2.6. Lipoprotein Particle Size Analysis

Non-denaturing polyacrylamide gradient gels (Alamo Gels, USA) were used for the separation of IDL and LDL (2–16% gradient) and HDL subclasses (4–30% gradient) within isolated samples. Aliquots of IDL (31.5 nm), LDL₁ (23.6 nm), and LDL₃ (20 nm), for which particle sizes were previously determined using electron microscopy [24], were also run on the 2–16% gels to generate a standard curve for the determination of LDL particle size. Standards run on the 4–30% gradient gels included latex beads (38 nm), thyroglobulin (17.1 nm) ferritin, (12.2 nm), lactate dehydrogenase (8.16 nm), and albumin (7.1 nm). Following electrophoresis, gels were stained, scanned, and analysed using Image J version 1.43 u software (National Institutes of Health, Bethesda, MD, USA). Peak particle diameter was quantified for LDL samples. HDL particle diameter was obtained from a logarithmic standard curve of the diameter of standards against their positions on the scanned gel. HDL sub-classes were defined as HDL_2b (9.9–12.0 nm), HDL_2a (8.8–9.9 nm), HDL_3a (8.2–8.8 nm), HDL_3b (7.8–8.2 nm), or HDL_3c (7.0–7.8 nm) [25]. For these subclasses, the relative distribution of cholesterol under each peak was calculated as a percentage of the total area for all HDL subclasses.