Chemically, GSH can also be derivatized by iodoacetamide (IAM, Fig. 14.1) to
form the corresponding GS–AM conjugate. The following is a modified
protocol using IAM for the analysis of individual GSH and GSSG
concentrations in freshly isolated rat hepatocytes.
14.3.2 Measurement of Intracellular GSH/GSSG in Hepatocytes
14.3.2.1 Rat Hepatocytes Rat hepatocytes are freshly isolated by perfusion
and are diluted with Krebs-bicarbonate buffer (pH 7.4) to 1 106 cells/mL.
Cell viability is analyzed by the trypan blue exclusion method.
14.3.2.2 The GS–AM Standard Curve IAM stock solution (10 mM, 100mL)
in 10 mM ammonium bicarbonate buffer (pH 10) is added to amber centrifuge
tubes, to which 100mL of various GSH stock solutions in Krebs-bicarbonate
buffer (pH 7.4) is added. The final GSH concentrations are 1, 5, 10, 25, 50, and
100 mM. All samples are vortexed and are placed in dark at room temperature
for 60 min. Derivatization reaction is quenched by the addition of 400mLof
acetonitrile. The resulting mixture is mixed with 25mLofg-Glu-Glu stock
solution (0.25 mM, internal standard) in water/acetonitrile (1:1). After
centrifugation, the supernatant (300mL) is transferred to a 96-well plate for
LC/MS/MS analysis.
14.3.2.3 The GSSG Standard Curve The procedures for preparation of the
GSSG standard curve are the same as described in the Section 14.3.2.2 (the GS–
AM standard curve), except that various GSSG stock solutions are used. The
final GSSG concentrations are 1, 5, 10, 25, 50, and 100mM.
14.3.2.4 GSH/GSSG Measurement in Rat Hepatocytes in the Presence of a Test
Compound IAM stock solution (10 mM, 100mL) in 10 mM ammonium
bicarbonate buffer (pH 10) is added to amber centrifuge tubes. Menadione
stock solutions (6 and 60 mM) and a test compound (2, 10, 20, and 60 mM) in
methanol are added to rat hepatocytes. The final concentrations are 30 and
300 mM for menadione and 10, 50, 100, and 300mM for a test compound.
Aliquots (100mL) of incubation samples are added to the above amber tubes at
various time points (0, 5, 10, 20, 30, and 60 min). All samples are vortexed and
are placed in the dark at room temperature for 60 min. Derivatization reaction
is quenched by addition of 400mL of acetonitrile. The resulting mixture is then
mixed with 25mLofg-Glu-Glu stock solution (0.25 mM, internal standard) in
water/acetonitrile (1:1). After centrifugation, the supernatant (300mL) is
transferred to a 96-well plate for LC/MS/MS analysis.
14.3.2.5 Instrumentation LC/MS/MS analysis is carried out on a Perkin-
Elmer SCIEX API 3000 triple quadrupole mass spectrometer (Toronto,
Canada) interfaced to an HPLC system consisting of a Perkin-Elmer Series 200
PROTOCOL FOR MEASUREMENT OF INTRACELLULAR GSH AND GSSG 469