Glutathione in redox research: measuring GSH and GSSG reliably

Cells keep glutathione mostly reduced, often at millimolar concentrations. Glutathione peroxidases use GSH to detoxify peroxides, producing GSSG, which glutathione reductase recycles using NADPH. A fall in GSH:GSSG is widely used as an index of oxidative stress in cell-culture and tissue studies.

GSSG is usually present at less than 1% of GSH. Even slight oxidation of GSH during sample handling can therefore inflate GSSG many-fold. Published protocols trap GSH immediately with a thiol-alkylating agent such as N-ethylmaleimide (NEM) at the point of lysis, before acidification. That step greatly improves accuracy, especially for GSSG.

The enzymatic recycling assay (Tietze, with DTNB and glutathione reductase) is sensitive and inexpensive. It needs careful masking of GSH to measure GSSG. HPLC with fluorescent derivatization and LC-MS/MS give more specific quantification and can measure related thiols at the same time. Genetically encoded redox sensors (such as Grx1-roGFP2) allow compartment-specific, real-time readouts in living cells.

Reporting the alkylation step, time to processing, normalization (protein or cell number) and recovery of spiked standards makes glutathione data far easier to interpret and compare.