A fluorometric assay for glutathione

Cohn, Victor; Lyle, Jerry

doi:10.1016/0003-2697(66)90286-7

Cited by 417 publications

(117 citation statements)

References 2 publications

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“…The method employed was that described by Klingenberg [12]. Glutathione content of 1 ml aliquots of the incubates was estimated by the fluorometric method described by Cohn and Lyle [13] using an AmincoBowman model spectrophotofluorometer.…”

Section: Methodsmentioning

confidence: 99%

Lipid Peroxidation in Isolated Hepatocytes

1975

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Intracellular lipid peroxidation was initiated by the addition of ADP-complexed ferric iron to isolated rat hepatocytes and the reaction monitored by the thiobarbituric acid method or by measurement of the formation of conjugated dienes. Both the production of malondialdehyde (thiobarbituricacid-reacting substances) and of conjugated dienes was dependent, on the ADP . Fe3+ concentration in a dose-related fashion. Malondialdehyde formation stopped spontaneously within 20 min after the initiation of the reaction and the plateau reached was also related to the ADP . Fe3+ concentration. Control experiments revealed that more than 90 % of the malondialdehyde accumulating during the incubation period could be ascribed to intracellular production. The cellular NADPH/NADP+ ratio was always high and only slightly decreased upon ADP . Fe3+-induced lipid peroxidation which, however, was associated with a marked decrease in the cellular glutat hione concentration.The rate of accumulation of malondialdehyde as well as the final level reached during ADP . Fe3 +-initiated lipid peroxidation was increased by the addition of chloral hydrate. This apparent stimulatory effect could, however, be ascribed to the inhibition of the mitochondria1 oxidation of the malondialdehyde formed during cellular lipid peroxidation, thus allowing more malondialdehyde to accumulate during the process.ADP . Fe3 +-induced cellular lipid peroxidation was associated with a decrease in the concentration of glutathione. Also, lowering of the intracellular glutathione level by the addition of diethyl maleate or by simply preincubating the hepatocytes (up to 50 min) promoted the ADP . Fe3 + malondialdehyde production and formation of conjugated dienes. Furthermore, when cellular glutathione concentration had been lowered by preincubation of the hepatocytes, significant malondialdehyde production could be observed even at ADP . Fe3+ concentrations which were too low to induce measurable lipid peroxidation in fresh hepatocytes. It is thus concluded that glutathione has an important role in the cell defence against lipid peroxidation and suggested that the isolated hepatocytes provide a suitable experimental model system for the characterization of this and other possible cellular defence mechanisms and how they are affected by the nutritional status of the donor animal.That peroxidation of the polyunsaturated lipids of intracellular membraneous structures can occur and is associated with the hepatotoxicity of several chemicals is well documented [I -41. Heretofore, investigations of lipid peroxidation have employed either isolated cellular organelles (mitochondria, microsomes or lysosomes) or isolated, perfused whole livers, or intact animals. Studies using isolated organelles, while providing an ability to measure the rapid, short-course events associated with lipid peroxidation, suffer from the elimination of potentially protective -~ intracellular systems. The significance of the observations in terms of real impact on the cell is thus open for interpreta...

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Section: Methodsmentioning

confidence: 99%

Lipid Peroxidation in Isolated Hepatocytes

1975

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“…Reduced and oxidized glutathione were measured in albumin-free samples as described by Cohn and Lyle [14]. The amount of cytochrome P-450 was determined as described by Kupfer and Orrenius [15].…”

