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Formation of a hydroperoxide during oxidation of urate by MPO in the presence of superoxide. A, urate (200 M ) was incubated with MPO (150 n M ) and xanthine oxidase ( XO ) in 50 m M phosphate buffer, pH 7.4.
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Urate and myeloperoxidase (MPO) are associated with adverse outcomes in cardiovascular disease. In this study, we assessed whether urate is a likely physiological substrate for MPO and if the products of their interaction have the potential to exacerbate inflammation. Urate was readily oxidized by MPO and hydrogen peroxide to 5-hydroxyisourate, whi...
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... B ). The observed first order rate constant for formation of compound II showed a linear dependence with respect to the concentration of urate and had a positive intercept on the y axis (Fig. 2 C ). The line of best fit revealed a slope of 4.6 Ϯ 0.4 ϫ 10 5 M Ϫ 1 s Ϫ 1 and an intercept of 5.8 Ϯ 0.5 s Ϫ 1 . This intercept corresponds to reduction of compound I by reactants other than urate. The reaction of urate with compound II was studied using the same conditions for compound I but was measured over a longer time scale (20 –200 s). Urate promoted the conversion of compound II to the ferric enzyme (Soret maximum 430 nm) (Fig. 3 A ). The clear isosbestic point at 441 nm suggests that no other intermediates of MPO were involved in this reaction. The decay in absorption of compound II at 456 nm was found to be sigmoidal in shape (Fig. 3 B ). The final section of the curve represents the reduction of compound II back to the ferric enzyme. It could be fitted by a single exponential decay as described previously (50). The first order rate constants obtained in this way were plotted against the initial concentration of urate (Fig. 3 C ). The slope of the line provides the second order rate constant for the reaction of urate with compound II to produce ferric MPO and has a value of 1.7 Ϯ 0.1 ϫ 10 4 M Ϫ 1 s Ϫ 1 . The intercept corresponds to the spontaneous decay of compound II and was found to have a value of 0.017 s Ϫ 1 . Chloride and Urate as Competing Substrates for MPO — Chloride is a major physiological substrate for MPO (22). Therefore, we determined how chloride and urate affected each other’s oxidation by MPO. The effect of urate on the chlorination activity of MPO was determined by measuring the initial rate of hydrogen peroxide consumption by the enzyme in the presence of 100 m M chloride and varying concentrations of urate (Fig. 4 A ). Urate decreased the initial rate of hydrogen peroxide consumption markedly, inhibiting by 50% (IC 50 ) at a concentration of only 9.3 M . However, it inhibited by a maximum of only 75% over the physiological range of urate concentrations. We also measured production of allantoin by MPO and determined how it was affected by chloride. Methionine was included in the buffer to scavenge hypochlorous acid and prevent it from reacting with urate to produce allantoin (51). When 200 M urate was oxidized by MPO and 50 M hydrogen peroxide in the absence of chloride, 20 M allantoin was produced over 1 h (Fig. 4 B ). Chloride inhibited formation of allantoin in a concentration-dependent manner, having an IC 50 of 9.6 m M . In combination, these results establish chloride and urate as competing substrates of MPO. They also indicate that at normal physiological concentrations of chloride and urate, hypochlorous acid would be the major product formed by the enzyme, and urate would be expected to be a minor substrate. Effects of Glutathione on the Oxidation of Urate by MPO —To provide additional evidence that the urate is oxidized by the classical peroxidation cycle to produce radical intermediates, glutathione (GSH) was added to the reaction system. This cys- teinyl tripeptide is known to reduce radicals generated by peroxidases and is converted to oxidized glutathione (GSSG) (52). GSH markedly slowed the oxidation of urate as assessed by monitoring the loss in absorbance at 283 nm (supplemental Fig. S4). We also determined whether the oxidation of urate by MPO could promote the consumption of GSH (Fig. 5). With 1 m M GSH, there was a small amount of oxidation of GSH by MPO or urate alone. In combination, urate and MPO promoted super-stoichiometric oxidation of GSH, i.e. with 50 M hydrogen peroxide, MPO and urate promoted the oxidation of ϳ 150 M GSH. Most of the GSH that was oxidized was accounted for by the formation of GSSG. Even greater loss of GSH occurred with tyrosine as the reducing substrate for MPO as found previously (52). This result demonstrates that, like tyrosyl radical, urate radical promotes a chain reaction with GSH, resulting in formation of hydrogen peroxide that fuels further oxidation of GSH. Oxidation of Urate in the Presence of Superoxide —Urate radicals react with superoxide at diffusion-controlled rates (53). The product of this reaction is not known. Electron transfer from superoxide to urate radical would regenerate urate. Alter- natively, addition of superoxide would produce urate hydroperoxide as occurs with tyrosine, serotonin, and melatonin (54 –57). To determine whether urate is converted to a hydroperoxide by superoxide, we used MPO to oxidize urate, whereas superoxide and hydrogen peroxide were generated with xanthine oxidase. Hydroperoxides were detected using ferrous iron-catalyzed oxidation of xylenol orange (FOX assay) (46). The complete reaction system generated substantial levels of hydroperoxides (Fig. 6, A and B ). Their formation was blocked by omitting one of MPO, urate, xanthine oxidase, or acetaldehyde from the reaction system (Fig. 6 A ). Superoxide dismutase decreased hydroperoxide formation to control levels, and catalase inhibited by ϳ 50%. Incomplete inhibition by catalase most likely reflects competition of MPO and catalase for hydrogen peroxide. Production of hydroperoxides increased with increasing concentrations of urate to a maximum at 200 M urate (Fig. 6 B ). Superoxide dismutase inhibited hydroperoxide formation at all concentrations of urate indicat- ing the essential role of superoxide in their formation. To confirm that the hydroperoxide detected by the FOX assay was urate hydroperoxide, we used LC/MS with selected ion monitoring to identify [M Ϫ H] Ϫ ions with an m / z value of 199, i.e. 32 mass units greater than for urate ([M Ϫ H] Ϫ m / z 167). Two products with the expected mass were detected in the reaction mixture, and their formation was completely blocked by superoxide dismutase (Fig. 6 C ). These are likely to be isomers of urate hydroperoxide. The hydroperoxide detected by the FOX assay was reactive because it was eliminated by glutathione. In contrast, hydrogen peroxide reacted slowly with glutathione (Fig. 6 D ). Urate-dependent Consumption of Nitric Oxide by MPO — MPO can modulate vascular inflammatory responses by oxidiz- ing NO directly or via generation of radical intermediates that react with NO (32, 33). Therefore, we also tested the possibility that urate radicals generated by MPO react with nitric oxide (NO) in a similar fashion to tyrosyl radicals (32, 33). NO was generated by the NO donor NOC-9. The combination of MPO and hydrogen peroxide alone promoted consumption of NO, but this was enhanced by the addition of urate (Fig. 7 A ). Urate alone and in combination with either MPO (data not shown) or hydrogen peroxide (Fig. 7 A ) did not promote consumption of NO (data not shown). The consumption of NO was dependent on the concentration of urate over its physiological range (Fig. 7 B ). Urate was comparable with tyrosine in promoting consumption of NO. From these results, we conclude that urate promotes the NO oxidase activity of MPO. Production of Allantoin by Neutrophils in Plasma —To assess the potential for MPO to oxidize urate in vivo , we added isolated neutrophils to plasma. The cells were then stimulated with phorbol myristate acetate in the presence of cytochalasin B to promote degranulation and release of MPO. The formation of allantoin was followed using LC/MS. After a lag of ϳ 10 min, the concentration of allantoin began to increase over the 1-h incubation period (Fig. 8 A ). Formation of allantoin required stimulation of the cells and was inhibited by diphenyliodonium, which blocks the activity of the NADPH oxidase (Fig. 8 B ). Allantoin formation was also retarded by the heme poison azide as well as catalase and thiocyanate, which is a substrate for MPO. Superoxide dismutase inhibited formation of allantoin, which indicates that superoxide was involved in the oxidation of urate. Collectively, these results demonstrate that activated neutrophils oxidize urate in plasma in reactions that involve the NADPH oxidase, MPO and superoxide. We have demonstrated that urate should be considered as a physiological substrate for MPO that competes with chloride for oxidation and is converted to urate radicals. The fate of these radicals has considerable potential to influence oxidative stress during inflammation. Reaction with superoxide will produce a hydroperoxide, which will propagate oxidative reactions of both the constituent radicals. Reaction with NO will decrease the availability of this vasodilator and increase hypertension and endothelial dysfunction (58). When scavenged by thiols, such as glutathione and cysteine, urate radicals will promote chain reactions and also exacerbate oxidative stress. Even their dismutation to dehydrourate may be harmful because this species hydrolyzes to 5-hydroxyisourate (Scheme 2). This metab- olite, which was produced by MPO, has recently been shown to be associated with development of liver tumors in mice (59). Adverse reactions of urate radicals will be kept in check only when ascorbate is available at sufficient concentrations to reduce them back to urate (60, 61). Given the associations of both urate and MPO with numerous inflammatory diseases and their co-localization, our work indicates that interactions between the two should be considered when assessing the role of either in inflammation. Two previous studies have identified urate as a substrate for MPO. Originally, MPO was shown to oxidize urate to allantoin (19), and it was found that urate reacts favorably with compound II (62). Until our current work, however, the potential of urate to act as a physiological substrate for MPO had not been addressed. MPO is an unusual enzyme in that it oxidizes numerous substrates via a variety of mechanisms (22). In vivo, its dominant activity is the two-electron oxidation of chloride and thiocyanate (63). Bromide is also oxidized by this route in vivo ...
