Rapid colorimetric determination of reduced and oxidized glutathione using an end point coupled enzymatic assay

A simple and rapid colorimetric coupled enzymatic assay for the determination of glutathione is described. The proposed method is based on the specific reaction catalyzed by γ-glutamyltransferase, which transfers the γ-glutamyl moiety from glutahione to an acceptor, with the formation of the γ-glutamyl derivative of the acceptor and cysteinylglycine. The latter dipeptide is a substrate of leucyl aminopeptidase, which hydrolyzes cysteinylglycine to glycine and cysteine that can be easily measured spectrophotometrically. The proposed method was used to measure the content of glutathione in acid extracts of bovine lens, to follow the NADPH-dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) catalyzed by the enzyme glutathione reductase and to determine the glutathione content in human astrocytoma ADF cells subjected to oxidative stress. The results obtained showed that the method can be suitably used for the determination of GSH and GSSG in different biological samples and to monitor tissue or cell redox status under different conditions. It is also applicable for following reactions involving GSH and/or GSSG.

Measurement of thiols in human plasma using liquid chromatography with precolumn derivatization and fluorescence detection

A liquid chromatography (LC) method for the simultaneous measurement of the main low molecular mass thiols (i.e., cysteine, cysteinylglycine, homocysteine, and glutathione) in human plasma is described. The sample treatment consists of the reduction of disulfide bounds with tri-n-butylphosphine and protein precipitation with trichloroacetic acid followed by precolumn derivatization with a thiol-selective fluorogenic reagent (7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide). The structure of thiol derivatives is assessed using electrospray ionization-mass spectrometry (MS). The stability of resulting adducts in acidic medium (24 h at 10 degrees C) allows the automation of the technique and a high throughput of samples (approximately 50 per day). Separation is complete within 12 min using isocratic reversed-phase mode, and detection is operated by spectrofluorimetry (lambda ex = 385 nm and lambda em = 515 nm). Quantitation is performed by an internal standardization mode using thioglycolic acid. The LC method is fully validated, and homocysteine concentrations obtained in plasma samples are compared with values measured using either fluorescence polarization immunoassay or capillary gas chromatography-MS; a good correlation is observed between LC and both methods. The method has been applied in daily use to a large-scale study in a human healthy population, and some resulting data are discussed.     

Plasma thiols redox status by laser-induced fluorescence capillary electrophoresis

High concentrations of total plasma thiols such as cysteine and homocysteine are important risk factors for atherosclerosis and cardiovascular diseases. We have recently described a new laser-induced fluorescence capillary electrophoresis (CE-LIF) method to measure total plasma thiols, in which the baseline separation of cysteinylglycine, homocysteine, cysteine, and glutathione was achieved by adding the organic base N-methyl-D-glucamine to the run buffer. However, because the active fractions of homocysteine and cysteine responsible for vascular injuries are still unknown, research calls for a set up of methods able to analyze different forms of plasma thiols. In this paper, we present an improvement of our previous method that allows the measurement of different thiol forms. Total, reduced, and free thiols were measured by varying the order of disulfide reduction with tributylphosphine and proteins precipitation with 5-sulfosalicylic acid. After derivatization with 5-iodoacetamidofluorescein, samples were separated and measured by CE-LIF using a phosphate/borate buffer in the presence of 75 mmol/L N-methyl-D-glucamine. Oxidized thiols and protein bound thiols were calculated by difference, free minus reduced and total minus free form, respectively. Linearity, reproducibility, analytical recovery, and sensitivity were evaluated. The assay was used to measure the thiols redox status in 15 plasma samples from healthy volunteers.     

