Glutathione In Biological Samples Research Articles

Fluorescent Pyrazole Derivatives: An Attractive Scaffold for Biological Imaging Applications

Among the huge number of fluorescent compounds described recently, pyrazole derivatives could play a paramount role in the design of probes for bioimaging applications–an important and simple tool for modern biology because of their easy synthetic methodologies, remarkable optical properties and chelating points. In this mini-review, we highlighted some pyrazole derivatives that have shown remarkable performance in this area; from the detection of chromium (III) with limits of detection of 37 × 10<sub>-12</sub> M to sensing glutathione in biological samples, as well as small molecule labeling of drugs and identifying unhealthy cells such as HeLa and labeling subcellular organelles. Evidently, this important class of N-heterocyclic compounds is part of interesting applications.

Facile synthesis of biomass-derived sulfonated carbon microspheres and nanosheets for the electrochemical detection of glutathione in biological samples

Herein, we report a low cost and highly selective electrochemical sensor for the detection of glutathione (GSH) using sulfonated carbon microspheres and nanosheets (SCMN) modified glassy carbon electrode (GCE) with an ultra-low limit of detection of 24.91 pM along with excellent stability and reproducibility. The sensor is successful in the detection of GSH in biological fluids such as blood serum samples with excellent recovery percentages from ~92.97 to ~101.58%. Thus, SCMN/GCE makes a promising platform for developing low-cost, biocompatible electrodes for biosensing applications.

Design and applications of a novel fluorescent probe for detecting glutathione in biological samples

This study aimed to develop a novel and practical fluorescent method for GSH detection in complex biological samples. To this end, a series of coumarin-based fluorescent probes was designed and synthesized using various aliphatic halogens as the sensing group. By using a new evaluation method of GSH/Cys/Hcy coexisting conditions, the probe with chloropropionate (CBF3) showed a high selectivity, excellent sensitivity, good stability for GSH detection. The reaction mechanism is proposed as nucleophilic substitution/cyclization and intramolecular charge transfer (ICT), which was confirmed by LC-MS and NMR analysis, as well as density functional theory calculations. In addition, CBF3 was demonstrated to be competent not only for the quantitative detection of GSH in real serum samples, but also for sensing GSH changes in different oxidative stress models in living cells and nematodes. This study showed a practical strategy for constructing GSH-specific fluorescent probes, and provided a sensitive tool for real-time sensing of GSH in real biological samples. The findings would greatly facilitate further investigations on GSH-associated clinical diagnosis and biomedical studies.

Simultaneous Determination of 35 Organic Metabolites Including Amino Acids, Creatine, Creatinine, and Oxidized and Reduced Glutathione in Lung Tissue, Plasma and Urine Using Liquid Chromatography–Tandem Mass Spectrometry

A simple and rapid method for the simultaneous determination of 35 non-derivatized metabolites was developed using liquid chromatography – tandem mass spectrometry. Under the optimal conditions, linearity was achieved in the range from 0.05 to 10.0 ng (R2 ≥ 0.989). The repeatability provided percentage relative standard deviations ≤11.2 and a trueness expressed as percentage relative error from −10.7% to 12%. The recoveries ranged from 87.3% to 109.8% with good repeatability. When applied to mouse lung tissue, plasma, and urine samples, 30 amino acids, creatine, creatinine, and oxidized glutathione were positively identified and quantified. The described present method was useful for the simultaneous determination of amino acids, creatine, creatinine, and oxidized glutathione in biological samples, including tissue, plasma, and urine.

Analysis of Glutathione in Biological Samples by HPLC Involving Pre-Column Derivatization with o-Phthalaldehyde.

