液相色谱-质谱联用法测定U937细胞内胆固醇

Cholesterol in U937 foam cells assayed in liquid chromatographic-mass spectrometry

目的利用液相色谱-质谱联用法测定细胞内胆固醇及胆固醇酯,建立一种精确鉴定泡沫细胞的方法。方法用氧化修饰低密度脂蛋白(oxidized low density lipoprotein,oxLDL)干预U937巨噬细胞诱导泡沫细胞形成,然后用己烷/异丙醇(体积比3/2)抽提细胞内胆固醇及胆固醇酯,抽提液经真空干燥后再溶于甲醇,用液相色谱-质谱联用法直接测定细胞内游离胆固醇,胆固醇酯经胆固醇酯酶水解后测定总胆固醇,总胆固醇减去游离胆固醇即胆固醇酯。抽提脂质后的沉淀物溶于氢氧化钠溶液中,用BCA法测定蛋白含量,胆固醇及胆固醇酯用μg/mg细胞蛋白为单位来表示。结果氧化低密度脂蛋白干预后的巨噬细胞内存在高水平胆固醇及胆固醇酯的蓄积。在100μg的oxLDL处理组中,胆固醇酯与阴性对照组差异存在显著性[(25.733±2.970)μg/mg细胞蛋白vs (13.024±2.875)μg/mg细胞蛋白,P<0.01],同样胆固醇的水平差异也存在显著性[(18.675±1.315)μg/mg细胞蛋白vs (24.428±2.370)μg/mg细胞蛋白,P<0.01]。结论液相色谱-质谱联用法测定细胞内胆固醇是一种鉴定泡沫细胞简便而精确的方法。

Objective: To assess the merits of liquid chromatographic-mass spectrometry and to explore a useful and precise method to assay the cholesterol and cholesterol ester in foam cells. Methods: U937 cells incubated with oxidized LDL were induced to form foam cells, and the cell monolayer was extracted with hexane/isopropanol 3/2(v/v). Free and total cholesterol were assayed in liquid chromatographic-mass spectrometry. To assay total cholesterol, the cellular extracts were evaporated to dryness and the residue was hydrolyzed by cholesterol esterase. Cholesterol ester levels were abtained by subtracting free cholesterol from total cholesterol levels, cholesterol was used as an external standard. After the lipid extraction, the cell pellet was solubilized with sodium hydrate solutions, and then protein content was detemined. Results were normalized by cell protein. Results: Oxidized LDL caused a higher degree of cholesterol ester accummulation in the U937 macrophage-like cells [(25.733±2.970) μg/mg protein in cells incubated with oxidized LDL vs (13.024±2.875)μg/mg protein in cells without oxidized LDL,P<0.01]. The levels of free cholesterol in the same cells also had significant differences [(18.675±1.315) μg/mg protein vs (24.428±2.370) μg/mg protein,P<0.01]. Conclusions: The liquid chromatographic-mass spectrometry is a useful and precise method to identify foam cell.