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United States Department of Agriculture

Agricultural Research Service


Location: Jean Mayer Human Nutrition Research Center On Aging

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

item Sankaranarayanan, Sandhya
item Kellner-weibel, Ginny L.
item De La Llera-moya, Margarita
item Phillips, Michael C.
item Asztalos, Bela F.
item Bittman, Robert
item Rothblat, George H.

Submitted to: Journal of Lipid Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/12/2011
Publication Date: 9/27/2011
Citation: Sankaranarayanan, S., Kellner-Weibel, G., De La Llera-Moya, M., Phillios, M., Asztalos, B., Bittman, R., Rothblat, G. 2011. A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol. Journal of Lipid Research. 52(12):2332-2340.

Interpretive Summary: The efflux (expulsion) of cholesterol from macrophages in the arterial wall is thought to be an early step in the process termed reverse cholesterol transport (RCT). Efflux can be mediated by a number of pathways involving 1) aqueous diffusion, 2) scavenger receptor BI (SR-B1), 3) ABCG1, and 4) ABCA1. Although all of these pathways can contribute to the removal of free cholesterol (FC) from cells, it is ABCA1 that has been shown to play a major role in the maintenance of normal cholesterol levels in tissues, as demonstrated by the accumulation of large amounts of cholesterol in macrophages in individuals with genetic mutations in ABCA1 (Tangier disease) or in mouse models in which this protein is genetically eliminated. A link between the in vitro efflux of cholesterol from macrophages and hardening of the arteries has recently been established by studies demonstrating a negative correlation between cholesterol efflux from J774 mouse cells and coronary artery disease (CAD) as measured by either the thickness of certain layers of the carotid artery walls (cIMT) or angiographic measurements. We have substituted a fluorescent sterol, BODIPY-cholesterol, for the generally used radiolabeled cholesterol. BODIPY-cholesterol is a fluorescent analog of free cholesterol in which carbon-24 of the sterol side chain is linked directly to the dipyrromethene boron difluoride (“BODIPY”) moiety. In a series of experiments using J774 macrophages together with a variety of cholesterol acceptors, we have compared efflux of BODIPY-cholesterol and [3H]cholesterol. These studies have demonstrated that the use of the fluorescent-labeled sterol provides an efficient measurement of efflux when compared with radiolabeled cholesterol. We found that BODIPY-cholesterol is released from cells primarily via the ABCA1 pathway. Thus, the greater efficiency for efflux of the BODIPY-cholesterol compared with labeled cholesterol, together with the greater simplicity of the fluorescent assay, allows the development of efflux protocols suitable for rapid, high-throughput screening for efflux capacity of large numbers of sera or serum fractions.

Technical Abstract: Studies have shown a negative association between cellular cholesterol efflux and coronary artery disease (CAD). Standard protocol for quantifying 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 high-throughput assays needed to screen large numbers of serum as would be required in studying the link between efflux and CAD. In this study, 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, or 2% apoB-depleted human serum were used as acceptors. BODIPY-cholesterol efflux correlated significantly with [(3)H]cholesterol efflux (p less than 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 [(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, examine the relationship between cholesterol efflux and atherosclerosis.

Last Modified: 10/16/2017
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