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... of compound I by reactants other than urate. The reaction of urate with compound II was studied using the same conditions for compound I but was measured over a longer time scale (20 –200 s). Urate promoted the conversion of compound II to the ferric enzyme (Soret maximum 430 nm) (Fig. 3 A ). The clear isosbestic point at 441 nm suggests that no other intermediates of MPO were involved in this reaction. The decay in absorption of compound II at 456 nm was found to be sigmoidal in shape (Fig. 3 B ). The final section of the curve represents the reduction of compound II back to the ferric enzyme. It could be fitted by a single exponential decay as described previously (50). The first order rate constants obtained in this way were plotted against the initial concentration of urate (Fig. 3 C ). The slope of the line provides the second order rate constant for the reaction of urate with compound II to produce ferric MPO and has a value of 1.7 Ϯ 0.1 ϫ 10 4 M Ϫ 1 s Ϫ 1 . The intercept corresponds to the spontaneous decay of compound II and was found to have a value of 0.017 s Ϫ 1 . Chloride and Urate as Competing Substrates for MPO — Chloride is a major physiological substrate for MPO (22). Therefore, we determined how chloride and urate affected each other’s oxidation by MPO. The effect of urate on the chlorination activity of MPO was determined by measuring the initial rate of hydrogen peroxide consumption by the enzyme in the presence of 100 m M chloride and varying concentrations of urate (Fig. 4 A ). Urate decreased the initial rate of hydrogen peroxide consumption markedly, inhibiting by 50% (IC 50 ) at a concentration of only 9.3 M . However, it inhibited by a maximum of only 75% over the physiological range of urate concentrations. We also measured production of allantoin by MPO and determined how it was affected by chloride. Methionine was included in the buffer to scavenge hypochlorous acid and prevent it from reacting with urate to produce allantoin (51). When 200 M urate was oxidized by MPO and 50 M hydrogen peroxide in the absence of chloride, 20 M allantoin was produced over 1 h (Fig. 4 B ). Chloride inhibited formation of allantoin in a concentration-dependent manner, having an IC 50 of 9.6 m M . In combination, these results establish chloride and urate as competing substrates of MPO. They also indicate that at normal physiological concentrations of chloride and urate, hypochlorous acid would be the major product formed by the enzyme, and urate would be expected to be a minor substrate. Effects of Glutathione on the Oxidation of Urate by MPO —To provide additional evidence that the urate is oxidized by the classical peroxidation cycle to produce radical intermediates, glutathione (GSH) was added to the reaction system. This cys- teinyl tripeptide is known to reduce radicals generated by peroxidases and is converted to oxidized glutathione (GSSG) (52). GSH markedly slowed the oxidation of urate as assessed by monitoring the loss in absorbance at 283 nm (supplemental Fig. S4). We also determined whether the oxidation of urate by MPO could promote the consumption of GSH (Fig. 5). With 1 m M GSH, there was a small amount of oxidation of GSH by MPO or urate alone. In combination, urate and MPO promoted super-stoichiometric oxidation of GSH, i.e. with 50 M hydrogen peroxide, MPO and urate promoted the oxidation of ϳ 150 M GSH. Most of the GSH that was oxidized was accounted for by the formation of GSSG. Even greater loss of GSH occurred with tyrosine as the reducing substrate for MPO as found previously (52). This result demonstrates that, like tyrosyl radical, urate radical promotes a chain reaction with GSH, resulting in formation of hydrogen peroxide that fuels further oxidation of GSH. Oxidation of Urate in the Presence of Superoxide —Urate radicals react with superoxide at diffusion-controlled rates (53). The product of this reaction is not known. Electron transfer from superoxide to urate radical would regenerate urate. Alter- natively, addition of superoxide would produce urate hydroperoxide as occurs with tyrosine, serotonin, and melatonin (54 –57). To determine whether urate is converted to a hydroperoxide by superoxide, we used MPO to oxidize urate, whereas superoxide and hydrogen peroxide were generated with xanthine oxidase. Hydroperoxides were detected using ferrous iron-catalyzed oxidation of xylenol orange (FOX assay) (46). The complete reaction system generated substantial levels of hydroperoxides (Fig. 6, A and B ). Their formation was blocked by omitting one of MPO, urate, xanthine oxidase, or acetaldehyde from the reaction system (Fig. 6 A ). Superoxide dismutase decreased hydroperoxide formation to control levels, and catalase inhibited by ϳ 50%. Incomplete inhibition by catalase most likely reflects competition of MPO and catalase for hydrogen peroxide. Production of hydroperoxides increased with increasing concentrations of urate to a maximum at 200 M urate (Fig. 6 B ). Superoxide dismutase inhibited hydroperoxide formation at all concentrations of urate indicat- ing the essential role of superoxide in their formation. To confirm that the hydroperoxide detected by the FOX assay was urate hydroperoxide, we used LC/MS with selected ion monitoring to identify [M Ϫ H] Ϫ ions with an m / z value of 199, i.e. 32 mass units greater than for urate ([M Ϫ H] Ϫ m / z 167). Two products with the expected mass were detected in the reaction mixture, and their formation was completely blocked by superoxide dismutase (Fig. 6 C ). These are likely to be isomers of urate hydroperoxide. The hydroperoxide detected by the FOX assay was reactive because it was eliminated by glutathione. In contrast, hydrogen peroxide reacted slowly with glutathione (Fig. 6 D ). Urate-dependent Consumption of Nitric Oxide by MPO — MPO can modulate vascular inflammatory responses by oxidiz- ing NO directly or via generation of radical intermediates that react with NO (32, 33). Therefore, we also tested the possibility that urate radicals generated by MPO react with nitric oxide (NO) in a similar fashion to tyrosyl radicals (32, 33). NO was generated by the NO donor NOC-9. The combination of MPO and hydrogen peroxide alone promoted consumption of NO, but this was enhanced by the addition of urate (Fig. 7 A ). Urate alone and in combination with either MPO (data not shown) or hydrogen peroxide (Fig. 7 A ) did not promote consumption of NO (data not shown). The consumption of NO was dependent on the concentration of urate over its physiological range (Fig. 7 B ). Urate was comparable with tyrosine in promoting consumption of NO. From these results, we conclude that urate promotes the NO oxidase activity of MPO. Production of Allantoin by Neutrophils in Plasma —To assess the potential for MPO to oxidize urate in vivo , we added isolated neutrophils to plasma. The cells were then stimulated with phorbol myristate acetate in the presence of cytochalasin B to promote degranulation and release of MPO. The formation of allantoin was followed using LC/MS. After a lag of ϳ 10 min, the concentration of allantoin began to increase over the 1-h incubation period (Fig. 8 A ). Formation of allantoin required stimulation of the cells and was inhibited by diphenyliodonium, which blocks the activity of the NADPH oxidase (Fig. 8 B ). Allantoin formation was also retarded by the heme poison azide as well as catalase and thiocyanate, which is a substrate for MPO. Superoxide dismutase inhibited formation of allantoin, which indicates that superoxide was involved in the oxidation of urate. Collectively, these results demonstrate that activated neutrophils oxidize urate in plasma in reactions that involve the NADPH oxidase, MPO and superoxide. We have demonstrated that urate should be considered as a physiological substrate for MPO that competes with chloride for oxidation and is converted to urate radicals. The fate of these radicals has considerable potential to influence oxidative stress during inflammation. Reaction with superoxide will produce a hydroperoxide, which will propagate oxidative reactions of both the constituent radicals. Reaction with NO will decrease the availability of this vasodilator and increase hypertension and endothelial dysfunction (58). When scavenged by thiols, such as glutathione and cysteine, urate radicals will promote chain reactions and also exacerbate oxidative stress. Even their dismutation to dehydrourate may be harmful because this species hydrolyzes to 5-hydroxyisourate (Scheme 2). This metab- olite, which was produced by MPO, has recently been shown to be associated with development of liver tumors in mice (59). Adverse reactions of urate radicals will be kept in check only when ascorbate is available at sufficient concentrations to reduce them back to urate (60, 61). Given the associations of both urate and MPO with numerous inflammatory diseases and their co-localization, our work indicates that interactions between the two should be considered when assessing the role of either in inflammation. Two previous studies have identified urate as a substrate for MPO. Originally, MPO was shown to oxidize urate to allantoin (19), and it was found that urate reacts favorably with compound II (62). Until our current work, however, the potential of urate to act as a physiological substrate for MPO had not been addressed. MPO is an unusual enzyme in that it oxidizes numerous substrates via a variety of mechanisms (22). In vivo, its dominant activity is the two-electron oxidation of chloride and thiocyanate (63). Bromide is also oxidized by this route in vivo (64). These substrates reduce compound I of MPO to produce the respective hypohalous acids and regenerate the native ferric enzyme (Scheme 1). MPO also acts as a classical peroxidase where the substrate reduces compound I to compound II, which is in turn reduced back to the native ferric enzyme (Scheme 1). Each step involves a ...
