrote the manuscript and designed the study. NT critically reviewed the manuscript. ME evaluated the biochemical analyses. AZ and CS drafted the manuscript. None of the authors had any conflicts of interest.
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