Liu Li, Lu Yu-Han, Wang Min-Dan, Zhao Qun-Fei, Chen Xiu-Ping, Yin Hang, Feng Chen-Guo, Zhang Fang
The Research Center of Chiral Drugs, Shanghai Frontiers Science Center for TCM Chemical Biology, Innovation Research Institute of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.
Department of Thoracic Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, 200127, China.
J Pharm Anal. 2023 Nov;13(11):1365-1373. doi: 10.1016/j.jpha.2023.08.016. Epub 2023 Sep 22.
In this work, a new pyrylium derivatization-assisted liquid chromatography-mass spectrometry (LC-MS) method was developed for metabolite profiling of the glutathione anabolic pathway (GAP) in cancer tissues and cells. The pyrylium salt of 6,7-dimethoxy-3-methyl isochromenylium tetrafluoroborate (DMMIC) was used to label the amino group of metabolites, and a reductant of dithiothreitol (DTT) was employed to stabilize the thiol group. By combining DMMIC derivatization with LC-MS, it was feasible to quantify the 13 main metabolites on the GAP in complex biological samples, which had good linearity ( = 0.9981-0.9999), precision (interday precision of 1.6%-19.0% and intraday precision of 1.4%-19.8%) and accuracy (83.4%-115.7%). Moreover, the recovery assessments in tissues (82.5%-107.3%) and in cells (98.1%-118.9%) with GSH-C2, N, and Cys-N demonstrated the reliability of the method in detecting tissues and cells. Following a methodological evaluation, the method was applied successfully to investigate difference in the GAP between the carcinoma and para-carcinoma tissues of esophageal squamous cell carcinoma (ESCC) and the effect of -hydroxycinnamaldehyde (CMSP) on the GAP in KYSE-150 esophageal cancer cells. The results demonstrate that the developed method provides a promising new tool to elucidate the roles of GAP in physiological and pathological processes, which can contribute to research on drugs and diseases.
在本研究中，开发了一种新的吡喃鎓衍生化辅助液相色谱 - 质谱（LC - MS）方法，用于分析癌症组织和细胞中谷胱甘肽合成途径（GAP）的代谢物谱。使用6,7 - 二甲氧基 - 3 - 甲基异苯并呋喃鎓四氟硼酸盐（DMMIC）的吡喃鎓盐标记代谢物的氨基，并使用二硫苏糖醇（DTT）作为还原剂来稳定巯基。通过将DMMIC衍生化与LC - MS相结合，能够对复杂生物样品中GAP上的13种主要代谢物进行定量，其具有良好的线性（ = 0.9981 - 0.9999）、精密度（日间精密度为1.6% - 19.0%，日内精密度为1.4% - 19.8%）和准确度（83.4% - 115.7%）。此外，使用GSH - C2、N和Cys - N对组织（82.5% - 107.3%）和细胞（98.1% - 118.9%）进行的回收率评估证明了该方法在检测组织和细胞方面的可靠性。经过方法学评估后，该方法成功应用于研究食管鳞状细胞癌（ESCC）癌组织和癌旁组织之间GAP的差异，以及对羟基肉桂醛（CMSP）对KYSE - 150食管癌细胞中GAP的影响。结果表明，所开发的方法为阐明GAP在生理和病理过程中的作用提供了一种有前景的新工具，这有助于药物和疾病的研究。