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... of urate (Fig. 3 C ). The slope of the line provides the second order rate constant for the reaction of urate with compound II to produce ferric MPO and has a value of 1.7 Ϯ 0.1 ϫ 10 4 M Ϫ 1 s Ϫ 1 . The intercept corresponds to the spontaneous decay of compound II and was found to have a value of 0.017 s Ϫ 1 . Chloride and Urate as Competing Substrates for MPO — Chloride is a major physiological substrate for MPO (22). Therefore, we determined how chloride and urate affected each other’s oxidation by MPO. The effect of urate on the chlorination activity of MPO was determined by measuring the initial rate of hydrogen peroxide consumption by the enzyme in the presence of 100 m M chloride and varying concentrations of urate (Fig. 4 A ). Urate decreased the initial rate of hydrogen peroxide consumption markedly, inhibiting by 50% (IC 50 ) at a concentration of only 9.3 M . However, it inhibited by a maximum of only 75% over the physiological range of urate concentrations. We also measured production of allantoin by MPO and determined how it was affected by chloride. Methionine was included in the buffer to scavenge hypochlorous acid and prevent it from reacting with urate to produce allantoin (51). When 200 M urate was oxidized by MPO and 50 M hydrogen peroxide in the absence of chloride, 20 M allantoin was produced over 1 h (Fig. 4 B ). Chloride inhibited formation of allantoin in a concentration-dependent manner, having an IC 50 of 9.6 m M . In combination, these results establish chloride and urate as competing substrates of MPO. They also indicate that at normal physiological concentrations of chloride and urate, hypochlorous acid would be the major product formed by the enzyme, and urate would be expected to be a minor substrate. Effects of Glutathione on the Oxidation of Urate by MPO —To provide additional evidence that the urate is oxidized by the classical peroxidation cycle to produce radical intermediates, glutathione (GSH) was added to the reaction system. This cys- teinyl tripeptide is known to reduce radicals generated by peroxidases and is converted to oxidized glutathione (GSSG) (52). GSH markedly slowed the oxidation of urate as assessed by monitoring the loss in absorbance at 283 nm (supplemental Fig. S4). We also determined whether the oxidation of urate by MPO could promote the consumption of GSH (Fig. 5). With 1 m M GSH, there was a small amount of oxidation of GSH by MPO or urate alone. In combination, urate and MPO promoted super-stoichiometric oxidation of GSH, i.e. with 50 M hydrogen peroxide, MPO and urate promoted the oxidation of ϳ 150 M GSH. Most of the GSH that was oxidized was accounted for by the formation of GSSG. Even greater loss of GSH occurred with tyrosine as the reducing substrate for MPO as found previously (52). This result demonstrates that, like tyrosyl radical, urate radical promotes a chain reaction with GSH, resulting in formation of hydrogen peroxide that fuels further oxidation of GSH. Oxidation of Urate in the Presence of Superoxide —Urate radicals react with superoxide at diffusion-controlled rates (53). The product of this reaction is not known. Electron transfer from superoxide to urate radical would regenerate urate. Alter- natively, addition of superoxide would produce urate hydroperoxide as occurs with tyrosine, serotonin, and melatonin (54 –57). To determine whether urate is converted to a hydroperoxide by superoxide, we used MPO to oxidize urate, whereas superoxide and hydrogen peroxide were generated with xanthine oxidase. Hydroperoxides were detected using ferrous iron-catalyzed oxidation of xylenol orange (FOX assay) (46). The complete reaction system generated substantial levels of hydroperoxides (Fig. 6, A and B ). Their formation was blocked by omitting one of MPO, urate, xanthine oxidase, or acetaldehyde from the reaction system (Fig. 6 A ). Superoxide dismutase decreased hydroperoxide formation to control levels, and catalase inhibited by ϳ 50%. Incomplete inhibition by catalase most likely reflects competition of MPO and catalase for hydrogen peroxide. Production of hydroperoxides increased with increasing concentrations of urate to a maximum at 200 M urate (Fig. 6 B ). Superoxide dismutase inhibited hydroperoxide formation at all concentrations of urate indicat- ing the essential role of superoxide in their formation. To confirm that the hydroperoxide detected by the FOX assay was urate hydroperoxide, we used LC/MS with selected ion monitoring to identify [M Ϫ H] Ϫ ions with an m / z value of 199, i.e. 32 mass units greater than for urate ([M Ϫ H] Ϫ m / z 167). Two products with the expected mass were detected in the reaction mixture, and their formation was completely blocked by superoxide dismutase (Fig. 6 C ). These are likely to be isomers of urate hydroperoxide. The hydroperoxide detected by the FOX assay was reactive because it was eliminated by glutathione. In contrast, hydrogen peroxide reacted slowly with glutathione (Fig. 6 D ). Urate-dependent Consumption of Nitric Oxide by MPO — MPO can modulate vascular inflammatory responses by oxidiz- ing NO directly or via generation of radical intermediates that react with NO (32, 33). Therefore, we also tested the possibility that urate radicals generated by MPO react with nitric oxide (NO) in a similar fashion to tyrosyl radicals (32, 33). NO was generated by the NO donor NOC-9. The combination of MPO and hydrogen peroxide alone promoted consumption of NO, but this was enhanced by the addition of urate (Fig. 7 A ). Urate alone and in combination with either MPO (data not shown) or hydrogen peroxide (Fig. 7 A ) did not promote consumption of NO (data not shown). The consumption of NO was dependent on the concentration of urate over its physiological range (Fig. 7 B ). Urate was comparable with tyrosine in promoting consumption of NO. From these results, we conclude that urate promotes the NO oxidase activity of MPO. Production of Allantoin by Neutrophils in Plasma —To assess the potential for MPO to oxidize urate in vivo , we added isolated neutrophils to plasma. The cells were then stimulated with phorbol myristate acetate in the presence of cytochalasin B to promote degranulation and release of MPO. The formation of allantoin was followed using LC/MS. After a lag of ϳ 10 min, the concentration of allantoin began to increase over the 1-h incubation period (Fig. 8 A ). Formation of allantoin required stimulation of the cells and was inhibited by diphenyliodonium, which blocks the activity of the NADPH oxidase (Fig. 8 B ). Allantoin formation was also retarded by the heme poison azide as well as catalase and thiocyanate, which is a substrate for MPO. Superoxide dismutase inhibited formation of allantoin, which indicates that superoxide was involved in the oxidation of urate. Collectively, these results demonstrate that activated neutrophils oxidize urate in plasma in reactions that involve the NADPH oxidase, MPO and superoxide. We have demonstrated that urate should be considered as a physiological substrate for MPO that competes with chloride for oxidation and is converted to urate radicals. The fate of these radicals has considerable potential to influence oxidative stress during inflammation. Reaction with superoxide will produce a hydroperoxide, which will propagate oxidative reactions of both the constituent radicals. Reaction with NO will decrease the availability of this vasodilator and increase hypertension and endothelial dysfunction (58). When scavenged by thiols, such as glutathione and cysteine, urate radicals will promote chain reactions and also exacerbate oxidative stress. Even their dismutation to dehydrourate may be harmful because this species hydrolyzes to 5-hydroxyisourate (Scheme 2). This metab- olite, which was produced by MPO, has recently been shown to be associated with development of liver tumors in mice (59). Adverse reactions of urate radicals will be kept in check only when ascorbate is available at sufficient concentrations to reduce them back to urate (60, 61). Given the associations of both urate and MPO with numerous inflammatory diseases and their co-localization, our work indicates that interactions between the two should be considered when assessing the role of either in inflammation. Two previous studies have identified urate as a substrate for MPO. Originally, MPO was shown to oxidize urate to allantoin (19), and it was found that urate reacts favorably with compound II (62). Until our current work, however, the potential of urate to act as a physiological substrate for MPO had not been addressed. MPO is an unusual enzyme in that it oxidizes numerous substrates via a variety of mechanisms (22). In vivo, its dominant activity is the two-electron oxidation of chloride and thiocyanate (63). Bromide is also oxidized by this route in vivo (64). These substrates reduce compound I of MPO to produce the respective hypohalous acids and regenerate the native ferric enzyme (Scheme 1). MPO also acts as a classical peroxidase where the substrate reduces compound I to compound II, which is in turn reduced back to the native ferric enzyme (Scheme 1). Each step involves a one-electron reduction of the enzyme intermediates and is accompanied by the liber- ation of a substrate free radical. Peroxidation substrates include ascorbate, tyrosine, and serotonin as well as exoge- nous phenolics and aromatic amines (56, 65– 67). Collectively, our results indicate that urate is also oxidized by the classical peroxidase cycle shown in Scheme 1, i.e. compound II was the redox intermediate of the enzyme present during oxidation of urate, and urate readily reduced compounds I and II. Also, oxidation of urate promoted super-stoichiometric oxidation of GSH in a similar fashion to tyrosine. The urate and tyrosyl radical would oxidize glutathione to a radical, which then undergoes a chain reaction in which oxygen is consumed and reduced to hydrogen peroxide via the ...
