Lipid/lipoprotein measurements
SEPARATION
All blood sampling for the determination of lipid parameters were determined under fasting conditions (at least 8 h of fasting). Triglycerides and total cholesterol were assessed by an enzymatic colorimetric assay run on a Beckman–Coulter analyzer. HDL-C was determined by precipitation, which involved precipitating all non-HDL-C using 50 kDa dextran sulfate with magnesium ions as the precipitating agent, followed by the determination of HDL-C in the supernatant using an adapted method for determining total cholesterol on a Beckman–Coulter analyzer. LDL-C was determined by ultracentrifugation; after separation of the very-low-density lipoprotein/chylomicron sub-fraction by ultracentrifugation, LDL-C was determined as the cholesterol in the infranatant (performed by an enzymatic colorimetric assay) minus HDL-C. ApoB and ApoA1 were assessed by nephelometry using a Siemens BNII nephelometer. In addition, serial ultracentrifugation was performed to separate lipoprotein subfractions (chylomicrons, very-low-density lipoprotein, intermediate-density lipoprotein, LDL and HDL) and lipids (triglycerides, cholesterol and phospholipids) and proteins (for example, ApoB, ApoA1, ApoC2, ApoC3 and ApoC5) were measured in the fractions by established methods.