Section: Methodsmentioning

confidence: 99%

The Consequences of Lipid Peroxidation in Isolated Hepatocytes

Högberg

Moldéus

Orrenius

et al. 1975

European Journal of Biochemistry

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Lipid peroxidation was initiated by the addition of either ADP-complexed Fe3 + or cumene hydroperoxide to isolated rat hepatocytes and the resultant biochemical and morphological alterations investigated. As previously observed with microsomes, malonaldehyde formation was associated with the inactivation of glucose-6-phosphatase. Inhibition of microsomal oxidative drug metabolism was correlated with the release and subsequent inactivation of NADPH-cytochrome c reductase, whereas cytochrome P-450 destruction occurred only in the presence of high concentrations of the organic hydroperoxide which were associated with extensive malonaldehyde formation. Under these conditions there were also marked ultrastructural alterations in the hepatocytes which were not apparent after incubation in the presence ofiron (I 187pM Fe3+). The latter treatment was, however, associated with moderate biochemical effects such as glucose-6-phosphatase inactivation and increased membrane permeability. The cellular defence system against lipid peroxidation is discussed and it is concluded that the isolated liver cell system provides a valuable tool for the study of lipid peroxidation and its pathological implications. Because of its widespread toxic effects lipid peroxidation might have pathological implications as has been proposed in connection with iron toxicity [6] and CCI, toxicity [7]. However, the role of lipid peroxidation during the early stages of cell damage leading to cell necrosis has yet to be established.Isolated liver cells produce malonaldehyde, a metabolite of lipid peroxides, upon exposer to ADP-complexed iron or the organic peroxide cumene hydroEn:ymc.s. Collagenase (EC 3.4.24.3); Hyaluronidase or hyaluronate 4-glycanohydrolase (EC 3.2.1.35); NADPH-cytcchrome c reductase (EC 1.6.2.4); Glucose 6-phosphatase (EC 3.1.3.9).peroxide [8]. Malonaldehyde production, which is readily monitored in this isolated-cell system, is probably catalyzed by the drug-hydroxylating enzyme system located in the endoplasmic reticulum and is thus comparable with the enzyme-dependent lipid peroxidation reactions of isolated microsomes [9,10]. The isolated cell system, however, differs from a microsomal system in being fortified with natural inhibitors, which alter the response to agents that stimulate lipid peroxidation [8,11]. The purpose of this work was to characterize some early alterations in isolated hepatocytes in which malonaldehyde production had been induced. MATERIALS AND METHODSThe cell preparation and incubation techniques were the same as described previously [8,11]. Lipid peroxide formation was initiated by addition of ADPcomplexediron(stocksolution50mMADP + 1.87mM FeC1,) or an aqueous solution of cumene hydroperoxide (stock solution 30 mM). The reaction was followed by the withdrawal of aliquots (0.2 ml) of the whole incubation mixture for spectrophotometric measurements of thiobarbituric-acid-reacting substances at 535 nm (malonaldehyde) [12].

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“…It is based on the reaction of reduced glutathione with o-phthalaldehyde to generate a highly fluorescent adduct that is specific for determination of reduced glutathione in rat tissue at pH 8.0. 41 However, the original protocol developed by Hissin and Hilf requires a large amount of tissue samples. As such, it is not practical to routinely use the original protocol for glutathione determination in cultured neuronal cells especially under the conditions of using small volumes and microgram quantities of samples.…”

Section: Nucleusmentioning

confidence: 99%

Oxidative stress in spinal cord injury and antioxidant-based intervention

Jia

Zhu

Li³

et al. 2011

Spinal Cord

259

176

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Study design: Literature review. Objectives: Spinal cord injury (SCI) remains a major public health issue in developed countries as well as worldwide. The pathophysiology of SCI is characterized by an initial primary injury followed by secondary deterioration. Although the etiology and pathogenesis of SCI remain to be fully understood, it has been suggested that reactive oxygen species (ROS) and oxidative stress have a significant role in the pathophysiology of SCI. Thus, alleviating oxidative stress may be an effective strategy for therapeutic intervention of SCI. The aim of this review was to describe (i) the sources of ROS as well as the major antioxidant defenses with particular attention being paid to lipid peroxidation; (ii) the biomarkers of oxidative stress in SCI and (iii) the neuroprotective effects of various compounds with antioxidative properties in animal models of SCI. Methods: PubMed, one of the most comprehensive biomedical databases, was searched from 1976-2011. All relevant papers were read by title, abstract and full-length article. Results: Oxidative stress is considered a hallmark of injury of SCI. Thus, alleviating oxidative stress may be an effective way of therapeutic intervention of SCI. Two of these agents, the glucocorticoid steroid methylprednisolone and the non-glucocorticoid 21-aminosteroid tirilazad, have been shown to possess significant antioxidant activities and improve recovery of SCI patients in clinical trials. Other promising botanical compounds and their molecular targets and mechanisms of action with regard to potential protection against SCI were also described. These include carotenoids and phenolic compounds. Conclusion: ROS and oxidative stress have a significant role in the pathophysiology of SCI. Alleviating oxidative stress is be an effective strategy for therapeutic intervention of SCI. Extensive research over the past several decades has identified numerous bioactive compounds that have antioxidative stress benefits in animal models of SCI. Thus, continued studies on bioactive compounds with ROS-scavenging capacity may lead to the development of effective antioxidant-based modalities for treating SCI in human subjects.

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A fluorometric assay for glutathione

Cited by 417 publications

References 2 publications

Lipid Peroxidation in Isolated Hepatocytes

Lipid Peroxidation in Isolated Hepatocytes

The Consequences of Lipid Peroxidation in Isolated Hepatocytes

Oxidative stress in spinal cord injury and antioxidant-based intervention

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