[48] Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples

doi:10.1016/S0076-6879(81)77050-2

Methods in Enzymology

Volume 77, 1981, Pages 373-382

https://doi.org/10.1016/S0076-6879(81)77050-2 Get rights and content

Publisher Summary

This chapter presents the assay of glutathione (GSH), glutathione disulfide (GSSG), and glutathione mixed disulfides (GSSR). The sum of the reduced and oxidized forms of glutathione can be determined by using a kinetic assay in which catalytic amounts of GSH or GSSG and glutathione reductase bring about the continuous reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) by nicotinamide adenine dinucleotide phosphate (NADPH). The sensitivity of the assay may be enhanced by measuring NADPH fluorometrically. There are other two enzymatic assay available. In the first assay, the formation of the reduced glutathione using glyoxalase I can be monitored directly at 240 nm, or at very low levels of GSH, a dual wavelength spectrophotometer can be used at the wavelength-pair 240–270 nm with the same extinction coefficient. The second assay involves O-phthaldialdehyde, which forms a fluorescent complex with GSH and can be monitored at 350 nm–420 nm.

References (45)

A. Meister
N.S. Kosower et al.
Int. Rev. Cytol.
(1978)
T. Higashi et al.
Biochem. Biophys. Res. Commun.
(1976)
J. Isaacs et al.
Biochim. Biophys. Acta
(1977)
J. Isaacs et al.
Biochim. Biophys. Acta
(1977)
H. Sies et al.
Adv. Enzyme Regul.
(1980)
S.K. Srivastava et al.
Anal. Biochem.
(1968)
F. Tietze
Anal. Biochem.
(1969)
E. Beutler et al.
Anal. Biochem.
(1977)
M. Pace et al.
J. Chromatogr.
(1978)

P.J. Hissin et al.

Anal. Biochem.

(1976)

O.W. Griffith

Anal. Biochem.

(1980)

D.J. Reed et al.

Anal. Biochem.

(1980)

C.W. Tabor et al.

Anal. Biochem.

(1977)

H. Modig

Biochem. Pharmacol.

(1968)

K.R. Harrap et al.

Biochim. Biophys. Acta

(1973)

A.F.S.A. Habeeb

Anal. Biochem.

(1973)

G.L. Ellman

Arch. Biochem. Biophys.

(1959)

J.E. Brehe et al.

Anal. Biochem.

(1976)

E. Racker

J. Biol. Chem.

(1951)

E. Bernt et al.

V.H. Cohn et al.

Anal. Biochem.

(1966)

Cited by (1514)

