Determination of cholesterol in erythrocyte membranes by HPLC method and its clinical application

Objective To develop a high performance liquid chromatographic method (HPLC) for the analysis of of cholesterol in erythrocyte membranes. Methods The study included 167 consecutive chest pain patients who underwent coronary artery angiography in the Department of Cardiology, Nanjing General Hospital of Nanjing Command between September 2012 and February 2013. According to the clinical symptoms and t angiographic results，patients were divided into three groups: acute coronary syndrome (ACS) group（n=46），stable angina pectoris (SAP) group （n=76）and the control group（n=45）. After the erythrocyte sample was hypotonically lysed and washed, saponification was carried out in a polassium hydroxide solution at 70 ℃. After extraction by Hexane/isopropanol mixture, the sample was separated on a Lichrospher column and detected by ultraviolet absorbance at 208 nm. A mobile phase composed of acetonitrile-isopropyl alcohol was found to be the most suitable for this separation. Concentrations of cholesterol in erythrocyte membranes were tested. Analysis of variance with covariates (ANOVA) was used to evaluate differences in CEM levels among groups. The relationship between continuous variables was evaluated by Spearman′s correlation coefficient. Results Under the chromatographic conditions described, retention time of the cholesterol was approximately 6.1 min. Good separation and detectability of cholesterol in erythrocyte membranes were obtained. The method proved to be linear in the injection range of cholesterol from 0.05 g to 2.00 g. Cholesterol content in erythrocyte membranes were (87.0 μg/mg, 75.4-98.9 μg/mg), (92.9 μg/mg, 83.8-109.0 μg/mg) and (173.9 μg/mg, 140.0-188.8 μg/mg) in the control, SAP and ACS groups, respectively. Cholesterol content in erythrocyte membranes was significantly higher in ACS group than that in SAP and control groups (P<0.01). Conclusion We have successfully developed a method for the determination of cholesterol in erythrocyte membranes with good sensitivity, specificity and repeatability. （Chin J Lab Med，2014,37：179-183） Key words: Erythrocyte membrane; Cholesterol; Chromatography, high pressure liquid; Acute coronary syndrome; Atherosclerosis