title: Measurement of cholesterol bidirectional flux between cells and lipoproteins.
abstract: We developed an assay that quantitates bidirectional cholesterol flux between cells and lipoproteins. Incubating Fu5AH cells with increasing concentrations of human serum resulted in increased influx and efflux; however, influx was 2- to 3-fold greater at all serum concentrations. With apolipoprotein B (apoB)-depleted serum, the ratio of influx to efflux (I/E) was close to 1, indicating cholesterol exchange. The apoB fraction of serum induced influx and little efflux, with I/E > 1. Using block lipid transport-1 to block scavenger receptor class B type I (SR-BI)-mediated flux with different acceptors, we determined that 50% to 70% of efflux was via SR-BI. With HDL, 90% of influx was via SR-BI, whereas with LDL or serum, 20% of influx was SR-BI-mediated. Cholesterol-enriched hepatoma cells produced increased efflux without a change in influx, resulting in reduced I/E. The assay was applied to cholesterol-normal and -enriched mouse peritoneal macrophages exposed to serum or LDL. The enrichment enhanced efflux without shifts in influx. With cholesterol-enriched macrophages, HDL efflux was enhanced and influx was greatly reduced. With all lipoproteins, cholesterol enrichment of murine peritoneal macrophages led to a reduced I/E. We conclude that this assay can simultaneously and accurately quantitate cholesterol bidirectional flux and can be applied to a variety of cells exposed to isolated lipoproteins or serum.
authors.affiliation: Gastrointestinal and Nutrition Division, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
authors.forename: Francesca
authors.initials: F
authors.lastname: Zimetti
authors.forename: Ginny K
authors.initials: GK
authors.lastname: Weibel
authors.forename: Myngan
authors.initials: M
authors.lastname: Duong
authors.forename: George H
authors.initials: GH
authors.lastname: Rothblat
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods; D001692:Biological Transport / Q000502:physiology*; D002478:Cells, Cultured; D002784:Cholesterol / Q000032:analysis* / Q000097:blood; D006801:Humans; D008074:Lipoproteins / Q000378:metabolism*; D008075:Lipoproteins, HDL / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000378:metabolism; D051379:Mice; D051381:Rats; D013997:Time Factors
chemical_list: D008074:Lipoproteins; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D002784:Cholesterol