Glutathione (GSH) forms conjugates with polyamines in prokaryotes and eukaryotes. There is also evidence suggesting cross-talk between GSH and polyamines to regulate cellular homeostasis and function, particularly under the conditions of oxidative stress. Because of its versatile roles in cell metabolism and function, a number of high performance liquid chromatography (HPLC) methods have been developed for glutathione analysis. Here, we describe our rapid and sensitive method for the analysis of GSH and the oxidized form of glutathione (GS-SG) in animal tissues and cells by HPLC involving pre-column derivatization with o-phthalaldehyde (OPA). OPA reacts very rapidly (within 1min) with S-carboxymethyl-glutathione at room temperatures (e.g.,20-25°C) in an autosampler, and their derivatives are immediately injected into the HPLC column without any need for extraction. This method requires two simple steps (a total of 15min) before samples are loaded into the autosampler: (a) the conversion of GS-SG into GSH by 2-mercaptoethanol; and (b) the oxidation of GSH by iodoacetic acid to yield S-carboxymethyl-glutathione. The autosampler is programmed to mix S-carboxymethyl-glutathione with OPA for 1min to generate a highly fluorescent derivative for HPLC separation and detection (excitation wavelength 340nm and emission wavelength 450nm). The detection limit for GSH and GS-SG is 15pmol/ml or 375fmol/injection. The total time for chromatographic separation (including column regeneration) is 16min for each sample. Our routine HPLC technique is applicable for analyses of cysteine and cystine, as well as polyamines and GSH-polyamine conjugates in biological samples.

Colorimetric detection of biothiols based on aggregation of chitosan-stabilized silver nanoparticles

We have described a simple and reliable colorimetric method for the sensing of biothiols such as cysteine, homocysteine, and glutathione in biological samples. The selective binding of chitosan capped silver nanoparticles to biothiols induced aggregation of the chitosan–Ag NPs. But the other amino acids that do not have thiol group cannot aggregate the chitosan–Ag NPs. Aggregation of chitosan–Ag NPs has been confirmed with UV–vis absorption spectra, zeta potential and transmission electron microscopy images. Under optimum conditions, good linear relationships existed between the absorption ratios (at A500/A415) and the concentrations of cysteine, homocysteine, and glutathione in the range of 0.1–10.0μM with detection limits of 15.0, 84.6 and 40.0nM, respectively. This probe was successfully applied to detect these biothiols in biological samples (urine and plasma).

Sensitive analysis of reduced glutathione in bacteria and HaCaT cells by capillary electrophoresis via online pre-concentration of transient trapping combined with the dynamic pH junction mode

A new method of capillary electrophoresis was developed for the sensitive determination of glutathione in biological samples.

Sensitive and selective colorimetric detection of glutathione in human plasma with 2,2′–azino–bis(3–ethylbenzothiazoline–6–sulfonic acid) and Ag+ ion

Glutathione is of vital importance to human beings through involving in many cellular functions. Simple and sensitive methods capable of detecting glutathione in biological samples are significant to diagnosis and prevention of disease. Here a simple, label–free, and sensitive colorimetric method was developed for the determination of glutathione. It was observed that Ag+ ion could directly oxidize 2,2′–azino–bis(3–ethylbenzothiazoline–6–sulfonic acid) (ABTS), a commonly used peroxidase substrate, to produce a green solution, which possessed a maximum absorbance at 420nm. The presence of glutathione hindered the oxidation process and decreased the absorbance at 420nm owing to its ability to bind with Ag+ ion. The procedure allowed the measurement of 0.1–4.0μM glutathione with a detection limit of 59nM. The relative standard deviation was 1.8% in eleven replicated measurements of 1.0μM glutathione solution. The method was applied to the determination of glutathione in human plasma with satisfactory results.

A dopamine-modulated nitrogen-doped graphene quantum dot fluorescence sensor for the detection of glutathione in biological samples

A dopamine-modulated nitrogen-doped graphene quantum dot (N-GQD) system was explored to develop a fluorescent sensor based on a chemical redox mechanism for the facile, sensitive and selective detection of glutathione (GSH) in biological samples.