Profiling thiol metabolites and quantification of cellular glutathione using FT-ICR-MS spectrometry

We describe preparation and use of the quaternary ammonium-based α-iodoacetamide QDE and its isotopologue *QDE as reagents for chemoselective derivatization of cellular thiols. Direct addition of the reagents to live cells followed by adduct extraction into n-butanol and analysis by FT-ICR-MS provided a registry of matched isotope peaks from which molecular formulae of thiol metabolites were derived. Acidification to pH 4 during cell lysis and adduct formation further improves the chemoselectivity for thiol derivatization. Examination of A549 human lung adenocarcinoma cells using this approach revealed cysteine, cysteinylglycine, glutathione, and homocysteine as principal thiol metabolites as well as the sulfinic acid hypotaurine. The method is also readily applied to quantify the thiol metabolites, as demonstrated here by the quantification of both glutathione and glutathione disulfide in A549 cells at concentrations of 34.4?±?11.5 and 10.1?±?4.0 nmol/mg protein, respectively.

An improved method for simultaneous analysis of aminothiols in human plasma by high-performance liquid chromatography with fluorescence detection

Altered levels of aminothiols in biological fluids are thought to be an important risk indicator for several diseases, and reliable methods for the accurate determination of aminothiols concentrations in plasma are thus required. In this paper ammonium 5-bromo-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-BF) is proposed as a convenient fluorogenic derivatizating reagent for the determination of aminothiols (cysteine, cysteinylglycine, homocysteine and glutathione) by HPLC with fluorescence detection. The reactions of SBD-BF with aminothiols at room temperature are about three-times faster than those of ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (the most frequently employed reagent) at 60 °C. The derivatives of SBD-BF with cysteine, cysteinylglycine, homocysteine and glutathione are easily separated by HPLC and their calibration curves show excellent linearity over the range 0.05–20 μmol/L with excellent r² values for all analytes. SBD-BF reacts with thiols under mild conditions, i.e. at 25 °C over about 30 min, and is proposed as a suitable fluorogenic reagent for thiol derivatization to be introduced in analytical clinical chemistry. The detection limits of Cys, Cys-Gly, Hcy and GSH at a signal-to-noise ratio of 5 were 0.1 μM for Cys, 0.01 μM for Cys-Gly and Hcy, and 0.02 μM for GSH. Furthermore, validation parameters of the proposed method are quite satisfactory. As an application of this method the determination of thiol derivatives in human plasma was carried out on a number of samples.     

High-throughput capillary electrophoretic method for determination of total aminothiols in plasma and urine.

Increased interest in the analysis of aminothiols in body fluids during the last years results in a request for high-throughput analytical methods for their determination. We report here a novel, high-throughput method for the determination of total concentrations of biogenous aminothiols - homocysteine, cysteine, glutathione, cysteinylglycine, gamma-glutamylcysteine, and of penicilamine, mercaptopropionylglycine, and cysteamine, three compounds used to treat disorders of aminothiol metabolism in plasma and urine. Samples were reduced with tris(carboxyethyl)phosphine and labeled with 5-(bromomethyl)fluorescein. Capillary electrophoretic separations were performed in 60 mmol/L borate - 15 mmol/L sodium dodecyl sulfate - 2-amino-2-methyl-1-propanol, pH 10.0, with laser-induced fluorescence detection. Analysis time was less than 2 min. The assay is linear (r > 0.999) up to 500 micromol/L. Reproducibilities of migration times (coefficient of variation, CV) were < 0.5%. Interassay repeatabilities (CV, n = 10) were 5.08% and 6.09% for 5 micromol/L addition of homocysteine and 0.60% and 3.78% for 100 micromol/L addition of cysteine in plasma and urine, respectively. Recovery values were within 94-106% and sensitivity was better than 0.19 micromol/L for all analyzed compounds. Results agreed well with a standard high-performance liquid chromatography (HPLC) method. The diagnostic usefulness of the method has been proven on 79 samples of cystinuric patients and 12 samples of homocystinuric patients. We report here a novel method for the determination of aminothiols in body fluids by capillary electrophoresis (CE). Determination is fast and sensitive enough for diagnostic purposes.     

