ABCA1 protein, human

Cholesterol efflux capacity was quantified in blood samples from the cohort of healthy volunteers as described previously.17 (link) This assay quantifies total efflux mediated by pathways of known relevance in cholesterol efflux from macrophages (i.e., ATP-binding cassette transporter A1 [ABCA1] and G1 [ABCG1], scavenger receptor B1, and aqueous diffusion).17 (link) Each sample was run in triplicate, with a mean coefficient of variation of 4.3%. Values were normalized by dividing the efflux capacity of individual patients by the efflux capacity of a serum pool run with each assay.
Cholesterol efflux capacity in the coronary disease and pharmacologic-study cohorts was quantified with the use of a slightly modified method designed to increase throughput. J774 cells, derived from a murine macrophage cell line, were plated and radiolabeled with 2 μCi of ³H-cholesterol per milliliter. ABCA1 was up-regulated by means of a 6-hour incubation with 0.3 mM 8-(4-chlorophenylthio)-cyclic AMP. Subsequently, efflux mediums containing 2.8% apolipoprotein B–depleted serum were added for 4 hours. All steps were performed in the presence of the acyl–coenzyme A:cholesterol acyltransferase inhibitor CP113,818 (2 μg per milliliter). In a pilot study involving serum samples from 20 healthy volunteers, results from the original assay procedure17 (link) and the modified method were strongly correlated (r = 0.85).
Liquid scintillation counting was used to quantify the efflux of radioactive cholesterol from the cells. The quantity of radioactive cholesterol incorporated into cellular lipids was calculated by means of isopropanol extraction of control wells not exposed to patient serum. Percent efflux was calculated by the following formula: [(microcuries of ³H-cholesterol in mediums containing 2.8% apolipoprotein B–depleted serum – microcuries of ³H-cholesterol in serum-free mediums) ÷ microcuries of ³H-cholesterol in cells extracted before the efflux step] × 100. All assays were performed in duplicate. To correct for interassay variation across plates, a pooled serum control from five healthy volunteers was included on each plate, and values for serum samples from patients were normalized to this pooled value in subsequent analyses. Additional studies that were performed to validate the measurement of cholesterol efflux capacity are described in the Supplementary Appendix.

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.