Quantifying LDL Aggregation Susceptibility

Development of a method to quantify person-to-person variability in LDL aggregation-susceptibility is available in Supplementary material online, Methods and Figures S1–S3. In the assay we developed, LDL isolated from human plasma was extensively dialysed against 20 mM MES buffer, pH 5.5, containing 150 mM NaCl, 2 mM MgCl₂, 2 mM CaCl₂, and 50 µM ZnCl₂. The sample was diluted with the same buffer to give a final concentration of 0.2 mg of apoB-100/mL, then human recombinant secretory sphingomyelinase (hrSMase) was added to to give a final enzyme concentration of 75 µg/mL, and the mixture was incubated at +37°C. The size of LDL was determined immediately and the hourly up to 6 h. An aliquot was taken at the same time points for phosphorylcholine measurement. The degree of sphingomyelins (SM) hydrolysis was determined by measuring the phosphorylcholine content from the samples with Amplex Red reagent (Invitrogen).