Nonionic surfactant-capped gold nanoparticles as postcolumn reagents for high-performance liquid chromatography assay of low-molecular-mass biothiols

Gold nanoparticles (GNPs) have been stabilized with nonionic surfactant ligands, i.e., Brij 35, and their aggregation could be induced rapidly and selectively by biologically active low-molecular-mass thiols including sulphydryl-containing amino acids (cysteine and homocysteine) and small peptides (glutathione, cysteinylglycine, and glutamylcysteine). A new postcolumn detection method has been developed for high-performance liquid chromatography (HPLC) assay of these small biothiols based on the analyte-induced aggregation of the GNPs. Compared with conventional thiol-reactive probes, the GNP colloids are easier to prepare, much more stable in aqueous solution over a wide pH range and at ambient temperature, and exhibit relatively high selectivity toward small biothiols. The analysis of human urine samples demonstrated that the proposed method is promising in HPLC assay of the small thiol molecules in biological fluids.     

HPLC‐fluorescence determination of thiol compounds in the serum of human male and female subjects using HILIC‐mode column

High‐performance liquid chromatography–fluorescence detection using a hydrophilic interaction chromatography‐mode column (ZIC®‐HILIC) was used to determine four kinds of thiol compounds in human serum. Sera were obtained from 34 subjects for this study (17 male subjects aged 22–38 years and 17 female subjects aged 18–38 years). Serum cysteine, cysteinylglycine, glutathione, and γ‐glutamylcysteine, derivatized with ammonium 7‐fluoro‐2,1,3‐benzoxadiazole‐4‐sulfonate, were separated on the ZIC®‐HILIC column and quantified. The serum concentrations of cysteine, cysteinylglycine, glutathione and γ‐glutamylcysteine were 226 ± 4.7, 23.4 ± 1.3, 3.7 ± 0.2 and 3.2 ± 0.1 μm , respectively. In addition, the concentrations of serum thiol compounds from male subjects were significantly higher than those of the female subjects (p < 0.05). Copyright © 2014 John Wiley & Sons, Ltd.     

Amperometric argentimetric and mercurimetric titration of sulfhydryl: Argentimetric titration

Results of amperometric argentimetric titrations at the rotated platinum wire electrode in ammonia buffers of cysteine, glutathione, thioglycolic acid and 2-mercaptoethanol are presented. The method gives satisfactory results with glutathione and 2-mercaptoethanol. Thioglycolic acid gives good results in the presence of sulfite but high results in its absence. Its disulfide can be titrated accurately in the presence of sulfite. In agreement with other workers it was found that cysteine yields high results. The error increases with increasing cysteine concentration and is reduced in the presence of 0.1 M sulfite. It becomes equal to zero at cysteine concentrations equal to or smaller than 5.10^-5M.     

Capillary electrophoresis method for determination of related substances in glutathione reduced drug substance

Summary A sensitive capillary electrophoretic method using low pH with direct UV detection has been developed to evaluate the impurity profile of reduced glutathione obtained from its production and purification. The method is characterized by high detection sensitivity and selectivity. Validation has been performed with model mixtures consisting of the main known related substances—oxidized glutathione, glutamylcystein, cysteinylglycine and cysteine. The results from this study show that with respect to quantification criteria the method is acceptable for routine control of reduced glutathione for pharmaceutical application.     

Fluorosurfactant-prepared triangular gold nanoparticles as postcolumn chemiluminescence reagents for high-performance liquid chromatography assay of low molecular weight aminothiols in biological fluids

