Exposure of immature rats to lead acetate results in hemorrhagic encephalopathy of variable evolu... more Exposure of immature rats to lead acetate results in hemorrhagic encephalopathy of variable evolution. As the maintenance of adequate protection against peroxides may be critical in this condition, the activities of selenium-glutathione peroxidase and catalase in cerebrum and cerebellum of suckling rats poisoned with lead acetate were studied from day six to day sixteen post-exposure. Age-related decreases of glutathione peroxidase and catalase activities in both controls and lead poisoned animals were observed. An increase in catalase activity was observed in cerebrum and cerebellum of lead-treated rats compared to controls. Glutathione peroxidase activity did not change significantly in cerebrum over the period studied. By contrast, glutathione peroxidase activity in cerebellum of lead-treated rats remained at about twice the control level over most of the study period. This apparent increase in glutathione peroxidase activity may be due either to a slower ontogenic decrease of its specific activity or to enzyme induction in response to oxidant stress in cerebellum.
Glutathione (gamma-glutamyl-cysteinyl-glycine or GSH) is a cysteine-containing tripeptide with re... more Glutathione (gamma-glutamyl-cysteinyl-glycine or GSH) is a cysteine-containing tripeptide with reducing and nucleophilic properties which play an important role in cellular protection from oxidative damage of lipids, proteins and nucleic acids. GSH regulates the metabolism of proteins and their activities by means of thiol-disulfide exchange. During oxidative stress, GSH plays a key role of protection and detoxification as a cofactor of glutathione peroxidases and glutathione-S-transferases. There are synergistic interactions between GSH and other components of the antioxidant defense system such as vitamin C, vitamin E and superoxide dismutases.
Based on the assumption that glutathione peroxidase (GPx) activity might be limiting in preventin... more Based on the assumption that glutathione peroxidase (GPx) activity might be limiting in preventing peroxide-induced impairment of endothelial regulatory functions, we studied the effect of a series of new selenium-containing GPx mimics on endothelial cells exposed to an inflammatory stress. The two compounds that have the highest GPx activity, BXT-51072 and BXT-51077, were shown to be the most efficient inhibitors
Rats fed a vitamin E-deficient diet from age 3-10 weeks were either maintained on a vitamin E-def... more Rats fed a vitamin E-deficient diet from age 3-10 weeks were either maintained on a vitamin E-deficient diet or fed a vitamin E-enriched diet for 8 subsequent weeks. The content of vitamin E, endoperoxide-derived malonaldehyde, lipofluorescent material and polyunsaturated fatty acids, and the activities of catalase, glutathione reductase, and glutathione peroxidase were then measured in cerebral tissues, with or without intoxication with methyl ethyl ketone peroxide (MEKP). For this purpose, one half of the animals in each vitamin E group received an ip injection of 5 mg MEKP per kg of body weight, which was followed 44 hours later, i.e., 4 hours before sample collection, by a second ip injection of 15 mg MEKP per kg of body weight. Despite the fact that the vitamin E concentration was 12-times lower in the brain of vitamin E-deficient rats, no significant change in other cerebral parameters was found between the two groups of animals. In contrast, the activity of selenium-glutathione peroxidase was markedly decreased in the liver of 10-week old vitamin E-deficient rats. Unexpectedly, acute systemic intoxication with MEKP caused only a small, albeit significant, decrease in glutathione reductase activity in the brain of vitamin E-sufficient rats, while no significant change in other cerebral parameters was observed in either group of animals. These results suggest that the central nervous system (CNS) is still substantially protected when its vitamin E content has been decreased to 3 micrograms/g fresh weight, and that systemic intoxication with MEKP may not cause lipid peroxidation in the CNS.
