Oxidized Forms of Glutathione in Peripheral Blood as Biomarkers of Oxidative Stress

Abstract

Background: Reduced glutathione (GSH) and its redox forms, glutathione disulfide (GSSG) and glutathionylated proteins (PSSG), are biomarkers of oxidative stress, but methodologic artifacts can interfere with their measurement. We evaluated the importance of correct sample handling during the preanalytical phase for GSH, GSSG, and PSSG measurement. Methods: We used human blood for in vitro experiments with oxidants [tert-butylhydroperoxide (t-BOOH), diamide, and menadione]. For in vivo experiments, we used rats in which we cannulated the jugular and femoral veins for both oxidant administration and blood collection. We measured GSH, GSSG, and PSSG with HPLC with or without sample pretreatment with N-ethylmaleimide (NEM) to prevent artifacts. We also measured malondialdehyde (MDA) with HPLC, and protein carbonyls (PCO) with spectrophotometric procedures. Results: When methodologic artifacts were prevented by pretreatment with NEM, GSSG results increased up to 3-fold over the basal concentrations, even in the presence of 5 mol/L t-BOOH or diamide and 20 mol/L menadione. PSSG increased by ϳ50% at 20 mol/L t-BOOH or diamide and at 50 mol/L menadione. PCO and MDA remained unchanged. In vivo oxidation treatments elicited immediate and significant increases in GSSG and PSSG over basal values (up to 200-fold), whereas PCO and MDA showed only slight variation 120 or 180 min after treatment. Conclusions: With the use of artifact-free measurement methods, GSH, GSSG, and PSSG are potentially powerful and reliable biomarkers of oxidative stress status

Proteomic Analysis of Mouse Hypothalamus under Simulated Microgravity

“…Our results clearly showed a significant decrease in GSH amount in hypothalamus and this indicates generation of oxidative stress in simulated microgravity. The reduced GSH and its redox forms, glutathione disulfide (GSSG) and glutathionylated proteins are biomarkers of oxidative stress [21]. Therefore the oxidative stress generated in the hypothalamus in microgravity supports earlier findings showing generation of oxidative stress under similar conditions.…”

In vitro/in vivo screening of oxidative homeostasis and damage to DNA, protein, and lipids using UPLC/MS-MS

“…Thereby, MDA and GSH were determined after a reaction with DNPH [26,27] and NEM [28,13], respectively. To optimize the derivatization procedure, MDA and GSH solutions were prepared separately in PBS (1 μg/mL) and were analyzed after the reaction with the corresponding derivatization reagent at different concentrations (i.e., 0.25 to 2 mM DNPH and 2.5 to 50 mM NEM) (Electronic Supplementary Material (ESM) Fig.…”

“…To this end, a set of well-known biomarkers of OS was selected: (i) sulfur-containing substances, such as reduced glutathione (GSH), oxidized glutathione (GSSG) [13], S-(5-adenosyl)-Lmethionine chloride (SAM), and S-(adenosyl)-L-homocysteine (SAH) [14]; (ii) phenylalanine (Phe) [15] and tyrosines (3-iodo-L-tyrosine (I-Tyr), 3-nitro-tyrosine (N-Tyr), 3-chloro-Ltyrosine (Cl-Tyr), DL-o-tyrosine (o-Tyr), DL-m-tyrosine (mTyr), and L-tyrosine (p-Tyr)) as markers of oxidative protein damage [11,16]; (iii) 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 2-deoxyguanosine (2-dG), which indicate DNA damage [17]; and (iv) malondialdehyde (MDA) and 8-isoprostaglandin-F2α (8-IsoPGF), which are markers of lipid peroxidation [18,19]. Furthermore, a novel OS biomarker, like ophthalmic acid (Opht A) that indicates glutathione consumption, was also included in our set of OS biomarkers [20].…”

Effect of Using Aqueous Extract of Salvia officinalis L. Leaves on Some Antioxidants Status in Irradiated Rats

“…Assessment of GSH in biological samples is essential for evaluation of the redox homeostasis and detoxification status of cells in relation to its protective role against oxidative and free radical-mediated cell injury (Rossi et al, 2005). The present study, recorded a significant depletion of blood and liver tissues GSH concentration in animals exposed to γ-radiation, compared to that of control groups.…”