Background
Non-cholesterol sterols, as well as plant sterols, cross the blood-brain barrier and, thus, can be incorporated into cell membranes, affecting the cell's inflammatory response. The
Conclusion
By applying our newly developed and validated analytical protocol, we show that the non-CH sterol concentration is about 38% of the total sterol content in microglia membrane microdomains. Further investigations must clarify how changes in the non-sterol composition influence membrane fluidity and cell signaling.
Methods
A protocol for cell membrane isolation using OptiPrepTM gradient ultracentrifugation, in combination with a targeted mass spectrometry (LC-MS/MS)-based assay, was developed and validated for the quantitative analysis of free sterols in microglia cell membranes.
Results
Utilizing an established LC-MS/MS assay, cholesterol and seven non-cholesterol sterols were analyzed with a limit of detection from 0.001 to 0.05 mg/L. Applying the detergent-free isolation of SIM-A9 microglia cell membranes, cholesterol (CH), desmosterol (DE), lanosterol (LA) stigmasterol (ST), beta-sitosterol (SI) and campesterol (CA) were quantified with coefficients of variations between 6 and 29% (fractions 4-6, n = 5). The highest concentrations of non-CH sterols within the microglia plasma membranes were found in the microdomain region (DE>LA>SI>ST>CA), with ratios to CH ranging from 2.3 to 435 lower abundancies.