Laboratory measurements of lipoprotein and lipoprotein subspecies

The methodology of lipoprotein subspecies quantification has been described previously [24]. Briefly, plasma was thawed and incubated overnight at 4 °C in anti-apoC-III immuno-affinity columns to bind lipoproteins containing apoC-III. The unbound plasma fraction (CIII-) was eluted with phosphate-buffered saline and the bound lipoproteins (CIII+) were eluted with 3 M sodium thiocyanate. Very-low density lipoprotein (VLDL) was isolated from each fraction by ultracentrifugation at 4 °C and 25,000 rpm for 16 h. The combined intermediate-density lipoprotein (IDL) and low-density lipoprotein (LDL) fraction was then isolated following density adjustment with potassium bromide to d = 1.063 g/mL by ultracentrifugation at 4 °C and 25,000 rpm for 24 h. The remaining solution contained the HDL and other components of plasma. Therefore, six lipoprotein subspecies were generated: VLDL that contains or lacks apoC-III, IDL + LDL that contains or lacks apoC-III, and HDL that contains or lacks apoC-III. Among these subspecies, apoB, apoC-III, and apoE concentrations were further assayed using sandwich ELISAs (Academy Biomedical, Houston, TX), and cholesterol and triglyceride concentrations were determined using enzymatic assays (Thermo Electron Corp, Waltham, MA). The samples from the same participants were assayed in the same run by the same technicians in a random sequence, and any sample with an intra-assay CV > 15% was repeated.