Determination of glutathione disulfide levels in biological samples using thiol–disulfide exchanging agent, dithiothreitol

Ateş, Burhan; Erçal, Barış; Manda, Kalyan Reddy; Abraham, Linu; Erçal, Nuran

doi:10.1002/bmc.1083

Cited by 43 publications

(34 citation statements)

References 20 publications

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“…Moreover, the use of a small amount of tissue allows the determination of these compounds in the isolated structures of this organ. As shown in a few previous studies, occurring concentrations of GSH and GSSG in the rat brain can be determined by HPLC method with fluorescence detection [22]. Our research confirms that this thesis also indicates the greater degree of reliability in the independent determination of GSSG after blocking thiol groups by NEM.…”

Section: Discussionsupporting

confidence: 88%

“…Ates et al [22] in brain samples obtained from Sprague-Dawley rats determined the concentration of GSSG at 0.3-0.4 nmol mg −1 protein.…”

Section: Linearitymentioning

confidence: 99%

“…Determination of reduced glutathione (GSH) also requires derivatization with OPA, but without reduction step. A GSSG level is estimated after subtraction of GSH content from tGSH and by dividing the difference by two [20][21][22].…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 88%

“…Ates et al [22] in brain samples obtained from Sprague-Dawley rats determined the concentration of GSSG at 0.3-0.4 nmol mg −1 protein.…”

Section: Linearitymentioning

confidence: 99%

See 1 more Smart Citation

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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“…The samples were analyzed as detailed for the determination of GSH using reverse phase HPLC. Data from the original GSH levels and the total current GSH levels in each sample were subsequently used to calculate the levels of GSSG present in each sample [54].…”

Section: Total Glutathione and Glutathione Disulfide (Gssg) Measurementmentioning

confidence: 99%

N-acetylcysteine amide protects against dexamethasone-induced cataract related changes in cultured rat lenses

Tobwala¹,

Pınarcı²,

Maddirala³

et al. 2014

ABC

Self Cite

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Glucocorticoids (GCs) are one of the most widely used immunosuppressive and anti-inflammatory agents. However, their long term and systemic use is associated with adverse drug reactions including posterior subcapsular cataracts as one of its ocular complications. Balanced redox state is crucial for maintenance of lens transparency, and a high content of glutathione (GSH) in the lens is believed to play a key role in doing so. Depletion of GSH is implicated in the etiopathogenesis of dexamethasone-induced cataracts and, therefore, the present study was sought to evaluate the efficacy of a novel thiol antioxidant, N-acetylcysteine amide (NACA), in preventing dexamethasone-induced cataractogenesis. Cataract formation was induced by incubation of rat lenses with 5 µM dexamethasone. To assess whether NACA had a significant impact on dexamethasone-induced cataracts, the rat lenses were divided into four groups: 1) control group (Dulbecco's Modified Eagle Medium (DMEM), 2) dexamethasone group (DMEM with 5 µM dexamethasone), 3) NACA-only group (50 µM NACA solution), and 4) NACA pretreatment group (50 µM NACA for 6 hours followed by 5 µM dexamethasone only for 18 hours). Lenses were cultured for 7 days at 37˚C under 5% CO 2 . Lenses were evaluated daily using a dissecting microscope and photographed and graded for the development of opacity. The rat lenses in both the control and the NACA-only groups were clear, whereas all lenses within the dexamethasone-only group developed well-defined cataracts. Overall observations indicated that NACA inhibits cataract formation by limiting lipid peroxidation and increasing the ratio of GSH/GSSG in lens. Therefore, NACA can be developed into a potential adjunctive therapeutic option for patients undergoing therapy with GCs to inhibit glucocorticoid-induced cataracts.

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“…1). NPM is one of the most popular fluorogenic reagents for the LC analysis of thiols used in both postcolumn [23][24][25][26] and precolumn reactions; [27][28][29][30][31] however, excimer fluorescence detection has not been carried out until now. Dimercaprol (BAL) and dithiothreitol (DTT) were used as model polythiols to optimize the separation and derivatization conditions.…”

Section: Introductionmentioning

confidence: 99%

Highly Selective and Simple Method for Determination of Polythiols Based on Liquid Chromatography with Postcolumn Excimer Fluorescence Derivatization

et al. 2009

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An LC postcolumn derivatization method for determination of polythiols has been developed. This method involves separation using reversed-phase LC, postcolumn derivatization with N-(1-pyrenyl)maleimide, and excimer fluorescence detection. Analytes with a polythiol structure were converted into corresponding polypyrene-labeled derivatives, and the derivatives exhibited intramolecular excimer fluorescence (440 -520 nm). In this study, dimercaprol and dithiothreitol were used as model polythiols. This polythiol analysis method is simple; it is also highly selective and sensitive and yields good calibration curves.

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Determination of glutathione disulfide levels in biological samples using thiol–disulfide exchanging agent, dithiothreitol

Cited by 43 publications

References 20 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

N-acetylcysteine amide protects against dexamethasone-induced cataract related changes in cultured rat lenses

Highly Selective and Simple Method for Determination of Polythiols Based on Liquid Chromatography with Postcolumn Excimer Fluorescence Derivatization

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