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... to produce pseudo first-order conditions, and kinetic traces were collected at 456 nm to follow the appearance of compound II (Fig. 2 B ). The observed first order rate constant for formation of compound II showed a linear dependence with respect to the concentration of urate and had a positive intercept on the y axis (Fig. 2 C ). The line of best fit revealed a slope of 4.6 Ϯ 0.4 ϫ 10 5 M Ϫ 1 s Ϫ 1 and an intercept of 5.8 Ϯ 0.5 s Ϫ 1 . This intercept corresponds to reduction of compound I by reactants other than urate. The reaction of urate with compound II was studied using the same conditions for compound I but was measured over a longer time scale (20 –200 s). Urate promoted the conversion of compound II to the ferric enzyme (Soret maximum 430 nm) (Fig. 3 A ). The clear isosbestic point at 441 nm suggests that no other intermediates of MPO were involved in this reaction. The decay in absorption of compound II at 456 nm was found to be sigmoidal in shape (Fig. 3 B ). The final section of the curve represents the reduction of compound II back to the ferric enzyme. It could be fitted by a single exponential decay as described previously (50). The first order rate constants obtained in this way were plotted against the initial concentration of urate (Fig. 3 C ). The slope of the line provides the second order rate constant for the reaction of urate with compound II to produce ferric MPO and has a value of 1.7 Ϯ 0.1 ϫ 10 4 M Ϫ 1 s Ϫ 1 . The intercept corresponds to the spontaneous decay of compound II and was found to have a value of 0.017 s Ϫ 1 . Chloride and Urate as Competing Substrates for MPO — Chloride is a major physiological substrate for MPO (22). Therefore, we determined how chloride and urate affected each other’s oxidation by MPO. The effect of urate on the chlorination activity of MPO was determined by measuring the initial rate of hydrogen peroxide consumption by the enzyme in the presence of 100 m M chloride and varying concentrations of urate (Fig. 4 A ). Urate decreased the initial rate of hydrogen peroxide consumption markedly, inhibiting by 50% (IC 50 ) at a concentration of only 9.3 M . However, it inhibited by a maximum of only 75% over the physiological range of urate concentrations. We also measured production of allantoin by MPO and determined how it was affected by chloride. Methionine was included in the buffer to scavenge hypochlorous acid and prevent it from reacting with urate to produce allantoin (51). When 200 M urate was oxidized by MPO and 50 M hydrogen peroxide in the absence of chloride, 20 M allantoin was produced over 1 h (Fig. 4 B ). Chloride inhibited formation of allantoin in a concentration-dependent manner, having an IC 50 of 9.6 m M . In combination, these results establish chloride and urate as competing substrates of MPO. They also indicate that at normal physiological concentrations of chloride and urate, hypochlorous acid would be the major product formed by the enzyme, and urate would be expected to be a minor substrate. Effects of Glutathione on the Oxidation of Urate by MPO —To provide additional evidence that the urate is oxidized by the classical peroxidation cycle to produce radical intermediates, glutathione (GSH) was added to the reaction system. This cys- teinyl tripeptide is known to reduce radicals generated by peroxidases and is converted to oxidized glutathione (GSSG) (52). GSH markedly slowed the oxidation of urate as assessed by monitoring the loss in absorbance at 283 nm (supplemental Fig. S4). We also determined whether the oxidation of urate by MPO could promote the consumption of GSH (Fig. 5). With 1 m M GSH, there was a small amount of oxidation of GSH by MPO or urate alone. In combination, urate and MPO promoted super-stoichiometric oxidation of GSH, i.e. with 50 M hydrogen peroxide, MPO and urate promoted the oxidation of ϳ 150 M GSH. Most of the GSH that was oxidized was accounted for by the formation of GSSG. Even greater loss of GSH occurred with tyrosine as the reducing substrate for MPO as found previously (52). This result demonstrates that, like tyrosyl radical, urate radical promotes a chain reaction with GSH, resulting in formation of hydrogen peroxide that fuels further oxidation of GSH. Oxidation of Urate in the Presence of Superoxide —Urate radicals react with superoxide at diffusion-controlled rates (53). The product of this reaction is not known. Electron transfer from superoxide to urate radical would regenerate urate. Alter- natively, addition of superoxide would produce urate hydroperoxide as occurs with tyrosine, serotonin, and melatonin (54 –57). To determine whether urate is converted to a hydroperoxide by superoxide, we used MPO to oxidize urate, whereas superoxide and hydrogen peroxide were generated with xanthine oxidase. Hydroperoxides were detected using ferrous iron-catalyzed oxidation of xylenol orange (FOX assay) (46). The complete reaction system generated substantial levels of hydroperoxides (Fig. 6, A and B ). Their formation was blocked by omitting one of MPO, urate, xanthine oxidase, or acetaldehyde from the reaction system (Fig. 6 A ). Superoxide dismutase decreased hydroperoxide formation to control levels, and catalase inhibited by ϳ 50%. Incomplete inhibition by catalase most likely reflects competition of MPO and catalase for hydrogen peroxide. Production of hydroperoxides increased with increasing concentrations of urate to a maximum at 200 M urate (Fig. 6 B ). Superoxide dismutase inhibited hydroperoxide formation at all concentrations of urate indicat- ing the essential role of superoxide in their formation. To confirm that the hydroperoxide detected by the FOX assay was urate hydroperoxide, we used LC/MS with selected ion monitoring to identify [M Ϫ H] Ϫ ions with an m / z value of 199, i.e. 32 mass units greater than for urate ([M Ϫ H] Ϫ m / z 167). Two products with the expected mass were detected in the reaction mixture, and their formation was completely blocked by superoxide dismutase (Fig. 6 C ). These are likely to be isomers of urate hydroperoxide. The hydroperoxide detected by the FOX assay was reactive because it was eliminated by glutathione. In contrast, hydrogen peroxide reacted slowly with glutathione (Fig. 6 D ). Urate-dependent Consumption of Nitric Oxide by MPO — MPO can modulate vascular inflammatory responses by oxidiz- ing NO directly or via generation of radical intermediates that react with NO (32, 33). Therefore, we also tested the possibility that urate radicals generated by MPO react with nitric oxide (NO) in a similar fashion to tyrosyl radicals (32, 33). NO was generated by the NO donor NOC-9. The combination of MPO and hydrogen peroxide alone promoted consumption of NO, but this was enhanced by the addition of urate (Fig. 7 A ). Urate alone and in combination with either MPO (data not shown) or hydrogen peroxide (Fig. 7 A ) did not promote consumption of NO (data not shown). The consumption of NO was dependent on the concentration of urate over its physiological range (Fig. 7 B ). Urate was comparable with tyrosine in promoting consumption of NO. From these results, we conclude that urate promotes the NO oxidase activity of MPO. Production of Allantoin by Neutrophils in Plasma —To assess the potential for MPO to oxidize urate in vivo , we added isolated neutrophils to plasma. The cells were then stimulated with phorbol