Miguel A Martinez-Gonzalez

Dolores Corella

Jordi Salas-Salvado

Emilio Ros

Maria Isabel Covas

Miquel Fiol

Ramón Estruch

Miguel ; Martinez-Gonzalez

Martínez-González Ma

Covas Mi

Ruiz-Gutiérrez

Lamuela-Raventos Rm

Muñoz Ma

Martinez Ja
email: mamartinez@unav.es

Sáez G 15

Mitjavila Mt

Tur Ja

Portillo Mp

Prof Angel Miguel

MD, MPH Martinez-Gonzalez

Cohort profile: Design and methods of the PREDIMED study

Keywords: Diet, Mediterranean, Olive oil, Cardiovascular Diseases, Prevention

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

How did the PREDIMED study come about?

A call for grants was issued in 2002 by the Spanish Government (Instituto de Salud Carlos III). This call was specifically designed to initiate networking research among Spanish biomedical investigators. During 2002, Ramón Estruch -the leader of our initiative-contacted different Spanish investigators (the rest of us) working in nutrition from different perspectives. We applied together for a grant to start a large randomised trial to test the effectiveness of a Mediterranean diet (MeDiet) on the primary prevention of cardiovascular disease (CVD) and to continue the study as an observational cohort of high-risk participants to be followed-up in the long term. On January 6th, 2003 our project was funded.

From January to June 2003 we developed the protocol: logistics, manual of operations, instruments, forms, and data entry/management systems. The needed personnel (a minimum of a dietician and a nurse for each of the 11 field centres, FC) was then hired, trained and certified. Each FC contacted approximately 20 Primary Care Practices (PCP) to recruit participants. The recruitment of participants started in October 2003. The name PREDIMED (in Spanish: PREvención con DIeta MEDiterránea) was proposed by Dolores Corella. This name is applied to both the cohort study and the networking group.

Despite being an interventional study, the PREDIMED study provides a unique opportunity for conducting the long-term follow-up (after the completion of the trial) of a large observational cohort of high cardiovascular risk subjects in a Mediterranean setting. The MeDiet represents the dietary exposure in closest agreement with the Bradford Hill criteria for a potential causal protection against coronary heart disease (CHD) according to a recent systematic review. [START_REF] Mente | A systematic review of the evidence supporting a causal link between dietary factors and coronary heart disease[END_REF] This conclusion is mainly supported by observational cohort studies. A recent meta-analysis of these cohorts showed that adherence to the MeDiet was associated with reductions in total mortality and CHD mortality. [START_REF] Sofi | Accruing evidence on benefits of adherence to the Mediterranean diet on health: an updated systematic review and meta-analysis[END_REF] Subsequently, similar evidences have been collected for non-fatal CVD. [START_REF] Fung | Mediterranean diet and incidence of and mortality from coronary heart disease and stroke in women[END_REF][START_REF] Buckland | Adherence to the Mediterranean diet and risk of coronary heart disease in the Spanish EPIC cohort study[END_REF][START_REF] Martínez-González | Mediterranean diet and the incidence of cardiovascular disease: A Spanish cohort[END_REF] An increasing body of evidence is supporting also a benefit of the MeDiet against major cancers and neurodegenerative diseases. [START_REF] Benetou | Conformity to traditional Mediterranean diet and cancer incidence: the Greek EPIC cohort[END_REF][START_REF] Buckland | Adherence to a Mediterranean diet and risk of gastric adenocarcinoma within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort study[END_REF][START_REF] Scarmeas | Mediterranean diet and risk for Alzheimer's disease[END_REF][START_REF] Féart | Adherence to a Mediterranean diet, cognitive decline, and risk of dementia[END_REF][START_REF] Sánchez-Villegas | Association of the Mediterranean dietary pattern with the incidence of depression: the Seguimiento Universidad de Navarra/University of Navarra follow-up (SUN) cohort[END_REF] Our hypothesis was that two traditional MeDiets, one enriched with virgin olive oil (VOO) and another enriched with nuts, both high in total fat and unsaturated fat, would be superior to the usually recommended low-fat diet for the primary prevention of CVD in a high-risk population. This fit well into the paradigm of focusing on dietary patterns instead of isolated foods or nutrients. Overall patterns better represent dietary practices found in free-living populations, therefore providing useful epidemiological information with a high potential for acceptability, palatability and future compliance.

However, no randomised controlled trial has ever been conducted to test the MeDiet in the primary prevention of major chronic diseases. The only available clinical trial supporting a cardioprotective role of the MeDiet is the Lyon Diet Heart Study. [START_REF] De Lorgeril | Mediterranean diet, traditional risk factors, and the rate of cardiovascular complications after myocardial infarction: final report of the Lyon Diet Heart Study[END_REF] That trial, an important step forward the benefits of the Mediterranean diet, included only myocardial infarction survivors (i.e., it was a secondary prevention trial) and showed a remarkable 50-70% reduction in CHD event rates and mortality with a "MeDiet" (enriched with alphalinolenic acid, but not with olive oil). These results were criticized because no special consideration was given to olive oil, which is the major source of dietary fat in Mediterranean countries. [START_REF] Martinez-Gonzalez | The emerging role of Mediterranean diets in cardiovascular epidemiology: monounsaturated fats, olive oil, red wine or the whole pattern?[END_REF] Another problem was that dietary assessments at baseline and at the end of the study were reported for only 30% of the control group and 50% of the experimental group and no biochemical markers of adherence were obtained. Finally, concerns have been raised regarding the low number of observed endpoints (44 in the control group versus 14 in the treatment group), and the improbable contrast between the large reduction in risk and the lack of changes in most classical risk factors. The PREDIMED study attempts to overcome previous limitations and to provide the best quality of evidence to answer the question of whether the MeDiet, compared with the previously tested model of advice on a low-fat diet, provides a relatively higher protection for the prevention of chronic disease. In the face of the increasing global burden of CVD and cancer, the answer to this question is a major public health priority. The long tradition of adherence to this food pattern in Mediterranean countries, where CHD incidence is low despite high levels of cardiovascular risk factors [START_REF] Gabriel | Prevalence, geographic distribution, and geographic variability of major cardiovascular risk factors in Spain. Pooled analysis of data from population-based epidemiological studies: the ERICE Study[END_REF] , the diversity of mechanisms supporting the beneficial effects on cardiovascular health of olive oil [START_REF] López-Miranda | Olive oil and health: Summary of the II International Conference on Olive Oil and Health consensus report, Jaén and Córdoba (Spain)[END_REF][START_REF] Fernández-Jarne | Risk of first non-fatal myocardial infarction negatively associated with olive oil consumption: a case-control study in Spain[END_REF] or nuts [START_REF] Kris-Etherton | The role of tree nuts and peanuts in the prevention of coronary heart disease: multiple potential mechanisms[END_REF] ; and the higher palatability, acceptance and compliance of MeDiets in comparison with low-fat diets [START_REF] Mcmanus | A randomized controlled trial of a moderate-fat, low-energy diet compared with a low fat, low-energy diet for weight loss in overweight adults[END_REF][START_REF] Shai | Weight loss with lowcarbohydrate, Mediterranean and low-fat diet[END_REF] lend support to our hypothesis.

