Fasting blood samples (12 to 14 h) were collected from an arm vein while subjects were reclining in a blood sampling chair, into tubes containing EDTA-Na2 at a final concentration of 0.15%. Plasma was obtained by centrifugation at 1000 g for 25 min at 4°C. Plasma was assayed for TC and triglycerides using enzymatic methods (Boehringer Mannheim, Montreal Quebec); cholesterol was assayed using the cholesterol oxi-dase/p-aminophenazone method, and triglycerides were measured without free glycerol. A 5 mL aliquot of plasma from each sample was subjected to ultracentrifugation . Very low density lipoprotein cholesterol (VLDL) was collected by tube slicing, and the infranatant was carefully recovered and adjusted to 5 mL. Aliquots were taken for triglyceride, cholesterol, apoB and apoAI, and were stored at -20°C until assayed.
In order to rule out type III hyperlipidemia, the VLDL from the first sample obtained from each patient was assayed for apoE phenotype by analytical isoelectric focusing gel electrophoresis . HDL was determined after precipitation of plasma with dextran sulphate-magnesium chloride by the method of Warnick et al . LDL was determined by subtracting HDL from the infranatant cholesterol value. VLDL and triglycerides were determined as the difference between total plasma and infranatant values. The intra- and interassay coefficients of variation for TC were 2.2% and 3.0%, respectively, and for total and infranatant triglycerides were 2.2% and 3.0%, respectively. levitra super active plus
ApoB and apoAI were determined by immuno-turbidimetric assay using reagents obtained from Boehringer Mannheim. ApoB and apoAI were measured in the plasma in-franatant following ultracentrifugation at d 1.006. These values were taken as representing LDL and intermediate density lipoprotein cholesterol, apoB and, total apoAI, respectively. The intra- and interassay coefficients of variation were 4.5% and 8.0% for apoB, and 3.8% and 7.2% for apoAI, respectively. All assays were standardized to plasma secondary standards for which the target values were assigned at the Lipid Research Laboratory, University of Toronto, St Michael’s Hospital, Toronto, Ontario, which is standardized by the Centers for Disease Control and Prevention (Atlanta, Georgia).