Tea polyphenols protect against Flavobacterium columnare-induced gill injury via suppression of oxidative stress, inflammation, and apoptosis in grass carp
2024, International Journal of Biological Macromolecules
Flavobacterium columnare (F. columnare) is one of the deadliest fish pathogens causing bacterial gill rot disease in various freshwater fish species globally. Tea polyphenols (TPs) are an inexpensive product extracted from tea that have received much attention as a feed additive in aquaculture. The current study was designed to investigate the underlying mechanisms and protective effects of dietary TPs against F. columnare-induced gill injury via suppression of oxidative stress, apoptosis, and inflammation in grass carp. TPs were not supplemented to the diet (control) and were supplemented at 40, 80, 120, 160 or 200 mg/kg diet. The feeding experiment was carried out for 60 days, followed by a 3-Day F. columnare challenge test. The results showed that 120 mg/kg TPs in the diet exerted the following five protective effects in fish gill: (1) control gill-rot disease and improved histopathology, (2) inhibit excessive apoptosis, (3) enhance the activity of antioxidant enzymes and upregulate related gene expression via the Nrf2/Keap1 pathway, (4) increase the activity of immune enzymes, And (5) mediate inflammatory cytokine gene expression via the JAK/STAT3 pathway. Taken together, dietary supplementation with TPs is a compelling approach to protect the gill function of fish against F. columnare.
NOX2 control over energy metabolism plays a role in acute myeloid leukaemia prognosis and survival
2023, Free Radical Biology and Medicine
Acute myeloid leukaemia (AML) is a highly heterogeneous disease, however the therapeutic approaches have hardly changed in the last decades. Metabolism rewiring and the enhanced production of reactive oxygen species (ROS) are hallmarks of cancer. A deeper understanding of these features could be instrumental for the development of specific AML-subtypes treatments. NADPH oxidases (NOX), the only cellular system specialised in ROS production, are also involved in leukemic metabolism control. NOX2 shows a variable expression in AML patients, so patients can be classified based on such difference. Here we have analysed whether NOX2 levels are important for AML metabolism control. The lack of NOX2 in AML cells slowdowns basal glycolysis and oxidative phosphorylation (OXPHOS), along with the accumulation of metabolites that feed such routes, and a sharp decrease of glutathione. In addition, we found changes in the expression of 725 genes. Among them, we have discovered a panel of 30 differentially expressed metabolic genes, whose relevance was validated in patients. This panel can segregate AML patients according to CYBB expression, and it can predict patient prognosis and survival. In summary, our data strongly support the relevance of NOX2 for AML metabolism, and highlights the potential of our discoveries in AML prognosis.
Serum levels of oxidative stress biomarkers is changed in pharmacoresistant mesial temporal lobe epilepsy patients with or without psychiatric disorders
2023, Brain Disorders
The objective of the present study was to assess the levels of oxidative stress biomarkers (OSB) in patients with pharmacoresistant temporal lobe epilepsy and mesial temporal sclerosis (TLE-MTS) in order to investigate a possible role of oxidative stress (OS) in the pathophysiology of such disease. Ninety-eight participants were included and distributed in three groups: group 1 (G1) - 25 patients with pharmacoresistant TLE-MTS, without comorbid PD; Group 2 (G2) - 21 patients with pharmacoresistant TLE-MTS with PD; Group 3 (G3) - 52 healthy control subjects. Serum levels of catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), reduced glutathione plus oxidated glutathione (total GSH), glucose-6-phosphate dehydrogenase (G6PD), Trolox-equivalent antioxidant capacity (TEAC), and thiobarbituric acid reactive substances (TBARS) were analyzed through spectrometry. The significance level was set at P<0.05. Sociodemographic data were similar in all groups (P>0.05). A family history of epilepsy was more frequent among G1 and G2 groups when compared to G3 (P = 0.0002). Compared to controls, G1 and G2 presented higher CAT (P<0.0001) and TBARS levels (P = 0.001), as well as lower GPx (P<0.0001), G6PD (P = 0.005) and total GSH levels (P = 0.04). The presence of psychiatric disorders (PD), analyzed both separately and together, was not associated with significant differences in OSB levels (P>0.05). The lower levels of GPx, G6PD, and total GSH, as well as the higher CAT and TBARS levels observed in pharmaco-resistant TLE-MTS patients can indicate an imbalance between oxidizing agent production and elimination, supporting the hypothesis of a possible role of OS in the pathogenesis of this condition.
Microanatomy and behaviour of the date mussel's adductor and foot muscles after chronic exposure to bisphenol A, with inhibition of ATPase enzyme activities and DNA damage