The results of some observational studies have been subsequently refuted by evidence from clinical trials (i.e., the presumptive cardioprotective effects of postmenopausal hormone therapy [START_REF] Manson | Estrogen plus progestin and the risk of Coronary Heart disease[END_REF] or antioxidant supplements [START_REF] Bjelakovic | Mortality in randomized trials of antioxidant supplements for primary and secondary prevention. Systematic review and meta-analysis[END_REF] ). This highlights the need to obtain first level evidence before considering any global public health strategy. Dietary guidelines can be safely issued when consistency is found between observational and experimental studies.

What does the PREDIMED study cover?

A large cohort has been assembled for long-term follow-up. This cohort includes 7447 high-risk participants. The last participant was recruited on June 30 th , 2009. Trial closeout will take place by December 31 st , 2011. Subsequent follow-up will continue as an observational multi-purpose cohort to explore other hypotheses (i.e., the roles of different types of alcoholic beverages on cancer or CVD prevention) and to develop nested casecontrol analyses for studies of biomarkers and gene-nutrient interactions.

The primary aim of the trial is to assess the effects of two MeDiets on a composite endpoint of cardiovascular death, myocardial infarction and stroke (primary endpoint) in comparison with a low-fat, control diet. Secondary endpoints are death of any cause, incidence of heart failure, diabetes mellitus, dementia or other neurodegenerative disorders, and major cancers (colorectal, breast, lung, stomach, prostate). To better understand how dietary changes may modify the risk of clinical events, we also evaluate intermediate outcomes, including changes in blood pressure (BP), weight gain, fasting blood glucose, blood lipids, and markers of inflammation.

Who are the participants in the PREDIMED study?

Participants are men (55-80 year-old) or women (60-80 year-old) who were free of CVD at Potential participants were approached by PCPs by a telephone call or during their clinical visits. If candidates were interested in participating, a face-to-face interview was scheduled. During this interview, the purpose and characteristics of the study were explained, and a signed informed consent was obtained from willing participants. A brief explanation of the study, including the possibility that they might receive free allowances of VOO or nuts for the duration of the trial, was given in this first visit. Most (>70 %) candidates approached in this way agreed to return for the screening visit.

The ALLHAT trial included similar participants and observed a 8.9% cumulative rate for the primary outcome (fatal CHD + non-fatal myocardial infarction) after 4.9 years of followup. [START_REF]The Antihypertensive and Lipid-Lowering Treatment Prevent Heart Attack Trial[END_REF] Adapting this figure to a 6-year follow-up and including also stroke in the end-point definition, an 11% absolute risk in the control group could be conservatively assumed in our study. We expect a 25% relative risk reduction in both MeDiet groups. Under these assumptions, the total number of participants required was 5631 (1877 per group) for beta=0.2 and 2-tailed alpha=0.05. We included 7447 subjects to allow for both 10% losses during follow-up and a lower incidence than expected. Figure 1 shows the flow of participants.

Study participants were randomised to three equally-sized groups. Tables of random allocation were centrally elaborated. The study nurses in charge of the random allocation were independent of the nurse staff of the PCP. At baseline, General Practitioners (GP)

were not informed of the allocation of participants. This is consistent with CONSORT guidelines for randomised trials to prevent selection biases [START_REF] Altman | The revised CONSORT statement for reporting randomized trials explanation and elaboration[END_REF] .

Characteristics of participants according to group allocation are shown in Table 1 Besides being an excellent source of monounsaturated fat (MUFA), VOO also contains significant amounts of phenolic antioxidants and other phytochemicals (tocopherols, polyphenols) because it is obtained from the first pressing of the ripe fruit (i.e., it is an olive juice). In contrast, these phytochemicals are present to a lower extent in common refined olive oils (ROO). [START_REF] López-Miranda | Olive oil and health: Summary of the II International Conference on Olive Oil and Health consensus report, Jaén and Córdoba (Spain)[END_REF] ROO lose polyphenols and other elements in the refining process, although fatty acid composition is similar to that of VOO [START_REF] Owen | Phenolic compounds and squalene in olive oils: the concentration and antioxidant potential of total phenols, simple phenols, secoiridoids, lignans and squalene[END_REF] . When compared to ROO, VOO increases HDL cholesterol, total plasma antioxidant capacity and LDL resistance to oxidation. In vivo markers of lipid and LDL oxidation decrease in a dose-dependent manner with the phenolic content of the olive oil. [START_REF] Covas | The effect of polyphenols in olive oil on heart disease risk factors: a randomized trial[END_REF] Most nuts are rich in MUFA (mostly, oleic acid), whereas walnuts are high in polyunsaturated fatty acids (PUFAs, i.e. linoleic and alpha-linolenic acids). The dietary fiber content in nuts is also high. Nuts are good sources of arginine, potassium, vitamin E, and other bioactive compounds. This may help explain their beneficial health effects. [START_REF] Kris-Etherton | The role of tree nuts and peanuts in the prevention of coronary heart disease: multiple potential mechanisms[END_REF] The rationale for the free provision of these food items (VOO and nuts) is that they may contribute to a higher compliance with the overall MeDiet food pattern.