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

The ratio of glutathione disulfide (GSSG) to reduced glutathione (GSH) in biological samples is a frequently used parameter of oxidative stress. As a result, many methods are developed to measure GSSG. The most popular and convenient of these relies on enzymatic cycling following the chemical masking of GSH in the sample using 2-vinylpyridine (2VP). However, 2VP is a slow reactant and its use may result in artificially high GSSG values due to oxidation of the sample over time. Fast-reacting reagents such as N-ethylmaleimide (NEM) may provide more accurate results. We performed a direct comparison of methods using 2VP and NEM. With 2VP, the percentage of total glutathione (GSH + GSSG) in the oxidized form was significantly higher in all tested tissues (kidney, lung and liver) compared to the same procedure performed using NEM. We conclude that NEM, when coupled with a simple solid-phase extraction procedure, is more accurate for the determination of GSSG. We also tested the effects of various handling and storage conditions on GSSG. A detailed description and a discussion of other methods are also included.

Sensitive determination of glutathione in biological samples by capillary electrophoresis with green (515 nm) laser-induced fluorescence detection

A new sensitive capillary electrophoretic method with laser-induced fluorescence (LIF) was developed for quantitation of glutathione (GSH) in biological samples. Eosin-5-maleimide was used to label the GSH molecule and the formed conjugate was separated in a 15mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid electrolyte at pH 7.0 in less than 3min. The conjugate was detected with an in-house built LIF system, utilizing an inexpensive 515nm diode laser module. Studies were performed to optimize the derivatization (the ratio of reagent to analyte, the reaction time, pH, etc.) and separation conditions. Sensitive detection of GSH at concentrations as low as 0.18nM was obtained. The method was applied in the analysis of biological fluids (exhaled breath condensate, saliva) and was found to be suitable for determination of GSH in these samples at trace levels below 1nM. To the best of our knowledge, this is the first report on determination of GSH in exhaled breath condensate by capillary electrophoresis (CE).

Molecular speciated isotope dilution mass spectrometric methods for accurate, reproducible and direct quantification of reduced, oxidized and total glutathione in biological samples.

Novel protocols were developed to accurately quantify reduced (GSH), oxidized (GSSG) and total (tGSH) glutathione in biological samples using molecular speciated isotope dilution mass spectrometry (SIDMS). For GSH and GSSG measurement, the sample was spiked with isotopically enriched analogues of the analytes ((310)GSH and (616)GSSG), along with N-ethylmaleimide (NEM), and treated with acetonitrile to solubilize the endogenous analytes via protein precipitation and equilibrate them with the spikes. The supernatant was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the analytes were quantified with simultaneous tracking and correction for auto-oxidation of GSH to GSSG. For tGSH assay, a (310)GSH-spiked sample was treated with dithiothreitol (DTT) to convert disulfide-bonded glutathione to GSH. After removing the protein, the supernatant was analyzed by LC-MS/MS and the analyte was quantified by single-spiking isotope dilution mass spectrometry (IDMS). The mathematical relationships in IDMS and SIDMS quantifications are based on isotopic ratios and do not involve calibration curves. The protocols were validated using spike recovery tests and by analyzing synthetic standard solutions. Red blood cell (RBC) and saliva samples obtained from healthy subjects, and whole blood samples collected and shipped from a remote location were analyzed. The concentrations of tGSH in the RBC and whole blood samples were 2 orders of magnitude higher than those found in saliva. The fractions of GSSG were 0.2-2.2% (RBC and blood) and 15-47% (saliva) of the free glutathione (GSH + 2xGSSG) in the corresponding samples. Up to 3% GSH was auto-oxidized to GSSG during sample workup; the highest oxidations (>1%) were in the saliva samples.

