Blood collected from all the participants at baseline by means of venipuncture was placed into EDTA tubes, stored at 4°C for less than 4 hours, and centrifuged, and plasma was removed and stored at −70°C. Plasma lipids, including HDL cholesterol, were measured as described previously.11 (link) HDL particle concentration and size were measured by means of nuclear magnetic resonance spectroscopy (LipoScience).
Cholesterol efflux capacity was assessed by measuring the efflux of fluorescence-labeled cholesterol from J774 macrophages to apolipoprotein B–depleted plasma in study participants with the use of a previously described method.12 (link) This assay primarily evaluates cholesterol efflux as mediated by ATP-binding cassette transporter A1 (ABCA1). The fluorescence-labeled reagent, termed boron dipyrromethene difluoride (BODIPY) cholesterol, was used because it is more amenable to use in a large number of samples than radiolabeled cholesterol (details of the assay protocol are provided in the Supplementary Appendix). For comparison, we performed a parallel assessment of efflux capacity with the use of radiolabeled cholesterol in a limited number of plasma samples.9 (link)Cholesterol efflux capacity measured with the use of fluorescence-labeled cholesterol was moderately correlated with measurements performed with radiolabeled cholesterol (correlation coefficient for normalized cholesterol efflux, 0.54) (Fig. S1 in the Supplementary Appendix). The cholesterol efflux capacity did not change significantly when it was measured in samples obtained throughout a single day or 7 days apart (Fig. S2 in the Supplementary Appendix) or when samples underwent a freeze–thaw cycle (Fig. S3A and S3B in the Supplementary Appendix). However, as compared with 3-to-12-month storage at −70°C, parallel storage of plasma at −20°C reduced cholesterol efflux capacity measured with the use of either fluorescence-labeled cholesterol or radiolabeled cholesterol (Fig. S3C and S3D in the Supplementary Appendix). Measurements of cholesterol efflux capacity in this study were therefore performed with the use of the fluorescence-labeled cholesterol assay on plasma samples stored at −70°C.