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.     

Quantitation of oxidized and reduced glutathione in plasma by micellar electrokinetic capillary electrophoresis.

A method for the separation of reduced (GSH) and oxidized (GSSG) glutathione was optimized in terms of buffer concentration, sodium dodecyl sulfate concentration, buffer pH, detection wavelength, run voltage and injection volume. The method demonstrated good linearity (r2 > 0.999) and reproducibility (internal standard corrected peak area RSD < 2.3%) in the range of interest (16-81 microM GSH and 8-40 microM GSSG). A detection limit of less than 1 microM GSH and GSSG was obtained using a high sensitivity flow cell. When the optimized method was applied to plasma samples, concentrations of 1.6 microM GSH and 0.8 microM GSSG were easily detected without the need for derivatization. The on-capillary detection was calculated to be 38.6 fmol of GSH and 18.3 fmol of GSSG.     

Chip-based SALDI-MS for rapid determination of intracellular ratios of glutathione to glutathione disulfide

Alterations in the ratio of glutathione(GSH) to glutathione disulfide(GSSG) reveal the cell living state and are associated with a variety of diseases. In this study, an Au NPs grafted nanoporous silicon chip was used for surface assisted laser desorption ionization-mass spectrometry(SALDI-MS) detection of GSH. Due to the bond interaction between thiol of GSH and Au NPs modified on the chip surfaces, GSH could be captured from the complex cellular lysate. Meanwhile, the composite nanostructures of Au NPs grafted porous silicon surface presented good desorption/ionization efficiency for GSH detection. The GSH levels in different tumor cells were successfully detected. Chip-based SALDI-MS was optimized for quantification of intracellular GSH/GSSG ratio changing under drug stimulation in liver tumor cells, GSSG was reduced to GSH by reductant of tris(2-carboxyethyl)phosphine(TCEP) and isotope-labeling GSH was as an internal standard. It was found that the increasing concentration of drug irinotecan and hypoxia culture condition caused the rapid consumption of GSH and a decrease of GSH/GSSG ratio in liver tumor cells. The developed SALDI-MS method provided a convenient way to accurately measure and rapidly monitor cellular GSH value and the ratios of GSH/GSSG.     

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.     

A generic method to detect electrophilic intermediates using isotopic pattern triggered data-dependent high-resolution accurate mass spectrometry

A need still exists for a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method that can detect broad classes of glutathione (GSH) conjugates and provide characterization of their structures. We now describe the development of a method that multiplexes high-resolution accurate mass analysis with isotope pattern triggered data-dependent product ion scans, for simultaneous detection and structural elucidation of GSH conjugates within a single analysis using a LTQ/Orbitrap. This method was initially developed to detect GSH conjugates generated from incubating 10 microM test compound with pooled human liver microsomes fortified with NADPH-regenerating system and a 2:1 ratio of 5 mM glutathione and [(13)C(2) (15)N-Gly]glutathione. The GSH conjugates were detected by isotope search of mass defect filtered and control subtracted full scan accurate MS data using MetWorks software. This was followed by elucidation of reactive intermediate structures using chemical formulae for both protonated molecules and their product ions from accurate masses in a single analysis. The mass accuracies measured for the precursor and product ions by the Orbitrap were <2 ppm in external mass calibration mode. Successful detection and characterization of GSH conjugates of acetaminophen, tienilic acid, clozapine, ticlopidine and mifepristone validated this method. In each case, the detected GSH conjugates were within the top five hits by isotope search. This method also has a broader detection capability since it is independent of the collision-induced dissociation behavior of the GSH conjugates. Furthermore, this method is amenable to a broad class of reactive intermediate trapping agents as exemplified by the simultaneous detection and structural elucidation of the cyano-N-methylene iminium ion conjugates of verapamil and its O-desmethyl metabolites, which we report for the first time. In addition to the chemically tagged reactive intermediates, this method also provides information on stable metabolites from the full scan accurate MS data.     

