Direct Detection of Glutathione Biosynthesis, Conjugation, Depletion and Recovery in Intact Hepatoma Cells

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.