The PREDIMED dieticians are directly responsible for the dietary intervention. After 2 screening visits, participants randomised to each one of the 3 treatment arms have a face-to-face interview with the dietician and a group session (<20 subjects). A 14-point score of adherence to the MeDiet is a main tool to change dietary habits (Table 2). [START_REF] Estruch | Effects of a Mediterranean-style diet on cardiovascular risk factors. A randomized trial[END_REF][START_REF] Martinez-Gonzalez | Development of a short dietary intake questionnaire for the quantitative estimation of adherence to a cardioprotective Mediterranean diet[END_REF][START_REF] Zazpe | A large randomized individual and group intervention conducted by dieticians increased the adherence to Mediterranean-type diets: The PREDIMED study[END_REF] A similar 9-point score is used for the low-fat control group. For total fat intake, the recommendations given to participants in the low-fat diet group are opposite to those given to participants in the 2 MeDiet groups. The focus can be shifted from changing portion sizes, frequency of intake or cooking methods. We have reported an adequate effectiveness of the intervention after one year of follow-up. [START_REF] Zazpe | A large randomized individual and group intervention conducted by dieticians increased the adherence to Mediterranean-type diets: The PREDIMED study[END_REF] Because unsaturated fats like those contained in olive oil and nuts are still wrongly perceived as fattening, it has been particularly important to allay the fear of an eventual weight gain. Tactful exposition of recent scientific evidence [START_REF] Shai | Weight loss with lowcarbohydrate, Mediterranean and low-fat diet[END_REF][START_REF] Sacks | Comparison of weight-loss diets with different compositions of fat, protein, and carbohydrates[END_REF][START_REF] Bes-Rastrollo | Olive oil consumption and weight change: the SUN prospective cohort study[END_REF][START_REF] Bes-Rastrollo | Prospective study of nut consumption, long-term weight change, and obesity risk in women[END_REF] , together with the fact that body weight did not change after 3 months of MeDiet intervention in the pilot phase of the PREDIMED study [START_REF] Estruch | Effects of a Mediterranean-style diet on cardiovascular risk factors. A randomized trial[END_REF] have been instrumental for this aim.

The PREDIMED group sessions are organized separately for each of the 3 intervention groups. Participants are provided with written material (see: http://www.predimed.org and http://www.predimed.es) including descriptions of seasonal foods, shopping lists, weekly meal plans and cooking recipes. Olive oil and nut industry companies are committed to supply for free the food supplements used in the study until December, 2011. None of the investigators has any commercial interest with these food companies.

How often are cohort volunteers contacted?

Table 3 shows the frequency of contacts with participants. The individual and group visits are repeated every 3 months with the same contents, except that shopping lists and recipes vary with the season of the year. Each visit includes three steps: assessment, intervention, and future directions. Once a year, general medical and food frequency questionnaires (FFQ) are obtained, an electrocardiogram (ECG) is performed, and blood and urine samples are collected (Table 3). After the trial formally terminates (December, 2011), we will follow our cohort for occurrence of clinical events, ECG and measurement of weight and BP. We will continue to ascertain participants' vital status through yearly personal interviews by PREDIMED personnel, close contact with GPs who care for them and reviews of medical records. On a yearly basis, the Spanish official mortality index (Indice Nacional de Defunciones) is also reviewed.

What variables are measured?

Table 3 shows the variables collected in the PREDIMED study. The yearly-administered FFQ provide information about compliance with food and nutrient targets. This FFQ was previously validated in Spain. [START_REF] Boyle | Development and validation of a food frequency questionnaire in Spain[END_REF] We have performed a new validation of the FFQ with highrisk persons similar to PREDIMED participants [START_REF] Fernández-Ballart | Relative validity of a semiquantitative food-frequency questionnaire in an elderly Mediterranean population of Spain[END_REF] and have confirmed its reproducibility [START_REF] De La Fuente-Arrillaga | Reproducibility of a Food Frequency Questionnaire (FFQ) validated in Spain[END_REF] .

Biological markers of compliance (plasma oleic and alpha-linolenic acid proportions and urinary concentrations of tyrosol and hydroxytyrosol, resveratrol and ethanol) are measured in random subsets of participants from the three arms of the trial [START_REF] Zazpe | A large randomized individual and group intervention conducted by dieticians increased the adherence to Mediterranean-type diets: The PREDIMED study[END_REF] . Clinical evaluations are limited to yearly follow-up visits that include the same examinations performed at baseline, with the exception of the general questionnaire, which is substituted by a follow-up questionnaire, and a tolerance/adverse events questionnaire.

Although no intervention on physical activity is performed, the Minnesota physical activity questionnaire (validated Spanish version) [START_REF] Elosua | Validation of the Minnesota Leisure Time Physical Activity Questionnaire in Spanish men. The MARATHOM Investigators[END_REF][START_REF] Elosua | Validation of the Minnesota Leisure Time Physical Activity Questionnaire In Spanish Women. Investigators of the MARATDOM Group[END_REF] is completed each year.

Blood and urine samples are collected at baseline and years 1, 3, 5 and 6 (or final visit).

Tubes for EDTA plasma, citrate plasma, buffy coat, and serum are collected and aliquots are kept frozen (-80ºC). The Short-Form 36 to assess quality of life is completed by all [START_REF] Buil-Cosiales | Carotid intimamedia thickness is inversely associated with olive oil consumption[END_REF][START_REF] Buil-Cosiales | Dietary fibre intake is inversely associated with carotid intima-media thickness: a cross-sectional assessment in the PREDIMED study[END_REF][START_REF] Salas-Salvado | Components of the Mediterranean-type food pattern and serum inflammatory markers among patients at high risk for cardiovascular disease[END_REF] Outcomes are ascertained on a yearly basis by a Clinical Events Committee whose members are blinded to the intervention group.

What is the attrition?

A high retention rate is a major methodological requirement in follow-up studies. The attrition rate after 2 years follow-up for participants recruited before 2006 (n=4.381) was 9.3%. The highest retention rates occurred in the MeDiet with VOO group and the lowest retention rate was observed in the control (low-fat diet) group (Figure 1). The highest retention rate in the two groups allocated to MeDiets can be partly attributed to the free provision of food items (VOO and nuts). However, in the PREDIMED trial we will eventually be able to obtain a nearly complete follow-up for the main outcomes because participants represent a stable and well-defined population regularly attending GP visits in their PCPs. In addition, a comprehensive search for events is performed yearly through review of the medical records of participants in all the hospitals of the city where the respective FC is located.

