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(-1) glucose 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 H(2)O(2) and cell viability. However, the GSH/GSSG ratio in myotubes treated with 100, 250 and 500 mU mL(-1) glucose oxidase exhibited a dose-dependent decrease that reflected the increase in intracellular ROS.

Copper as a target for prostate cancer therapeutics: copper-ionophore pharmacology and altering systemic copper distribution

“…GSH and GSSG were measured by HPLC with permanganate chemiluminescence detection [72, 73] using a GloCel detector with serpentine flow-cell [74]. Cell pellets (1 × 10 6 cells) were homogenised (vortexed) in 300 μL of 0.1% formic acid and then centrifuged at 13000 rpm for 15 min at 4°C.…”

A direct comparison of methods used to measure oxidized glutathione in biological samples: 2-vinylpyridine and N -ethylmaleimide

“…The importance of sample handling, acidification reagent and GSH trapping agent has been shown in previous studies (Jones et al, 1998; Rossi et al, 2002; McDermott et al, 2011a,b; Giustarini et al, 2013). In general, the results of these studies agree with ours and show that samples should be quickly processed, kept cold and derivatized with NEM instead of other trapping reagents.…”

“…Detailed reviews have been published elsewhere (Monostori et al, 2009; Iwasaki et al, 2009). The most popular of these techniques make use of high-performance liquid chromatography (HPLC) separation with UV absorbance (Reed et al, 1980; Katrusiak et al, 2001; Bald and Glowacki, 2005), chemiluminescence (McDermott et al, 2011a,b), fluorescence (Imai et al, 1983; Martin and White, 1991; Paroni et al, 1995; Yang et al, 1995; Kand’ar et al, 2007), electrochemical (Rodriguez-Ariza et al, 1994; Rose and Bode, 1995; Lakritz et al, 1997; Potesil et al, 2005; Sequellerio et al 2012), or mass spectrometry-based (Lafaye et al, 2005; Bouligard et al, 2006; Iwasaki et al, 2009; Squellerio et al, 2012; Moore et al, 2013; Zhang et al, 2015; Fahrenholz et al, 2015) detection. Even nanosensors for GSH and GSSG are now being developed (Xu et al, 2012; Moore et al, 2013; Ni et al, 2015).…”

Effect of High‐Dose Cysteine Supplementation on Erythrocyte Glutathione

“…First, cysteine and GSH could have been rapidly oxidized or used for other unmeasured processes, or hepatic GSH stores were adequate to offset intracellular cysteine deficiencies. To decrease the risk for oxidation, which could result in artificially elevated GSSG concentrations, samples were treated with the reducing agent, 4‐vinylpyridine 25 . In addition, while the liver represents the main source of GSH, obtaining hepatic tissue in this study would not have been possible without adding potential morbidity.…”