Association Between HDL Functions and Atherosclerotic Burden in Healthy Individuals

Selection: Study participants are initially selected trough the evaluation of consecutive lipid profiles from individuals who spontaneously seek governmental primary care centers of the city of Campinas, Sao Paulo, Brazil. Telephone-based screening interviews are performed, followed by in-person clinical evaluation and blood exams.

Biochemical analyses: Glucose, triglycerides, HDL-C and c-reactive protein (CRP) are measured in an automated Modular® Analytics Evo (Roche Diagnostics, Burgess Hill, West Sussex, UK), using Roche Diagnostics® reagents (Mannheim, Germany). LDL-cholesterol is calculated by the Friedewald formula. Serum insulin levels are measured using ELISA (Millipore, Massachusetts, USA). The Homeostasis Model Assessment (HOMA) Calculator version 2.2 (University of Oxford, UK) is used to estimate insulin sensitivity. Apolipoproteins A-I and B-100 and lipoprotein (a) are determined by nephelometry in an automated system and reagents from Dade-Behring® (Marburg, Germany).

Carotid artery ultrasound: Carotid artery atherosclerosis is estimated by using high-resolution B-mode ultrasound, at the posterior wall of the common carotid artery.

Lipoprotein isolation: HDL from each study participant is isolated from plasma, through ultracentrifugation (Beckman Coulter Inc., Palo Alto, USA).

HDL chemical composition: Using commercially available enzymatic kits, HDL content of total proteins, total cholesterol, free cholesterol, phospholipids, triglycerides and apolipoprotein A-I are measured. Cholesteryl ester (CE) is calculated as the difference between total cholesterol and free cholesterol times 1.67. HDL molar concentration is estimated based on particle total mass and molecular weight.

HDL physical-chemical characterization: HDL particle size is determined using dynamic light scattering, and zeta potential using laser Doppler micro-electrophoresis.

Determination of proteins involved in HDL metabolism: Cholesteryl ester transfer protein, phospholipids transfer protein, lipoprotein lipase, hepatic lipase and lecithin:cholesterol acyl transferase activities are determined trough radiometric exogenous assays.

HDL functions: Cholesterol efflux capacity, antioxidant activity, susceptibility to oxidation, anti-inflammatory activity and platelet aggregation inhibition are measured using straightforward and consolidated methodologies.

Statistical Analyses: Differences between groups are evaluated using ANOVA or Kruskal-Wallis, with Bonferroni's post-hoc multiple comparison analysis, according to variable distribution. Chi-Square is used to compare categorical data. Analysis of covariance (ANCOVA) adjusted for confounding variables is also used, after checking variables with histograms, normality plots, and residual scatter plots that tested for linearity, normality, and variance. Pearson's correlation test is used to assess the relationships between variables. A two-sided p-value of 0.05 is considered significant.

Inclusion criteria

• glucose <100 mg/dL
• Urea <71 mg/dL
• Creatinine < 1.20 mg/dL
• Uric acid <7.0 mg/dL
• Alanine aminotransferase <50 U/L
• Aspartate aminotransferase < 33 U/L
• Gamma-glutamyl transferase <71 U/L
• Alkaline phosphatase <129 U/L
• Thyroid stimulating hormone between 0.41 and 4.50 μUI/mL
• Free thyroxin between 0.9 and 1.8 ng/dL

Exclusion criteria

• Symptomatic cardiovascular disease
• LDL-cholesterol ≥130 mg/dL
• Triglycerides ≥150 mg/dL
• Metabolic syndrome
• BMI ≥ 30 kg/m2
• Smoking habit
• Daily intake of alcohol >14g
• Regular use of medical treatments