Abstract
Objective To develop a safe and reliable method for the measurement of cholesterol net flux between macrophage cells and human serum by high performance liquid chromatography,and to investigate the relationship between cholesterol net flux and traditional risk factors of atherosclerosis. Methods J774 (murine cell line) cells were plated in growth medium containing 5% individual human serum and cell cholesterol before and after incubation were measured by HPLC.Serum induced cell cholesterol net influx was calculated.Fasting blood samples of 75 apparently healthy volunteers (38 males and 37 females) from Oct to Dec 2010 were collected.Cholesterol net flux was measured by the newly established method.Spearman correlation analysis was used to evaluate the relationship between cholesterol net flux and lipid,lipoprotein,highsensitivity C-reactive protein (hsCRP) and other CVD risk factors. Results When J774 cells were incubated with individual serum,cell cholesterol levels were elevated with the increase of serum concentrations and incubation time.J774 cells were incubated with medium containing 5% serum for 8 hours,the within assay and total CV s for the net movement of cellular cholesterol measured by HPLC were 0.81%-4.03% and 4.25%-6.58%,respectively.The average cholesterol net efflux of 75 healthy subjects was 10.27μg/mg,with a standard deviation of 2.69μg/mg protein,and a median of 10.12μg/mgprotein.Cholesterol net efflux was significantly positively correlated with total cholesterol (TC),triglycerides (TG), hsCRP, apolipoprotein (apo) CⅡ, apoCⅢ, small-dense low density lipoprotein cholesterol (LDLb-C),molar esterification rate of HDL cholesterol (MERHDL)(P<0.001),fractional esterification rate of HDL cholesterol (FERHDL) and Pre β-HDL (P<0.01).Cholesterol net efflux was not related with HDL as well as its subfractions. Conclusions A new method for the measurement of the net movement of cellular cholesterol between cells and serum by HPLC has been established.This method is safe,precise and reliable and may be used in the assessment of HDL function and cardiovascular diseases risks.(Chin J Lab Med,2013,36:151-155) Key words: Macrophages; Cholesterol; Chromatography; high pressure liquid; Aoherosclerosis
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