Subclinical Atherosclerosis: Biomarkers of Early Disease

This study cross-sectionally evaluated consecutive patients at the outpatient clinic of Dyslipidemia and of Hypertension and Nephrology Medical Sections of Dante Pazzanese Institute of Cardiology, a cardiologic tertiary hospital in São Paulo - Brazil. After they fulfilled inclusion criteria and signed an informed consent, a brief medical history and anthropometric data (abdominal circumference, height and weight for body mass index calculation) were collected. Within of a month after inclusion, all patients underwent blood sample collection for dosing fast plasma glucose and insulin and 2 hours after 75g of dextrose test glucose and insulin, fast serum triglycerides, total cholesterol, HDL cholesterol, creatinine, high sensitive c-reactive protein (hs-CRP), HbA1c, thyroid stimulating hormone (TSH), free thyroxin (fT4), creatine kinase, adiponectin, resistin, leptin, tumor necrosis factor alpha (TNF-α), lipoprotein associated phospholipase A2 (Lp-PLA2). Blood sample was also stored for DNA (rs2383206, rs10757274, rs10757278, rs1051931, rs16874954 and rs1799724) and RNA PLA2G7 (2-ΔCT), TNF-α (2-ΔCT), LEP (2-ΔCT), LEPR (2-ΔCT) extraction and analyses.

Subclinical atherosclerosis was evaluated by four methods: carotid doppler ultrasound, cardiac computed tomography, exercise stress test and ankle-brachial index (ABI). All above tests were performed at the same day.

Carotid Doppler ultrasound was performed by using high-resolution Vivid 7 ultrasound (GE, USA) and high-frequency linear transducer type 9 MHz with automated measurement. Carotid Intima media thickness (cIMT) was evaluated 1 cm distal of the posterior wall of the left and right common carotid arteries and the presence of atherosclerotic plaques in common, internal and external carotid arteries was assessed. Carotid plaque was defined by the presence of focal wall thickening at least 50% greater than that of the surrounding vessel wall, or as a focal region with IMT greater than 1.5 mm, which protrude into the lumen and that is distinct from the adjacent boundary.

Cardiac Computed Tomography was performed to determine coronary calcium score (CAC) and calcium distribution. Hepatic density was performed to evaluate the presence of hepatic steatosis with Aquilion apparatus (Toshiba Medical, Tochigi, Japan) with 64 rows of detectors. The acquisition of tomographic data was represented by images of average thickness (3 mm) and low intensity (50 milliamps, 120 kV) and coupled to the electrocardiogram. Calcium score was considered present when its density in the coronary artery was above 130 Housfield units (HU) for at least 3 continuous pixels (> 1 mm2) of the same coronary artery. Calcium score comprised the sum of individual scores of the left and right coronary arteries.

Exercise stress test was performed following under one of following protocols: Bruce, modified Bruce or Ellestad treadmill. The test was interrupted if maximum heart rate was reached or if clinical symptoms installed, including exhaustion. The exam was considered positive when there were: negative horizontal/descending deflection of ST segment measured at the J point ≥1 mm, pain suggestive of coronary artery disease, complex and sustained ventricular arrhythmias, plateau or drop in systolic blood pressure in effort and/or functional capacity lower than 5 METS.