A quantitative LC-MS/MS method for analysis of mitochondrial-specific oxysterol metabolism
A quantitative LC-MS/MS method for analysis of mitochondrial-specific oxysterol metabolism
复制标题
用于分析线粒体特异性氧甾醇代谢的定量 LC-MS/MS 方法
DOI：
10.1016/j.redox.2020.101595
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发表时间：
2020-09-01
期刊：
影响因子：
11.4
通讯作者：
Griffiths, Helen R.
中科院分区：
文献类型：
--
作者：
Borah, Khushboo;Rickman, Olivia J.;Griffiths, Helen R.
氧化甾醇是细胞内炎症和胆固醇代谢的关键调节剂。它们是胆固醇的氧化产物，在亚细胞室和生物液中可能有不同的代谢。需要新的分析方法来提高我们对氧固醇运输以及维持胆固醇/氧固醇稳态所需的细胞间隔之间的分子相互作用的理解。本文介绍了一种用于组织、细胞和线粒体的固相萃取和液-质联用定量分析方法。采用反相色谱LC-MS/MS联用技术分析了5种单羟基甾醇：24(S)-羟基胆固醇、25-羟基胆固醇、27-羟基胆固醇、7α-羟基胆固醇、7酮胆固醇和3种二羟基甾醇7α-24(S)二羟基胆固醇、7α-25二羟基胆固醇、7α-27二羟基胆固醇。我们的新方法，使用Triton和DMSO提取，显示了从细胞基质中提取氧甾醇的效率和回收率的提高。我们通过重复测量小鼠脑组织中的氧化甾醇来验证我们的方法，结果表明，喂食高脂饮食的小鼠的24S/25diOHC、27diOHC和7ketoOHC的水平显著降低。我们测量了外周血单核细胞线粒体中的氧固醇，并强调了快速分离细胞的重要性，以最大限度地减少处理和储存条件对临床样本中氧固醇组成的影响。此外，THP-1单核细胞和神经元样SH-SH5Y细胞的体外细胞培养系统显示线粒体特异的氧固醇代谢和谱系特异性。总之，我们描述了一种稳健和可重复性的方法，该方法在回收率、定量线性和检测、重复性和选择性方面得到了验证，用于细胞氧固醇分析。这种方法能够监测亚细胞氧固醇代谢，并可广泛应用于各种生物和临床样品。
Oxysterols are critical regulators of inflammation and cholesterol metabolism in cells. They are oxidation products of cholesterol and may be differentially metabolised in subcellular compartments and in biological fluids. New analytical methods are needed to improve our understanding of oxysterol trafficking and the molecular interplay between the cellular compartments required to maintain cholesterol/oxysterol homeostasis. Here we describe a method for isolation of oxysterols using solid phase extraction and quantification by liquid chromatography-mass spectrometry, applied to tissue, cells and mitochondria.We analysed five monohydroxysterols; 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 7 alpha-hydroxycholesterol, 7 ketocholesterol and three dihydroxysterols 7 alpha-24(S)dihydroxycholesterol, 7 alpha-25dihydroxycholesterol, 7 alpha-27dihydroxycholesterol by LC-MS/MS following reverse phase chromatography. Our new method, using Triton and DMSO extraction, shows improved extraction efficiency and recovery of oxysterols from cellular matrix. We validated our method by reproducibly measuring oxysterols in mouse brain tissue and showed that mice fed a high fat diet had significantly lower levels of 24S/25diOHC, 27diOHC and 7ketoOHC. We measured oxysterols in mitochondria from peripheral blood mononuclear cells and highlight the importance of rapid cell isolation to minimise effects of handling and storage conditions on oxysterol composition in clinical samples. In addition, in vitro cell culture systems, of THP-1 monocytes and neuronal-like SH-SH5Y cells, showed mitochondrial-specific oxysterol metabolism and profiles were lineage specific. In summary, we describe a robust and reproducible method validated for improved recovery, quantitative linearity and detection, reproducibility and selectivity for cellular oxysterol analysis. This method enables sub cellular oxysterol metabolism to be monitored and is versatile in its application to various biological and clinical samples.