Effect of glutathione on peroxyoxalate chemiluminescence of hypericin as the fluorophore

Herein, the effect of amino acid Glutathione (GSH) on Peroxyoxalate Chemiluminescence was studied for the first time. Hypericin (HYP) was employed as the efficient fluorophore. The investigated parameters included rise and fall rate constant for the chemiluminescence burst, theoretical and experimental maximum intensity, the time-needed to reach maximum intensity and the total light yield emission which theoretically was evaluated using the pooled intermediate model by a computerized non-linear least-squares curve fitting program (KINFIT). Furthermore, based on observed quenching effect of GSH, the Stern–Volmer plot in quencher concentration range of 2.8×10−6 to 3.4×10−5M with KQ value of 1.59×104 was calculated. The bimolecular quenching rate constant (Kq) was also estimated about 2.8×1012 and M−1S−1. Moreover the system was applied successfully to determine glutathione in biological samples.

Recent advances in analysis of glutathione in biological samples by high-performance liquid chromatography: a brief overview

Glutathione (GSH) is a tri-peptide that plays an important role in protecting cells and tissues against oxidative stress. So far a lot of analytical methods of glutathione have been reported. This brief review presents an overview of the analysis of glutathione in biological samples by high-performance liquid chromatography (HPLC) in recent five years, focusing on the sample pretreatment, derivatization and mass spectrometric detection.

PREPARATION OF GLUTATHIONE IMPRINTED POLYMER

Glutathione imprinted polymer was prepared using 1-vinyl imidazole and ethylene glycol dimethacrylate as the functional monomer and crosslinker, respectively, in dimethyl sulfoxide. The adsorption selectivity of glutathione-imprinted polymer was tested by reduced glutathione, oxidized glutathione, and L-Gly-Leu-Tyr in 30% phosphate buffer (0.01 M, pH 5.0)–70% acetonitrile and binding affinity values were compared. Reusability of molecularly imprinted polymer particles was also investigated. Molecularly imprinted polymer particles were found to be stable and to maintain glutathione adsorption capacity at 95% when washed with methanol–acetic acid (10%) after seven usages. Functional monomer 1-vinyl imidazole and cross linker ethylene glycol dimethacrylate-based glutathione imprinted polymer could be used as solid phase extraction material for recognition of glutathione in biological samples.

Determination of reduced and oxidized glutathione in biological samples using liquid chromatography with fluorimetric detection

A HPLC method for determination of both reduced (GSH) and oxidized (GSSG) glutathione in plasma, whole blood and rat hepatocytes has been developed and evaluated. Reduced glutathione reacts with orthophthaldehyde (OPA) to form a stable, highly fluorescent tricyclic derivate at pH 8, while GSSG reacts with OPA at pH 12. At measurement of GSSG, GSH was complexed to N-ethylmaleimide. For the separation, reverse phase column Discovery C 18, 150 mm × 4 mm, 5 μm, was used. The mixture of methanol and 25 mM sodium hydrogenphosphate (15:85, v/v), pH 6.0, was used as mobile phase. The analytical performance of this method is satisfactory for both GSH and GSSG. The intra-assay coefficients of variation were 1.8 and 2.1% for whole blood, 2.0 and 1.9% for rat hepatocytes, 4.3 and 5.2% for plasma. The inter-assay coefficients of variation were 5.8 and 6.2% for whole blood, 6.6 and 7.1% for rat hepatocytes, 6.9 and 7.8% for plasma. The recoveries were as follows: 98.2% (CV 3.5%) and 101.5% (CV 4.2%) for whole blood, 99.1% (2.5%) and 102.3 (4.4%) for rat hepatocytes, 94.1% (CV 7.5%) and 103.5 (CV 8.5%) for plasma. The calibration curve was linear in the whole range tested. The limit of detection was 14.0 and 5.6 fmol, respectively. The preliminary reference ranges of reduced and oxidized glutathione in a group of blood donors are (4.69 ± 0.93) and (0.28 ± 0.12) μmol/g Hb for whole blood, (1.82 ± 0.55) and (0.154 ± 0.044) μM for plasma.