Archives of Biochemistry and Biophysics, Jul 1, 1992
Selenocystamine (RSe-SeR) was shown to catalyze the oxygen-mediated oxidation of excess GSH to gl... more Selenocystamine (RSe-SeR) was shown to catalyze the oxygen-mediated oxidation of excess GSH to glutathione disulfide, at neutral pH and ambient PO2. This glutathione oxidase activity required the heterolytic reduction of the diselenide bond, which produced two equivalents of the selenolate derivative selenocysteamine (RSe-), via the transient formation of a selenenylsulfide intermediate (RSe-SG). Formation of RSe- was the only reaction observed in anaerobic conditions. At ambient PO2, the kinetics and stoichiometry of GSSG production as well as that of GSH and oxygen consumptions demonstrated that RSe- performed a three-step reduction of oxygen to water. The first step was a one-electron transfer from RSe- to dioxygen, yielding superoxide and a putative selenyl radical RSe., which decayed very rapidly to RSe-SeR. In the second step, RSe- reduced superoxide to hydrogen peroxide through a much faster one-electron transfer, also associated with the decay of RSe. to RSe-SeR. The third step was a two-electron transfer from RSe- to hydrogen peroxide, again much faster than oxygen reduction, which resulted in the production of RSe-SG, presumably via a selenenic acid intermediate (RSeOH) which was trapped by excess GSH. This third step was studied on exogenous hydroperoxide in anaerobic conditions, and it could be eliminated from the glutathione oxidase cycle in the presence of excess catalase. The role of RSe- as a one- and two-electron reductant was confirmed by competitive carboxymethylation with iodoacetate. RSe- was able to rapidly reduce ferric cytochrome c to its ferrous derivative. The overall rate of catalytic glutathione oxidation was GSH concentration dependent and oxygen concentration independent. Excess glutathione reductase and NADPH increased the catalytic oxidation of GSH, probably by switching the rate-limiting step from selenylsulfide to diselenide cleavage. When GSH was substituted for dithiothreitol, it was shown to reduce RSe-SeR to RSe- in a fast and quantitative reaction, and selenocystamine behaved as a dithiothreitol oxidase, whose catalytic cycle was dependent on oxygen concentration. The oxidase cycle of glutathione was inhibited by mercaptosuccinate, while that of dithiothreitol was not affected. When mercaptosuccinate was substituted for GSH, a stable selenenylsulfide was formed. These observations suggest that electrostatic interactions affect the reductive cleavage of diselenide and selenenylsulfide linkages. This study illustrates the ease of one-electron transfers from RSe- to a variety of reducible substrates. Such free radical mechanisms may explain much of the cytotoxicity of alkylselenols, and they demonstrate that selenocystamine is a poor catalytic model of the enzyme glutathione peroxidase.
A new spectrophotometric assay of superoxide dismutase (SOD) activity is described. The assay is ... more A new spectrophotometric assay of superoxide dismutase (SOD) activity is described. The assay is based on the SOD-mediated increase in the rate of autoxidation of 5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzo[c]fluorene (BXT-01050) in aqueous alkaline solution. This autoxidation yields a chromophore with a maximal absorbance wavelength of 525 nm. The optimized assay of SOD activity is performed at pH 8.8, 37 degrees C, in 50 mM air-saturated 2-amino-2-methyl-1,3- propanediol buffer containing 3 mM boric acid and 0.1 mM diethylenetriaminepentaacetic acid. The kinetic measurement of 525-nm absorbance is performed for 1 min upon addition of BXT-01050. BXT-01050 is stabilized in stock solution acidified at pH 1.5. The SOD activity is determined from the Vs/Vc ratio of the autoxidation rates measured in the presence (Vs) and in the absence (Vc) of sample. One SOD activity unit (U-525) has been defined as the activity that doubles the autoxidation background (Vs/Vc = 2). The equation that fits the standard curve is the same with all studied SODs. Another reagent, 1,4,6-trimethyl-2-vinylpyridinium trifluoromethanesulfonate, directly eliminates interference due to sample mercaptans such as glutathione by means of a very fast alkylation reaction. A fast and reproducible measurement of SOD activity requires only a single determination per sample. At pH 8.8, an optimal assay sensitivity is achieved without strongly affecting the activity of known SODs such as Cu/Zn-, Mn-, or Fe-SOD.
