radical generated from above redox cycling of quinones can oxidize cysteine
residues of proteins, causing oxidative stress in cells.
Glutathione, an endogenous tripeptide (Glu-Cys-Gly), is the most abundant
nonprotein sulfhydryl compound in the human body. It exists mainly as the
reduced form (GSH) with a little portion as the oxidized glutathione disulfide
form (GSSG)in vivo. In addition to its role as a nucleophile to react with
reactive electrophile intermediates (e.g.,p-quinones) to form corresponding
GSH-hydroquinone adducts (Fig. 14.7), GSH also plays a critical role in the
detoxication of reactive oxygen species (e.g., hydrogen peroxide) by the
reduction of these species. As a consequence, GSH is oxidized by reactive
oxygen species to GSSG.
Collectively, the above protective mechanisms result in a decrease in cellular
GSH concentrations and an increase in GSSG concentrations. Therefore,
changes of GSH and GSSG concentrations in cells may be used as biomarkers
for oxidative stress (Afzal et al., 2002). Freshly isolated rat hepatocytes are
commonly used forin vitroevaluation of oxidative stress potential of a test
compound. Menadione (Fig. 14.1), a quinone compound that can induce
oxidative stress in cells (Smith et al., 1985), might be used as a positive control.
A negative control that does not use any test compound is also warranted, due
to the fact that GSH also can be oxidized to GSSG by molecular oxygen. By
comparing the concentrations of GSH and GSSG or the ratios of GSH/GSSG
of the test compound with those of menadione as well as the negative control, a
relative potential of a compound to induce oxidative stress in cells might be
assessed.
Several chemical, enzymatic, and chromatographic methods have been
reported for the measurement of GSH and GSSH contents in biological
samples. The enzymatic method for total GSH (GSH+GSSG) involves both
oxidation of GSH by 5, 5^0 -dithiobis(2-nitrobenzoic acid) (DTNB) to form
GSSG with stoichiometric formation of 5-thio-2-nitrobenzoic acid (TNB) and
reduction of GSSG by GSSG reductase to GSH (Anderson, 1985). The total
GSH concentration is calculated based on the rate of TNB formation
monitored at 412 nm. Chromatographic methods for total GSH
(GSH+GSSG) involves initial derivatization of GSH with 2-vinylpyridine,
followed by comparison with GSH standard samples in ion-exchange
chromatography or high performance liquid chromatography (Anderson,
1985). Total GSH concentration can also be measured by converting GSSG to
GSH with NaBH 4 , followed by derivatization with monobromobimane and
fluorescence detection (Svardal et al., 1990). The major disadvantage of these
assays is the inability to conveniently measure both GSH and GSSG. Afzal
et al. 2002 developed a rapid HPLC–UV method for the quantification of GSH
and GSSG in biological samples. It has been reported that both GSH and
GSSG concentrations in hepatocytes were measured through LC/MS/MS
analysis (Loughlin et al., 2001). In this assay, GSH was derivatized by
iodoacetic acid to form S-carboxymethyl derivative (GS–CM). The GS–CM
conjugate, together with GSSG, were subjected to direct LC/MS/MS analysis.
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