Direct spectrofluorimetric determination of glutathione in biological samples using 5-maleimidyl-2-( m-methylphenyl) benzoxazole

A new thiol fluorescence probe, 5-maleimidyl-2-( m-methylphenyl)benzoxazole (MMPB) has been developed for the direct determination of reduced glutathione (GSH) in real samples. Compared to the reported N-substituted maleimide type of thiol reagents, the main advantage of MMPB is its rather high selectivity for GSH to cysteine (Cys), which often coexists with GSH in biological samples. Under mild conditions similar to the physiological environment, MMPB reacted with GSH to give a highly fluorescent derivative with the excitation and emission wavelengths of 299.2 and 355.8 nm, respectively. In the presence of 0.40-fold (molar ratio) of Cys, a linear relationship was found in the range of 0–1.62×10 −7 mol l −1 with the detection limit (3 σ) of 3.23×10 −10 mol l −1 for GSH determination. Many other amino acids (100-fold) did not interfere with the determination. Since the molar ratio of Cys to GSH in mammalian tissues and blood does not exceed the value of 0.40:1, the proposed method has been used in the direct determination of GSH in these kinds of biological samples, such as human blood, pig’s liver and heart with the recoveries of 94.3–104.5%

Gas chromatographic–mass spectrometric analysis of stable isotopes of cysteine and glutathione in biological samples

A gas chromatographic–mass spectrometric (GC–MS) procedure for the determination of stable isotope labelled glutathione has been applied to animal and human samples. The method, based on preparation of the N, S-ethoxycarbonyl methyl ester derivative of the intact peptide, is rapid and requires little or minor tissue treatment. The same method was applied to cysteine. The method was found to be reliable in terms of within-day and between-day precision, accuracy and linearity. The procedure was applied in humans and animals to determine in vivo the glutathione fractional synthesis rate using labelled cysteine infusion. The glutathione fractional synthesis rate was found to be 22.5%/day in blood from a healthy volunteer and 337±29%/day in rat liver.

An Improved Simultaneous Measurement of Oxidized and Reduced Glutathione in Biological Samples by High-Performance Liquid Chromatography Following Derivatization with Dansyl Chloride

Reduced (GSH) and oxidized glutathione (GSSG) concentrations in biological samples can be determined simultaneously utilizing high-performance liquid chromatography (HPLC) following derivatization with dansyl chloride. In the present study, two points were investigated in detail to improve upon their method : 1) pH in the derivatization reaction and 2) the method of deproteinization prior to dansylation. Dansylation of standard GSH after S-carboxymethylation and of GSSG both gave the highest relative fluorescence at pH 8.5.Degradation of GSSG to GSH was not detected when GSSG was dansylated at pH 8.5 or more acidic pHs but was apparent at pH 9.0 and even more obvious at pH 10.0. The highest fluorescence values for dansylated GSH and GSSG were also obtained in tissue homogenates following the derivatization at pH 8.5. Although the value for dansylated GSH in the homogenates was also high at pH 9.0, this probably was due to degradation of intrinsic GSSG to GSH. Deproteinization with trichloroacetic acid (TCA) was found to give considerably higher relative fluorescence of dansylated GSSG than that with perchloric acid (PCA), whereas the dansylated-GSH concentrations obtained with the two acids were similar. Moreover, dansylated GSSG was more stable in TCA than in PCA, although there was no difference between these acids in relation to stability of dansylated GSH. In the livers and kidneys of rats, the GSH and GSSG concentrations obtained by the present method were consistent with those obtained by the recycling enzymatic method.

Different efficacy of iodoacetic acid and N-ethylmaleimide in high-performance liquid chromatographic measurement of liver glutathione

The widely used high-performance liquid chromatography (HPLC) procedure to determine glutathione in biological samples utilizing iodoacetic acid as thiol quenching agent and 1-fluoro-2,4-dinitrobenzene for derivatization has been modified regarding tissue sample processing and storage of the working solutions. The modified procedure compared with the original method reduces artifactual oxidation in rat liver glutathione measurement (1.47±0.8% vs. 2.84±0.69%, respectively). In both HPLC procedures, an increase in artifactual oxidation was found in both standard glutathione solutions and hepatic samples when N-ethylmaleimide instead of iodoacetic acid was used for thiol trapping.