Our recent study demonstrates the synthesized triangular gold nanoparticles (AuNPs) by trisodium citrate reduction of HAuCl(4) in the presence of nonionic fluorosurfactant (FSN) could display stronger catalytic activity towards luminol-chemiluminescence (CL) than spherical AuNPs. Ultratrace aminothiols may cause a great decrease in CL intensity of the triangular AuNPs-luminol CL system. In this article, we utilize the as-prepared triangular AuNPs as novel postcolumn CL reagents to explore a simple high-performance liquid chromatography (HPLC)-CL method for the determination of low molecular weight aminothiols (i.e., cysteine, homocysteine, glutathione, cysteinylglycine and glutamylcysteine). The as-prepared triangular AuNPs were easier to synthesize, stable at a wider pH range and high ionic strength, and highly selective and sensitive towards reduced aminothiols. The detection limits at a signal-to-noise ratio of 3 for cysteine, homocysteine, glutathione, cysteinylglycine and glutamylcysteine were 0.016, 0.08, 0.1, 0.04 and 0.1pmol, respectively. Recoveries from spiked urine and plasma samples were 95.7-104.3%. The applicability of the proposed method has been validated by determining these low molecular weight aminothiols in human urine and plasma samples with satisfactory results, and thus it will have great potential application in clinical diagnosis.     

Synthesis of Peptide Cysteine Dimedone Using Fmoc-Cys(Dmd)-OH: Glutathione Cysteine Dimedone as a Probe in Investigating the Sulfenic Acid Mediated Oxidation of Glutathione

Dimedone is the most widely used chemical probe for detection of cysteine sulfenic acid in peptides and proteins. The reaction of dimedone with cysteine sulfenic acid results in the formation of unique cysteine dimedone motif containing thioether bridge. Based on the structure of cysteine dimedone residue in polypeptide, a new building block of Fmoc-Cys(Dmd)-OH was developed for solid phase synthesis of peptide cysteine dimedone. Mass spectrometric sequencing of synthetic peptides have confirmed successful incorporation of cysteine dimedone in peptide chain using HBTU/HOBt as a coupling agent. The new method permits synthesis of peptides containing both cysteine thiol and cysteine dimedone in the same sequence which was difficult to achieve by conventional methods. The synthetic peptide of glutathione cysteine dimedone was used as a standard in probing the air-mediated oxidation of thiol to disulfide form of glutathione. The co-elution of standard peptide and reaction mixture of oxidation of glutathione in presence of dimedone using RP-HPLC have confirmed the formation of glutathione cysteine sulfenic as an intermediate in the air-mediated oxidation of glutathione. The synthetic peptides of cysteine dimedone may find application in the field of redox proteomics and generation of antibodies against modified cysteine residue.     

Quantification of Intracellular Thiols by HPLC-Fluorescence Detection

Biothiols, such as cysteine and glutathione, play important roles in various intracellular reactions represented by the redox equilibrium against oxidative stress. In this study, a method for intracellular thiol quantification using HPLC-fluorescence detection was developed. Thiols were derivatized with a thiol-specific fluorescence derivatization reagent, viz. ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), followed by reversed-phase separation on an InertSustain AQ-C18 column. Six different SBD-thiols (homocysteine, cysteine, cysteinylglycine, γ-glutamylcysteine, glutathione, and N-acetylcysteine as an internal standard) were separated within 30 min using a citric buffer (pH 3.0)/MeOH mobile phase. The calibration curves of all the SBD-thiols had strong linearity (R² > 0.999). Using this developed method, the thiol concentrations of human chronic myelogenous leukemia K562 cell samples were found to be 5.5–153 pmol/1 × 10⁶ cells. The time-dependent effect of a thiol scavenger, viz. N-ethyl maleimide, on intracellular thiol concentrations was also quantified. This method is useful for elucidating the role of intracellular sulfur metabolism.     

Distribution of low-density lipoprotein-bound low-molecular-weight thiols: a new analytical approach

