Profiling the Concentration of Reduced and Oxidized Glutathione in Rat Brain Using HPLC/DAD Chromatographic System

This study aimed to develop a HPLC/DAD method in order to determine and quantify the reduced glutathione (GSH) and oxidized glutathione (GSSG) levels in rat brain. Due to the presence of the thiol group (-SH), GSH can interact with the Ellman's reagent (DTNB), with which it forms a reaction product through which the level of GSH can be quantified, using the DAD detection system. Chromatographic separation was achieved after a derivatization process by using a mobile phase acetonitrile (A) and phosphate buffer (20 mM, pH = 2.5) (B). The compounds of interest were detected at 330 nm using a chromatographic C8 column. The method of determination met the validation criteria, specified by the regulatory bodies. The applicability of the method was demonstrated in a chronic toxicology study of central nervous system (CNS), following different treatment regimens with haloperidol.

Glutathione system activity in the blood of overweight postmenopausal women

One of the important components of the antioxidant defense system is the glutathione system, the activity of which, when overweight, changes direction depending on gender and ethnicity. The results of studies involving overweight menopausal women are mixed. The study involved 61 postmenopausal women, who, after clinical and anamnestic examination, were divided into 2 groups: control (BMI = 19-24.9 kg / m2) and overweight group (BMI = 25-29.9 kg/m2). The use of hormone replacement therapy; the use of antioxidant drugs; diseases of endocrine genesis; exacerbation of chronic diseases; premature early menopause; surgical menopause was the exclusion criteria for women from the study. The lipid profile parameters with the calculation of the atherogenic coefficient; reduced and oxidized glutathione levels with the calculation of their ratio, the glutathione S-transferase and glutathione reductase activities were determined in the blood. Overweight women showed an increase in the triacylglycerols (p = 0.041) and cholesterol in very low density lipoproteins levels (p = 0.044). When assessing the glutathione system activity in women of the main group, compared with the control, an increase in the glutathione-S-transferase (p = 0.023) and glutathione reductase (p = 0.022) activities was noted, however, the reduced and oxidized glutathione levels, as well as their ratio did not differ from the control values. The results obtained indicate the activation of the glutathione system enzymatic link in response to changes in lipid status in postmenopausal women with overweight.

Dithiophosphate-Induced Redox Conversions of Reduced and Oxidized Glutathione

Phosphorus species are potent modulators of physicochemical and bioactive properties of peptide compounds. O,O-diorganyl dithiophoshoric acids (DTP) form bioactive salts with nitrogen-containing biomolecules; however, their potential as a peptide modifier is poorly known. We synthesized amphiphilic ammonium salts of O,O-dimenthyl DTP with glutathione, a vital tripeptide with antioxidant, protective and regulatory functions. DTP moiety imparted radical scavenging activity to oxidized glutathione (GSSG), modulated the activity of reduced glutathione (GSH) and profoundly improved adsorption and electrooxidation of both glutathione salts on graphene oxide modified electrode. According to NMR spectroscopy and GC–MS, the dithiophosphates persisted against immediate dissociation in an aqueous solution accompanied by hydrolysis of DTP moiety into phosphoric acid, menthol and hydrogen sulfide as well as in situ thiol-disulfide conversions in peptide moieties due to the oxidation of GSH and reduction of GSSG. The thiol content available in dissolved GSH dithiophosphate was more stable during air oxidation compared with free GSH. GSH and the dithiophosphates, unlike DTP, caused a thiol-dependent reduction of MTS tetrazolium salt. The results for the first time suggest O,O-dimenthyl DTP as a redox modifier for glutathione, which releases hydrogen sulfide and induces biorelevant redox conversions of thiol/disulfide groups.

Low-molecular-mass labile metal pools in Escherichia coli: advances using chromatography and mass spectrometry

Labile low-molecular-mass (LMM) transition metal complexes play essential roles in metal ion trafficking, regulation, and signalling in biological systems, yet their chemical identities remain largely unknown due to their rapid ligand-exchange rates and weak M–L bonds. Here, an Escherichia coli cytosol isolation procedure was developed that was devoid of detergents, strongly coordinating buffers, and EDTA. The interaction of the metal ions from these complexes with a SEC column was minimized by pre-loading the column with 67ZnSO4 and then monitoring 66Zn and other metals by inductively coupled plasma mass spectrometry (ICP-MS) when investigating cytosolic ultrafiltration flow-through-solutions (FTSs). Endogenous cytosolic salts suppressed ESI-MS signals, making the detection of metal complexes difficult. FTSs contained ca. 80 µM Fe, 15 µM Ni, 13 µM Zn, 10 µM Cu, and 1.4 µM Mn (after correcting for dilution during cytosol isolation). FTSs exhibited 2–5 Fe, at least 2 Ni, 2–5 Zn, 2–4 Cu, and at least 2 Mn species with apparent masses between 300 and 5000 Da. Fe(ATP), Fe(GSH), and Zn(GSH) standards were passed through the column to assess their presence in FTS. Major LMM sulfur- and phosphorus-containing species were identified. These included reduced and oxidized glutathione, methionine, cysteine, orthophosphate, and common mono- and di-nucleotides such as ATP, ADP, AMP, and NADH. FTSs from cells grown in media supplemented with one of these metal salts exhibited increased peak intensity for the supplemented metal indicating that the size of the labile metal pools in E. coli is sensitive to the concentration of nutrient metals.