ABSTRACT This chapter discusses the nonenzymatic colorimetric assay of glutathione in the presenc... more ABSTRACT This chapter discusses the nonenzymatic colorimetric assay of glutathione in the presence of other mercaptans. Direct assays of total mercaptans are based on chromogenic reactions of sulfhydryl groups with electrophilic reagents. Such assays have obvious limitations in terms of specificity. Other methods are time-consuming and are based on glutathione reductase-coupled assays, with interferences of mixed disulfides of glutathione and enzyme inhibitors or on chromatographic techniques. Reliable chromatographic methods are relatively complex and require expensive materials. Such observations underline the need for faster and easier methodology, especially when large series of biological samples must be processed on a daily basis. The chapter describes a colorimetric procedure that takes advantage of a glutathione (GSH)-specific reaction in the absence of a coupling enzyme. The chapter discusses the advantages and limitations of this methodology. The assay—which is based on the colorimetric measurement of 7-trifluoromethyl-4-thioquinolone at the 400-nm wavelength—has the unique advantage to be glutathione specific in the absence of enzyme. Another advantage is that a colorimetric estimation of total sulfhydryl groups can be obtained at the 356-nm wavelength, before the addition of sodium hydroxide to the mixture of thioethers produced at pH 7.8. The sensitivities of the two assays are very similar and given the stability of chromophoric products, large series of samples can be processed within 20–30 min for subsequent absorbance measurement.
Under specific acidic conditions, both malondialdehyde (1, MDA) and 4-hydroxynonenal (2, 4-HNE) r... more Under specific acidic conditions, both malondialdehyde (1, MDA) and 4-hydroxynonenal (2, 4-HNE) react with N-methyl-2-phenylindole (3) to give the same chromophoric cyanine 4 with maximal absorbance at 586 nm. Under such conditions, the reaction of 3 with 4-HNE (2) as well as with alkanals yields a second chromophoric cyanine 10 with maximal absorbance at 505 nm. The influence of different acids, iron(III), and oxygen on the reaction of 3 with such aldehydes was studied in detail. Under anaerobic conditions, the acid-induced reaction of 4-HNE with 3 afforded three rapidly interconverting intermediates, 5-7. Their subsequent fragmentation to 4 and hexanal in the presence of iron(III) and oxygen is consistent with the tandem beta-fragmentation of an indolyl radical cation. 1-Indolylalkenes were identified as essential intermediates in the acid-induced reaction of 3 with alkanals. A very mild iron(III)-catalyzed fragmentation of these intermediates afforded the corresponding 3-formylindole 11 as the direct precursor of the 505 nm chromophore 10. Such reactions were markedly influenced by the nature of the acid. Contrary to the rapid chromogenic reaction of 4-HNE which was observed in the presence of methanesulfonic acid, the HCl-induced reaction of 4-HNE with 3 did not afford the 586 nm chromophore. Furthermore, hexanal did not yield the 505 nm chromophore 10 upon reaction with 3 in the presence of HCl, again in contrast with the rapid chromogenic reaction which was observed in the presence of methanesulfonic acid. Comparison of the reaction mixtures under the two assay conditions confirmed that the same intermediates were formed. We conclude that the nature of the acid plays a crucial role in the oxidative fragmentation of intermediates into chromophores, allowing the selective assay of MDA in the presence of 4-HNE, using HCl acidic conditions.
New selenium-containing compounds behave as GPx mimics and protect endothelial cells (HUVEC) from... more New selenium-containing compounds behave as GPx mimics and protect endothelial cells (HUVEC) from damage upon exposure to 55 microM linoleic acid hydroperoxide or to 200 microM hydrogen peroxide. The simultaneous presence of the GPx mimic and the hydroperoxyde is not necessary, since a pre-treatment of endothelial monolayers with 1 to 10 microM of such compounds, preserves their morphology, their cell density and their longer-term viability. The compounds which are most efficient in this model of oxidative stress also protect endothelial monolayers which have been incubated with an excess (10:1) of polymorphonuclear neutrophils (PMN) and with 1 ng/ml of TNF-alpha, if such monolayers are pre- and co-treated (10 microM). They inhibit the adhesion of activated neutrophils which show-up as polymorphous and very dense particles, in the vicinity of which endothelial alterations can be seen. The inhibition of leucocyte adhesion and that of endothelial activation/alteration have been quantified by means of immunoassays of myeloperoxidase and von Willebrand factor (vWf). The lead-compound BXT-51072 is not a direct inhibitor of the NADPH oxidase of PMN. TNF-alpha alone induces the endothelial release of Interleukin-8 (Il-8) as well as the expression of P- and E-selectin. The extent and the kinetics of inhibition of such processes by compound BXT-51072 would explain several of the effects observed in the presence of PMN. The GPx mimics also inhibit the endothelial production of Il-8 which is induced by Interleukin-1 alpha. Finally, compound BXT-51072 inhibits the endothelial expression of the adhesion factor VCAM-1 which is more slowly induced by TNF-alpha. Such antioxidant catalysts therefore protect endothelial cells from the toxic effects of TNF-alpha through mechanisms which include a down-regulation of cytokines and cell-adhesion factors.