Determination of intracellular glutathione and cysteine using HPLC with a monolithic column after derivatization with monobromobimane

An optimized high‐performance liquid chromatography (HPLC) method is used to show that, as myoblasts differentiate into multinucleated muscle fibers, there is a shift to a more oxidized cell redox state. The HPLC method incorporated derivatization with monobromobimane for the determination of the reduced (GSH) and oxidized (GSSG) forms of glutathione and the reduced (Cys) and oxidized (CysSS) forms of cysteine. The derivatization was optimized to improve the sensitivity of the approach; the limits of detection for glutathione and cysteine were 3 × 10^?8 and 5 × 10^?8 M , respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

基于谷胱甘肽抑制纳米金催化3,3’,5,5’-四甲基联苯胺与过氧化氢反应比色检测谷胱甘肽

采用具有类似过氧化物酶活性的金纳米粒子(AuNPs)催化四甲基联苯胺(TMB)-H2O2反应,氧化产物(oxTMB)被谷胱甘肽(GSH)还原成TMB,导致吸光度下降,颜色由蓝色变为无色。利用上述现象,设计了一种超灵敏检测谷胱甘肽的比色传感器。在10 pmol/L~10μmol/L范围内,吸光度随GSH浓度呈良好的线性降低关系,检出限为7.5 pmol/L。该方法可以定量检测人血清中的谷胱甘肽。     

Application of capillary isotachophoresis to the analysis of glutathione conjugates

An analytical capillary isotachophoretic method has been applied for the quantitative assay of reduced glutathione (GSH) conjugates produced by the cytosolic enzyme GSH S-transferase. The donor molecule GSH in reduced and oxidized (GSSG) forms and the GSH conjugates of at least two electrophilic acceptors can be assayed in a single analysis. This technique also permits the quantitative assay of further metabolites of GSH conjugates including mercapturic acids. The total analysis time was of the order of 30 min. The sensitivity of the method is sufficient for the accurate detection of 0.15 nmol GSH conjugate of 1-chloro-2,4-dinitrobenzene and p-nitrobenzyl chloride, 0.2 nmol GSH conjugate of 1,2-epoxy-3-(p-nitrophenoxy)-propane and 0.15 nmol GSH. The present method is simple, accurate and does not require radioactively labelled compounds or separate analytical procedures.     

Disposable Amperometric Sensors for Thiols with Special Reference to Glutathione

The antioxidant ‘reduced glutathione’ tripeptide is conventionally called glutathione (GSH). The oxidized form is a sulfur‐sulfur linked compound, known as glutathione disulfide (GSSG). Glutathione is an essential cofactor for antioxidant enzymes; it provides protection also for the mitochondria against endogenous oxygen radicals. The ratio of these two forms can act as a marker for oxidative stress. The majority of the methods available for estimation of both the forms of glutathione are based on colorimetric and electrochemical assays. In this study, electrochemical sensors were developed for the estimation of both GSH and GSSG. Two different types of transducers were used: i) screen‐printed three‐electrode disposable sensor (SPE) containing carbon working electrode, carbon counter electrode and silver/silver chloride reference electrode; ii) three‐electrode disposable system (CDE) consisting of three copper electrodes. 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB) was used as detector element for estimation of total reduced thiol content. The enzyme glutathione reductase along with a co‐enzyme reduced nicotinamide adenine dinucleotide phosphate was used to estimate GSSG. By combining the two methods GSH can also be estimated. The detector elements were immobilized on the working electrodes of the sensors by bulk polymerization of acrylamide. The responses were observed amperometrically. The detection limit for thiol (GSH) was less than 0.6 ppm when DTNB was used, whereas for GSSG it was less than 0.1 ppm.     

微流控芯片电泳/电化学检测人单个肝癌细胞中的谷胱甘肽

采用微流控装置结合电化学检测研究了测定人单个血红细胞中谷胱甘肽(GSH)的方法。在该方法中,细胞的进样、定位、溶膜以及细胞中谷胱甘肽的转移和检测都在配有通道端安培检测器的双T形芯片中完成。单个细胞用液压导入到双T的交界面,在电泳缓冲液中毛地黄皂苷的作用下,细胞膜被穿孔。再施加直流电压,细胞被溶膜。释放出来的GSH被此直流电压电迁移至通道端并在Au/Hg电极上被检测。用校正曲线法可以定量测定单个细胞中的GSH。     

Study of Oxidation of Glutathione Treated with Hypochlorous Acid by Capillary Electrophoresis

Glutathione (GSH) protects human and animal's cells by the exchange with reversible oxidized glutathione (GSSG) when cells meet with oxidants, such as hydrogen peroxide (H2O2) and lipid peroxide. But when GSH meets with hypochlorous acid (HOCl), via the myeloperoxidase-catalysed oxidation of chloride by H2O2, the amount of GSSG formed does not account for all the GSH lost, which is quite different from that we has investigated in the reaction of GSH with H2O21. Prutz proposed the fo…     

A highly sensitive assay for spectrofluorimetric determination of reduced glutathione using organic nano-probes

