Guo Zhiting, Yu Huiyan, Yang Kexin, Feng Wenjing, Liu Miao, Wang Tao, Xiao Rong
School of Public Health, Capital Medical University, Beijing 100069, China.
Int J Mol Sci. 2024 Dec 25;26(1):77. doi: 10.3390/ijms26010077.
Oxysterols, as metabolites of cholesterol, play a key role in cholesterol homeostasis, autophagosome formation, and regulation of immune responses. Disorders in oxysterol metabolism are closely related to the pathogenesis of neurodegenerative diseases. To systematically investigate the profound molecular regulatory mechanisms of neurodegenerative diseases, it is necessary to quantify oxysterols and their metabolites in central and peripheral biospecimens simultaneously and accurately. However, there are a lot of unsolved problems with the existing methods, such as the hindrance of applying a single method to different biological specimens or the challenge of simultaneous quantification due to differential groups on the ends of the oxysterol side chains. Herein, according to the physicochemical properties and structure of oxysterols, an optimized liquid chromatography-tandem mass spectrometry method for the quantification of oxysterols was established by optimizing the sample preparation process, chromatographic conditions, mobile phase pH, and solvent selection. Seven oxysterols were detected by this method, including 27-hydroxycholesterol, 7α-hydroxycholesterol, 7α,27-dihydroxycholesterol, 7-dehydrocholesterol, 7α-hydroxy-3-oxo-4-cholestenoic acid, 3-hydroxy-5-cholestenoic acid, and 24(S)-hydroxycholesterol. Non-derivatization extraction with methyl tert-butyl ether was used for different biospecimens, followed by simultaneous chromatographic separation of oxysterols on a phenyl hexyl column. By repeated validation, this method exhibited satisfactory linearity, precision, recovery, sensitivity, repeatability, and stability, and it was successfully applied to the detection of oxysterols in the plasma, cerebral cortex, and liver of mouse. In summary, our optimized method enables concurrent analysis and quantification of oxysterols and their metabolites in various biospecimens, presenting a broad range of applicability.
氧化甾醇作为胆固醇的代谢产物，在胆固醇稳态、自噬体形成和免疫反应调节中发挥关键作用。氧化甾醇代谢紊乱与神经退行性疾病的发病机制密切相关。为了系统地研究神经退行性疾病的深层分子调控机制，有必要同时准确地定量中枢和外周生物样本中的氧化甾醇及其代谢产物。然而，现有方法存在许多未解决的问题，例如将单一方法应用于不同生物样本时存在障碍，或者由于氧化甾醇侧链末端的差异基团导致同时定量存在挑战。在此，根据氧化甾醇的物理化学性质和结构，通过优化样品制备过程、色谱条件、流动相pH值和溶剂选择，建立了一种优化的液相色谱-串联质谱法用于氧化甾醇的定量分析。该方法检测到了七种氧化甾醇，包括27-羟基胆固醇、7α-羟基胆固醇、7α,27-二羟基胆固醇、7-脱氢胆固醇、7α-羟基-3-氧代-4-胆甾烯酸、3-羟基-5-胆甾烯酸和24(S)-羟基胆固醇。采用甲基叔丁基醚进行非衍生化提取用于不同的生物样本，随后在苯基己基柱上对氧化甾醇进行同时色谱分离。通过反复验证，该方法具有令人满意的线性、精密度、回收率、灵敏度、重复性和稳定性，并成功应用于小鼠血浆、大脑皮层和肝脏中氧化甾醇的检测。总之，我们优化后的方法能够同时分析和定量各种生物样本中的氧化甾醇及其代谢产物，具有广泛的适用性。
