Research on the determination of cholesterol efflux from foam cells mediated by apoA-1 using HPLC analysis / 重庆医学

ABSTRACT

Objective To establish a high performance liquid chromatography (HPLC) method for determining the choles‐terol efflux from macrophage‐derived foam cells mediated by apolipoprotein A‐1(apoA‐1) .Methods Human THP‐1 monocytic cells ,pre‐treated with 160 nmol/L phorbol‐12‐myristate acetate (PMA) for 24 h to differentiate into adherence macrophages ,then incubated with 50 μg/mL acetylated low density lipoprotein (ac‐LDL) for 48 h to induce foam cells formation ,then added apoA‐1 for 24 h .THP‐1‐derived macrophage foam cells were identified by oil red O‐staining ,and the cellular cholesterol content by meas‐ured by HPLC method .Cholesterol efflux from macrophage foam cells was determined by HPLC analysis and liquid scintillation counting ,respectively .Results Oil red O‐stainable lipid droplet accumulation were observed in entire cytoplasm of THP‐1‐derived macrophage foam cells .Measuring cellular cholesterol content showed that free cholesterol ,total cholesterol and cholesterol ester content in macrophage foam cells were increased remarkably than PMA group macrophages (P<0 .01) .After treated with ac‐LDL for 48 h ,the macrophage foam cells accumulated 80 .25 μg/mg cell protein and 47 .65 μg/mg cell protein respectively ,and the cho‐lesterol ester accounted for 59 .38% of the cellular total cholesterol (P<0 .01) .The ratio of cholesterol efflux reached 5 .63% and 7 .08% respectively by HPLC analysis and liquid scintillation counting using apoA‐1 mediation (P<0 .01) .Conclusion Combina‐tion of an enzymatic catalysis and HPLC method for determining cholesterol efflux from foam cells is successfully established in this study , thus providing a technical foundation for the further study of cellular lipid homeostasis .