Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC, 20007, USA.
Department of Oncology, Georgetown University Medical Center, Washington, DC, 20007, USA.
J Transl Med. 2022 May 10;20(1):199. doi: 10.1186/s12967-022-03414-7.
Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine.
3 EV isolation methods were compared: ultracentrifugation, magnetic bead-based, and size-exclusion chromatography from 0.5 mL or 1 mL of rat and human urine. EV yield and mass spectrometry signals (Q-ToF and Triple Quad) were evaluated from each method. Metabolomic profiling was performed on EVs isolated from the urine of rats exposed to ionizing radiation 1-, 14-, 30- or 90-days post-exposure, and human urine from patients receiving thoracic radiotherapy for the treatment of lung cancer pre- and post-treatment.
Size-exclusion chromatography is the preferred method for EV isolation from 0.5 mL of urine. Mass spectrometry-based metabolomic analyses of EV cargo identified biochemical changes induced by radiation, including altered nucleotide, folate, and lipid metabolism. We have provided standard operating procedures for implementation of these methods in other laboratories.
We demonstrate that EVs can be isolated from small volumes of urine and analytically investigated for their biochemical contents to detect radiation induced metabolomic changes. These findings lay a groundwork for future development of methods to monitor response to radiotherapy and can be extended to an array of molecular phenotyping studies aimed at characterizing EV cargo.
尿细胞外囊泡 (EVs) 是生物标志物的来源，具有广泛的临床研究应用潜力，包括监测辐射暴露。其实施的一个关键限制是 EV 分离和分析方法的标准化程度低。此外，大多数尿 EV 分离方案需要大量的样本。本研究旨在比较和优化从小体积尿液中分离和分析 EV 的方法。
比较了 3 种 EV 分离方法：超速离心、基于磁珠和大小排阻色谱法，从 0.5 mL 或 1 mL 的大鼠和人尿中分离 EV。从每种方法中评估 EV 的产量和质谱信号（Q-ToF 和 Triple Quad）。对暴露于电离辐射 1、14、30 或 90 天后的大鼠尿液以及接受肺癌胸部放疗治疗前后的人类尿液中分离的 EV 进行代谢组学分析。
大小排阻色谱法是从小体积尿液中分离 EV 的首选方法。基于质谱的 EV 货物代谢组学分析鉴定了辐射引起的生化变化，包括核苷酸、叶酸和脂质代谢的改变。我们已经为在其他实验室实施这些方法提供了标准操作程序。
我们证明可以从小体积尿液中分离 EV，并对其生化内容进行分析以检测辐射引起的代谢组学变化。这些发现为开发监测放疗反应的方法奠定了基础，并可以扩展到一系列旨在表征 EV 货物的分子表型研究。