myristate acetate in the presence of cytochalasin B to promote degranulation and release of MPO. The formation of allantoin was followed using LC/MS. After a lag of ϳ 10 min, the concentration of allantoin began to increase over the 1-h incubation period (Fig. 8 A ). Formation of allantoin required stimulation of the cells and was inhibited by diphenyliodonium, which blocks the activity of the NADPH oxidase (Fig. 8 B ). Allantoin formation was also retarded by the heme poison azide as well as catalase and thiocyanate, which is a substrate for MPO. Superoxide dismutase inhibited formation of allantoin, which indicates that superoxide was involved in the oxidation of urate. Collectively, these results demonstrate that activated neutrophils oxidize urate in plasma in reactions that involve the NADPH oxidase, MPO and superoxide. We have demonstrated that urate should be considered as a physiological substrate for MPO that competes with chloride for oxidation and is converted to urate radicals. The fate of these radicals has considerable potential to influence oxidative stress during inflammation. Reaction with superoxide will produce a hydroperoxide, which will propagate oxidative reactions of both the constituent radicals. Reaction with NO will decrease the availability of this vasodilator and increase hypertension and endothelial dysfunction (58). When scavenged by thiols, such as glutathione and cysteine, urate radicals will promote chain reactions and also exacerbate oxidative stress. Even their dismutation to dehydrourate may be harmful because this species hydrolyzes to 5-hydroxyisourate (Scheme 2). This metab- olite, which was produced by MPO, has recently been shown to be associated with development of liver tumors in mice (59). Adverse reactions of urate radicals will be kept in check only when ascorbate is available at sufficient concentrations to reduce them back to urate (60, 61). Given the associations of both urate and MPO with numerous inflammatory diseases and their co-localization, our work indicates that interactions between the two should be considered when assessing the role of either in inflammation. Two previous studies have identified urate as a substrate for MPO. Originally, MPO was shown to oxidize urate to allantoin (19), and it was found that urate reacts favorably with compound II (62). Until our current work, however, the potential of urate to act as a physiological substrate for MPO had not been addressed. MPO is an unusual enzyme in that it oxidizes numerous substrates via a variety of mechanisms (22). In ...
Context 5
... the reduction of compound II back to the ferric enzyme. It could be fitted by a single exponential decay as described previously (50). The first order rate constants obtained in this way were plotted against the initial concentration of urate (Fig. 3 C ). The slope of the line provides the second order rate constant for the reaction of urate with compound II to produce ferric MPO and has a value of 1.7 Ϯ 0.1 ϫ 10 4 M Ϫ 1 s Ϫ 1 . The intercept corresponds to the spontaneous decay of compound II and was found to have a value of 0.017 s Ϫ 1 . Chloride and Urate as Competing Substrates for MPO — Chloride is a major physiological substrate for MPO (22). Therefore, we determined how chloride and urate affected each other’s oxidation by MPO. The effect of urate on the chlorination activity of MPO was determined by measuring the initial rate of hydrogen peroxide consumption by the enzyme in the presence of 100 m M chloride and varying concentrations of urate (Fig. 4 A ). Urate decreased the initial rate of hydrogen peroxide consumption markedly, inhibiting by 50% (IC 50 ) at a concentration of only 9.3 M . However, it inhibited by a maximum of only 75% over the physiological range of urate concentrations. We also measured production of allantoin by MPO and determined how it was affected by chloride. Methionine was included in the buffer to scavenge hypochlorous acid and prevent it from reacting with urate to produce allantoin (51). When 200 M urate was oxidized by MPO and 50 M hydrogen peroxide in the absence of chloride, 20 M allantoin was produced over 1 h (Fig. 4 B ). Chloride inhibited formation of allantoin in a concentration-dependent manner, having an IC 50 of 9.6 m M . In combination, these results establish chloride and urate as competing substrates of MPO. They also indicate that at normal physiological concentrations of chloride and urate, hypochlorous acid would be the major product formed by the enzyme, and urate would be expected to be a minor substrate. Effects of Glutathione on the Oxidation of Urate by MPO —To provide additional evidence that the urate is oxidized by the classical peroxidation cycle to produce radical intermediates, glutathione (GSH) was added to the reaction system. This cys- teinyl tripeptide is known to reduce radicals generated by peroxidases and is converted to oxidized glutathione (GSSG) (52). GSH markedly slowed the oxidation of urate as assessed by monitoring the loss in absorbance at 283 nm (supplemental Fig. S4). We also determined whether the oxidation of urate by MPO could promote the consumption of GSH (Fig. 5). With 1 m M GSH, there was a small amount of oxidation of GSH by MPO or urate alone. In combination, urate and MPO promoted super-stoichiometric oxidation of GSH, i.e. with 50 M hydrogen peroxide, MPO and urate promoted the oxidation of ϳ 150 M GSH. Most of the GSH that was oxidized was accounted for by the formation of GSSG. Even greater loss of GSH occurred with tyrosine as the reducing substrate for MPO as found previously (52). This result demonstrates that, like tyrosyl radical, urate radical promotes a chain reaction with GSH, resulting in formation of hydrogen peroxide that fuels further oxidation of GSH. Oxidation of Urate in the Presence of Superoxide —Urate radicals react with superoxide at diffusion-controlled rates (53). The product of this reaction is not known. Electron transfer from superoxide to urate radical would regenerate urate. Alter- natively, addition of superoxide would produce urate hydroperoxide as occurs with tyrosine, serotonin, and melatonin (54 –57). To determine whether urate is converted to a hydroperoxide by superoxide, we used MPO to oxidize urate, whereas superoxide and hydrogen peroxide were generated with xanthine oxidase. Hydroperoxides were detected using ferrous iron-catalyzed oxidation of xylenol orange (FOX assay) (46). The complete reaction system generated substantial levels of hydroperoxides (Fig. 6, A and B ). Their formation was blocked by omitting one of MPO, urate, xanthine oxidase, or acetaldehyde from the reaction system (Fig. 6 A ). Superoxide dismutase decreased hydroperoxide formation to control levels, and catalase inhibited by ϳ 50%. Incomplete inhibition by catalase most likely reflects competition of MPO and catalase for hydrogen peroxide. Production of hydroperoxides increased with increasing concentrations of urate to a maximum at 200 M urate (Fig. 6 B ). Superoxide dismutase inhibited hydroperoxide formation at all concentrations of urate indicat- ing the essential role of superoxide in their formation. To confirm that the hydroperoxide detected by the FOX assay was urate hydroperoxide, we used LC/MS with selected ion monitoring to identify [M Ϫ H] Ϫ ions with an m / z value of 199, i.e. 32 mass units greater than for urate ([M Ϫ H] Ϫ m / z 167). Two products with the expected mass were detected in the reaction mixture, and their formation was completely blocked by superoxide dismutase (Fig. 6 C ). These are likely to be isomers of urate hydroperoxide. The hydroperoxide detected by the FOX assay was reactive because it was eliminated by glutathione. In contrast, hydrogen peroxide reacted slowly with glutathione (Fig. 6 D ). Urate-dependent Consumption of Nitric Oxide by MPO — MPO can modulate vascular inflammatory responses by oxidiz- ing NO directly or via generation of radical intermediates that react with NO (32, 33). Therefore, we also tested the possibility that urate radicals generated by MPO react with nitric oxide (NO) in a similar fashion to tyrosyl radicals (32, 33). NO was generated by the NO donor NOC-9. The combination of MPO and hydrogen peroxide alone promoted consumption of NO, but this was enhanced by the addition of urate (Fig. 7 A ). Urate alone and in combination with either MPO (data not shown) or hydrogen peroxide (Fig. 7 A ) did not promote consumption of NO (data not shown). The consumption of NO was dependent on the concentration of urate over its physiological range (Fig. 7 B ). Urate was comparable with tyrosine in promoting consumption of NO. From these results, we conclude that urate promotes the NO