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

被引：0

作者：

Brkovic, Ilijana Begcevic ^{[1
]}

Reinicke, Madlen ^{[1
]}

Chey, Soroth ^{[1
]}

Bechmann, Ingo ^{[2
]}

Ceglarek, Uta ^{[1
]}

机构：

[1] Univ Leipzig Med Ctr, Inst Lab Med, Clin Chem & Mol Diagnost, D-04103 Leipzig, Germany

[2] Univ Leipzig, Inst Anat, Fac Med, D-04103 Leipzig, Germany

来源：

CELLS | 2023年 / 12卷 / 07期

关键词：

sterols; targeted mass spectrometry; plasma membrane microdomains; microglia; plant sterols; cholesterol precursors; BETA-SITOSTEROL; PLANT STEROLS; LIPID RAFTS; HUMAN SERUM; RECEPTOR; SUPPLEMENTATION; QUANTIFICATION; PHYTOSTEROLS; PLASMA; BRAIN;

D O I：

10.3390/cells12070974

中图分类号：

Q2 [细胞生物学];

学科分类号：

071009 ; 090102 ;

摘要：

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.

引用

页数：13

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