What has been found so far?

The pilot study of the PREDIMED trial (n=772) suggested that a MeDiet was a safe strategy to reduce the levels of major cardiovascular risk factors after a 3-month followup. [START_REF] Estruch | Effects of a Mediterranean-style diet on cardiovascular risk factors. A randomized trial[END_REF] Inverse baseline associations with inflammatory markers for cereals, fruits, nuts and VOO were found. [START_REF] Salas-Salvado | Components of the Mediterranean-type food pattern and serum inflammatory markers among patients at high risk for cardiovascular disease[END_REF] In the first 3204 participants, the 14-point score was able to predict the prevalence of diabetes, hypertension and obesity or the joint presence of metabolic conditions. [START_REF] Sanchez-Tainta | Adherence to a Mediterranean-type diet and reduced prevalence of clustered cardiovascular risk factors in a cohort of 3204 high-risk patients[END_REF] In a 3-month longitudinal study we found a favourable effect of the MeDiet interventions on LDL oxidation [START_REF] Fitó | Effect of a traditional Mediterranean diet on lipoprotein oxidation: a randomized controlled trial[END_REF] and cellular and serum inflammatory biomarkers related to atherosclerosis. [START_REF] Mena | Inhibition of circulating immune cell activation: a molecular anti-inflammatory effect of the Mediterranean diet[END_REF] After a 12-month follow-up of the first 1224 participants, the prevalence of metabolic syndrome was reduced in all groups, but it was more marked in the MeDiet groups, especially in the MeDiet+nuts group. [START_REF] Salas-Salvadó | Effect of a Mediterranean diet supplemented with nuts on metabolic syndrome status[END_REF] Other studies have assessed dietary associations with hypertension [START_REF] Guxens | Hypertensive status and lipoprotein oxidation in an elderly population at high cardiovascular risk[END_REF][START_REF] Toledo | Low-fat dairy products and blood pressure: follow-up of 2290 older persons at high cardiovascular risk participating in the PREDIMED study[END_REF][START_REF] Barceló | Mediterranean-style diet effect on the structural properties of the erythrocyte cell membrane of hypertensive patients: the Prevencion con Dieta Mediterranea Study[END_REF] and gene-nutrient interactions in obesity and weight gain. [START_REF] Razquin | A 3-year intervention with a Mediterranean diet modified the association between the rs9939609 gene variant in FTO and body weight changes[END_REF][START_REF] Razquin | A 3 years follow-up of a Mediterranean diet rich in virgin olive oil is associated with high plasma antioxidant capacity and reduced body weight gain[END_REF][START_REF] Razquin | The Mediterranean diet protects against waist circumference enlargement in 12Ala carriers for the PPARgamma gene: 2 years' follow-up of 774 subjects at high cardiovascular risk[END_REF] In 2009 more than 30 papers derived from the PREDIMED study have been either published or are accepted for publication in peer-reviewed journals. In 2010 a substantial effect of both MeDiets in the reduction of type-2 diabetes risk after a median follow-up of 4.0 years was reported from a nested analysis conducted in one of the centres [START_REF] Salas-Salvadó | Reduction in the Incidence of Type 2-Diabetes with the Mediterranean Diet: Results of the PREDIMED-Reus Nutrition Intervention Randomized Trial[END_REF] .

Funding

What are the main strengths and weaknesses?

The strengths are the randomised design, the large sample size, the storing of abundant biological samples, the objective assessment of compliance with biomarkers, and the close monitoring of participants. The main weakness is the difficulty to change longestablished dietary habits and increase adherence to a low-fat diet in participants allocated the control group.

From a public health perspective, a behavioural intervention coupled with an easy (free) access to representative healthy foods is a realistic test of the effectiveness to be attained with official policies and health promotion activities. The PREDIMED trial attempts to obtain relevant information for public health use, because the nutritional intervention is undertaken in free-living persons who receive information, motivation, support and empowerment to modify their food habits in a real-life context, i.e., they continue to buy their foods and cook their meals. Such an intervention provides a real life scenario that may be easily applied to public health policies.

Where can

  Design and methods of the PREDIMED study

  baseline. Inclusion criteria were to have either type 2 diabetes or ≥3 major cardiovascular risk factors, out of the following: current smoking (>1 cig/day during the last month); hypertension (systolic BP ≥140 mm Hg or diastolic BP ≥90 mm Hg or antihypertensive medication); LDL-cholesterol ≥160 mg/dl or lipid-lowering therapy; HDL-cholesterol ≤40 in men or ≤50 mg/dl in women; body mass index ≥25 kg/m 2 ; and family history of premature CHD.Exclusion criteria were previous history of CVD (i.e. a previous medical diagnosis of CHD, stroke or peripheral arterial disease), any severe chronic illness, immunodeficiency or human immunodeficiency virus (HIV) positive status, illegal drug or alcohol abuse, history of allergy to olive oil or nuts, and low predicted likelihood of changing dietary habits according to the Prochaska and DiClemente stages of change model.[START_REF] Nigg | Stages of change across ten health risk behaviors for older adults[END_REF] Figure 1 provides further details on the selection procedure. The selection process started by extracting names of potential participants from the records of the PCPs. Most PCPs participating in the study have computer-based records of patients, making the selection relatively simple. The clinical records of these persons were then individually reviewed to exclude those who did not meet eligibility criteria.

  . The Institutional Review Board (IRB) of Hospital Clinic (Barcelona, Spain) approved the study protocol on July 2002. This IRB is accredited by the US Department of Health and Human Services (DHHS). Later, the IRBs of all other centres also approved the protocol. The trial is registered (http://www.controlled-trials.com/ISRCTN35739639).What are the interventions in the PREDIMED study?Participants were randomly assigned to 3 interventions: MeDiet with VOO, MeDiet with mixed nuts, or control group (low-fat diet). The two groups allocated MeDiets receive intensive education to follow the MeDiet and supplemental foods at no cost. VOO (1 l/wk) is provided to the first group and 30 g/d of mixed nuts (15 g walnuts, 7.5 g hazelnuts, and almonds) to the second group. In the control group participants do not receive education on the MeDiet, but are given advice to follow a low-fat diet.