oxidase activity of MPO. Production of Allantoin by Neutrophils in Plasma —To assess the potential for MPO to oxidize urate in vivo , we added isolated neutrophils to plasma. The cells were then stimulated with phorbol myristate acetate in the presence of cytochalasin B to promote degranulation and release of MPO. The formation of allantoin was followed using LC/MS. After a lag of ϳ 10 min, the concentration of allantoin began to increase over the 1-h incubation period (Fig. 8 A ). Formation of allantoin required stimulation of the cells and was inhibited by diphenyliodonium, which blocks the activity of the NADPH oxidase (Fig. 8 B ). Allantoin formation was also retarded by the heme poison azide as well as catalase and thiocyanate, which is a substrate for MPO. Superoxide dismutase inhibited formation of allantoin, which indicates that superoxide was involved in the oxidation of urate. Collectively, these results demonstrate that activated neutrophils oxidize urate in plasma in reactions that involve the NADPH oxidase, MPO and superoxide. We have demonstrated that urate should be considered as a physiological substrate for MPO that competes with chloride for oxidation and is converted to urate radicals. The fate of these radicals has considerable potential to influence oxidative stress during inflammation. Reaction with superoxide will produce a hydroperoxide, which will propagate oxidative reactions of both the constituent radicals. Reaction with NO will decrease the availability of this vasodilator and increase hypertension and endothelial dysfunction (58). When scavenged by thiols, such as glutathione and cysteine, urate radicals will promote chain reactions and also exacerbate oxidative stress. Even their dismutation to dehydrourate may be harmful because this species hydrolyzes to 5-hydroxyisourate (Scheme 2). This metab- olite, which was produced by MPO, has recently been shown to be associated with development of liver tumors in mice (59). Adverse reactions of urate radicals will be kept in check only when ascorbate is available at sufficient concentrations to reduce them back to urate (60, 61). Given the associations of both urate and MPO with numerous inflammatory diseases and their co-localization, our work indicates that interactions between the two should be considered when assessing the role of either in inflammation. Two previous studies have identified urate as a substrate for MPO. Originally, MPO was shown to oxidize urate to allantoin (19), and it was found that urate reacts favorably with compound II (62). Until our current work, however, the potential of urate to act as a physiological substrate for MPO had not been addressed. MPO is an unusual enzyme in that it oxidizes numerous substrates via a variety of mechanisms (22). In vivo, its dominant activity is the two-electron oxidation of chloride and thiocyanate (63). Bromide is also oxidized by this route in vivo (64). These substrates reduce compound I of MPO to produce the respective hypohalous acids and regenerate the native ferric enzyme (Scheme 1). MPO also acts as a classical peroxidase where the substrate reduces compound I to compound II, which is in turn reduced back to the native ferric enzyme (Scheme 1). Each step involves a one-electron reduction of the enzyme intermediates and is accompanied by the liber- ation of a substrate free radical. Peroxidation substrates include ascorbate, tyrosine, and serotonin as well as exoge- nous phenolics and aromatic amines (56, 65– 67). Collectively, our results indicate that urate is also oxidized by the classical peroxidase cycle shown in Scheme 1, i.e. compound II was the redox intermediate of the enzyme present during oxidation of urate, and urate readily reduced compounds I and II. Also, oxidation of urate promoted super-stoichiometric ...
Similar publications
Urate and myeloperoxidase (MPO) are associated with adverse outcomes in cardiovascular disease. In this study, we assessed
whether urate is a likely physiological substrate for MPO and if the products of their interaction have the potential to exacerbate
inflammation. Urate was readily oxidized by MPO and hydrogen peroxide to 5-hydroxyisourate, whi...
Citations
... The outcome of HOX production is determined by the differences in availability of the three anions as well as the distinct selectivity of MPO for each (pseudo-)halide. Approximately 70%-80% of all H 2 O 2 consumed by MPO yields HOCl and the remainder is accounted for by HOSCN, as the formation of HOBr is negligibly small due to the low bromide concentration available (8). ...
To eradicate bacterial pathogens, neutrophils are recruited to the sites of infection, where they engulf and kill microbes through the production of reactive oxygen and chlorine species (ROS/RCS). The most prominent RCS is the antimicrobial oxidant hypochlorous acid (HOCl), which rapidly reacts with various amino acid side chains, including those containing sulfur and primary/tertiary amines, causing significant macromolecular damage. Pathogens like uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, have developed sophisticated defense systems to protect themselves from HOCl. We recently identified the RcrR regulon as a novel HOCl defense strategy in UPEC. Expression of the rcrARB operon is controlled by the HOCl-sensing transcriptional repressor RcrR, which is oxidatively inactivated by HOCl resulting in the expression of its target genes, including rcrB . The rcrB gene encodes a hypothetical membrane protein, deletion of which substantially increases UPEC’s susceptibility to HOCl. However, the mechanism behind protection by RcrB is unclear. In this study, we investigated whether (i) its mode of action requires additional help, (ii) rcrARB expression is induced by physiologically relevant oxidants other than HOCl, and (iii) expression of this defense system is limited to specific media and/or cultivation conditions. We provide evidence that RcrB expression is sufficient to protect E. coli from HOCl. Furthermore, RcrB expression is induced by and protects from several RCS but not from ROS. RcrB plays a protective role for RCS-stressed planktonic cells under various growth and cultivation conditions but appears to be irrelevant for UPEC’s biofilm formation.
IMPORTANCE
Bacterial infections pose an increasing threat to human health, exacerbating the demand for alternative treatments. Uropathogenic Escherichia coli (UPEC), the most common etiological agent of urinary tract infections (UTIs), are confronted by neutrophilic attacks in the bladder, and must therefore be equipped with powerful defense systems to fend off the toxic effects of reactive chlorine species. How UPEC deal with the negative consequences of the oxidative burst in the neutrophil phagosome remains unclear. Our study sheds light on the requirements for the expression and protective effects of RcrB, which we recently identified as UPEC’s most potent defense system toward hypochlorous acid (HOCl) stress and phagocytosis. Thus, this novel HOCl stress defense system could potentially serve as an attractive drug target to increase the body’s own capacity to fight UTIs.
... Ozone formation in both liver and blood contributes to the exercise-mediated allantoin formation. In the blood, allantoin can also be formed independently of ozone, through myeloperoxidase-catalyzed reaction of hydrogen peroxide, superoxide anion and uric acid [202]. ...
Inhalation of tropospheric ozone increases the risk of respiratory diseases and the metabolic syndrome (MS). On the other hand, medical ozone therapy is used in the management of many chronic diseases including components of MS. However, medical ozone has not gained universal acceptance because the mechanisms involved therein are not fully understood. Ozone has also been reported to be endogenously formed in cells and organisms. Like medical ozone, endogenous ozone has not been fully embraced, due to limited understanding of the mechanisms of its formation. This review seeks to improve our understanding of the mechanisms of endogenous ozone formation by outlining previously proposed mechanisms, and suggesting new pathways based on reactions that have been reported to be involved in tropospheric ozone formation and electrochemical ozone production from water. New perspectives on the mechanisms of the harms of ozone inhalation and the benefits of medical ozone are discussed. It is hypothesized that endogenous ozone is involved in the harmful effects of particulate matter and ozone inhalation, as well as the benefits of medical ozone, nutraceuticals and physical activity. Thus, endogenous ozone should be regarded as a mainstream reactive oxygen species in redox biology.
