Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples

Baker, Margaret; Cerniglia, George J.; Zaman, Aziza

doi:10.1016/0003-2697(90)90208-q

Cited by 843 publications

(455 citation statements)

References 8 publications

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“…The contralateral hemisphere was used as control. Total (GSx) and oxidized (GSSG) glutathione concentrations (nmol/mg of protein) were measured as described by Baker et al (1990), with minor modifications. Tissue was sonicated in 3.3% 5-sulfosalicylic acid (1 mL per 200 mg of tissue) and centrifuged at 12,000 g for 30 mins at 41C.…”

Section: Determination Of Glutathione Concentrationmentioning

confidence: 99%

Antioxidant CR-6 Protects against Reperfusion Injury after a Transient Episode of Focal Brain Ischemia in Rats

Pérez-Asensio

Rosa

Jiménez‐Altayó

et al. 2009

J Cereb Blood Flow Metab

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Oxidative and nitrosative stress are targets for intervention after ischemia/reperfusion. The aim of this study was to explore the effect of CR-6, a vitamin-E analogue that is antioxidant and scavenger of nitrogen-reactive species. Sprague-Dawley rats had the middle cerebral artery (MCA) occluded either for 90 mins or permanently. Cortical perfusion was continuously monitored by laser-Doppler flowmetry. CR-6 (100 mg/kg) was administered orally either at 2 and 8 h after MCA occlusion, or at 2 h only. Infarct volume, neurological deficit, and signs of reperfusion injury were evaluated. CR-6 was detected in plasma and brain by HPLC. CR-6 reduced glutathione consumption in the ischemic brain and superoxide generation in the isolated MCA. CR-6 decreased infarct volume and attenuated the neurological deficit at 1 and 7 days after ischemia/reperfusion, but not after permanent ischemia. Immediately after reperfusion, cortical blood flow values returned to their baseline ( ± 20%) in several animals, whereas others showed hyper-perfusion ( > 20% of baseline). Reactive hyperemia was associated with adverse events such as increased cortical BBB leakage, edema, protein nitrotyrosination, COX-2 expression, and neutrophil accumulation; and with a poorer outcome, and CR-6 attenuated these effects. In conclusion, oral CR-6 administration after transient ischemia protects the brain from reperfusion injury.

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Section: Determination Of Glutathione Concentrationmentioning

confidence: 99%

Antioxidant CR-6 Protects against Reperfusion Injury after a Transient Episode of Focal Brain Ischemia in Rats

Pérez-Asensio

Rosa

Jiménez‐Altayó

et al. 2009

J Cereb Blood Flow Metab

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“…To determine GSH and oxidized glutathione levels, we used an enzymatic recycling assay (glutathione assay kit, Cayman Chemicals, Ann Arbor, MI) in the presence of glutathione reductase and spectophotometrically determined 5-thio-2-nitrobenzoic acid generation in deproteinated cells. 32 To do so, we used 30 g of total cell lysate protein for each sample, an amount determined by bicinchoninic acid protein assay before deproteinization. 32 Conditioned media PP i was determined radiometrically following centrifugation at 20,000 ϫ g for 10 minutes to remove cellular debris, and samples were normalized per DNA concentration.…”

Section: Pantetheinase Activity Gsh and Pp I Assaysmentioning

confidence: 99%

“…32 To do so, we used 30 g of total cell lysate protein for each sample, an amount determined by bicinchoninic acid protein assay before deproteinization. 32 Conditioned media PP i was determined radiometrically following centrifugation at 20,000 ϫ g for 10 minutes to remove cellular debris, and samples were normalized per DNA concentration. 18 Alkaline phosphatase specific activity was determined as described.…”

Section: Pantetheinase Activity Gsh and Pp I Assaysmentioning

confidence: 99%

Vanin-1 Pantetheinase Drives Increased Chondrogenic Potential of Mesenchymal Precursors in ank/ank Mice