  2007. Toenails are collected at baseline and the final visit. In two centres (Barcelona-North and Pamplona) carotid intima-media thickness has been measured in subsets of participants.

The

  Spanish Ministry of Health -Instituto de Salud Carlos III (ISCIII) funded the project for the period 2003-2005 (RTIC G03/140). In 2006 a new funding modality was established by ISCIII through the CIBER Fisiopatología de la Obesidad y Nutrición (CIBERobn), which is providing funding for 7 of the original research groups, while the other 12 were funded by a new research network (RTIC RD 06/0045). Other official funds from Spanish government agencies have been obtained for subprojects related to intermediate outcomes (lipoproteins, inflammatory markers, vascular imaging, genomic and proteomic studies, etc.).

  donation by food companies of all the VOO and mixed nuts needed throughout the duration of the study is a substantial contribution. None of these companies (Patrimonio Comunal Olivarero, California Walnut Commission, Borges, La Morella Nuts and Hojiblanca) played or will play any role in the design, collection, analysis, or interpretation of the data or in the decision to submit manuscripts for publication.

I find out more?

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Table 1 . Description of participants in the PREDIMED study at baseline according to intervention group. Characteristics at baseline

 1

		MeDiet + VOO	MeDiet + nuts	Contol (low-fat)
		(n=2543)	(n=2454)	(n=2450)
	Age (mean, SD)	67 (6)	67 (6)	67 (6)
	Sex (% women)	58.7	54.0	59.7
	Diabetes (%)	50.2	46.5	48.4
	Hypertension (%)	82.1	82.4	83.7
	Current smokers (%)	13.9	14.5	13.8
	Former smokers (%)	24.3	25.8	23.8
	High blood cholesterol (%)	71.6	73.3	71.9
	Family history of CHD* (%)	22.7	21.7	22.8
	BMI (mean, SD)	30.0 (3.7)	29.7 (3.8)	30.2 (4.0)
	Waist circumference (mean, SD)	100 (10)	100 (11)	101 (11)
	Adherence to MeDiet** (mean, SD)	8.7 (2.0)	8.7 (2.0)	8.4 (2.1)
	Spanish region (% participants)
	North (Navarra and Basque country)	23.4	22.9	22.2
	North-East (Catalonia)	31.3	33.2	31.5
	East (Valencia and Balearic Islands)	23.0	24.2	23.1
	South (Andalusia and Canary Islands)	22.3	19.7	23.1
	Mediet: Mediterranean diet; VOO: virgin olive oil; SD: standard deviation; CHD: coronary heart disease
	* Definite myocardial infarction or sudden death before 55 years in male first-degree relatives or before 65 years in
	female first-degree relatives
	** 14-point score of adherence to Mediet

Table 3 . Measurements in the PREDIMED study.

 3 BP: blood pressure; MeDiet: Mediterranean diet; CAGE: 4-item screening test for alcohol dependence; ECG: electrocardiogram; SF-36: short-form 36, 36-item questionnaire for quality of life. *Only for participants recruited before 2005. **Includes direct measurements of weight, height, waist circumference, BP, and ankle-brachial blood pressure index.

	Measurements		Description of measurements		Number of repeated measurements
		Number	Content	Baseline Year	Year	Year	Year	Year	Year	Year
		of items			1	2	3	4	5	6	7*
	Eligibility questionnaire	33	Socio-demographic, inclusion and	1
			exclusion criteria, smoking
	General questionnaire**	77	Marital status, job, BP and anthropometry,	1
			medical conditions, medications, CAGE
	14-item MeDiet questionnaire	14	MeDiet adherence (intervention tool)	1	2-5	6-9	10-13 14-17 18-21 22-25 26-29
	Food frequency questionnaire	137	32-34 Previously validated	1	2	3	4	5	6	7	8
	Physical activity questionnaire	67	35-36 Validated Minnesota questionnaire	1	2	3	4	5	6	7	8
	Follow-up questionnaire**	75	Risk factors, symptoms and conditions,		1	2	3	4	5	6	7
			job, BP, anthropometry, medication
	Tolerance questionnaire	6	Potential adverse events		1	2	3	4	5	6	7
	Abandonment questionnaire***	21	Reasons for terminating study		1	2	3	4	5	6	7
	ECG			1	2	3	4	5	6	7	8
	Blood chemistry	14	Lipids, glucose, renal function,	1	2	3	4	5	6	7	8
			transaminases, blood count and others
	Blood sample		9 tubes (38.5 ml)	1	2	(o)	3	(o)	4	5	6
			~40 aliquots (-80ºC)
	Urine sample		16 aliquots (-80ºC)	1	2	(o)	3	(o)	4	5	6
	Toenail sample		A clip of each toenail	1					2
	SF36****	36	Quality of life	1	2	3	4	5

***Only if applicable. ** Only for participants recruited after 2007. (o)-Optional collection.

Figure 1.

Flow chart of participants in the PREDIMED study.

		8713 assessed for eligibility
		1266 excluded
		973 refused to participate
		108 did not meet inclusion criteria
		51 had prior cardiovascular disease
		45 had difficulties with follow-up
		39 were unwilling to change dietary habits
		20 had excessive alcohol intake
		12 had food allergies
		10 had other chronic diseases
		8 had other exclusion criteria
		7447 randomised
	2543 assigned to	2454 assigned to	2450 assigned
	Mediterranean	Mediterranean	to low-fat diet
	diet with virgin	diet with mixed	(AHA
	olive oil	nuts	guidelines)
	2-y retention rate*	2-y retention rate*	2-y retention rate*
	96.2%	92.1%	82.7%
	*Calculated only among participants recruited during 2003-2005

F o r R e v i e w O n l y

List of original publications and other information can be found at www.predimed.es (or www.predimed.org). Collaboration with national and international studies is welcome and can be proposed to restruch@clinic.ub.es.

Conflict of interest statement

The authors declare that they do not have any conflict of interest.


A Tosenberger
email: tosenberger@math.univ-lyon1.fr

V Salnikov

N Bessonov

E Babushkina

V Volpert

Particle Dynamics Methods of Blood Flow Simulations

Keywords: molecular dynamics, dissipative particle dynamics, blood flows, erythrocyte dynamics AMS subject classification: 76Z05, 92C05

Various particle methods are widely used to model dynamics of complex media. In this work molecular dynamics and dissipative particles dynamics are applied to model blood flows composed of plasma and erythrocytes. The properties of the homogeneous particle fluid are studied. Capillary flows with erythrocytes are investigated.

