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 [(3)H]cholesterol and measuring release of the labeled sterol. Using [(3)H]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 [(3)H]cholesterol in J774 macrophages. Fractional efflux of BODIPY-cholesterol was significantly higher than that of [(3)H]cholesterol when apo A-I, HDL(3), or 2% apoB-depleted human serum were used as acceptors. BODIPY-cholesterol efflux correlated significantly with [(3)H]cholesterol efflux (p < 0.0001) when apoB-depleted sera were used. The BODIPY-cholesterol efflux correlated significantly with preβ-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 [(3)H]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.
Highlights
ABCG1 does not release cholesterol to lipid-free apolipoproteins whereas apo A-I serves as a primary ligand for the ABCA1 efflux pathway
The observation that cAMP treatment enhanced efflux of BODIPY-cholesterol to apo A-I is consistent with a substantial amount of efflux of the fluorescent sterol occurring via the ABCA1 pathway
This recent study used apoB-depleted human serum as the acceptor; this total HDL preparation has been used in several studies because the presence of apoB-containing lipoproteins can confound the measurement of cellular cholesterol flux [19]
Summary
Our initial experiment compared the efflux of BODIPY-cholesterol and [3H]cholesterol from J774 macrophage cells treated with or without cAMP to 10 μg/ml of lipid-free apo A-I, 25 μg/ml human HDL3, and 2% apoB-depleted pooled human serum. For both fluorescence and radiolabel, the % efflux values can be determined by direct measurement of either the loss of the BODIPY-cholesterol/[3H]cholesterol from the cell monolayer or by the accumulation of these labeled sterols in the incubation medium.
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