... Uric acid, the end product of purine metabolism in humans, is present in the blood in plasma in concentrations ranging from 50 to 500 µM in healthy individuals mainly in the dissociated form as urate (pK a of uric acid is 5.4). Urate can be oxidized by myeloperoxidase and lactoperoxidase to generate urate free radical and urate hydroperoxide [124,125]. Urate hydroperoxide oxidizes methionine and cysteine and reacts with glutathione at a rate constant of 13.8 M −1 s −1 [126]. Urate hydroperoxide was found to be a good substrate of Prdx2; the second-order rate constant for the reaction was 2.3 × 10 6 M −1 s −1 , making Prdx2 the main reactant for this compound in the erythrocyte. ...
Peroxiredoxin 2 (Prdx2) is the third most abundant erythrocyte protein. It was known previously as calpromotin since its binding to the membrane stimulates the calcium-dependent potassium channel. Prdx2 is present mostly in cytosol in the form of non-covalent dimers but may associate into doughnut-like decamers and other oligomers. Prdx2 reacts rapidly with hydrogen peroxide (k > 107 M−1 s−1). It is the main erythrocyte antioxidant that removes hydrogen peroxide formed endogenously by hemoglobin autoxidation. Prdx2 also reduces other peroxides including lipid, urate, amino acid, and protein hydroperoxides and peroxynitrite. Oxidized Prdx2 can be reduced at the expense of thioredoxin but also of other thiols, especially glutathione. Further reactions of Prdx2 with oxidants lead to hyperoxidation (formation of sulfinyl or sulfonyl derivatives of the peroxidative cysteine). The sulfinyl derivative can be reduced by sulfiredoxin. Circadian oscillations in the level of hyperoxidation of erythrocyte Prdx2 were reported. The protein can be subject to post-translational modifications; some of them, such as phosphorylation, nitration, and acetylation, increase its activity. Prdx2 can also act as a chaperone for hemoglobin and erythrocyte membrane proteins, especially during the maturation of erythrocyte precursors. The extent of Prdx2 oxidation is increased in various diseases and can be an index of oxidative stress.
... When the body's high level of urate reaches a certain threshold, it will induce the body's inflammatory response and trigger AGA [13]. Glutathione can reduce the damage of oxidative stress to the body. ...
Background
Acute gouty arthritis (AGA) is a metabolic disease with acute arthritis as its main manifestation. However, the pathogenesis of asymptomatic hyperuricemia (HUA) to AGA is still unclear, and metabolic markers are needed to early predict and diagnose. In this study, gas chromatography (GC)/liquid chromatography (LC)–mass spectrometry (MS) was used to reveal the changes of serum metabolites from healthy people to HUA and then to AGA, and to find the pathophysiological mechanism and biological markers.
Methods
Fifty samples were included in AGA, HUA, and healthy control group, respectively. The metabolites in serum samples were detected by GC/LC–MS. According to the statistics of pairwise grouping, the statistically significant differential metabolites were obtained by the combination of multidimensional analysis and one-dimensional analysis. Search the selected metabolites in KEGG database, determine the involved metabolic pathways, and draw the metabolic pathway map in combination with relevant literature.
Results
Using metabonomics technology, 23 different serum metabolic markers related to AGA and HUA were found, mainly related to uric acid metabolism and inflammatory response caused by HUA/AGA. Three of them are completely different from the previous gout studies, nine metabolites with different trends from conventional inflammation.
Conclusions
In conclusion, we analyzed 150 serum samples from AGA, HUA, and healthy control group by GC/LC–MS to explore the changes of these differential metabolites and metabolic pathways, suggesting that the disease progression may involve the changes of biomarkers, which may provide a basis for disease risk prediction and early diagnosis.
... We have focused on its reactions with other radicals and with myeloperoxidase-the neutrophil's most abundant anti-microbial enzyme. [13][14][15][16][17][18] On the basis of our work and that of numerous other groups, we will describe how superoxide resembles a chemical chameleon that adopts different reactivities, which depend on the situation and the presence of other reactants. Superoxide can act as a mild reductant and oxidant and as a nucleophile that adds to free radicals to form hydroperoxides. ...
The burst of superoxide produced when neutrophils phagocytose bacteria is the defining biochemical feature of these abundant immune cells. But 50 years since this discovery, the vital role superoxide plays in host defense has yet to be defined. Superoxide is neither bactericidal nor is it just a source of hydrogen peroxide. This simple free radical does, however, have remarkable chemical dexterity. Depending on its environment and reaction partners, superoxide can act as an oxidant, a reductant, a nucleophile, or an enzyme substrate. We outline the evidence that inside phagosomes where neutrophils trap, kill, and digest bacteria, superoxide will react preferentially with the enzyme myeloperoxidase, not the bacterium. By acting as a cofactor, superoxide will sustain hypochlorous acid production by myeloperoxidase. As a substrate, superoxide may give rise to other forms of reactive oxygen. We contend that these interactions hold the key to understanding the precise role superoxide plays in neutrophil biology. State‐of‐the‐art techniques in mass spectrometry, oxidant‐specific fluorescent probes, and microscopy focused on individual phagosomes are needed to identify bactericidal mechanisms driven by superoxide. This work will undoubtably lead to fascinating discoveries in host defense and give a richer understanding of superoxide's varied biology.
... Serum urate, or uric acid, is thought to be involved in the biological processes of oxidative stress and inflammation, with particular action in endothelial cells causing vascular damage-mechanisms that may explain its role in increasing SBP and the development of cardiovascular diseases. 34,35 With little evidence to currently support a direct causal role for maternal urate lowering the mean offspring birthweight, it is possible that higher mean urate levels seen in pregnancies with small babies reflect the impact of higher urate on blood pressure and of higher blood pressure on lower birthweight. Maternal pre-existing high blood pressure and hypertensive disorders of pregnancy are related to placental vascular malperfusion and dysfunction, which may then exert greater impact on fetal growth than urate might influence placental function through direct mechanisms. ...
Background
Higher urate levels are associated with higher systolic blood pressure (SBP) in adults, and in pregnancy with lower offspring birthweight. Mendelian randomization (MR) analyses suggest a causal effect of higher urate on higher SBP and of higher maternal SBP on lower offspring birthweight. If urate causally reduces birthweight, it might confound the effect of SBP on birthweight. We therefore tested for a causal effect of maternal urate on offspring birthweight.
Methods
We tested the association between maternal urate levels and offspring birthweight using multivariable linear regression in the Exeter Family Study of Childhood Health (EFSOCH; n = 872) and UK Biobank (UKB; n = 133 187). We conducted two-sample MR to test for a causal effect of maternal urate [114 single-nucleotide polymorphisms (SNPs); n = 288 649 European ancestry] on offspring birthweight (n = 406 063 European ancestry; maternal SNP effect estimates adjusted for fetal effects). We assessed a causal relationship between urate and SBP using one-sample MR in UKB women (n = 199 768).
Results
Higher maternal urate was associated with lower offspring birthweight with similar confounder-adjusted magnitudes in EFSOCH [22 g lower birthweight per 1-SD higher urate (95% CI: –50, 6); P = 0.13] and UKB [–28 g (95% CI: –31, –25); P = 1.8 × 10–75]. The MR causal effect estimate was directionally consistent, but smaller [–11 g (95% CI: –25, 3); PIVW = 0.11]. In women, higher urate was causally associated with higher SBP [1.7 mmHg higher SBP per 1-SD higher urate (95% CI: 1.4, 2.1); P = 7.8 × 10–22], consistent with that previously published in women and men.
Conclusion
The marked attenuation of the MR result of maternal urate on offspring birthweight compared with the multivariable regression result suggests previous observational associations may be confounded. The 95% CIs of the MR result included the null but suggest a possible small effect on birthweight. Maternal urate levels are unlikely to be an important contributor to offspring birthweight.
... Additional potential mechanisms to consider include UA consumption by the increased free radicals and one electron oxidation by hemoglobin and heme-peroxidases such as myeloperoxidase (MPO) [78]. Urate is a known physiological substrate for MPO and urate free radical generated by such reaction can lead to production of urate hydroperoxide which is strong oxidant and a putative intermediate in urate oxidation during inflammatory and vascular diseases [79][80][81]. Both urate and MPO are associated with adverse outcomes in cardiovascular disorders [82][83][84]. ...
Background
The pathophysiologic significance of redox imbalance is unquestionable as numerous reports and topic reviews indicate alterations in redox parameters during corona virus disease 2019 (COVID-19). However, a more comprehensive understanding of redox-related parameters in the context of COVID-19-mediated inflammation and pathophysiology is required.
