title: The measurement of high-density lipoprotein mediated cholesterol efflux from macrophage cells by liquid chromatography tandem mass spectrometry.
abstract: BACKGROUND
  Studies have shown a negative association between macrophage cholesterol efflux and atherosclerotic cardiovascular diseases (CVD). However, the current methods for measuring cholesterol efflux require a radioactive tracer and involve a variety of cell treatments, making the measurement of macrophage cholesterol efflux impractical for use in clinical laboratories. In this study, we developed a non-radioactive and precise LC/MS/MS method for the measurement of high-density lipoprotein (HDL) mediated cholesterol efflux from J774 macrophages.
  
  
  METHODS
  J774 cells were seeded on 12-well plates at a density of 1.5×10(5) cells/ml in H-DMEM medium, and when the cells were approximately 80% confluent, they were incubated with H-DMEM medium containing 2% FBS, 0.5 μg/ml ACAT inhibitor Sandoz 58-035, and 20 μg/ml [3,4-(13)C]cholesterol for 6 h. After washing and equilibrating the cells, HDL samples were added at a final concentration of 7% and incubated for 8 h. The cells were lysed, and [3,4-(13)C]cholesterol and cholesterol were measured by LC/MS/MS. Cholesterol efflux was expressed as the percent decrease of cell [3,4-(13)C]cholesterol mass during the incubation.
  
  
  RESULTS
  When incubated with [3,4-(13)C]cholesterol enriched J774 cells, HDL mediated higher cell cholesterol efflux than influx compared to serum and isolated LDL; therefore, HDL was used as the extracellular acceptor. The results from healthy volunteers showed that the rate of cholesterol efflux was negatively correlated with weight, BMI, blood pressure, and FERHDL and positively correlated with HDL-C, HDL2-C, and apoAI levels.
  
  
  CONCLUSIONS
  A LC/MS/MS method for the measurement of HDL mediated cholesterol efflux from macrophage cells has been established. This method is non-radioactive, precise and reliable and is potentially useful for the assessment of HDL function and cardiovascular disease risks.
authors.affiliation: Graduate School of Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China.
authors.forename: Mo
authors.initials: M
authors.lastname: Wang
authors.forename: Hanbang
authors.initials: H
authors.lastname: Guo
authors.forename: Siming
authors.initials: S
authors.lastname: Wang
authors.forename: Ruiyue
authors.initials: R
authors.lastname: Yang
authors.forename: Hongxia
authors.initials: H
authors.lastname: Li
authors.forename: Haijian
authors.initials: H
authors.lastname: Zhao
authors.forename: Shu
authors.initials: S
authors.lastname: Wang
authors.forename: Jun
authors.initials: J
authors.lastname: Dong
authors.forename: Wenxiang
authors.initials: W
authors.lastname: Chen
mesh_terms: D000328:Adult; D000577:Amides; D000818:Animals; D050197:Atherosclerosis / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D002784:Cholesterol / Q000302:isolation & purification / Q000378:metabolism*; D002853:Chromatography, Liquid; D006801:Humans; D008075:Lipoproteins, HDL / Q000302:isolation & purification / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism / Q000473:pathology; D051379:Mice; D017646:Organosilicon Compounds; D053719:Tandem Mass Spectrometry
chemical_list: D000577:Amides; D008075:Lipoproteins, HDL; D017646:Organosilicon Compounds; C040256:SAN 58035; D002784:Cholesterol