Example: Tracing cystine into glutathione
Stable isotope tracingRedoxTargeted LC-HRMSUnpublished data, groups and model masked
Do treated cells channel more cystine into glutathione than control cells, and what is their true redox state?
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What we did
Cells were fed heavy-labeled cystine ([13C6,15N2]-L-cystine, 25 µM, 4 h). Cells and medium were harvested straight into an extraction mix containing NEM, which locks free thiols at once, so glutathione cannot oxidize during extraction and the measured GSH : GSSG ratio is the true one. Two LC-HRMS methods then measured about 40 metabolites and their labeled forms in the same extract.
What we found
Treated cells took up more of the labeled cystine (94% vs 61% in 4 h) and carried 7.2 times more labeled cysteine and 1.6 times more labeled glutathione. Relative to their cysteine labeling, glutathione labeling was only slightly higher (36% vs 32%): the extra glutathione comes mostly from more fuel, not faster synthesis. Their glutathione was also more reduced (GSH/GSSG 1.5 times higher), and their central carbon pools were much smaller.
In collaboration with Dr. Yaacov Lawrence and Dr. Ariel Sabag, Sheba Medical Center.
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