title: Importance of evaluating cell cholesterol influx with efflux in determining the impact of human serum on cholesterol metabolism and atherosclerosis.
abstract: OBJECTIVE
  Cholesterol efflux relates to cardiovascular disease but cannot predict cellular cholesterol mass changes. We asked whether influx and net flux assays provide additional insights.
  
  
  RESULTS
  Adapt a bidirectional flux assay to cells where efflux has clinical correlates and examine the association of influx, efflux, and net flux to serum triglycerides (TGs). Apolipoprotein B-depleted (high-density lipoprotein-fraction) serum from individuals with unfavorable lipids (median [interquartile range]; high-density lipoprotein-cholesterol=39 [32-42], low-density lipoprotein-cholesterol=109 [97-137], TGs=258 [184-335] mg/dL; n=13) promoted greater ATP-binding cassette transporter A1-mediated [1,2-(3H)] cholesterol efflux (3.8±0.3%/4 hour versus 1.2±0.4%/4 hour; P<0.0001) from cyclic 3',5'-amp(CTP-amp)-treated J774 macrophages than from individuals with favorable lipids (high-density lipoprotein-cholesterol=72 [58-88], low-density lipoprotein-cholesterol=111 [97-131], TGs=65 [56-69] mg/dL; n=10). Thus, high TGs associated with more ATP-binding cassette transporter A1 acceptors. Efflux of cholesterol mass (μg free cholesterol/mg cell protein per 8 hour) to serum was also higher (7.06±0.33 versus 5.83±0.48; P=0.04). However, whole sera from individuals with unfavorable lipids promoted more influx (5.14±0.65 versus 2.48±0.85; P=0.02) and lower net release of cholesterol mass (1.93±0.46 versus 3.36±0.47; P=0.04). The pattern differed when mass flux was measured using apolipoprotein B-depleted serum rather than serum. Although individuals with favorable lipids tended to have greater influx than those with unfavorable lipids, efflux to apolipoprotein B-depleted serum was markedly higher (6.81±0.04 versus 2.62±0.14; P<0.0001), resulting in an efflux:influx ratio of ≈3-fold. Thus both serum and apolipoprotein B-depleted serum from individuals with favorable lipids promoted greater net cholesterol mass release despite increased ATP-binding cassette transporter A1-mediated efflux in samples of individuals with high TGs/unfavorable lipids.
  
  
  CONCLUSIONS
  When considering the efficiency of serum specimens to modulate cell cholesterol content, both influx and efflux need to be measured.
authors.affiliation: From the Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition, The Children's Hospital of Philadelphia, PA (G.L.W., D.D.-S., D.K.S., G.H.R., M.d.l.L.-M.); Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia (M.P.R.); and School of Public Health and School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa (A.N.W.).
authors.forename: Ginny L
authors.initials: GL
authors.lastname: Weibel
authors.forename: Denise
authors.initials: D
authors.lastname: Drazul-Schrader
authors.forename: Debra K
authors.initials: DK
authors.lastname: Shivers
authors.forename: Alisha N
authors.initials: AN
authors.lastname: Wade
authors.forename: George H
authors.initials: GH
authors.lastname: Rothblat
authors.forename: Muredach P
authors.initials: MP
authors.lastname: Reilly
authors.forename: Margarita
authors.initials: M
authors.lastname: de la Llera-Moya
mesh_terms: D064286:ATP Binding Cassette Transporter 1 / Q000097:blood / Q000378:metabolism; D000368:Aged; D000818:Animals; D001055:Apolipoproteins B / Q000097:blood; D050197:Atherosclerosis / Q000097:blood*; D001692:Biological Transport; D002460:Cell Line; D002784:Cholesterol / Q000097:blood*; D008076:Cholesterol, HDL / Q000097:blood; D008078:Cholesterol, LDL / Q000097:blood; D050171:Dyslipidemias / Q000097:blood*; D005260:Female; D006801:Humans; D007700:Kinetics; D008264:Macrophages / Q000378:metabolism*; D008297:Male; D051379:Mice; D008875:Middle Aged; D014280:Triglycerides / Q000097:blood
chemical_list: D064286:ATP Binding Cassette Transporter 1; D001055:Apolipoproteins B; D002784:Cholesterol; D008076:Cholesterol, HDL; D008078:Cholesterol, LDL; D014280:Triglycerides; C577836:ABCA1 protein, human; C577833:Abca1 protein, mouse
keywords: coronary artery disease; high-density lipoprotein-1