In this study, the new nanometer-sized fluorescent particles (1-pyrenemethylamine nanoparticles) have been prepared by reprecipitation method under ultrasonic radiation. These nanoparticles have the potential to overcome problems encountered by organic small molecules by combining the advantages of high photobleaching threshold, high quantum yield, long fluorescence lifetime, good chemical stability, and wide excitation spectral properties. These nanoparticles will be able to be directly used as fluorescent nanoparticles probe without modification. A new fluorimetric method for the determination of reduced glutathione (GSH) has been developed with these nanoparticles. Under optimal conditions, the organic nanoparticles reacted with GSH and o-phthalaldehyde (OPA) to give a highly fluorescent derivative in Na2CO3-HCl buffer (pH=9.0). The fluorescence excitation and emission wavelengths of fluorescent derivative were located at 345 and 400 nm, respectively. The relative fluorescence intensity (RF) was linear in the range of the GSH concentration from 8.0x10(-7) to 1.1x10(-4)moll(-1). Limit of detection of 7.1x10(-8)moll(-1) was achieved for the reduced glutathione. The method was validated and applied to the analysis of three synthetic samples containing reduced glutathione.     

Ring‐disc Microelectrodes towards Glutathione Electrochemical Detection

An alternative approach for space‐resolved glutathione (GSH) detection using a ring‐disc microelectrode and an appropriate electroactive probe (acetaminophen) is reported. Acetaminophen is electrochemically oxidized at one of the electrodes and a fraction of the reaction product (N‐acetyl‐p‐quinoneimine) diffuses to the other, where it is detected. The collection efficiency value is dependent on the concentration of glutathione in solution, which consumes N‐acetyl‐p‐quinoneimine during its transit from the disc to the ring. Collection efficiency values close to 100 % were obtained by confining the electroactive species in a gap (<2 μm) that resembles a thin layer cell in a SECM configuration. The proposed indirect method was used to image the transport of GSH across an impermeable membrane in a SECM experiment. The method proved to be useful as a proof of concept for space‐resolved GSH electrochemical detection and a topography independent electrochemical image was acquired.     

Determination of glutathione in human plasma using high-performance liquid chromatography with electrochemical detection with a carbon-epoxy resin composite electrode chemically modified with cobalt phthalocyanine

High-performance liquid chromatography with electrochemical detection (LCEC), incorporating a novel carbon-epoxy resin working electrode modified with cobalt phthalocyanine, has been employed for preliminary studies directed towards the determination of normal circulating levels of reduced glutathione (GSH) in human plasma. The mobile phase consisted of 0.05 M phosphate buffer (pH 3) containing 0.1% m/m ethylenediaminetetraacetic acid (EDTA); the calibration graph was linear in the range 0.24-30.7 ng of GSH injected. The mean recovery of GSH added to a control serum over the physiological concentration range (0.38-3.07 ng ml-1) was 99%; this was achieved following a simple sample pre-treatment method, prior to LCEC, involving chelation of divalent cations with EDTA and subsequent acidification with orthophosphoric acid. Using the LCEC method, the mean circulating level of GSH in plasma, found in three normal subjects, was 2.69 microM, GSH; this indicates that the method might be applicable to the determination of depressed circulating levels of GSH.     

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.     

Substrate Profiling of Glutathione S‐transferase with Engineered Enzymes and Matched Glutathione Analogues

The identification of specific substrates of glutathione S‐transferases (GSTs) is important for understanding drug metabolism. A method termed bioorthogonal identification of GST substrates (BIGS) was developed, in which a reduced glutathione (GSH) analogue was developed for recognition by a rationally engineered GST to label the substrates of the corresponding native GST. A K44G‐W40A‐R41A mutant (GST‐KWR) of the mu‐class glutathione S‐transferases GSTM1 was shown to be active with a clickable GSH analogue (GSH‐R1) as the cosubstrate. The GSH‐R1 conjugation products can react with an azido‐based biotin probe for ready enrichment and MS identification. Proof‐of‐principle studies were carried to detect the products of GSH‐R1 conjugation to 1‐chloro‐2,4‐dinitrobenzene (CDNB) and dopamine quinone. The BIGS technology was then used to identify GSTM1 substrates in the Chinese herbal medicine Ganmaocongji.     