Methods
COVID-19 subjects (n = 64) and control subjects (n = 19) were enrolled, and blood was drawn within 72 hours of diagnosis. Serum multiplex assay and buffy coat cell mRNA sequencing was performed. Oxidant/free radical (electron paramagnetic resonance (EPR) spectroscopy, nitrite-nitrate assay) and antioxidant (ferrous reducing ability of serum assay and high-performance liquid chromatography) were performed. Multivariate analyses were performed to evaluate potential of indicated parameters to predict clinical outcome.
Results
Significantly greater levels of multiple inflammatory and vascular markers were quantified in the subjects admitted to the ICU compared to non-ICU subjects. Gene set enrichment analyses indicated significant enhancement of oxidant related pathways and biochemical assays confirmed a significant increase in free radical production and uric acid reduction in COVID-19 subjects. Multivariate analyses confirmed a positive association between serum levels of VCAM-1, ICAM-1 and a negative association between the abundance of one electron oxidants (detected by ascorbate radical formation) and mortality in COVID subjects while IL-17c and TSLP levels predicted need for intensive care in COVID-19 subjects.
Conclusion
Herein we demonstrate a significant redox imbalance during COVID-19 infection affirming the potential for manipulation of oxidative stress pathways as a new therapeutic strategy COVID-19. However, further work is requisite for detailed identification of oxidants (O2•-, H2O2 and/or circulating transition metals such as Fe or Cu) contributing to this imbalance to avoid the repetition of failures using non-specific antioxidant supplementation.
... When investigating a causal association of maternal urate on offspring birthweight using MR, the effect estimate was directionally consistent with the observational estimates, though imprecise and including the null. The association between maternal SBP and lower birthweight, has previously been established as causal, Serum urate, or uric acid, is thought to be involved in the biological processes of oxidative stress and inflammation, with particular action in endothelial cells causing vascular damage, mechanisms which may explain its role in increasing SBP and the development of cardiovascular diseases 29,30 . With little evidence to currently support a direct causal role for maternal urate lowering mean offspring . ...
Background
Higher urate levels associate with higher systolic blood pressure (SBP) in adults, and in pregnancy, with lower offspring birthweight. Mendelian randomization (MR) analyses suggest a causal effect of higher urate on higher SBP and of higher maternal SBP on lower offspring birthweight. If urate causally reduces birthweight, it might confound the effect of SBP on birth weight. We therefore tested for a causal effect of maternal urate on offspring birthweight.
Methods
We tested the association between maternal urate levels and offspring birthweight using multivariable linear regression in UK Biobank (UKB; n =133,187) and Exeter Family Study of Childhood Health (EFSOCH; n =872). We conducted two-sample MR to test for a causal effect of maternal urate (114 single nucleotide polymorphisms [SNPs]; n =288,649 European-ancestry) on offspring birthweight ( n =406,063 European-ancestry; maternal SNP effect estimates adjusted for fetal effects). Using one-sample MR ( n =199,768 UKB women), we also tested for a causal relationship between urate and SBP.
Results
Higher maternal urate was associated with lower offspring birthweight in UKB (28g lower birthweight per 1-SD higher urate [95% CI: -31, -25]; P =1.8×10 ⁻⁷⁵ ), with a similar effect estimate in EFSOCH (22g [95%CI: -50, 6]; P =0.13). The MR causal effect estimate was directionally consistent, but smaller (−11g [95% CI: -25, 3]; P IVW =0.13). In women, higher urate was causally associated with higher SBP (1.7 mmHg higher SBP per 1-SD higher urate [95% CI: 1.4, 2.1]; P =7.8×10 ⁻²² ) consistent with that previously published in women and men.
Conclusions
The marked attenuation of the MR result of maternal urate on offspring birthweight, compared to the multivariable regression result suggests previous observational associations may be confounded. The 95% CIs of the MR result included the null but suggest a possible weak effect on birthweight. Maternal urate levels are unlikely to be an important contributor to offspring birthweight.
Key Messages
Previous research suggests higher maternal serum urate in pregnancy is associated with lower offspring birthweight, and Mendelian randomization studies suggest a causal relationship between urate and systolic blood pressure (SBP), and SBP and birthweight; a causal effect of urate on birthweight has not yet been estimated, and thus it is also unknown whether it confounds maternal SBP-birthweight effects.
The causal effect estimate of urate on offspring birthweight was directionally consistent, but weaker than, observational estimates; the estimate had 95% confidence intervals which included the null.
This study confirmed a causal association between serum urate and higher SBP in women consistent with that published from a sample of both women and men.
Maternal urate is unlikely to be a major determinant of birthweight or an important confounder of the causal relationship between SBP and lower birthweight.
... Neutrophils are unique in generating secondary oxidants by means of MPO. This peroxidase is primarily expressed in neutrophils and metabolises hydrogen peroxide (H2O2) to produce a range of non-radical oxidants, namely hypochlorous (HOCl) and hypothiocyanous acid, and certain radical products (Figure 1-1) (Meotti et al. 2011, Love et al. 2016. Notably, despite being collectively referred to as ROS, the different species of oxidants have diverse reactivities and antimicrobial functions (Winterbourn et al. 2016). ...
Sepsis is a complex and severe pathological condition that is characterized by hyper-inflammation followed by leukocyte dysfunction and excessive tissue injury. The mechanisms controlling immune dysregulation remain poorly understood. In this thesis, we show that fungal capture in spleen promotes inflammation and DAMPs-mediated immune dysfunction during systemic candidiasis. We identify the molecules and cellular players and define the order of events that link immune dysfunction between lymphocytes and neutrophils. SIGNR1+ marginal zone macrophages (MZMΦs) capture Candida albicans (C. albicans) and enable fungal colonization of the spleen marginal zone (MZ), triggering aberrant lymphocyte death and release of cell-free chromatin, that synergizes with fungal hyphae to stimulate G-CSF production by CD169+ marginal metallophilic macrophages (MMMΦs). G-CSF and extracellular chromatin selectively reduce the lifespan of mature Ly6Ghigh neutrophils, leading to severe neutropenia. ROS-deficient immature Ly6Glow neutrophils are mobilised and become the predominant peripheral neutrophil population, causing impaired fungal clearance and severe pathology. SIGNR1-blockade effectively limits fungal colonization in the MZ and increases survival. Similarly, T cell- deficiency or neutralization of G-CSF, chromatin or histones consistently reduces inflammation, neutrophil dysfunction and pathology. The release of cell-free actin by dying splenocytes and tissue damage further enhances pathology by interfering with extracellular chromatin clearance. These findings demonstrate that PAMPs and DAMPs mediate inflammation and neutrophil dysregulation, causing a detrimental positive feedback-loop that impairs fungal clearance and increases sepsis pathology.
... The list of potential one-electron substrates of Compound I is long. The important one-electron substrates for MPO are selected polyphenols, urate, tyrosine, tryptophan, sulfhydryls, indole derivatives, nitrogen oxide, nitrite, H 2 O 2 , and superoxide anion radicals [53][54][55][56][57][58]. ...
... As a result of this restriction, heme peroxidases can accumulate as inactive Compound II in the absence of substrates that are well oxidized by Compound II. For MPO and LPO, efficient substrates for Compound II are superoxide anion radicals, urate, tyrosine, serotonin, nitrite, and selected flavonoids [53,[57][58][59][60][61][62][63]. In the presence of these substrates, an accumulation of Compound II can be avoided and the halogenation activity can be enhanced. ...
In our organism, mucous surfaces are important boundaries against the environmental milieu with defined fluxes of metabolites through these surfaces and specific rules for defense reactions. Major mucous surfaces are formed by epithelia of the respiratory system and the digestive tract. The heme peroxidases lactoperoxidase (LPO), myeloperoxidase (MPO), and eosinophil peroxidase (EPO) contribute to immune protection at epithelial surfaces and in secretions. Whereas LPO is secreted from epithelial cells and maintains microbes in surface linings on low level, MPO and EPO are released from recruited neutrophils and eosinophils, respectively, at inflamed mucous surfaces. Activated heme peroxidases are able to oxidize (pseudo)halides to hypohalous acids and hypothiocyanite. These products are involved in the defense against pathogens, but can also contribute to cell and tissue damage under pathological conditions. This review highlights the beneficial and harmful functions of LPO, MPO, and EPO at unperturbed and inflamed mucous surfaces. Among the disorders, special attention is directed to cystic fibrosis and allergic reactions.
