Abstract
Cultured mouse peritoneal macrophages accumulate large quantities of cholesteryl (C-)ester deposits when incubated with acetylated low density lipoprotein (A-LDL). Accordingly, macrophages have been used as models in atherosclerosis research. A rapid method for the quantification of unesterified and esterified cholesterols (FC and CE) in human plasma lipoprotein fractions is achieved by high-performance liquid chromatography (HPLC). In this study we examined whether HPLC can be applicable in quantifying cellular FC and CE.
To determine the content of cellular FC and CE, macrophages were incubated with A-LDL (50μg protein/ml) and 0.1mM oleate albumin complex for 12 hours. Macrophages were harvested, and cellular lipid were isolated by the Folch method. The composition of the fatty acyl components of CE was determined by reversed-phase HPLC. The mobile phase was acetonitrile/isopropanol (75/25) mixture, and the elute was monitored at 205nm UV absorption detector.
The composition of the fatty acyl components of cellular CE indicated the presence of C-linolenate, C-arachidonate, C-linoleate (containing C-palmitoleate peak), C-oleate, and C-palmitate. Absolute quantification of FC, C-linoleate, and C-oleate was obtained by using an internal standard (C-heptadecanoate). The analysis of cellular FC and CE was successfully achieved by reversephase HPLC.
