Chromatographic and mass spectrometric analysis of glutathione in biological samples

Iwasaki, Yusuke; Saito, Yûsuke; Nakano, Yüki; Mochizuki, Kimihiro; Sakata, Osami; Ito, Rie; Saito, Koichi; Nakazawa, Hiroyuki

doi:10.1016/j.jchromb.2009.07.001

Cited by 100 publications

(85 citation statements)

References 187 publications

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“…However, the used methods of determination of GSSG and GSH in different tissues described by different authors are varied, and it is difficult to compare results [27].…”

Section: Linearitymentioning

confidence: 99%

Optimization of determination of reduced and oxidized glutathione in rat striatum by HPLC method with fluorescence detection and pre-column derivatization

Krzyżanowska¹,

Gawlik²,

Filip³

2014

Acta Chromatographica

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Summary.A high-performance liquid chromatography (HPLC) method was used to assess the concentration of reduced and oxidized glutathione (GSH and GSSG) in rat striatum. Following decapitation, striatum was isolated from the male Wistar rat brain and immediately homogenized with double distilled water. GSH level was determined after pre-column derivatization with o-phthalaldehyde (OPA). The optimal incubation time with OPA was tested. The concentration of GSSG was determined after blockage the thiol groups of GSH by N-ethylmaleimide (NEM). The useful time for incubation with NEM was optimized. Next, disulfide bounds of GSSG were reduced by dithiothreitol (DTT), and released GSH is derivatized with OPA. The total glutathione, tGSH (sum of free and bound GSH, GSSG, and other low-molecular-mass aminothiols), was determined after reduction with DTT and then derivatization with OPA. The level of GSSG was calculated of the difference in concentrations of tGSH and GSH, but we showed that the calculated concentration of GSSG was within the range of standard deviation of the mean concentration of tGSH or GSH. Finally, the concentration of GSH was determined after 5-min incubation with OPA and the concentration of GSSG after 30-s incubation with NEM and 5-min incubation with DTT and OPA. The relative standard deviation (RSD) values obtained for the assay of GSH and GSSG were lower than 10%. The values obtained for accuracy for GSH (50-500 nM) and GSSG (0.5-5 nM) were within limits regarded as acceptable for analysis of biological samples (percent of recovery: 95-105%). Mean absolute recovery of GSH and GSSG was ranged from 97.1% to 99%. Limit of detection for GSH was 2.7 nM, and limit of quantification was 8.2 nM. Limit of detection (LOD) for GSSG is twice the value for GSH. Described method allows to determine GSH and GSSG levels in isolated rat brain structures with high level of reliability.

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“…However, the used methods of determination of GSSG and GSH in different tissues described by different authors are varied, and it is difficult to compare results [27].…”

Section: Linearitymentioning

confidence: 99%

Optimization of determination of reduced and oxidized glutathione in rat striatum by HPLC method with fluorescence detection and pre-column derivatization

Krzyżanowska¹,

Gawlik²,

Filip³

2014

Acta Chromatographica

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“…For many years, GSH has been measured by several analytical methods. In particular, high performance liquid chromatography (HPLC) with various detection techniques including ultraviolet (UV) absorbance and fluorescence detection, mass spectrometry and/or electrochemical detection (ED) are commonly used for determination of GSH and GSSG concentrations (46)(47)(48)(49). Each method has its advantages and limitations and may serve a particular need in analysis (50).…”

