Glutathione (GSH) acts as a co-factor for the enzyme glutathione peroxidase, reducing toxic peroxides and oxidizing itself to GSSG in the process. The GSH:GSSG ratio can be used as proxy for addressing cellular oxidative stress exposure but common methods suffer from artificial thiol oxidation during sample preparation and low quantitative power for GSSG.
We developed an optimized sample collection protocol (patented) to minimize GSH artificial oxidation and a method which greatly improves sensitivity for GSSG, dramatically reducing starting material requirement. In our two-step alkylation approach, all GSH is initially blocked with N-ethylmaleimide (NEM), after which all small thiols are reduced (including GSSG). Newly released GSH is then re-alkylated with deuterated NEM (d5-NEM). A third isotopologue can be used as isotopic standard for absolute quantification. With all isotopologues having similar chromatographic properties, matrix effects can only impact method sensitivity but not quantification accuracy.
Experts:Dr. Matthias Schittmayer-Schantl
Joint Research Project, Contract Research