There's an HPLC method that's specific for GSH and GSSG; see Anal. Biochem.
144 (1985) 553-562. It's a bit complicated to set up but easy to use. You
separate GSH and GSSG isocratically by anion-exchange (I used a TSK DEAE-5PW
column), then pump in glutathione reductase, NADPH and Ellman's reagent (DTNB)
post-column. After a minute in a post-column reactor, you monitor 412 nm to
detect the TNB generated from reduction of the DTNB. The reaction is as
follows:
NADPH ----> NADP
---------------------------------
/ Glutathione reductase \
/ \
GSSG 2 GSH
\ /
\ /
--------------------------------
2 TNB <------ DTNB
This is a recycling reaction. Every turn of the cycle regenerates the GSSG at
the expense of the DTNB. Thus, GSH and GSSG become catalysts, and you can
detect them with the sensitivity and selectivity you'd normally associate with
an enzyme assay. Typically, the detection is about 20x more sensitive than
for a stoichiometric thiol reaction. Everything else in tissue extracts drops
out of the profile. GSH and GSSG don't interfere with each other's assay
because you've separated them at the anion-exchange step.
There are two caveats to keep in mind:
1) The ratio GSH/GSSG in vivo is usually higher than 100 (I've measured it
as high as 800 using this method). It doesn't take much oxidation of GSH to
produce a dramatic decrease in the measured ratio. The method we worked out
to prevent this was to freeze-clamp the tissue involved, homogenize it in cold
perchloric acid containing 100 m N-ethyl maleimide (NEM), then neutralize the
perchloric acid with a premeasured amount of MES buffer. The NEM reacts with
the GSH before it can oxidize to GSSG. Thus, you need to make two
chromatography runs per sample: One run without NEM workup in order to get
the GSH concentration and a second run with the sample worked up with NEM in
order to get the GSSG cooncentration.
2) The assay also responds to any other substrates of glutathione
reductase. These include CoASSG and the spermine and spermidine adducts of
GSH (present in E. coli).
3) Maintenance of the GSH/GSSG levels in vivo requires a supply of NADPH
for glutathione reductase. NADPH is degraded by light, which in turn affects
the measured GSH/GSSG. Thus, keep your culture plates, tissue homogenates and
NADPH solutions covered with aluminum foil.
Regards,
Andy Alpert
PolyLC Inc.
Columbia, MD
tel: (410) 992-5400 FAX: (410) 730-8340