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

Kanďár, Roman; Žáková, Pavla; Lotková, Halka; Kučera, Otto; Červinková, Z

doi:10.1016/j.jpba.2006.11.028

Cited by 130 publications

(80 citation statements)

References 60 publications

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“…GSH-N-ethylmaleimide conjugate was detected by UV chromatography at a wavelength of 265 nm (Shimadzu SPD-20A) (71). For GSSG derivatization, 150 μl of the cell homogenate was deproteinized in 200 μl of 15% perchloric acid, vortexed, and then centrifuged for 5 minutes at 20,000 g. The resulting supernatant (200 μl) was next diluted in 1 ml of 0.1 M NaOH twice to pH 12 before reacting with 0.1% o-phthaladehyde to form a fluorescent product detectable at excitation/emission wavelengths of 350/420 nm (Shimadzu RF-20A xs) (70). Derivatized GSSG samples were processed using a 25 mM sodium phosphate buffer containing 15% HPLC-grade methanol at pH 6 and run through a Shimadzu Prominence HPLC system equipped with the same column described above at flow rate of 0.5 ml/min.…”

Section: Methodsmentioning

confidence: 99%

“…The 500-μl cell suspension was snap frozen for lysis. The cell homogenate was thawed on wet ice and split into 2 derivatization pathways for the measurement of GSH and GSSG using HPLC (70,71). For GSH derivatization, 150 μl of the homogenate was added to 100 μl of 60% trichloroacetic acid to deproteinize the sample, vortexed, and then centrifuged for 5 minutes at 20,000 g. The supernatant was transferred to an autosampler vial for HPLC.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Isocitrate-to-SENP1 signaling amplifies insulin secretion and rescues dysfunctional β cells

Ferdaoussi

Dai

Jensen

et al. 2015

Journal of Clinical Investigation

160

232

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Isocitrate-to-SENP1 signaling amplifies insulin secretion and rescues dysfunctional β cells

Ferdaoussi

Dai

Jensen

et al. 2015

Journal of Clinical Investigation

160

232

View full text Add to dashboard Cite

“…Both reduced (GSH) and oxidized (GSSG) glutathione in cells were measured according to Kand'ár R. et al (2007) with slight modifications. Cold 10% trichloroacetic acid (400 lL) was added into the collected cells resuspended in 200 lL of PBS.…”

Section: Measurement Of Intracellular Gsh and Gssg Contentsmentioning

confidence: 99%

Enhanced cytotoxicity of pentachlorophenol by perfluorooctane sulfonate or perfluorooctanoic acid in HepG2 cells

et al. 2013

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h i g h l i g h t sPFOS and PFOA could enhance the cytotoxity of PCP to HepG2 cells. PFOS displayed stronger synergistic cytotoxicity with PCP than PFOA. Cytotoxic enhancement might be via strengthening the phosphorylation uncoupling of PCP. PFOS and PFOA might cause membrane disruption and improve the uptake of PCP. a r t i c l e i n f o t r a c tChlorinated phenols and perfluoroalkyl acids (PFAAs) are two kinds of pollutants which are widely present in the environment. Considering liver is the primary toxic target organ for these two groups of chemicals, it is interesting to evaluate the possible joint effects of them on liver. In this work, the combined toxicity of pentachlorophenol (PCP) and perfluorooctane sulfonate (PFOS) or perfluorooctanoic acid (PFOA) were investigated using HepG2 cells. The results indicated that PFOS and PFOA could strengthen PCP's hepatotoxicity. Further studies showed that rather than intensify the oxidative stress or promote the biotransformation of PCP, PFOS (or PFOA) might lead to strengthening of the oxidative phosphorylation uncoupling of PCP. By measuring the intracellular PCP concentration and the cell membrane properties, it was suggested that PFOS and PFOA could disrupt the plasma membrane and increase the membrane permeability. Thus, more cellular accessibility of PCP was induced when they were co-exposed to PCP and PFOS (or PFOA), leading to increased cytotoxicity. Further research is warranted to better understand the combined toxicity of PFAAs and other environmental pollutants.

