Joint Department of Biomedical Engineering, Chapel Hill and North Carolina State University, University of North Carolina, Raleigh, NC 27695, USA.
Int J Mol Sci. 2022 Apr 25;23(9):4733. doi: 10.3390/ijms23094733.
Nuclear magnetic resonance (NMR) spectroscopy was used to monitor glutathione metabolism in alginate-encapsulated JM-1 hepatoma cells perfused with growth media containing [3,3′-13C2]-cystine. After 20 h of perfusion with labeled medium, the 13C NMR spectrum is dominated by the signal from the 13C-labeled glutathione. Once 13C-labeled, the high intensity of the glutathione resonance allows the acquisition of subsequent spectra in 1.2 min intervals. At this temporal resolution, the detailed kinetics of glutathione metabolism can be monitored as the thiol alkylating agent monobromobimane (mBBr) is added to the perfusate. The addition of a bolus dose of mBBr results in rapid diminution of the resonance for 13C-labeled glutathione due to a loss of this metabolite through alkylation by mBBr. As the glutathione resonance decreases, a new resonance due to the production of intracellular glutathione-bimane conjugate is detectable. After clearance of the mBBr dose from the cells, intracellular glutathione repletion is then observed by a restoration of the 13C-glutathione signal along with wash-out of the conjugate. These data demonstrate that standard NMR techniques can directly monitor intracellular processes such as glutathione depletion with a time resolution of approximately < 2 min.
利用核磁共振（NMR）光谱法监测含有[3,3′-13C2]-胱氨酸的生长培养基灌注的藻酸盐包被 JM-1 肝癌细胞中的谷胱甘肽代谢。用标记的培养基灌注 20 小时后，13C NMR 谱主要由 13C 标记的谷胱甘肽信号主导。一旦被 13C 标记，谷胱甘肽共振的高强度允许以 1.2 分钟的间隔采集后续光谱。在这种时间分辨率下，可以监测硫醇烷化剂单溴双马来酰亚胺（mBBr）添加到灌流液中时谷胱甘肽代谢的详细动力学。添加 mBBr 的大剂量会导致 13C 标记的谷胱甘肽共振迅速减少，因为该代谢物通过 mBBr 烷化而丢失。随着谷胱甘肽共振的降低，可以检测到由于细胞内谷胱甘肽-双马来酰亚胺缀合物的产生而产生的新共振。在用 mBBr 清除细胞中的剂量后，通过恢复 13C-谷胱甘肽信号以及缀合物的洗出，观察到细胞内谷胱甘肽的再补充。这些数据表明，标准 NMR 技术可以直接监测细胞内过程，如谷胱甘肽耗竭，时间分辨率约为<2 分钟。
