stigmasterol and pyridine

A mass spectrometric method for the quantification of free cholesterol in cells and subcellular membranes is presented. The method is based on a simple one-step chemical derivatization of cholesterol to cholesterol-3-sulfate by a sulfur trioxide-pyridine complex. Quantification is performed by nano-electrospray ionization tandem mass spectrometry (nanoESI-MS/MS) using a stable isotope labeled internal standard. The determination of free cholesterol is demonstrated in about 250 cells of a Chinese hamster ovary (CHO) cell line. With this method a molar ratio of free cholesterol to total phospholipids of 0.34 mol/mol in CHO cells was determined. In a subcellular membrane fraction enriched in Golgi membranes, a molar ratio of free cholesterol to total phospholipids of 0.57 mol/mol was determined. The method should be of value for quantification of other sterols as demonstrated for ergosterol and stigmasterol.

Topics: Animals; CHO Cells; Cholesterol; Cricetinae; Ergosterol; Fluorescence; Golgi Apparatus; Indicators and Reagents; Intracellular Membranes; Mass Spectrometry; Microchemistry; Phospholipids; Pyridines; Reference Standards; Sensitivity and Specificity; Stigmasterol; Sulfur Oxides