Introduction

Dissipative particle dynamics is a method to simulate systems of particles. It originates from molecular dynamics where each particle moves according to Newton's second law. The difference between two methods is in the form of forces acting between the particles. In molecular dynamics, it is a pairwise potential force which depends on the distance between each two particles. In dissipative particle dynamics, there are two additional forces, dissipative and random. Their form depends on the medium which we want to describe. In the case of a homogeneous Newtonian fluid, dissipative and random forces are also pairwise and have a special form [START_REF] Espanol | Statistical mechanics of dissipative particle dynamics[END_REF], [START_REF] Groot | Dissipative particle dynamics: Bridging the gap between atomistic and mesoscopic simulation[END_REF], [START_REF] Karttunen | A novel methods in soft matter simulations[END_REF]. For properly chosen values of parameters, this approach is equivalent to Navier-Stokes equations. However, from the computational point of view, it is more expensive. Its application becomes justified to model complex media, in particular blood flows. Conventional approaches, where blood is considered as a homogeneous non-Newtonian fluid, is widely used and allows one to study numerous problems of hemodynamics (see, e.g., [START_REF] Galdi | Hemodynamics flow. Modeling, analysis, and simulations[END_REF]). However it has its limitations when we need to describe in more detail cell-cell interaction in the flow or cell-vessel wall interaction. In this case we need to take into account individual cells with their geometry and structure (elastic cell membrane, intracellular medium). Several approaches are developed in order to study blood flows considered as a heterogeneous medium composed of plasma and blood cells: lattice Boltzmann method [START_REF] Tsubota | A particle method for blood flow simulation, application to flowing red blood cells and platelets[END_REF], smoothed particle dynamics [START_REF] Hosseini | A particle-based model for the transport of erythrocytes in capillaries[END_REF], dissipative particle dynamics [START_REF] Fedosov | A multiscale red blood cell model with accurate mechanics, rheology, and dynamics[END_REF], immerse boundary method [START_REF] Fogelson | em Cell-based models of blood clotting[END_REF] and other methods to approximate the membrane [START_REF] Bui | Dynamics of red blood cells in 2D[END_REF]. We will briefly present some results for molecular dynamics (MD) and dissipative particles dynamics (DPD).

Molecular dynamics and dissipative particle dynamics 2.1. MD description

The first approach is based on Molecular Dynamics (MD) simulations. We introduce a conservative force in a form similar to Lennard-Jones potential but simpler from the computational point of view:

F C ij = F C (r ij )r ij , (2.1)
where F ij is the force between the particles i and j, acting on the particle i,

r ij = r i -r j , r ij = |r ij |, rij = r ij /r ij ,
and the function F is given by the equality

F C (r ij ) = k r c r ij -1 nr c r ij -1 . (2.2)
Here r c is the force cut-off radius, which determines the distance of force influence, and n is the parameter which determines the second root of the function F . If the the roots of the conservative force function are equal to each other, then there is no attraction between the particles. In addition to the conservative force, on each particle a force of fixed amplitude, but random direction is applied. This random force, denoted F R i , acts as an energy source. The motion of the particles is governed by Newton's second law of motion, in which the artificial viscosity term is introduced in order to adjust dynamic viscosity of a simulated fluid, but also to stabilize the system of particles, if needed:

dr i = v i dt, dv i = 1 m i F R i + j =i F C ij -cv i dt, (2.3)
where r i is the position vector and v i is the velocity vector of particle i. The coefficient c is the strength of the artificial viscosity (dampening).

DPD description

The second approach considered in this work is Dissipative Particle Dynamics (DPD). We use it in the form described in literature [START_REF] Groot | Dissipative particle dynamics: Bridging the gap between atomistic and mesoscopic simulation[END_REF], [START_REF] Karttunen | A novel methods in soft matter simulations[END_REF], [START_REF] Fedosov | Multiscale Modeling of Blood Flow and Soft Matter[END_REF]. The DPD is governed by three equations describing conservative, dissipative and random force acting between each two particles:

F C ij = F C ij (r ij )r ij , ( 2.4)
F D ij = -γω D (r ij )(v ij • rij )r ij , ( 2.5)
F R ij = σω R (r ij ) ξ ij √ dt rij , (2.6)
where r i is the vector of position of the particle i,

r ij = r i -r j , r ij = |r ij |, rij = r ij /r ij , and v ij = v i -v j
is the difference between velocities of two particles. γ and σ are coefficients which determine strength of the dissipative and the random force respectively, while ω D and ω R are weight functions; ξ ij is a normally distributed random variable with zero mean, unit variance, and ξ ij = ξ ji . The conservative force is given by the equality

F C ij (r ij ) =    a ij (1 -r ij /r c ) for r ij ≤ r c , 0 for r ij > r c , (2.7)
where a ij is the conservative force coefficient between particles i and j, and r c is the cutoff radius.

The random and dissipative forces form a thermostat. If the following two relations are satisfied, system will preserve its energy and maintain the equilibrium temperature:

ω D (r ij ) = ω R (r ij ) 2 , σ 2 = 2γk B T, (2.8)
where k B is the Boltzmann constant and T is the temperature. The weight functions are determined by:

ω R (r ij ) =    (1 -r ij /r c ) k for r ij ≤ r c , 0 for r ij > r c , (2.9)
where k = 1 for the original DPD method, but it can be also scaled in order to change the dynamic viscosity of the simulated fluid. Similar to the previous method, the motion of particles is determined by Newton's second law of motion:

dr i = v i dt, dv i = 1 m i j =i F C ij dt + F D ij dt + F R ij √ dt .
(2.10)

Boundary conditions

A practical way to verify the correctness of MD and DPD is to compare the velocity and density profiles in the Poiseuille flow with the corresponding solution of the Navier-Stokes equations. Velocity and density profiles are averaged through a period of time which length depends on parameters, and it begins once the system is near its stable solution. The stability of the solution can be evaluated by the stability of the system energy.

