Determination of cellular redox status by stable isotope dilution liquid chromatography/mass spectrometry analysis of glutathione and glutathione disulfide.

Oxidation of glutathione (GSH) to glutathione disulfide (GSSG) occurs during cellular oxidative stress. The redox potential of the 2GSH/GSSG couple, which is determined by the Nernst equation, provides a means to assess cellular redox status. It is difficult to accurately quantify GSH and GSSG due to the ease with which GSH is oxidized to GSSG during sample preparation. To overcome this problem, a stable isotope dilution liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) method has been developed using 4-fluoro-7-sulfamoylbenzofurazan (ABD-F) derivatization. ABD-F derivatization of the GSH thiol group was rapid, quantitative, and occurred at room temperature. The LC/MRM-MS method, which requires no sample clean-up, was validated within the calibration ranges of 5 to 400 nmol/mL in cell lysates for GSH and 0.5 to 40 nmol/mL in cell lysates for GSSG. Calibration curves prepared by adding known concentrations of GSH and GSSG to cell lysates were parallel to the standard curve prepared in buffers. GSH and GSSG concentrations were determined in two monocyte/macrophage RAW 267.4 cell lines with or without 15-LOX-1 expression (R15LO and RMock cells, respectively) after treatment with the bifunctional electrophile 4-oxo-2(E)-nonenal (ONE). R15LO cells synthesized much higher concentrations of the lipid hydroperoxide, 15(S)-hydroperoxyeicosatetraenoic acid (15-HPETE), which undergoes homolytic decomposition to ONE. GSH was depleted by ONE treatment in both RMock and R15LO cells, leading to significant increases in their redox potentials. However, R15LO cells had higher GSH concentrations (most likely through increased GSH biosynthesis) and had increased resistance to ONE-mediated GSH depletion than RMock cells. Consequently, R15LO cells had lower reduction potentials at all concentrations of ONE. GSSG concentrations were higher in R15LO cells after ONE treatment when compared with the ONE-treated RMock cells. This suggests that increased expression of 15(S)-HPETE modulates the activity of cellular GSH reductases or the transporters involved in removal of GSSG.