The role of glutathione peroxidase in the oxidative metabolism and recent advances in the demonst... more The role of glutathione peroxidase in the oxidative metabolism and recent advances in the demonstration of the consequences of the desequilibrium in the proxidant/antioxidant balance on biological molecules oxidation, intracellular signals transduction, apoptosis and necrosis, have led to new approach in the knowledge of many pathological processes. Methods for determining antioxidant capacity have been developed. The measurement of glutathione peroxidase activity is a key step in the study of oxidative stress. Its determination in clinical biology needs optimal conditions for standardised assays which will be used for epidemiological studies aimed to evaluate the role of nutritional factors involved in the pathogeny of diseases caused or accompanied by oxidative stress.
Archives of Biochemistry and Biophysics, Nov 1, 2009
Anthocyanidin reductase from Vitis vinifera catalyzes an NADPH-dependent double reduction of anth... more Anthocyanidin reductase from Vitis vinifera catalyzes an NADPH-dependent double reduction of anthocyanidins. At pH 7.5 and 30 degrees C, steady-state kinetics support a hyperbolic and rapid-equilibrium ordered mechanism, with NADPH binding first, K(M(cyan))=2.82+/-0.66microM and K(i(NADPH))=111+/-23microM. The chromatographic method of Hummel and Dreyer was used for binding-equilibrium studies of NADPH, NADP(+) and catechin, at pH 7. This confirmed hyperbolic binding of NADPH and NADP(+) to the free enzyme, with a single binding site each and with dissociation constants K(NADPH)=45.9+/-2microM and K(NADP+)=83+/-5microM. There was no significant binding of catechin. We conclude (i) that the most likely mechanism is sequential ordered Bi Uni Uni Bi, with NADPH binding first and NADP(+) released last, and (ii) that internal conversion of the first ternary complex, i.e. that associated with the first hydride transfer, is rate-limiting.
Exposure of immature rats to lead acetate results in hemorrhagic encephalopathy of variable evolu... more Exposure of immature rats to lead acetate results in hemorrhagic encephalopathy of variable evolution. As the maintenance of adequate protection against peroxides may be critical in this condition, the activities of selenium-glutathione peroxidase and catalase in cerebrum and cerebellum of suckling rats poisoned with lead acetate were studied from day six to day sixteen post-exposure. Age-related decreases of glutathione peroxidase and catalase activities in both controls and lead poisoned animals were observed. An increase in catalase activity was observed in cerebrum and cerebellum of lead-treated rats compared to controls. Glutathione peroxidase activity did not change significantly in cerebrum over the period studied. By contrast, glutathione peroxidase activity in cerebellum of lead-treated rats remained at about twice the control level over most of the study period. This apparent increase in glutathione peroxidase activity may be due either to a slower ontogenic decrease of its specific activity or to enzyme induction in response to oxidant stress in cerebellum.
Glutathione (gamma-glutamyl-cysteinyl-glycine or GSH) is a cysteine-containing tripeptide with re... more Glutathione (gamma-glutamyl-cysteinyl-glycine or GSH) is a cysteine-containing tripeptide with reducing and nucleophilic properties which play an important role in cellular protection from oxidative damage of lipids, proteins and nucleic acids. GSH regulates the metabolism of proteins and their activities by means of thiol-disulfide exchange. During oxidative stress, GSH plays a key role of protection and detoxification as a cofactor of glutathione peroxidases and glutathione-S-transferases. There are synergistic interactions between GSH and other components of the antioxidant defense system such as vitamin C, vitamin E and superoxide dismutases.