One of the problems that raises when working with particle simulation methods, like MD and DPD, is the implementation of boundary conditions. Choosing a too simple implementation of a no-slip boundary condition can result in distortion of overall velocity and density profiles (Figure 1). Various implementations that try to arrange these problems can be found in literature [START_REF] Fedosov | Velocity limit in DPD simulations of wallbounded flows[END_REF]- [START_REF] Fedosov | Multiscale Modeling of Blood Flow and Soft Matter[END_REF].

For computational purposes, in our simulations we have tried to use as simple implementations as possible, while keeping the velocity and density profiles sufficiently precise. Therefore, we have started from a simple and straightforward implementation of no-slip boundary conditions where particles rebound from the boundary, but in the moment when a center of a particle is in contact with the wall the tangential to the boundary velocity is set zero. With no-slip boundary conditions, implemented like that, both measured profiles, velocity and density, have a deviation from the analytical solution in the region close to the boundary (Figure 1). These deviations can be decreased directly by increasing the number density while reducing the cutoff radius, but this makes the method much less robust. To correct them, the problem of velocity and density profiles can be studied separately. Density oscillations appear near the no-slip boundary because there is no particles on the other side of the wall, so the forces acting on the particles at the distance less than r c from the boundary are not in balance.

To solve this problem we consider two approaches. The first approach, which we call halfperiodic boundary condition, consists of allowing the particles which are next to one wall to feel the particles which are close to the opposite wall, just as it is case with periodic boundary conditions, but particles are still rebounded from the boundary. Interaction of particles on the opposite parts of the domain is done by taking into account only the part of forces between them which is orthogonal to the wall. The result is a correct density profile (Figure 1). For simple rectangular domains halfperiodic boundary conditions are effective and elegant solution for the density problem. However, it can not be easily generalized on the more complex domains. Therefore, for complex domains it is better to measure the average force density and to apply missing part on each particle which is near the boundary [START_REF] Fedosov | Multiscale Modeling of Blood Flow and Soft Matter[END_REF].

To solve the velocity profile distortions near the boundary, tangential part of velocity needs to be corrected. In the simplest approach, a parabolic velocity profile can be imposed in a narrow boundary layer. More precise and sophisticated methods are also described in literature (see, e.g., [START_REF] Fedosov | Velocity limit in DPD simulations of wallbounded flows[END_REF]).

Fluid and flow properties: density, viscosity, velocity

Once we have sufficiently precise velocity and density profiles it is very important to know how the properties of the fluid depend on the choice of method and on parameters. One of the most The Poiseuille flow can be organized with a body force applied to the fluid or with a gradient of pressure. In our simulations we have used the body force. This rises the question about the viscosity dependence on the body force. For the purposes of our work we simulate blood plasma, which can be considered as a Newtonian fluid. Therefore, dynamic viscosity should not depend on the body force. In the MD method we observe that this is not the case, and that velocity depends of body force (Figure 2, left). However, in the DPD method the viscosity remains practically the same (Figure 2, right), except for very small value of body force. This indicates that the DPD method can be more suitable for simulations of Newtonian fluids. In MD we can vary the cutoff radius (and/or the number density), the amplitude of the random force and the value of artificial viscosity. Cutoff radius and artificial viscosity can be used to make large changes in the viscosity of the fluid (Figure 3). However, increasing the artificial viscosity too much results in velocity profiles different from parabolic (Figure 2). On the other hand, random force can be used to scale the dynamic viscosity on a finer scale. In the DPD method, there are more parameters which have to be adjusted: coefficients of conservative a ij and dissipative force γ (random force coefficient σ is determined via equation (2.8)), exponent k in the equation (2.9), number density n and cutoff radius r c . A large viscosity variation can be obtained by variation of all parameters, except for the coefficient of the conservative force which can be used to adjust viscosity more precisely. However, for very large changes in viscosity, it is better not to use extreme values of parameters because it can influence the velocity profile, prolong the period of time needed to achieve the stable flow, or just make the simulation more complex to compute. Viscosity dependence on parameters is presented in Figure 4. One more significant difference between MD and DPD is that DPD converges faster to a stable flow.

Erythrocyte in capillary flows

Erythrocyte model

After simulating blood plasma with particle dynamics, the next step is to introduce erythrocytes in the model. It is known that erythrocytes have a viscoelastic membrane which can have a specific biconcave shape. This shape is a result of minimisation of surface energy. In 2D, we can take a membrane with perimeter 2rπ surrounding an area 3 5 r 2 π, and we will obtain a 2D ∞-shape. In order to model this behaviour, we take a n-sided regular polygon with particles at its vertices, and define three equations which govern the forces acting between vertices of the polygon. All three equations are based on the Hook's law (Figure 5). The first one defines the forces between each two neighbouring vertices and describes the membrane elongation:

F L i = k L 1 - l i l 0 , (4.1)
where k L is force strength coefficient, l 0 is equilibrium distance between two neighbouring vertices, and l i is the distance between vertices i and i + 1. The second equation describes the forces originating from local bending of the membrane:

F B i = k B 1 - α i α 0 , ( 4.2)
where k B is force strength coefficient, α 0 is the equilibrium angle between two neighbouring sides of the polygon, and α i is the angle between vertices i -1, i and i + 1. Since the membrane tends to become flat, the equilibrium angle α 0 is set to π. The third equation describes the pressure and it is responsible for preservation of the polygon area:

F P = k P 1 - A A 0 , (4.3)
where k P is force strength coefficient, A 0 is the equilibrium and A the current area of the polygon. As a result, particle i representing one polygon vertex feels the following forces:

F L i = 1 2 F L i-1 ri-1,i -F L i ri,i+1 , (4.4)
F B i = F B i-1 -F B i l i-1 ni-1,i + F B i+1 -F B i l i ni,i+1 , ( 4.5)
F P i = 1 2 F P (l i-1 ni-1,i + l i ni,i+1 ) , ( 4.6)
where

r i-1,i = r i -r i-1 , r i-1,i = |r i-1,i |, ri-1,i = r i-1,i /r i-1,i
, and ni-1,i is the unit vector normal to ri-1,i in the direction outside of the polygon. Then the movement of each particle is simply determined by the sum of these forces according to the Newton's second law of motion. In simulations we used large coefficients k L and k P to preserve polygon perimeter and area, while k B was significantly lower. Starting from a regular polygon of area A, for the choice of parameters so that the perimeter is preserved, the equilibrium angle equals π and the equilibrium area equals 0.6A, we obtain the characteristic biconcave shape of the membrane (Figure 5). However, because the model is basically the system of springs, to stabilise this system dampening has to be applied. This can be done directly, by introducing dampening expression in each of equations (4.1), (4.2) and (4.3). But once we put the erythrocytes in a flow simulated by MD or DPD it is not necessary to apply any additional dampening, because the artificial viscosity in MD or dissipative forces acting between particles in DPD will stabilise the membrane. Interaction between particles in MD or DPD with the erythrocyte is again point-wise, defined via vertices, and obeys the rules of MD or DPD.

