Department of General Surgery, Division of Vascular Surgery, Medical University of Vienna and University Hospital Vienna, Waehringer Guertel 18-20, 1090, Vienna, Austria.
Institute of Physiology, Pathophysiology and Biophysics, Unit of Physiology and Biophysics, University of Veterinary Medicine, 1210, Vienna, Austria.
Free Radic Biol Med. 2023 Sep;206:94-105. doi: 10.1016/j.freeradbiomed.2023.06.014. Epub 2023 Jun 21.
There is accumulating evidence that pro-inflammatory features are inherent to mitochondrial DNA and oxidized DNA species. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is the most frequently studied oxidatively generated lesion. Modified DNA reaches the circulation upon cell apoptosis, necrosis or neutrophil extracellular trap (NET) formation. Standard chromatography-based techniques for the assessment of 8-oxodGuo imply degradation of DNA to a single base level, thus precluding the attribution to a nuclear or mitochondrial origin. We therefore aimed to establish a protocol for the concomitant assessment of oxidized mitochondrial and nuclear DNA from human plasma samples. We applied immunoprecipitation (IP) for 8-oxodGuo to separate oxidized from non-oxidized DNA species and subsequent quantitative polymerase chain reaction (qPCR) to assign them to their subcellular source. The IP procedure failed when applied directly to plasma samples, i.e. isotype control precipitated similar amounts of DNA as the specific 8-oxodGuo antibody. In contrast, DNA isolation from plasma prior to the IP process provided assay specificity with little impact on DNA oxidation status. We further optimized sensitivity and efficiency of qPCR analysis by reducing amplicon length and targeting repetitive nuclear DNA elements. When the established protocol was applied to plasma samples of abdominal aortic aneurysm (AAA) patients and control subjects, the AAA cohort displayed significantly elevated circulating non-oxidized and total nuclear DNA and a trend for increased levels of oxidized mitochondrial DNA. An enrichment of mitochondrial versus nuclear DNA within the oxidized DNA fraction was seen for AAA patients. Regarding the potential source of circulating DNA, we observed a significant correlation of markers of neutrophil activation and NET formation with nuclear DNA, independent of oxidation status. Thus, the established method provides a tool to detect and distinguish the release of oxidized nuclear and mitochondrial DNA in human plasma and offers a refined biomarker to monitor disease conditions of pro-inflammatory cell and tissue destruction.
越来越多的证据表明，促炎特征是线粒体 DNA 和氧化 DNA 物种所固有的。8-氧代-7,8-二氢-2'-脱氧鸟苷（8-oxodGuo）是研究最广泛的氧化生成损伤。修饰后的 DNA 会在细胞凋亡、坏死或中性粒细胞细胞外陷阱（NET）形成时进入血液循环。评估 8-oxodGuo 的基于标准色谱的技术需要将 DNA 降解到单个碱基水平，从而排除其来源于核或线粒体的可能性。因此，我们旨在建立一种从人血浆样本中同时评估氧化的线粒体和核 DNA 的方案。我们应用免疫沉淀（IP）来分离氧化的和非氧化的 DNA 物质，然后应用定量聚合酶链反应（qPCR）将它们分配到它们的亚细胞来源。该 IP 程序直接应用于血浆样本时失败，即同种型对照沉淀的 DNA 量与特异性 8-oxodGuo 抗体相似。相比之下，在 IP 过程之前从血浆中分离 DNA 可提供检测特异性，对 DNA 氧化状态的影响很小。通过减少扩增子长度并靶向重复的核 DNA 元件，我们进一步优化了 qPCR 分析的灵敏度和效率。当将建立的方案应用于腹主动脉瘤（AAA）患者和对照受试者的血浆样本时，AAA 组显示出循环的非氧化和总核 DNA 水平显著升高，并且氧化的线粒体 DNA 水平呈升高趋势。在 AAA 患者中，氧化 DNA 部分中的线粒体与核 DNA 的丰度存在富集。关于循环 DNA 的潜在来源，我们观察到中性粒细胞活化和 NET 形成的标志物与核 DNA 之间存在显著相关性，与氧化状态无关。因此，该建立的方法提供了一种检测和区分人血浆中氧化的核和线粒体 DNA 释放的工具，并提供了一种更精细的生物标志物来监测促炎细胞和组织破坏的疾病状况。
