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 theSupplementary Appendix .
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