Development and Validation of HPLC Method for Simultaneous Estimation of Reduced and Oxidized Glutathione in Bulk Pharmaceutical Formulation

The objective of this study was the development, optimization, and validation of a RP-HPLC method for the quantification of reduced glutathione (GSH) and oxidized glutathione (GSSG) in pharmaceutical formulations The separation utilized a C18 column at room temperature with absorption wavelength 210nm. The mobile phase was an isocratic flow of a 95:5 (v/v) mixture of 25mM phosphate buffer (pH 2.7) and methanol with flow rate at 1.0 mL/min. Validation of the method assessed with the methods ability in seven categories: linearity, range, limit of detection, limit of quantification, accuracy, precision, and selectivity. The method show an acceptable degree of linearity with r²=0.9994 and 0.999 over a concentration range of 10-200 μg/mL for GSH and GSSG respectively. The detection limit and quantification limit for GSH 20.7μg/mL and 69.24μg/mL and for GSSG 17.22μg/mL and 57.42μg/mL respectively. The percent recovery of the method was 99.98-100.93 %. Following validation, the method was employed in the determination of glutathione in pharmaceutical formulations in the form of a liposome. The proposed method offers a simple, accurate, and inexpensive way to quantify reduced glutathione.

Teratogenic, Oxidative Stress and Behavioural Outcomes of Three Fungicides of Natural Origin (Equisetum arvense, Mimosa tenuiflora, Thymol) on Zebrafish (Danio rerio)

The improper use of synthetic fungicides has raised public concerns related to environmental pollution and animal health. Over the years, plant-derived antifungals have been investigated as safer alternatives, although little scientific evidence of its neurodevelopmental effects exist. The main objective of this study was to explore the effects of three alternative natural extracts (Equisetum arvense, Mimosa tenuiflora, Thymol) with antifungal properties during the early development of zebrafish by evaluating different teratogenic, oxidative stress and behavioural outcomes. Following the determination of the 96 h-LC50, exposure to sublethal concentrations showed the safety profile of both E. arvense and M. tenuiflora. However, following 96-h exposure to Thymol, increased lethality, pericardial oedema, yolk and eye deformations, and decreased body length were observed. The reduced and oxidized glutathione (GSH:GSSG) ratio was increased, and the glutathione-s-transferase activity in the group exposed to the highest Thymol concentration. Overall, these results support a more reducing environment associated with possible effects at the cellular proliferation level. In addition, the disruption of behavioural states (fear- and anxiety-like disorders) were noted, pointing to alterations in the c-Jun N-terminal kinase developmental signalling pathway, although further studies are required to explore this rationale. Notwithstanding, the results provide direct evidence of the teratogenic effects of Thymol, which might have consequences for non-target species.

Biomarker model for cancer: Development of fast LC-MS/MS method for reduced and oxidized glutathione

In recent years, there have been significant efforts devoted to countering the challenge of detecting cancer in early stages. Reduced glutathione (GSH) plays an important role in the antioxidant system and is required for the maintenance of the redox status of the cell, defense against free radicals and detoxification of toxic compounds. GSH may be converted to oxidized glutathione (GSSG) during the oxidative stress that it regularly undergoes when combating cancer cells. Therefore, the ratio of GSH to the total amount of glutathione can be an extremely useful biomarker for detecting cancer. However, there has yet to be an effective method of detecting and quantifying glutathione in cells, making it extremely difficult to use as a biomarker. In this study, we have created an effective method of detecting both forms of glutathione, utilizing high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The analysis time took less than 1 minute, and we were able to quantify both GSH and GSSG in one method. The limit of quantitation is 1 ng/mL, and we ran three trials, each examining a range of concentrations, from 1 to 500 ng/ml of GSH and GSSG. Results were calculated using peak area ratios, using HPLC-MS/MS technology, we were able to determine both the amounts of GSH and GSSG in a single method, creating a fast, reliable, non-invasive, and cost-effective method of testing early stages of cancer.

An roGFP2-Based Bacterial Bioreporter for Redox Sensing of Plant Surfaces

The reduction-oxidation (redox) environment of the phytobiome (i.e., the plant–microbe interface) can strongly influence the outcome of the interaction between microbial pathogens, commensals, and their host. We describe a noninvasive method using a bacterial bioreporter that responds to reactive oxygen species and redox-active chemicals to compare microenvironments perceived by microbes during their initial encounter of the plant surface. A redox-sensitive variant of green fluorescent protein (roGFP2), responsive to changes in intracellular levels of reduced and oxidized glutathione, was expressed under the constitutive SP6 and fruR promoters in the epiphytic bacterium Pantoea eucalypti 299R (Pe299R/roGFP2). Analyses of Pe299R/roGFP2 cells by ratiometric fluorometry showed concentration-dependent responses to several redox active chemicals, including hydrogen peroxide (H2O2), dithiothreitol (DTT), and menadione. Changes in intracellular redox were detected within 5 min of addition of the chemical to Pe299R/roGFP2 cells, with approximate detection limits of 25 and 6 μM for oxidation by H2O2 and menadione, respectively, and 10 μM for reduction by DTT. Caffeic acid, chlorogenic acid, and ascorbic acid mitigated the H2O2-induced oxidation of the roGFP2 bioreporter. Aqueous washes of peach and rose flower petals from young blossoms created a lower redox state in the roGFP2 bioreporter than washes from fully mature blossoms. The bioreporter also detected differences in surface washes from peach fruit at different stages of maturity and between wounded and nonwounded sites. The Pe299R/roGFP2 reporter rapidly assesses differences in redox microenvironments and provides a noninvasive tool that may complement traditional redox-sensitive chromophores and chemical analyses of cell extracts.