2023, Comparative Biochemistry and Physiology Part - C: Toxicology and Pharmacology
Environmental contaminants with estrogenic activity have recently received attention due to the potential harm they could cause to humans and wildlife. To assess the toxic effects of bisphenol A (BPA) on marine mussels, Lithophaga lithophaga were exposed for 4 weeks to 0, 0.25, 1, 2, and 5 μg/L BPA. Aside from DNA damage, a behavioural study including valve closure duration (VCD), valve opening duration (VOD), levels of malondialdehyde (MDA), and total glutathione, as well as superoxide dismutase (SOD) and ATPase activities in adductor muscle extracts, and histopathological examination of the adductor muscle and foot were performed. The behavioural response was marked by an increase in the percentage of VCD and a decrease in the percentage of VOD during 8 h. Furthermore, BPA treatments resulted in a significant concentration-dependent increase in muscle MDA and total glutathione levels. However, when compared to controls, SOD and ATPase activity was significantly reduced in the adductor muscles of BPA treatments. Histological examination of the adductor and foot muscles revealed qualitatively distinct abnormalities. DNA damage was strongly induced in a concentration-dependent manner. Our findings suggested that BPA exposure altered detoxification, antioxidation, ATPase activity, histopathological characteristics, and DNA damage, which resulted in behavioural changes. The multi-biomarker approach used suggests that clear relationships exist between genotoxic and higher-level effects in some cases, which could be used as an integrated tool to evaluate various long-term toxic effects of BPA.
Aqueous extract of pummelo pulp (Citrus maxima) improves the biochemical profile and reduces the inflammation process in Wistar rats with non-alcoholic fatty liver disease
2023, Food and Chemical Toxicology
This study investigated the effect of pummelo extract (Citrus maxima) on biochemical, inflammatory, antioxidant and histological changes in NAFLD rats. Forty male Wistar rats divided into four groups were used: (1) control group; (2) fructose associated with high-fat diet - DHF; (3) normal diet + pummelo extract (50 mg/kg); and (4) FHD + pummelo extract. This was administered at dose of 50 mg/kg of the animal's weight, by gavage, for 45 days. Significant improvement in lipid profile, liver and kidney function, inflammation, oxidative stress markers was identified in group 4 compared to group 2. Regarding TNF-α and IL-1β, group 2 showed higher values (respectively 142, 5 ± 0.7 and 560.5 ± 2.7 pg/mg protein) compared to group 4 (respectively 91.4 ± 0.9 and 402.1.4 ± 0.9 pg/mg protein), p < 0.05. Significant increases were found in SOD and CAT activities, respectively 0.10 ± 0.06 and 8.62 ± 1.67 U/mg protein for group 2 and respectively 0.28 ± 0.08 and 21.52 ± 2.28 U/mg of protein for group 4. Decreases in triglycerides, hepatic cholesterol and fat droplets in hepatic tissue were observed in group 4 compared to group 2. Results highlight that pummelo extract may be useful for prevent the development of NAFLD.
The effects of pentoxifylline and caffeic acid phenethyl ester on TNF-α and lung histopathology in D-galactosamine-induced pulmonary injury in rats
2023, Tissue and Cell
In this study, we aimed to investigate the effects of pentoxifylline [PTX] and caffeic acid phenethyl ester [CAPE] in D-galactosamine [D-GAL]-induced pulmonary injury in rats. The rats were randomly divided into six groups: control, D-GAL, D-GAL+PTX, D-GAL+CAPE, PTX and CAPE. Each group included eight animals. Lung sections from the control, PTX and CAPE groups had a normal histological appearance. The D-GAL group showed histopathological changes in lung tissue, including haemorrhage, oedema, inter-alveolar septal thickening and widespread infiltration of inflammatory lymphocytes and macrophages. Administration of PTX and CAPE significantly reduced histopathological damage scores in the D-GAL+PTX and D-GAL+CAPE groups compared with the D-GAL group. PTX and CAPE treatment also significantly decreased malondialdehyde levels, increased levels of reduced GSH and increased catalase and superoxide dismutase activity in lung tissue samples. These results indicate that the destructive effects of D-GAL-induced inflammation in the rat lung are significantly reduced following administration of PTX and CAPE.

View all citing articles on Scopus

View full text

[48] Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples

Publisher Summary

Int. Rev. Cytol.

Biochem. Biophys. Res. Commun.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Adv. Enzyme Regul.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

J. Chromatogr.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Biochem. Pharmacol.

Biochim. Biophys. Acta

Anal. Biochem.

Arch. Biochem. Biophys.

Anal. Biochem.

J. Biol. Chem.

Anal. Biochem.