We have recently demonstrated that low-density lipoprotein (LDL) apoprotein is able to bind the most concentrated plasma thiols such as cysteine, cysteinylglycine, and homocysteine by disulfide linkage. However, the LIF CE assay employed to measure linked thiols was not sensitive enough to verify whether low concentrated plasma thiols as glutathione and glutamylcysteine are also linked to apoprotein. By modifying sample treatment and electrophoretic parameters we set up a new method with an LOQ of about 1.5 nmol/L, by which we demonstrate that LDL apoprotein binds all physiological plasma thiols. The increased sensitivity was obtained by drying released apoB thiols after reduction treatment, dissolving them directly in a low volume of derivatization buffer and decreasing the dilution factor of derivatized sample before CE injection. Moreover, by increasing the concentration of the electrolyte buffer, we improved the selectivity of peaks, in particular between glutathione (GSH) and the impurity peak derived from unreacted 5-iodoacetamidofluorescein, which in the previous electrophoretic conditions were overlapped. The method optimization, reached by searching the best combination between sample matrix and CE run buffer, is fully described. Given the potential pathologic significance of protein thiolation, the proposed method may be useful to understand the mechanisms and the balances that regulate the interaction between thiols and -SH free groups of proteins.     

Effect of cysteine on bovine serum albumin (BSA) denaturation induced by solar ultraviolet (UVA, UVB) irradiation.

Long-term exposure to natural sun-light (UVA, UVB) induced fluorescence and caused disulfide bond formation in bovine serum albumin (BSA). The addition of cysteine enhanced the bond formation to such an extent that a solution of BSA was transformed into an insoluble gel. The disulfide bonds in the gels are derived from internal-SH groups of protein. This reaction occurred even if cysteine was added after exposure to ultraviolet (UV)-irradiation. Fluorescent substances seem to be involved in this reaction. On the other hand, low concentrations of cysteine (less than 5 mM) inhibited both fluorescence and disulfide bond formation. The addition of glutathione to BSA produced the same effect as that of cysteine. The addition of thiourea to BSA solution inhibited fluorescence, but did not inhibit disulfide bond formation. We assume that external-SH compounds such as cysteine and glutathione, which have high reactivity with hydroxyl radicals (.OH), act not only as free-radical scavengers, but also as radical mediators in the polymerization of protein through disulfide cross-links induced by UV-irradiation. Solar UVA as well as UVB irradiation are shown to have the same effect on the protein polymerization.     

高效液相色谱-电化学检测法测定鼠脑微透析液中谷胱甘肽和半胱氨酸

采用高效液相色谱法,用电化学检测手段成功地测定了沙土鼠脑纹状体微透析液内的谷胱甘肽（ＧＳＨ）和半胱氨酸（Ｃｙｓ）。检测前用电化学自清洗方式能有效地提高玻璃碳电极的检测水平,使其具有较好的重现性和回收率。Ｃｙｓ和ＧＳＨ的回收率分别为９１．４％和８７．３％。电化学检测的工作电压为０．９Ｖ,用外标法定量。由微透析探针获取的纹状体透析液Ｃｙｓ和ＧＳＨ的浓度分别为６８．５９和６５．５４ｎｍｏｌ／Ｌ,验证了方法的实用性和可靠性。     

Quantification of glutathione and glutathione disulfide in human plasma and tobacco leaves by capillary electrophoresis with laser-induced fluorescence detection

A new capillary electrophoresis (CE) method with laser-induced fluorescence (LIF) detection was developed for the rapid separation and sensitive detection of glutathione (GSH) and glutathione disulfide (GSSH) after derivatization by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl). The derivatization and separation conditions were investigated in detail and the optimums were obtained. Under the optimum experiment conditions, linear relationships between the peak height and concentrations of the analytes in normal and second-derivative electrophoregrams were obtained (0.22-45.00 μM). The detection limits for glutathione and glutathione disulfide in normal and second-derivative electrophoregrams were 0.046 and 0.012 μM and 0.046 and 0.014 μM, respectively. The method was applied to the analysis of glutathione and glutathione disulfide in human plasma and tobacco leaves with satisfactory results.     

