Zhou Zihang, Zong Jiangyu, Xu Ning
Institute of Drug Development and Chemical Biology, College of Pharmaceutical Science, Zhejiang University of Technology, Huzhou, 313200, Zhejiang, People's Republic of China.
Anal Sci. 2025 Aug 27. doi: 10.1007/s44211-025-00842-5.
Imbalances in cellular cholesterol homeostasis are associated with various diseases, and accurate determination of cholesterol levels and distribution is essential for a thorough understanding of cellular physiopathology. In this review, we comprehensively analyzed various techniques for cellular cholesterol determination. They include indirect methods based on SREBP2 activity monitoring, gas chromatography-liquid chromatography coupling, enzyme analysis, improved Abell-Kendall method, cholesterol-specific probes such as filipin III and cholesterol-dependent cytolysin, and cholesterol analogs such as dehydroergosterol, BODIPY-cholesterol, label-free as well as labeling Raman assays and mass spectrometry imaging techniques such as matrix-assisted laser desorption/ionization mass spectrometry imaging, desorption electrospray ionization mass spectrometry imaging, and nanoscale secondary ion mass spectrometry technology. Principles, advantages, and limitations of each technique are discussed in detail, their characteristics in terms of sensitivity, spatial resolution, and temporal resolution are compared in detail. Finally, suggestions for selecting the technique for different experimental objectives are also given. These findings will help researchers choose the most suitable method according to their own needs, provide strong support for cellular cholesterol research, and promote the development of related fields to better elucidate the significance of cellular cholesterol in normal biologic activities and also its intrinsic relationship with diseases.
细胞胆固醇稳态失衡与多种疾病相关，准确测定胆固醇水平和分布对于全面了解细胞生理病理学至关重要。在本综述中，我们全面分析了多种细胞胆固醇测定技术。它们包括基于SREBP2活性监测的间接方法、气相色谱-液相色谱联用、酶分析、改良的阿贝尔-肯德尔方法、诸如菲律宾菌素III和胆固醇依赖性细胞溶素等胆固醇特异性探针、诸如脱氢麦角固醇、硼二吡咯-胆固醇等胆固醇类似物、无标记以及标记拉曼测定法以及诸如基质辅助激光解吸/电离质谱成像、解吸电喷雾电离质谱成像和纳米级二次离子质谱技术等质谱成像技术。详细讨论了每种技术的原理、优点和局限性，详细比较了它们在灵敏度、空间分辨率和时间分辨率方面的特点。最后，还给出了针对不同实验目的选择技术的建议。这些发现将有助于研究人员根据自身需求选择最合适的方法，为细胞胆固醇研究提供有力支持，并促进相关领域的发展，以更好地阐明细胞胆固醇在正常生物活动中的意义及其与疾病的内在关系。