Based on the assumption that glutathione peroxidase (GPx) activity might be limiting in preventin... more Based on the assumption that glutathione peroxidase (GPx) activity might be limiting in preventing peroxide-induced impairment of endothelial regulatory functions, we studied the effect of a series of new selenium-containing GPx mimics on endothelial cells exposed to an inflammatory stress. The two compounds that have the highest GPx activity, BXT-51072 and BXT-51077, were shown to be the most efficient inhibitors
Rats fed a vitamin E-deficient diet from age 3-10 weeks were either maintained on a vitamin E-def... more Rats fed a vitamin E-deficient diet from age 3-10 weeks were either maintained on a vitamin E-deficient diet or fed a vitamin E-enriched diet for 8 subsequent weeks. The content of vitamin E, endoperoxide-derived malonaldehyde, lipofluorescent material and polyunsaturated fatty acids, and the activities of catalase, glutathione reductase, and glutathione peroxidase were then measured in cerebral tissues, with or without intoxication with methyl ethyl ketone peroxide (MEKP). For this purpose, one half of the animals in each vitamin E group received an ip injection of 5 mg MEKP per kg of body weight, which was followed 44 hours later, i.e., 4 hours before sample collection, by a second ip injection of 15 mg MEKP per kg of body weight. Despite the fact that the vitamin E concentration was 12-times lower in the brain of vitamin E-deficient rats, no significant change in other cerebral parameters was found between the two groups of animals. In contrast, the activity of selenium-glutathione peroxidase was markedly decreased in the liver of 10-week old vitamin E-deficient rats. Unexpectedly, acute systemic intoxication with MEKP caused only a small, albeit significant, decrease in glutathione reductase activity in the brain of vitamin E-sufficient rats, while no significant change in other cerebral parameters was observed in either group of animals. These results suggest that the central nervous system (CNS) is still substantially protected when its vitamin E content has been decreased to 3 micrograms/g fresh weight, and that systemic intoxication with MEKP may not cause lipid peroxidation in the CNS.
Archives of Biochemistry and Biophysics, Jul 1, 1992
Selenocystamine (RSe-SeR) was shown to catalyze the oxygen-mediated oxidation of excess GSH to gl... more Selenocystamine (RSe-SeR) was shown to catalyze the oxygen-mediated oxidation of excess GSH to glutathione disulfide, at neutral pH and ambient PO2. This glutathione oxidase activity required the heterolytic reduction of the diselenide bond, which produced two equivalents of the selenolate derivative selenocysteamine (RSe-), via the transient formation of a selenenylsulfide intermediate (RSe-SG). Formation of RSe- was the only reaction observed in anaerobic conditions. At ambient PO2, the kinetics and stoichiometry of GSSG production as well as that of GSH and oxygen consumptions demonstrated that RSe- performed a three-step reduction of oxygen to water. The first step was a one-electron transfer from RSe- to dioxygen, yielding superoxide and a putative selenyl radical RSe., which decayed very rapidly to RSe-SeR. In the second step, RSe- reduced superoxide to hydrogen peroxide through a much faster one-electron transfer, also associated with the decay of RSe. to RSe-SeR. The third step was a two-electron transfer from RSe- to hydrogen peroxide, again much faster than oxygen reduction, which resulted in the production of RSe-SG, presumably via a selenenic acid intermediate (RSeOH) which was trapped by excess GSH. This third step was studied on exogenous hydroperoxide in anaerobic conditions, and it could be eliminated from the glutathione oxidase cycle in the presence of excess catalase. The role of RSe- as a one- and two-electron reductant was confirmed by competitive carboxymethylation with iodoacetate. RSe- was able to rapidly reduce ferric cytochrome c to its ferrous derivative. The overall rate of catalytic glutathione oxidation was GSH concentration dependent and oxygen concentration independent. Excess glutathione reductase and NADPH increased the catalytic oxidation of GSH, probably by switching the rate-limiting step from selenylsulfide to diselenide cleavage. When GSH was substituted for dithiothreitol, it was shown to reduce RSe-SeR to RSe- in a fast and quantitative reaction, and selenocystamine behaved as a dithiothreitol oxidase, whose catalytic cycle was dependent on oxygen concentration. The oxidase cycle of glutathione was inhibited by mercaptosuccinate, while that of dithiothreitol was not affected. When mercaptosuccinate was substituted for GSH, a stable selenenylsulfide was formed. These observations suggest that electrostatic interactions affect the reductive cleavage of diselenide and selenenylsulfide linkages. This study illustrates the ease of one-electron transfers from RSe- to a variety of reducible substrates. Such free radical mechanisms may explain much of the cytotoxicity of alkylselenols, and they demonstrate that selenocystamine is a poor catalytic model of the enzyme glutathione peroxidase.
