Tom Heinze, Franziska Ebert, Christiane Ott, Judith Nagel, Carola Eberhagen, Hans Zischka, Tanja Schwerdtle
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To address this, a novel method to isolate an enriched mitochondria fraction (EMF) from frozen tissue was adapted from already established protocols. Validation of manganese (Mn), iron (Fe), and copper (Cu) quantification via inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) showed sufficiently low quantification limits for EMF TE analysis. Successful mitochondrial enrichment from frozen liver samples was confirmed via immunoblots and transmission electron microscopy (TEM) revealed sufficient structural integrity of the EMFs. No significant differences in EMF TEs between frozen and fresh tissue were evident for Mn and Cu and only slight decreases in EMF Fe. Consequently, EMF TEs were highly comparable for isolates from both tissue states. In application, this method effectively detected dietary differences in EMF Fe of a murine feeding study and identified the disease status in a Wilson disease rat model based on drastically increased EMF Cu. 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引用次数: 0
摘要
从器官到亚细胞器,微量元素(TE)的平衡是许多生理过程的基础。虽然微量元素失衡在早期阶段经常被忽视,但它会对健康造成严重后果,尤其是在衰老或病理情况下。在线粒体水平监测 TE 浓度可识别细胞器特异性失衡,有助于进行有针对性的诊断和实现更健康的衰老过程。然而,从冷冻组织中分离线粒体具有挑战性,因为这样做会带来细胞器因冷冻损伤而损失 TE 的风险,但却能大大简化常规实验室工作。为了解决这个问题,我们根据已有的方案改良了一种从冷冻组织中分离富集线粒体部分(EMF)的新方法。通过电感耦合等离子体串联质谱法(ICP-MS/MS)对锰(Mn)、铁(Fe)和铜(Cu)的定量进行验证,结果表明 EMF TE 分析的定量限足够低。通过免疫印迹技术从冷冻肝脏样本中成功富集线粒体,透射电子显微镜(TEM)显示 EMF 具有足够的结构完整性。锰和铜的EMF TE在冷冻组织和新鲜组织中没有明显差异,铁的EMF TE仅略有下降。因此,来自两种组织状态的分离物的 EMF TEs 具有很高的可比性。在应用中,该方法有效地检测了小鼠饲养研究中膳食中 EMF 铁的差异,并根据 EMF 铜的急剧增加确定了威尔逊病大鼠模型的疾病状态。总之,本方法适用于未来的应用,有利于样本储存和线粒体 TE 的高通量分析。
Subzero project: comparing trace element profiles of enriched mitochondria fractions from frozen and fresh liver tissue.
From organs to subcellular organelles, trace element (TE) homeostasis is fundamental for many physiological processes. While often overlooked in early stages, manifested TE disbalance can have severe health consequences, particularly in the context of aging or pathological conditions. Monitoring TE concentrations at the mitochondrial level could identify organelle-specific imbalances, contributing to targeted diagnostics and a healthier aging process. However, mitochondria isolation from frozen tissue is challenging, as it poses the risk of TE losses from the organelles due to cryodamage, but would significantly ease routine laboratory work. To address this, a novel method to isolate an enriched mitochondria fraction (EMF) from frozen tissue was adapted from already established protocols. Validation of manganese (Mn), iron (Fe), and copper (Cu) quantification via inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) showed sufficiently low quantification limits for EMF TE analysis. Successful mitochondrial enrichment from frozen liver samples was confirmed via immunoblots and transmission electron microscopy (TEM) revealed sufficient structural integrity of the EMFs. No significant differences in EMF TEs between frozen and fresh tissue were evident for Mn and Cu and only slight decreases in EMF Fe. Consequently, EMF TEs were highly comparable for isolates from both tissue states. In application, this method effectively detected dietary differences in EMF Fe of a murine feeding study and identified the disease status in a Wilson disease rat model based on drastically increased EMF Cu. In summary, the present method is suitable for future applications, facilitating sample storage and high-throughput analyses of mitochondrial TEs.
期刊介绍:
Analytical and Bioanalytical Chemistry’s mission is the rapid publication of excellent and high-impact research articles on fundamental and applied topics of analytical and bioanalytical measurement science. Its scope is broad, and ranges from novel measurement platforms and their characterization to multidisciplinary approaches that effectively address important scientific problems. The Editors encourage submissions presenting innovative analytical research in concept, instrumentation, methods, and/or applications, including: mass spectrometry, spectroscopy, and electroanalysis; advanced separations; analytical strategies in “-omics” and imaging, bioanalysis, and sampling; miniaturized devices, medical diagnostics, sensors; analytical characterization of nano- and biomaterials; chemometrics and advanced data analysis.