This observational, cross-sectional study was carried out in Palermo, the largest city in Sicily, Italy, with a population of 663,173. From March 28th to April 10th, 2011, groups composed of physicians (n = 5) and dieticians (n = 13) alternated their presence inside the Forum, a shopping mall in Palermo, from 9:00 a.m. until 9:00 p.m. and investigated those customers who asked to participate in the investigations proposed in an announcement presented on posters placed inside the Forum.
The Forum is the largest shopping center in Palermo, and customers come from all parts of the city, suburbs and neighboring areas. Data provided by the Forum administration show that the characteristics of their habitual customers were heterogeneous in terms of gender (females 65%, males 35%), age (10–54 years 50%, > 55 years 50%), place of residence (Palermo 62%, outside of Palermo 38%), education (college graduates = 14%, high school graduates = 37%, lower secondary school = 32%, primary school = 17%), and employment status (housewife = 40%, retired = 23%, employed = 19%, student = 8%, unemployed = 6%, manager/professional = 4%,).
Inclusion criteria were participants aged ≥ 18, and with residence in Palermo. There was no incentive provided to the participants. In order to promote the participation of people of younger age without known cardiovascular, metabolic or nutritional diseases an echographic check of the thyroid was also proposed to the customers of the mall. Further details about patient recruitment procedures have been presented elsewhere .
Participants were asked to come to the Laboratory of Clinical Nutrition of the Department of Internal and Specialized Medicine of the University of Palermo in the following weeks, up until July 15, 2011, to undergo blood sampling for assessment of blood chemistry and hormonal parameters. A blood sample was frozen and stored at -80°C, and a sample was treated and stored for subsequent measurements.
The institutional ethics committee approved the study protocol. Each participant signed an approved informed consent form.
Participants were administered a questionnaire on demographic characteristics, the presence of chronic diseases and pharmacologic treatment, physical activity, including items concerning the level of physical activity and its weekly frequency, daily time watching television, on the computer, and playing video games. Food intake was assessed and included questions on the usual frequency of fish consumption. Concerning habitual fish consumption, the following specific questions were used:
Question: Do you habitually eat fish or shell-fish (referred to the last 12 months)?
Answer (sign the one of the following): never, seldom (less than once a week), yes
Question: If yes, how many times a week do you estimate you eat fish or shell-fish?
Answer: ______ times a week.
Question: If you habitually eat fish, what percentage (from 1% to 100%) of each of the following modalities of stored fish do you eat (please observe that the sum of all items must add up to 100)?
Answer: A) Frozen fish ____% B) Local fresh fish ____% C) Canned fish ____% (A + B + C = 100%).
Habitual fish intake was categorized as follows: no habitual consumption or less than 1 serving a week = 0, 1 serving a week = 1, more than 1 serving a week = 2. Data requested referred to the last year.
Height and body weight were measured with participants lightly dressed and without shoes (SECA); the body mass index (BMI) was calculated as body weight (kg)/height2 (m2). Body circumferences were measured at the umbilicus (waist circumference) and at the most prominent buttock level (hip circumference); the ratio (waist-to-hip ratio) was used as an indirect index of body fat distribution.
Systolic and diastolic arterial blood pressure (two measurements obtained at 5-minute intervals in seated position) and heart rate were measured by physicians or dietitians according to standardized procedures (Omron M6; Omron Healthcare Co; Matsusaka, Mie, Japan) after 15 minutes of rest in sitting position; pulse pressure was calculated as the difference between systolic and diastolic blood pressure.
Carotid intima-media thickness
Images of the right and left extracranial carotid artery walls were obtained in several projections by using a high-resolution ultrasonographic 10-MHz linear array probe (Sonoline G50; Siemens, Germany). The end-diastolic c-IMT of the far wall of both common carotid arteries was measured 10 mm caudal to the bulb, using two-dimensional longitudinal sections of the vessel and the distance from the first echogenic line to the second echogenic line (three values for each carotid artery using antero-posterior, laterolateral and postero-anterior scans); the highest value was considered for calculations . Two physicians were responsible for carrying out the carotid ultrasonographic examination, and were blinded to participants’ characteristics. The intra-observer coefficient of variations were, respectively, 1.2 and 1.1%; the inter-observer coefficient of variation was 2.9%. Previously unknown asymptomatic carotid atherosclerosis was diagnosed in the presence of c-IMT ≥ 0.9 mm and/or plaques in common carotid, carotid bifurcation, extra-cranial internal and external carotid arteries. Carotid plaque was defined as a focal thickness of >1.2 mm .
Capillary blood glucose concentrations were randomly assessed using a glucose reflectometer (Glucocard G meter; Menarini Diagnostics; Florence, Italy). Fasting plasma glucose (FPG), total cholesterol, high-density lipoproteins (HDL) cholesterol, triglicerides, uric acid and creatinine concentrations were ascertained with standard clinical chemistry methods (Glucosio HK UV; Colesterolo tot. Mod P/D; Colesterolo HDL gen 3 mod P/917; Trigliceridi; Acido urico MOD P/917; Creatinina enzimatica; Roche diagnostics, Monza, Italy). Basal insulin concentrations (Elecsys insulina; Roche diagnostics; Monza, Italy) and glycated hemoglobin (HbA1c; HbA1c gen.3; Roche diagnostics; Monza, Italy) were also measured. Low-density lipoprotein (LDL) cholesterol concentration was calculated by means of Friedewald’s formula .
Glomerular filtration rate (GFR) was calculated according to modification of diet in renal disease study (MDRD)  and Cockcroft-Gault  equations. The HOMA-IR was calculated as described by Matthews et al. . The quantitative insulin sensitivity check index (QUICKI) was calculated as described by Katz et al. .
Participant characteristics were grouped in three classes according to fish intake (no habitual consumption or less than 1 serving a week = 0; 1 serving a week = 1; and more than 1 serving a week = 2). Since the three groups were heterogeneous in terms of age, comparisons were done with a generalized linear ANCOVA model for binary, multinomial, and continuous variables that were adjusted for age. Data were therefore reported as estimated means ± SEE for continuous variables, and estimated percentages for categorical ones. Dietary patterns were defined with an a posteriori approach by means of cluster analysis, as described elsewhere (18). Briefly, this procedure is based on the intercorrelations among food groups or nutrients, and is not biased because it does not require as a starting point any technical decision on which foods or nutrients are or are not healthy.
Multivariate logistic regression analyses were done to evaluate factors associated with asymptomatic carotid atherosclerosis (plaques and/or increased c-IMT). The following baseline covariates were tested: age (y), gender (male, female), smoking status (former, current or never a smoker), frequency of fish intake (<1, 1, or ≥2 servings/week), physical activity level (light, moderate/heavy or none), use of statins (yes or no), hypertension on treatment (yes or no), and pulse pressure (mmHg). Results of the logistic models are expressed as adjusted odds ratios (ORs) with their 95% confidence intervals. A two-tailed P value of < 0.05 was considered significant. All statistical analyses were done using SAS version 9.2 (SAS Institute Inc; Cary, NC, US).