Figure 5: Erythrocyte: scheme of membrane elongation and bending forces (left), pressure (middle), and the obtained stable ∞-shape (right)

Capillary flow

In order to test the behavior of the erythrocyte model coupled with MD and DPD we simulated flow in a capillary of diameter 10µm with one erythrocyte starting in a ∞-shape turned orthogonally to the flow. It is known ( [START_REF] Hosseini | A particle-based model for the transport of erythrocytes in capillaries[END_REF]) that in such situations erythrocyte goes from its natural ∞-shape to a so called parachute shape following the parabolic velocity profile. With our model we have obtained the described behavior (Figure 6) and the erythrocyte remained in the parachute shape and position orthogonal to the flow for a quite long time. However this was not the stable state of the erythrocyte, and if the simulations are ran for long enough time, the erythrocyte would eventually turn in the direction of the flow and regain its ∞-shape.

3D simulations

In this section we briefly describe blood flow simulations in 3D. We use the same approaches as described above to model homogeneous Newtonian fluids. Erythrocyte model, though similar to the 2D case, becomes more complex in realization. The main difference is related to the triangulation of the surface. Four forces govern behavior of the membrane, instead of three forces in the 2D case. They depend on the volume of the whole body, on the surface area of each triangle, on the angle between neighboring triangles and on the distance between neighboring vertices. The detailed description of the algorithms will be presented elsewhere. Let us note that similar approaches are used in literature [START_REF] Fedosov | Multiscale Modeling of Blood Flow and Soft Matter[END_REF]. With these values of parameters, we take the polyhedron as initial configuration. The form of the membrane converges to the parachute shape (Figure 7). We take this new shape as an initial condition and vary the value of bending coefficient. For each new simulation we take the result of the previous simulation as initial condition. The results are summarized in the diagram in Figure 7. If we decrease k b , then the shape of the membrane remains qualitatively the same. For larger values of this coefficient we observe transition to the biconcave shape. It persists in a rather wide range of values of k b . When it become sufficiently small, transition to asymmetric biconcave shape occurs. Taking this new shape as initial condition, we find the interval of values for which it exists. For a larger value of the coefficient, the shape changes back to symmetric biconcave.

Thus, three different shapes of the membrane are observed. For some values of parameters two of them or even all three can coexist.

Consider next behavior of a single erythrocyte in flow in the case where its equilibrium volume equals 0.6, when the shape of the membrane without flow can be either biconcave or parachute. It depends on various parameters including the radius of the cylinder R c and the ball R b (initial membrane shape in the simulations). Figure 8 shows the case where R c = 0.4 and R b = 0.35. When the flow change the elastic body to the parachute shape, its diameter becomes larger than that of the cylinder and it touches the walls. We impose slip boundary conditions for the elastic body and no-slip for the particles of the fluid (Section 2.3). After some time the elastic body reaches it steady shape and moves along the cylinder with a constant speed. In a larger cylinder (R c = 0.5), the erythrocyte behavior can be different. For other fixed parameters, it depends on the applied body force, that is on the velocity of the flow. If this velocity is large enough, then the erythrocyte moves along the cylinder and, at the same time, rotates along the internal surface of the cylinder in a periodic way (Figure 9). For a smaller flow velocity, erythrocyte can have a small oscillations near the axis of the cylinder or have a steady position. Finally, for even smaller flow velocity, it comes to the surface and slips along it parallel to the axis of the cylinder (Figure 10). We recall that it does not slip to the wall in our model. We finish this section with an example of flow simulation with many erythrocytes (Figure 11). As it is known, they are not uniformly distributed in the cross section of the cylinder concentrating more close to the axis, and the velocity distribution becomes more flat (Figure 12).

Figure 1 :

 1 Figure 1: Distortion in velocity and density profiles (left two figures), correct velocity and density profiles (right two figures). Here and in the following figures dimensionless parameters are used.

Figure 2 :

 2 Figure 2: Viscosity dependance on body force in MD (left) and DPD (middle), and MD velocity profile with too large artificial viscosity (right).

Figure 3 :

 3 Figure 3: MD (from left to right): dynamic viscosity dependance on random force, cutoff radius, number density and artificial viscosity.

Figure 4 :

 4 Figure 4: DPD (from left to right): dynamic viscosity dependance on conservative force coefficient a ij , coefficient of dissipative force σ, exponent k and cutoff radius r c .

Figure 6 :

 6 Figure 6: Single erythrocyte in a Poiseuille flow: development of the parachute shape.

Figure 7 :

 7 Figure 7: Bifurcation diagram. Existence of different forms of erythrocyte depending on the value of bending coefficient. Different shapes of membrane can coexist for the same values of parameters (see the explanation in the text).

Figure 8 :

 8 Figure 8: Single erythrocyte in a narrow Poiseuille flow. View from the side (left) and along the axis of the cylinder (right).

Figure 9 :

 9 Figure 9: Single erythrocyte in the capillary flow. View along the axis. Erythrocyte rolls around the internal wall of the cylinder.

Figure 10 :

 10 Figure 10: Single erythrocyte in the capillary flow. Erythrocyte slips along the internal wall of the cylinder.

Figure 11 :

 11 Figure 11: Flow with many erythrocytes.

Figure 12 :

 12 Figure 12: Velocity (left) and density (right) distributions in the flow with many erythrocytes.

Acknowledgements

This work was partially supported by the projects PICS "Mathematical modelling of blood diseases" (France-Russie), RFFI project No. 10-01-91055, ANR-10-BLAN 0107. The authors are