CZE of Sulfur-Containing Amino Acids and Peptides and Its Application to the Quantitative Study of Heavy Metal-Caused Thiol Oxidations

The redox-active, sulfur-containing amino acids cysteine and methionine, the tripeptide glutathione and their oxidized counterparts cystine, methionine sulfoxide, and glutathione disulfide were separated as anions by capillary zone electrophoresis (CZE) in a 72?cm long fused silica capillary filled with 100?mM phosphate buffer, pH 8.0, at a voltage of +30?kV in 20?min. The optimized CZE method was suited for the implementation of quantitative metal interaction studies of the biomolecules in a biologically relevant concentration range (μM–mM). Decreasing peak areas of the reduced forms of cysteine and glutathione and simultaneously increasing peak areas of the oxidized forms after incubation of the reduced biomolecules with divalent heavy metal cations indicated redox reactions which could be responsible for toxic metal actions in biological systems. CZE measurements revealed that a 50?% oxidation grade of cysteine was achieved at a molar metal:cysteine ratio of 0.85 in case of Zn(II) addition and of 0.11 in case of Cu(II) addition, respectively. Cu(II) oxidized 50?% of the initial glutathione at a molar Cu:peptide ratio of 0.036, whereas the 50?% oxidation grade was not reached after incubation with Co(II) up to a molar ratio of Co:peptide of 0.25.     

Characterization of dansylated glutathione, glutathione disulfide, cysteine and cystine by narrow bore liquid chromatography/electrospray ionization mass spectrometry

A method using reversed phase high performance liquid chromatography/electrospray ionization-mass spectrometry (RP-LC/ESI-MS) has been developed to confirm the identity of dansylated derivatives of cysteine (C) and glutathione (GSH), and their respective dimers, cystine (CSSC) and glutathione disulfide (GSSG). Cysteine, GSH, CSSC and GSSG are present at low concentrations in rainbow trout (Oncorhynchus mykiss) liver cells. Initially, hepatic cells were sampled from a suspension culture and disrupted upon addition of 10% perchloric acid. The reduced thiols present in the cell extracts were acetylated to prevent dimerization and then the C and GSH species were derivatized with dansyl chloride for fluorescence detection. An LC system using a weak anion exchange column (AE) with fluorescence detection (FLD) was used for sensitive routine analysis; however, it produced peaks of unknown origin in addition to the expected analytes. Analytes were then separated on a C18 RP-LC system using a water/acetonitrile gradient with 0.2% formic acid, and detected using LC/ESI-MS at 3.5 KV which produced an intense ion with a minimum limit of detection of less than 0.5 pmole injected (>10:1 signal-to-noise (S/N). Subsequently, fractions of effluent from the AE-LC/FLD system were analyzed by LC/ESI-MS to confirm the presence of the target analytes in routine cell extracts. Monodansylated GSSG was identified as a product that could possibly affect the quantification of GSH and GSSG.     

A new HPLC method for the simultaneous determination of ascorbic acid and aminothiols in human plasma and erythrocytes using electrochemical detection

A new, simple, economical and validated high-performance liquid chromatography linked with electrochemical detector (HPLC-ECD) method has been developed and optimized for different experimental parameters to analyze the most common monothiols and disulfide (cystine, cysteine, homocysteine, methionine, reduced (GSH) and oxidized glutathione (GSSG)) and ascorbic acid present in human plasma and erythrocytes using dopamine as internal standard (IS). Complete separation of all the targets analytes and IS at 35 °C on Discovery HS C18 RP column (250 mm × 4.6 mm, 5 μm) was achieved using 0.05% TFA:methanol (97:3, v/v) as a mobile phase pumped at the rate of 0.6 ml min⁻¹ using electrochemical detector in DC mode at the detector potential of 900 mV. The limits of detection (3 S/N) and limits of quantification (10 S/N) of the studied compounds were evaluated using dilution method. The proposed method was validated according to standard guidelines and optimization of various experimental parameters and chromatographic conditions was carried out. The optimized and validated HPLC-ECD method was successfully applied for the determination of the abovementioned compounds in human plasma and erythrocytes. The method will be quite suitable for the determination of plasma and erythrocyte profile of ascorbic acid and aminothiols in oxidative stress and other basic research studies.