A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol

Studies have shown a negative association between cellular cholesterol efflux and coronary artery disease (CAD). Standard protocol for quantitating cholesterol efflux involves labeling cells with [H-3] cholesterol and measuring release of the labeled sterol. Using [H-3] cholesterol is not ideal for the development of a high-throughput assay to screen large numbers of serum as would be required in studying the link between efflux and CAD. We compared efflux using a fluorescent sterol (boron dipyrromethene difluoride linked to sterol carbon-24, BODIPY-cholesterol) with that of [H-3] cholesterol in J774 macrophages. Fractional efflux of BODIPY-cholesterol was significantly higher than that of [H-3] cholesterol when apo A-I, HDL3, or 2% apoB-depleted human serum were used as acceptors. BODIPY-cholesterol efflux correlated significantly with [H-3] cholesterol efflux (p < 0.0001) when apoB-depleted sera were used. The BODIPY-cholesterol efflux correlated significantly with pre beta-1 (r(2) = 0.6) but not with total HDL-cholesterol. Reproducibility of the BODIPY-cholesterol efflux assay was excellent between weeks (r(2) = 0.98, inter-assay CV = 3.31%). These studies demonstrate that BODIPY-cholesterol provides an efficient measurement of efflux compared with [H-3] cholesterol and is a sensitive probe for ABCA1-mediated efflux. The increased sensitivity of BODIPY-cholesterol assay coupled with the simplicity of measuring fluorescence results in a sensitive, high-throughput assay that can screen large numbers of sera, and thus establish the relationship between cholesterol efflux and atherosclerosis.-Sankaranarayanan, S., G. Kellner-Weibel, M. de la Llera-Moya, M. C. Phillips, B. F. Asztalos, R. Bittman, and G. H. Rothblat. A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol. J. Lipid Res. 2011. 52: 2332-2340.