Assay of glutathione in must and wines using capillary electrophoresis and laser-induced fluorescence detection. Changes in concentration in dry white wines during alcoholic fermentation and aging

Glutathione (GSH) was assayed in must and wine using capillary electrophoresis coupled with laser-induced fluorescence (LIF) detection. Sample preparation involved conjugating thiols with monobromobimane (MBB) in a 2-(N-cyclohexylamino)ethanesulfonic acid [CHES] buffer (179mM). The electrophoretic conditions were 30kV with a capillary length of 105cm from the inlet to the detector (120cm total length) and a 50microm inner diameter. Under these conditions, the complete separation from the other main non-volatile thiols took less than 20min. We also described the optimum conditions for derivatizing wine samples with MBB to increase eletrophoretic sensitivity. The detection limit for glutathione assay is 65nmol/L. This simple, sensitive method provides a specific assay of glutathione in reduced form, as the sample preparation technique does not modify the balance of oxidized and reduced forms. We used this method to monitor changes in the reduced glutathione content of a white wine during alcoholic fermentation and barrel aging.     

Voltammetric Separation and Determination of Glutathione and L‐tyrosine with Chlorogenic Acid as an Electrocatalytic Mediator

A novel and sensitive voltammetric method was proposed for separation and determination of glutathione (GSH) and L‐tyrosine (Tyr) at acetylene black and chitosan modified glassy carbon electrode (AB‐CS/GCE). By introducing chlorogenic acid (CGA) as a new electrocatalytic mediator, GSH could be detected at much lower potential with symmetric peak shape. Acetylene black and chitosan composite served as current signal amplifier for sensitive detection. The electrochemical behavior of GSH and Tyr in the presence of CGA was studied at AB‐CS/GCE and complete separation of anodic peaks was achieved. Under the optimum conditions, the electrocatalytic oxidation peak current of GSH showed a linear dependence on its concentration in the ranges of 2.0×10⁻⁷‐4.0×10⁻⁵ M with the detection limit of 5.8×10⁻⁸ M (S/N=3), while the oxidation peak current of Tyr was linear to its concentration from 2.5×10⁻⁶ to 4.3×10⁻⁴ M with the detection limit of 9.2×10⁻⁷ M (S/N=3) by differential pulse voltammetry (DPV). The established method has been applied to the simultaneous determination of GSH and Tyr in human urine with satisfactory results.     

High-performance liquid chromatography of glutathione conjugates

Summary An ion-exchange high-performance liquid chromatographic method is described for the quantitative assay of glutathione (GSH) conjugates derived from endogenous electrophilic substances as well as xenobiotics. GSH (reduced and oxidized forms) and GSH conjugates were condensated with o-phthaldialdehyde to highly fluorescent derivatives and monitored at 338 nm excitation and 450 nm emission wavelengths after separation by ion-exchange HPLC on a 60-5NH₂ Polygosil analytical column. The detection limit was 2 pmol for the GSH conjugate of cholesterol epoxide and 6 pmol for the GSH conjugate of oleic acid epoxide. This method allows sensitive determination of all GSH conjugates independent of the chromatographic and spectrophotometric properties of the electrophilic substrates. Using this method we could show for the first time that the endogenous compound oleic acid epoxide is a specific substrate for the cytosolic rat liver GSH S-transferase. The method is applied to the determination of GSH S-transferase activity towards oleic acid epoxide and cholesterol epoxide.     

Smartphone-Assisted Colorimetric Detection of Glutathione and Glutathione Reductase Activity in Human Serum and Mouse Liver Using Hemin/G-Quadruplex DNAzyme

Abnormal levels of reduced glutathione (GSH) and glutathione reductase (GR) are usually related to a variety of diseases, so it is of great significance to determine the GSH concentration and GR activity. We herein develop a smartphone-assisted colorimetric biosensor for the detection of GSH and GR activity in human serum and mouse liver using hemin/G-quadruplex DNAzyme. Firstly, an obvious color change from colorless to green can be observed, owing to the high peroxidase-like activity of hemin/G-quadruplex DNAzyme toward 2,2′-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS). With the addition of GSH or GR, the H₂O₂-mediated oxidation of ABTS catalyzed by hemin/G-quadruplex DNAzyme is significantly inhibited, resulting in remarkable color fading. Therefore, the detection of GSH and GR activity can be achieved by observing the color transition or measuring the absorbance at 420 nm. The detection limit was estimated to be as low as 0.1 μM and 10 μU/mL for GSH and GR, respectively. More interestingly, the RGB values of the sensing system can be identified by the smartphone application (APP, color collect), which makes it an ideal format for on-site determination and point-of-care testing (POCT). In addition, the proposed method shows excellent selectivity and acceptable applicability for the determination of GSH concentration and GR activity in human serum samples and mouse liver tissues, which might hold great application potential in clinical diagnosis and drug screening.