title: 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 [(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.
authors.affiliation: Department of Pediatrics (Division of Gastroenterology, Hepatology, and Nutrition), The Children's Hospital of Philadelphia, Philadelphia, PA 19104.
authors.forename: Sandhya
authors.initials: S
authors.lastname: Sankaranarayanan
authors.affiliation: Department of Pediatrics (Division of Gastroenterology, Hepatology, and Nutrition), The Children's Hospital of Philadelphia, Philadelphia, PA 19104.
authors.forename: Ginny
authors.initials: G
authors.lastname: Kellner-Weibel
authors.affiliation: Department of Pediatrics (Division of Gastroenterology, Hepatology, and Nutrition), The Children's Hospital of Philadelphia, Philadelphia, PA 19104.
authors.forename: Margarita
authors.initials: M
authors.lastname: de la Llera-Moya
authors.affiliation: Department of Pediatrics (Division of Gastroenterology, Hepatology, and Nutrition), The Children's Hospital of Philadelphia, Philadelphia, PA 19104.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Phillips
authors.affiliation: Lipid Metabolism Laboratory (B.F.A.), Tufts University, Boston, MA 02111; and Department of Chemistry and Biochemistry (R.B).
authors.forename: Bela F
authors.initials: BF
authors.lastname: Asztalos
authors.affiliation: Department of Chemistry and Biochemistry (R.B), Queens College of The City University of New York, Flushing, NY 11367-1597.
authors.forename: Robert
authors.initials: R
authors.lastname: Bittman
authors.affiliation: Department of Pediatrics (Division of Gastroenterology, Hepatology, and Nutrition), The Children's Hospital of Philadelphia, Philadelphia, PA 19104. Electronic address: rothblat@email.chop.edu.
authors.forename: George H
authors.initials: GH
authors.lastname: Rothblat
mesh_terms: D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000328:Adult; D000368:Aged; D000818:Animals; D016632:Apolipoprotein A-I / Q000378:metabolism; D001055:Apolipoproteins B / Q000172:deficiency; D001692:Biological Transport / Q000187:drug effects; D001896:Boron Compounds / Q000378:metabolism*; D002460:Cell Line; D002784:Cholesterol / Q000097:blood / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008875:Middle Aged; D010743:Phospholipids / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D013997:Time Factors; D055815:Young Adult
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D016632:Apolipoprotein A-I; D001055:Apolipoproteins B; D001896:Boron Compounds; D005456:Fluorescent Dyes; D010743:Phospholipids; D002784:Cholesterol; D000242:Cyclic AMP