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

Abstract

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 [3H]cholesterol and measuring release of the labeled sterol. Using [3H]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 [3H]cholesterol in J774 macrophages. Fractional efflux of BODIPY-cholesterol was significantly higher than that of [3H]cholesterol when apo A-I, HDL3, or 2% apoB-depleted human serum were used as acceptors. BODIPY-cholesterol efflux correlated significantly with [3H]cholesterol efflux (p < 0.0001) when apoB-depleted sera were used. The BODIPY-cholesterol efflux correlated significantly with preβ-1 (r2= 0.6) but not with total HDL-cholesterol. Reproducibility of the BODIPY-cholesterol efflux assay was excellent between weeks (r2= 0.98, inter-assay CV = 3.31%). These studies demonstrate that BODIPY-cholesterol provides an efficient measurement of efflux compared with [3H]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.

Keywords

• BODIPY-cholesterol (boron dipyrromethene difluoride linked to sterol carbon -24) efflux
• ABCA1
• coronary artery disease
• atherosclerosis
• apo A-I
• apoB-depleted serum