4.6 Article

Characterization of Non-Cholesterol Sterols in Microglia Cell Membranes Using Targeted Mass Spectrometry

Journal

CELLS
Volume 12, Issue 7, Pages -

Publisher

MDPI
DOI: 10.3390/cells12070974

Keywords

sterols; targeted mass spectrometry; plasma membrane microdomains; microglia; plant sterols; cholesterol precursors

Categories

Ask authors/readers for more resources

An analytical protocol for the quantitative assessment of sterol composition in microglia cell membrane microdomains was developed and validated. It was found that non-cholesterol sterols accounted for about 38% of the total sterol content in microglia membrane microdomains. Further studies are needed to clarify the impact of non-sterol composition changes on membrane fluidity and cell signaling.
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 aim of our work was to develop an analytical protocol for a quantitative assessment of the sterol composition within the membrane microdomains of microglia. Methods: A protocol for cell membrane isolation using OptiPrep (TM) 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. 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.

Authors

I am an author on this paper
Click your name to claim this paper and add it to your profile.

Reviews

Primary Rating

4.6
Not enough ratings

Secondary Ratings

Novelty
-
Significance
-
Scientific rigor
-
Rate this paper

Recommended

No Data Available
No Data Available