A new spectrophotometric assay of superoxide dismutase (SOD) activity is described. The assay is ... more A new spectrophotometric assay of superoxide dismutase (SOD) activity is described. The assay is based on the SOD-mediated increase in the rate of autoxidation of 5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzo[c]fluorene (BXT-01050) in aqueous alkaline solution. This autoxidation yields a chromophore with a maximal absorbance wavelength of 525 nm. The optimized assay of SOD activity is performed at pH 8.8, 37 degrees C, in 50 mM air-saturated 2-amino-2-methyl-1,3- propanediol buffer containing 3 mM boric acid and 0.1 mM diethylenetriaminepentaacetic acid. The kinetic measurement of 525-nm absorbance is performed for 1 min upon addition of BXT-01050. BXT-01050 is stabilized in stock solution acidified at pH 1.5. The SOD activity is determined from the Vs/Vc ratio of the autoxidation rates measured in the presence (Vs) and in the absence (Vc) of sample. One SOD activity unit (U-525) has been defined as the activity that doubles the autoxidation background (Vs/Vc = 2). The equation that fits the standard curve is the same with all studied SODs. Another reagent, 1,4,6-trimethyl-2-vinylpyridinium trifluoromethanesulfonate, directly eliminates interference due to sample mercaptans such as glutathione by means of a very fast alkylation reaction. A fast and reproducible measurement of SOD activity requires only a single determination per sample. At pH 8.8, an optimal assay sensitivity is achieved without strongly affecting the activity of known SODs such as Cu/Zn-, Mn-, or Fe-SOD.
ABSTRACT This chapter discusses the nonenzymatic colorimetric assay of glutathione in the presenc... more ABSTRACT This chapter discusses the nonenzymatic colorimetric assay of glutathione in the presence of other mercaptans. Direct assays of total mercaptans are based on chromogenic reactions of sulfhydryl groups with electrophilic reagents. Such assays have obvious limitations in terms of specificity. Other methods are time-consuming and are based on glutathione reductase-coupled assays, with interferences of mixed disulfides of glutathione and enzyme inhibitors or on chromatographic techniques. Reliable chromatographic methods are relatively complex and require expensive materials. Such observations underline the need for faster and easier methodology, especially when large series of biological samples must be processed on a daily basis. The chapter describes a colorimetric procedure that takes advantage of a glutathione (GSH)-specific reaction in the absence of a coupling enzyme. The chapter discusses the advantages and limitations of this methodology. The assay—which is based on the colorimetric measurement of 7-trifluoromethyl-4-thioquinolone at the 400-nm wavelength—has the unique advantage to be glutathione specific in the absence of enzyme. Another advantage is that a colorimetric estimation of total sulfhydryl groups can be obtained at the 356-nm wavelength, before the addition of sodium hydroxide to the mixture of thioethers produced at pH 7.8. The sensitivities of the two assays are very similar and given the stability of chromophoric products, large series of samples can be processed within 20–30 min for subsequent absorbance measurement.
Under specific acidic conditions, both malondialdehyde (1, MDA) and 4-hydroxynonenal (2, 4-HNE) r... more Under specific acidic conditions, both malondialdehyde (1, MDA) and 4-hydroxynonenal (2, 4-HNE) react with N-methyl-2-phenylindole (3) to give the same chromophoric cyanine 4 with maximal absorbance at 586 nm. Under such conditions, the reaction of 3 with 4-HNE (2) as well as with alkanals yields a second chromophoric cyanine 10 with maximal absorbance at 505 nm. The influence of different acids, iron(III), and oxygen on the reaction of 3 with such aldehydes was studied in detail. Under anaerobic conditions, the acid-induced reaction of 4-HNE with 3 afforded three rapidly interconverting intermediates, 5-7. Their subsequent fragmentation to 4 and hexanal in the presence of iron(III) and oxygen is consistent with the tandem beta-fragmentation of an indolyl radical cation. 1-Indolylalkenes were identified as essential intermediates in the acid-induced reaction of 3 with alkanals. A very mild iron(III)-catalyzed fragmentation of these intermediates afforded the corresponding 3-formylindole 11 as the direct precursor of the 505 nm chromophore 10. Such reactions were markedly influenced by the nature of the acid. Contrary to the rapid chromogenic reaction of 4-HNE which was observed in the presence of methanesulfonic acid, the HCl-induced reaction of 4-HNE with 3 did not afford the 586 nm chromophore. Furthermore, hexanal did not yield the 505 nm chromophore 10 upon reaction with 3 in the presence of HCl, again in contrast with the rapid chromogenic reaction which was observed in the presence of methanesulfonic acid. Comparison of the reaction mixtures under the two assay conditions confirmed that the same intermediates were formed. We conclude that the nature of the acid plays a crucial role in the oxidative fragmentation of intermediates into chromophores, allowing the selective assay of MDA in the presence of 4-HNE, using HCl acidic conditions.