Johnson

Yao

Lane

et al. 2008

The American Journal of Pathology

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Widespread endochondral and intramembranous ectopic bone formation is mediated by extracellular PP i deficiency that develops in ank/ank mice. Herein we report on the rapid condensation into chondrogenic nodules of cultured ank/ank bone marrow stromal cells (BMSCs). We compared the roles of increased chondrogenic potential versus altered osteoblast function in the ank/ank phenotype. To do so, we crossbred ank/ank mice with mice lacking Vanin-1 pantetheinase, which inhibits synthesis of the chondrogenesis regulator glutathione, since we observed increased Vanin-1 expression and pantetheinase activity and decreased glutathione in ank/ank BMSCs.

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“…Protein concentrations in cell lysates were determined by the bicinochoninic method with bovine serum albumin as the standard. Total GSH concentrations were determined in acidified 10,000 × g supernatants of HepG2 cells by the method of Baker et al (Baker et al, 1990) using a 96-well microplate reader (Spectramax 250, Molecular Devices, Sunnyvale CA). Standard curve values were obtained from serial dilutions of GSH standards.…”

Section: Biochemical and Molecular Characterization Of Transfected Cellsmentioning

confidence: 99%

Transfection of HepG2 cells with hGSTA4 provides protection against 4-hydroxynonenal-mediated oxidative injury

Gallagher

Huisden

Gardner

2007

Toxicology in Vitro

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Abstract4-hydroxynonenal (4-HNE) is a mutagenic α,β-unsaturated aldehyde produced during oxidative injury that is conjugated by several glutathione S-transferase (GST) isoforms. The alpha class human GSTA4-4 enzyme (hGSTA4-4) has a particularly high catalytic efficiency toward 4-HNE conjugation. However, hGST4-4 expression is low in most human cells and there are other aldehyde metabolizing enzymes that detoxify 4HNE. In the current study, we determined the effect of overexpression of hGSTA4 mRNA on the sensitivity of HepG2 cells to 4-HNE injury. HepG2 cells transfected with an hGSTA4 vector construct exhibited high steady-state hGSTA4 mRNA, high GST-4HNE catalytic activities, but lower basal glutathione (GSH) concentrations relative to insertfree vector (control) cells. Exposure to 4-HNE elicited an increase in GSH concentrations in the control and hGSTA4 cells, although the dose-response of GSH induction differed among the two cell types. Specifically, hGSTA4 cells had significantly higher GSH concentrations when exposed to 5-15 μM 4-HNE, but not at 20 μM 4-HNE, suggesting extensive GSH utilization at high concentrations of 4-HNE. The hGSTA4 cells exhibited a significant growth advantage relative to control cells in the absence of 4-HNE, and a trend towards increased growth at low dose exposures to 4-HNE. However, the hGSTA4 cells did not exhibit a growth advantage relative to control cells at higher 4-HNE exposures associated with increased GSH utilization. As expected, the hGSTA4 cells showed resistance to 4-HNE-stimulated lipid peroxidation at all 4-HNE doses. In summary, our data indicates that over-expression of hGSTA4 at levels conferring high GST-4-HNE conjugating activity confers a partial growth advantage to HepG2 cells and protects against 4-HNE oxidative injury. However, the loss of proliferative capacity of hGSTA4 cells challenged with levels of 4-HNE associated with severe oxidative stress indicates a role of other aldehyde metabolizing enzymes, and/ or GSH-electrophile transporter proteins, in providing full cellular protection against 4-HNE toxicity.

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Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples

Cited by 843 publications

References 8 publications

Antioxidant CR-6 Protects against Reperfusion Injury after a Transient Episode of Focal Brain Ischemia in Rats

Antioxidant CR-6 Protects against Reperfusion Injury after a Transient Episode of Focal Brain Ischemia in Rats

Vanin-1 Pantetheinase Drives Increased Chondrogenic Potential of Mesenchymal Precursors in ank/ank Mice

Transfection of HepG2 cells with hGSTA4 provides protection against 4-hydroxynonenal-mediated oxidative injury

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