Chromatographic separation of γ-GG, GSH, and GSSG. GSH and GSSG standards were prepared and injected (50 μl) onto a 3-aminopropyl column for HPLC separation and fluorescence detected at 315 nm excitation/585 nm emission. Depicted are representative chromatograms for (A) GSH and (B) GSSG. γ-GG was used as an internal standard to monitor derivatization efficiency. (C) Baseline separation of microsomal GSH and GSSG by HPLC with fluorescence detection. Microsomes (6 mg protein) were acidified with PCA and acid-soluble thiols derivatized as described. Results show that baseline separation of the analytes is easily achieved using this method.

GSH oxidation during microsomal isolation. Microsomes (6 mg protein) were isolated in the presence or absence of IAA and GSH and GSSG monitored by HPLC and quantified relative to standards. The presence of IAA resulted in significantly (A) higher GSH and (B) lower GSSG than observed when IAA was omitted. (C) Calculation of the GSH:GSSG ratio revealed that IAA results in an ~ 5-fold higher GSH redox ratio in microsomes containing IAA versus non-IAA-treated control samples. Results are expressed as mean ± SEM; n = 11 animals per group. Asterisks indicate p < 0.01 versus non-IAA-treated samples.

Small changes in GSH redox state markedly affect disulfide bond reduction by PDI. PDI reductase activity was monitored using the insulin turbidity assay of Holmgren (16). PDI (0.25 μM), insulin (1 mg/ml), and glutathione (5 mM) concentrations were kept constant while altering the GSH:GSSG ratio (1:1 to 15:1) and changes in turbidity was monitored spectrophotometrically at 650 nm. (A) Kinetic traces of PDI reduction of insulin intramolecular disulfide bonds. (B) Rates of PDI reductase activity were calculated and plotted against varying GSH:GSSG ratios.

Minimal glutathione is present as mixed disulfides with proteins in rat liver microsomes. Acid precipitated protein pellets were re-solubilized and incubated with DTT (1 mM) to liberate any glutathione bound to proteins. (A) Results show the distribution of free versus protein-bound GSH. Only minimal glutathione was observed bound to protein regardless of the presence or absence of IAA. (B) Western blot analysis of microsomal glutathionylated proteins. When compared to the DTT control, very little glutathione is bound to proteins regardless of IAA treatment. Furthermore, incubation of microsomes with 1 mM GSSG resulted in minimal glutathionylation and only addition of supraphysiological GSSG concentrations (10 mM GSSG) resulted in extensive glutathione-protein mixed disulfides. (n = 3 animals per group).

Ex vivo GSH oxidation occurs very rapidly during microsomal isolation. Rat liver homogenates or microsomal preparations were treated with IAA at different times during the isolation of microsomes and GSH redox state monitored by HPLC. (A) Schematic representation of the microsomal isolation procedure and the times at which IAA (70 mM, final concentration) was added [at perfusion (Per), homogenization, or at 2 h intervals]. Bars under the timeline indicate the length of time that a particular sample was incubated with IAA. (B) The GSH redox ratio falls rapidly when liver homogenates are not immediately treated with IAA. Results show that the GSH:GSSG ratio was highest when liver tissue was either directly perfused or immediately homogenized in IAA-containing buffer (see Per and 6 h timepoints). However, significantly lower (p < 0.05) ratios were observed in all samples that were not initially treated with IAA (see 4–0 h timepoints). (C) Results show loss of GSH within the first 2 h of the microsomal isolation procedure when IAA is not present which becomes significant at the 2 and 0 h timepoints (p < 0.05). (D) The initial loss of GSH corresponds to a significant increase in the levels of GSSG (p < 0.05). (E) Nonprotein-bound total glutathione (GSH + 2GSSG) levels become lower in the last timepoint (0 h) versus longer IAA treatment (6 h timepoint) (p < 0.05). Results are expressed as mean ± SEM; n = 3 animals for perfusion group and n = 6 animals for all other groups. Different letters indicate statistical significance (p < 0.05).