New selenium-containing compounds behave as GPx mimics and protect endothelial cells (HUVEC) from... more New selenium-containing compounds behave as GPx mimics and protect endothelial cells (HUVEC) from damage upon exposure to 55 microM linoleic acid hydroperoxide or to 200 microM hydrogen peroxide. The simultaneous presence of the GPx mimic and the hydroperoxyde is not necessary, since a pre-treatment of endothelial monolayers with 1 to 10 microM of such compounds, preserves their morphology, their cell density and their longer-term viability. The compounds which are most efficient in this model of oxidative stress also protect endothelial monolayers which have been incubated with an excess (10:1) of polymorphonuclear neutrophils (PMN) and with 1 ng/ml of TNF-alpha, if such monolayers are pre- and co-treated (10 microM). They inhibit the adhesion of activated neutrophils which show-up as polymorphous and very dense particles, in the vicinity of which endothelial alterations can be seen. The inhibition of leucocyte adhesion and that of endothelial activation/alteration have been quantified by means of immunoassays of myeloperoxidase and von Willebrand factor (vWf). The lead-compound BXT-51072 is not a direct inhibitor of the NADPH oxidase of PMN. TNF-alpha alone induces the endothelial release of Interleukin-8 (Il-8) as well as the expression of P- and E-selectin. The extent and the kinetics of inhibition of such processes by compound BXT-51072 would explain several of the effects observed in the presence of PMN. The GPx mimics also inhibit the endothelial production of Il-8 which is induced by Interleukin-1 alpha. Finally, compound BXT-51072 inhibits the endothelial expression of the adhesion factor VCAM-1 which is more slowly induced by TNF-alpha. Such antioxidant catalysts therefore protect endothelial cells from the toxic effects of TNF-alpha through mechanisms which include a down-regulation of cytokines and cell-adhesion factors.
The role of glutathione peroxidase in the oxidative metabolism and recent advances in the demonst... more The role of glutathione peroxidase in the oxidative metabolism and recent advances in the demonstration of the consequences of the desequilibrium in the proxidant/antioxidant balance on biological molecules oxidation, intracellular signals transduction, apoptosis and necrosis, have led to new approach in the knowledge of many pathological processes. Methods for determining antioxidant capacity have been developed. The measurement of glutathione peroxidase activity is a key step in the study of oxidative stress. Its determination in clinical biology needs optimal conditions for standardised assays which will be used for epidemiological studies aimed to evaluate the role of nutritional factors involved in the pathogeny of diseases caused or accompanied by oxidative stress.
Archives of Biochemistry and Biophysics, Nov 1, 2009
Anthocyanidin reductase from Vitis vinifera catalyzes an NADPH-dependent double reduction of anth... more Anthocyanidin reductase from Vitis vinifera catalyzes an NADPH-dependent double reduction of anthocyanidins. At pH 7.5 and 30 degrees C, steady-state kinetics support a hyperbolic and rapid-equilibrium ordered mechanism, with NADPH binding first, K(M(cyan))=2.82+/-0.66microM and K(i(NADPH))=111+/-23microM. The chromatographic method of Hummel and Dreyer was used for binding-equilibrium studies of NADPH, NADP(+) and catechin, at pH 7. This confirmed hyperbolic binding of NADPH and NADP(+) to the free enzyme, with a single binding site each and with dissociation constants K(NADPH)=45.9+/-2microM and K(NADP+)=83+/-5microM. There was no significant binding of catechin. We conclude (i) that the most likely mechanism is sequential ordered Bi Uni Uni Bi, with NADPH binding first and NADP(+) released last, and (ii) that internal conversion of the first ternary complex, i.e. that associated with the first hydride transfer, is rate-limiting.
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