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“…Reduced form of glutathione (GSH) was analysed by reverse-phase high-performance liquid chromatography (Shimadzu, Japan), using a slightly modified method of Hissin and Hilf (1976) by Kanďár et al (2007). The separation was performed on reverse-phase column Discovery C18, 15 cm × 4 mm, 5 µm (Supelco, USA) followed by fluorimetric detection (excitation wavelength 350 nm, emission wavelength 420 nm).…”

Section: Biochemical Assaysmentioning

confidence: 99%

Effect of S-adenosylmethionine on Acetaminophen-induced Toxic Injury of Rat Hepatocytes in vitro

et al. 2009

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Acetaminophen (AAP) overdose causes severe liver injury and is the leading cause of acute liver injury in humans. The mechanisms participating in its toxic effect are glutathione depletion, oxidative stress and mitochondrial dysfunction. S-adenosylmethionine (SAMe) is the principal biological methyl donor and is also a precursor of glutathione. In our previous studies we have documented a protective action of SAMe against various toxic injuries of rat hepatocytes in primary cultures. The aim of this study was to evaluate a possible protective effect of SAMe against AAP-induced toxic injury of primary rat hepatocytes. Hepatocytes were exposed to AAP (2.5 mM) or AAP together with SAMe at the final concentrations of 5, 25 or 50 mg/l for 24 h. Incubation of hepatocytes with AAP caused a significant increase of the leakage of lactate dehydrogenase (LDH) (p < 0.001) and decline of the activity of cellular dehydrogenases (WST-1) (p < 0.001). Co-incubation of hepatocytes with SAMe at any dose did not improve these markers of cellular integrity. The functional indicators improved in hepatocytes co-cultured with SAMe -urea production was significantly increased when using the highest dose of SAMe (p < 0.05); albumin synthesis was higher in all cultured hepatocytes exposed to SAMe (p < 0.05). SAMe did not influence AAP-induced decrease of cellular content of glutathione. Mitochondrial respiration of harvested digitonin-permeabilized hepatocytes was measured; Complex II was more sensitive to toxic action of AAP, respiration was decreased by 20%. This decrease was completely abolished by SAMe. Hepatotoxicity, hepatoprotective effect, mitochondrial membrane potential, urea, albumin, glutathione, LDHAcetaminophen (AAP) is a widely used, relatively safe analgesic/antipyretic drug. Nevertheless, AAP overdose causes centrilobular necrosis of the liver and is the leading cause of acute liver injury in humans in the United States and most of Europe (Lee 2007). The primary metabolic pathway for AAP is glucuronidation and sulphation in the liver; this yields relative non-toxic metabolites, which are excreted into bile (Mitchell et al. 1973). AAP hepatotoxicity is dependent on another metabolic pathway. The remaining part of AAP dose, which is not directly conjugated with the hydrophylic group, is biotransformed by the cytochrome P450 family to an electrophilic, highly reactive metabolite N-acetylp-benzoquinone imine (NAPQI), which is detoxified by glutathione (Jaeschke and Bajt 2006). However, if the formation of NAPQI exceeds the capacity of liver glutathione (GSH), this reactive metabolite forms covalent adducts primarily with cysteine residues on various cellular proteins (Nelson and Bruschi 2003; Allameh and Alikhani 2006). The most important mechanism of AAP-induced cell death seems to be a change in the function of critical cellular proteins due to covalent bindings. Numerous cytosolic, mitochondrial, ribosomal, microsomal, and nuclear proteins bound to AAP have been identified. Nevertheless, the primary cellular targe...

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

Cited by 130 publications

References 60 publications

Isocitrate-to-SENP1 signaling amplifies insulin secretion and rescues dysfunctional β cells

Isocitrate-to-SENP1 signaling amplifies insulin secretion and rescues dysfunctional β cells

Enhanced cytotoxicity of pentachlorophenol by perfluorooctane sulfonate or perfluorooctanoic acid in HepG2 cells

Effect of S-adenosylmethionine on Acetaminophen-induced Toxic Injury of Rat Hepatocytes in vitro

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