Nishihara Tatsuya, Nishida Koki, Tsukame Rika, Sugawara Yumika, Nagano Shunya, Tanabe Kazuhito
Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University 5-10-1 Fuchinobe, Chuo-ku Sagamihara Kanagawa 252-5258 Japan
RSC Adv. 2025 Jul 23;15(32):26302-26307. doi: 10.1039/d5ra03353e. eCollection 2025 Jul 21.
The distribution of glutathione (GSH) in liver tissue is an important indicator for disease assessment. GSH depletion increases the susceptibility to oxidative stress and is involved in many diseases. Although mass spectrometry imaging (MSI) is a powerful tool for analysing the distribution of metabolites, it is difficult to image the distribution of reactive metabolites, such as the GSH bearing thiol group. In addition, MSI needs high-end instruments and takes a long time. In this study, we aimed to construct a methodology to trace GSH in frozen tissue slices onto a thin layer chromatography (TLC) plate by labelling with a fluorescent dye . When a frozen tissue slice was attached to the functional TLC plate, the thiol-specific fluorescence labelling agent for TLC imaging (tFLAT) immediately reacted with GSH in the frozen liver tissue slice to produce a high-polarity GSH adduct (tFLAT-GSH). After the separation of unreacted tFLAT on the TLC plate by expansion using a low-polarity solvent, fluorescence signals derived from tFLAT-GSH were observed . We also monitored the decrease in GSH in liver tissue slices when mice were treated with acetaminophen (APAP), which causes acute liver inflammation. The present method successfully detected GSH in the mouse liver tissue slices. This study provides a rational strategy for tracing target metabolites within frozen tissue slices on TLC plates for the first time.
谷胱甘肽（GSH）在肝组织中的分布是疾病评估的重要指标。GSH耗竭会增加对氧化应激的易感性，并与多种疾病有关。尽管质谱成像（MSI）是分析代谢物分布的强大工具，但难以对活性代谢物的分布进行成像，例如带有硫醇基团的GSH。此外，MSI需要高端仪器且耗时较长。在本研究中，我们旨在构建一种方法，通过用荧光染料标记，将冷冻组织切片中的GSH追踪到薄层色谱（TLC）板上。当将冷冻组织切片附着到功能性TLC板上时，用于TLC成像的硫醇特异性荧光标记剂（tFLAT）立即与冷冻肝组织切片中的GSH反应，生成高极性的GSH加合物（tFLAT-GSH）。使用低极性溶剂展开，在TLC板上分离未反应的tFLAT后，观察到来自tFLAT-GSH的荧光信号。我们还监测了用对乙酰氨基酚（APAP）处理小鼠时肝组织切片中GSH的减少情况，APAP会引起急性肝脏炎症。本方法成功检测到了小鼠肝组织切片中的GSH。本研究首次为在TLC板上追踪冷冻组织切片中的目标代谢物提供了合理策略。