Section: Introductionmentioning

confidence: 99%

Redox status expressed as GSH:GSSG ratio as a marker for oxidative stress in paediatric tumour patients

et al. 2012

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Abstract.Oxidative stress causes profound alterations of various biological structures, including cellular membranes, lipids, proteins and nucleic acids, and it is involved in numerous malignancies. Reduced glutathione (GSH) is considered to be one of the most important scavengers of reactive oxygen species (ROS), and its ratio with oxidised glutathione (GSSG) may be used as a marker of oxidative stress. The main aim of this study was to determine GSH:GSSG ratio in the blood serum of paediatric cancer patients to use this ratio as a potential marker of oxidative stress. The whole procedure was optimised and the recoveries for both substances were greater than 80% under the optimised conditions. We analysed a group of paediatric patients (n=116) with various types of cancer, including neuroblastoma, anaplastic ependymoma, germ cell tumour, genital tract tumour, lymphadenopathy, rhabdomyosarcoma, nephroblastoma, Ewing's sarcoma, osteosarcoma, Hodgkin's lymphoma, medulloblastoma and retinoblastoma. We simultaneously determined the levels of reduced and oxidised glutathione, and thus, its ratio in the blood serum of the patients. The highest ratio was observed in retinoblastoma patients and the lowest in anaplastic ependymoma. We were able to distinguish between the diagnoses based on the results of the obtained GSH:GSSG ratio.

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“…Short Characteristic of Glutathione γ-L-Glutamyl-L-cysteinylglycine is an important intracellular tripeptide whose content changes in different tissues and achieves concentrations of between 1 and 10 mM (Ji and Leeuwenburgh 1995;Iwasaki et al 2009). Some authors note GSH in a concentration of 12 mM, where 1 % is an oxidized form of glutathione (Camera and Picardo 2002), apart from the mitochondria, where GSSG accounted for up to 25 % of GSH (Camera and Picardo 2002).…”

Section: Introductionmentioning

confidence: 99%

“…GSH is connected with xenobiotics, such as free radicals or hydrogen peroxides, which are responsible for oxidative stress, and they are excreted as mercapturic acids and derivatives of glutathione. Glutathione conjugates are connected with steroids, prostaglandins, and leukotrienes (Iwasaki et al 2009). The highest concentration of glutathione is described in the eye retina (10 mM) and the lowest in blood plasma (0.05 mM) (Lomaestro and Malone 1995;Ji and Leeuwenburgh 1995).…”

Section: Introductionmentioning

confidence: 99%

The Use of Different MS Techniques to Determine Glutathione Levels in Marine Tissues

2012

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Several techniques were used, mainly mass spectrometry connected with gas chromatography, matrixassisted laser desorption with ionization time-of-flight and mass spectrometry connected with liquid chromatography. A major problem was encountered in determining glutathione, which can be conditioned by the pH and selected reducing agents. The GSH form can be oxidized through derivatization, and a small glutathione amount in the biological samples may hinder the determination process. Another problem is the existence of a metal ion in the tested organism; therefore, often a reagent with a chelating function is added to the sample and the mobile phase in liquid chromatography is applied with appropriate polarity for GSH and GSSG. We determined the concentrations of total, reduced, and oxidized glutathione in the liver, hepatopancreas, muscle, and gonad tissues of brown shrimp (Crangon crangon) and fish (Psetta maxima and Clupea harengus membras). The highest concentrations of tGSH were recorded in the shrimp hepatopancreas (7.21 ± 0.011 μmol g −1 wet weight), in herring liver (2.85±0.025 μmol g −1 ), and in turbot liver (1.86±0.063 μmol g −1). In turn, the highest concentrations were reported for GSSG in the muscle of shrimp (0.140±0.000204 μmol g ). We also investigated seasonal changes in the concentrations of glutathione in the muscle of C. crangon shrimp in the annual cycle. The lowest values of total glutathione were recorded during spring and autumn, which could be correlated with the increase in lipid peroxidation and oxidative stress.

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Chromatographic and mass spectrometric analysis of glutathione in biological samples

Cited by 100 publications

References 187 publications

Optimization of determination of reduced and oxidized glutathione in rat striatum by HPLC method with fluorescence detection and pre-column derivatization

Optimization of determination of reduced and oxidized glutathione in rat striatum by HPLC method with fluorescence detection and pre-column derivatization

Redox status expressed as GSH:GSSG ratio as a marker for oxidative stress in paediatric tumour patients

The Use of Different MS Techniques to Determine Glutathione Levels in Marine Tissues

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