BSHA is a cohort study of healthy elderlies enrolled voluntarily. It has been ongoing since December 2008. And the purpose of this study is to assess clinical and biological markers of cardiovascular risk in very elderly participants.

Non-institutionalized outpatients under preventive care were voluntarily enrolled into the study. Participants were aged of 80 years or over, have never manifested myocardial infarction, stroke or peripheral arterial disease. Enrolled participants underwent a detailed clinical examination, including assessment of anthropometric measures, quality of life by the WHOQOL-BREF, psychological test by Geriatric Depression Scale (GDS) and cognitive function by the mini-mental state examination (MMSE). After 12h of overnight fasting, the study participants underwent to blood sampling collection for biochemical analysis and freezing of plasma, serum and DNA. Immediately after blood collection, participants underwent to cardiovascular ultrasound evaluations and computed tomography (CT) scans during the week following the initial assessment. After baseline measurements, all participants were referred to the study as outpatient clinic for prospective medical follow-up evaluations.

Anthropometric measures: body weight, height, waist circumference and skinfold thickness (triceps, biceps, suprailiac and subscapular) were measured. Body mass index (BMI) was calculated. Skinfold thickness was the mean of triplicate measurements at the right side, using a skinfold caliper (WCS Plus®, Cardiomed, Curitiba, Brazil).

Biochemical analysis: After collection, EDTA blood was centrifuged at 5 ◦ C, 4500 rpm for 15min to separate plasma and carrying out the following measurements: glucose (Glucose GOD-PAP, Roche Diagnostics, Mannheim, USA), total cholesterol (CHOD-PAP, Roche Diagnostics, Mannheim, USA), triglycerides (GPO-PAP, Roche Diagnostics, Mannheim, USA), HDL-C (HDL cholesterol without pretreatment, Roche Diagnostics, Mannheim, USA), C-reactive protein (highly sensitive, CardioPhase, Dade Behring, Marburg, USA), urea and creatinine (GLDH, Hitachi, Tokyo, Japan), fibrinogen (Sysmex CA 1500, Siemens, Munich, Germany), interleukin (IL) 10 and tumor necrosis factor type alpha (TNF-alpha) (eBioscience, San Diego, CA, USA). Parathyroid hormone (PTH) and calcitonin (Immulite 2000, Siemens, Los Angeles, CA, USA), bone fraction of alkaline phosphatase (Hitachi Autoanalyzer, Tokyo Japan), calcium (Hitachi Autoanalyzer, Tokyo, Japan), apoA and apoB (Behring Nephelometer BNII, Dade Behring, Marburg, Germany) were also measured.

Carotid ultrasound: The evaluation of intima-media thickness (IMT) and presence of carotid plaques were assessed using high-resolution B-mode ultrasound (Philips, model IE 33, 3-9 MHz linear transducer, Philips Medical Systems, Andover, MA, USA) according to the protocols of the American Association of Echocardiography. Bilateral measurements were made at the posterior wall of the bulb of the common carotid artery and at the internal carotid artery through a program of automatic edge detection (QLAB version 6.0 software). Carotid plaque was defined as the presence of focal thickening of at least 50% higher than surrounding areas or as focal region with IMT > 1.5 mm and distinct adjacent edges.

Cardiac computed tomography: Computed tomography was performed in a 64-slice scanner (Aquillion 64, Toshiba, Ottawara, Japan). Axial slices of 3 mm thickness with 3 mm table-feed were acquired at 70% of R-R interval with prospective ECG triggering. Coronary artery calcification was defined as a minimum of 3 contiguous pixels with a peak Hounsfield unit density > 130. Coronary artery calcifications were scored by a certified radiologist. The Agatston score was used to express the value of CAC.

Inclusion Criteria:

• Aged of 80 years or over

Exclusion Criteria:

• Manifested atherosclerotic disease (MI, stroke or peripheral arterial disease) according to medical evaluation, electrocardiogram and echocardiogram
• Functional dependence or institutionalization
• Cognitive impairment assessed by mini-mental state examination (< 13 points)
• Use of any anti-inflammatory in the last 30 days
• Current or previous diagnosis of neoplastic or immune inflammatory disease
• Chronic obstructive pulmonary disease
• Glomerular filtration rate < 25 mL/min/1.73 m2
• Hepatic disease (ALT or AST ≥ 1.5 upper reference limit)
• Chronic infectious disease (≥ 3 months)
• Left ventricular ejection fraction < 50% on echocardiography
• Manifested neoplasia at admission or until the first year after enrollment

Non-institutionalized outpatients under preventive care were voluntarily enrolled into the study. Participants were aged of 80 years or over, have never manifested myocardial infarction, stroke or peripheral arterial disease.