Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

Abstract

Measurement of glutathione (GSH) and glutathione disulfide (GSSG) is a crucial tool to assess cellular redox state. Herein we report a direct approach to determine intracellular GSH based on a rapid chromatographic separation coupled with acidic potassium permanganate chemiluminescence detection, which was extended to GSSG by incorporating thiol blocking and disulfide bond reduction. Importantly, this simple procedure avoids derivatisation of GSH (thus minimising auto-oxidation) and overcomes problems encountered when deriving the concentration of GSSG from ‘total GSH’. The linear range and limit of detection for both analytes were 7.5 × 10⁻⁷ to 1 × 10⁻⁵ M, and 5 × 10⁻⁷ M, respectively. GSH and GSSG were determined in cultured muscle cells treated for 24 h with glucose oxidase (0, 15, 30, 100, 250 and 500 mU mL⁻¹), which exposed them to a continuous source of reactive oxygen species (ROS). Both analyte concentrations were greater in myotubes treated with 100 or 250 mU mL⁻¹glucose oxidase (compared to untreated controls), but were significantly lower in myotubes treated with 500 mU mL⁻¹ (p < 0.05), which was rationalised by considering measurements of H₂O₂ and cell viability. However, the GSH/GSSG ratio in myotubes treated with 100, 250 and 500 mU mL⁻¹glucose oxidase exhibited a dose-dependent decrease that reflected the increase in intracellular ROS.