Urate Hydroperoxide Research Articles

Uric Acid Reacts with Peroxidasin, Decreases Collagen IV Crosslink, Impairs Human Endothelial Cell Migration and Adhesion.

Uric acid is considered the main substrate for peroxidases in plasma. The oxidation of uric acid by human peroxidases generates urate free radical and urate hydroperoxide, which might affect endothelial function and explain, at least in part, the harmful effects of uric acid on the vascular system. Peroxidasin (PXDN), the most recent heme-peroxidase described in humans, catalyzes the formation of hypobromous acid, which mediates collagen IV crosslinks in the extracellular matrix. This enzyme has gained increasing scientific interest since it is associated with cardiovascular disease, cancer, and renal fibrosis. The main objective here was to investigate whether uric acid would react with PXDN and compromise the function of the enzyme in human endothelial cells. Urate decreased Amplex Red oxidation and brominating activity in the extracellular matrix (ECM) from HEK293/PXDN overexpressing cells and in the secretome of HUVECs. Parallelly, urate was oxidized to 5-hydroxyisourate. It also decreased collagen IV crosslink in isolated ECM from PFHR9 cells. Urate, the PXDN inhibitor phloroglucinol, and the PXDN knockdown impaired migration and adhesion of HUVECs. These results demonstrated that uric acid can affect extracellular matrix formation by competing for PXDN. The oxidation of uric acid by PXDN is likely a relevant mechanism in the endothelial dysfunction related to this metabolite.

Peroxiredoxin AhpC1 protects Pseudomonas aeruginosa against the inflammatory oxidative burst and confers virulence

Pseudomonas aeruginosa is an opportunistic bacterium in patients with cystic fibrosis and hospital acquired infections. It presents a plethora of virulence factors and antioxidant enzymes that help to subvert the immune system. In this study, we identified the 2-Cys peroxiredoxin, alkyl-hydroperoxide reductase C1 (AhpC1), as a relevant scavenger of oxidants generated during inflammatory oxidative burst and a mechanism of P. aeruginosa (PA14) escaping from killing. Deletion of AhpC1 led to a higher sensitivity to hypochlorous acid (HOCl, IC50 3.2 ± 0.3 versus 19.1 ± 0.2 μM), hydrogen peroxide (IC50 91.2 ± 0.3 versus 496.5 ± 6.4 μM) and the organic peroxide urate hydroperoxide. ΔahpC1 strain was more sensitive to the killing by isolated neutrophils and less virulent in a mice model of infection. All mice intranasally instilled with ΔahpC1 survived as long as they were monitored (15 days), whereas 100% wild-type and ΔahpC1 complemented with ahpC1 gene (ΔahpC1 attB:ahpC1) died within 3 days. A significantly lower number of colonies was detected in the lung and spleen of ΔahpC1-infected mice. Total leucocytes, neutrophils, myeloperoxidase activity, pro-inflammatory cytokines, nitrite production and lipid peroxidation were much lower in lungs or bronchoalveolar liquid of mice infected with ΔahpC1. Purified AhpC neutralized the inflammatory organic peroxide, urate hydroperoxide, at a rate constant of 2.3 ± 0.1 × 106 M−1s−1, and only the ΔahpC1 strain was sensitive to this oxidant. Incubation of neutrophils with uric acid, the urate hydroperoxide precursor, impaired neutrophil killing of wild-type but improved the killing of ΔahpC1. Hyperuricemic mice presented higher levels of serum cytokines and succumbed much faster to PA14 infection when compared to normouricemic mice. In summary, ΔahpC1 PA14 presented a lower virulence, which was attributed to a poorer ability to neutralize the oxidants generated by inflammatory oxidative burst, leading to a more efficient killing by the host. The enzyme is particularly relevant in detoxifying the newly reported inflammatory organic peroxide, urate hydroperoxide.

Urate hydroperoxide oxidizes endothelial cell surface protein disulfide isomerase-A1 and impairs adherence

BackgroundExtracellular surface protein disulfide isomerase-A1 (PDI) is involved in platelet aggregation, thrombus formation and vascular remodeling. PDI performs redox exchange with client proteins and, hence, its oxidation by extracellular molecules might alter protein function and cell response. In this study, we investigated PDI oxidation by urate hydroperoxide, a newly-described oxidant that is generated through uric acid oxidation by peroxidases, with a putative role in vascular inflammation. MethodsAmino acids specificity and kinetics of PDI oxidation by urate hydroperoxide was evaluated by LC-MS/MS and by stopped-flow. Oxidation of cell surface PDI and other thiol-proteins from HUVECs was identified using impermeable alkylating reagents. Oxidation of intracellular GSH and GSSG was evaluated with specific LC-MS/MS techniques. Cell adherence, detachment and viability were assessed using crystal violet staining, cellular microscopy and LDH activity, respectively. ResultsUrate hydroperoxide specifically oxidized cysteine residues from catalytic sites of recombinant PDI with a rate constant of 6 × 103 M−1 s−1. Incubation of HUVECs with urate hydroperoxide led to oxidation of cell surface PDI and other unidentified cell surface thiol-proteins. Cell adherence to fibronectin coated plates was impaired by urate hydroperoxide, as well as by other oxidants, thiol alkylating agents and PDI inhibitors. Urate hydroperoxide did not affect cell viability but significantly decreased GSH/GSSG ratio. ConclusionsOur results demonstrated that urate hydroperoxide affects thiol-oxidation of PDI and other cell surface proteins, impairing cellular adherence. General significanceThese findings could contribute to a better understanding of the mechanism by which uric acid affects endothelial cell function and vascular homeostasis.

Conjugation of urate-derived electrophiles to proteins during normal metabolism and inflammation

Urate is often viewed as an antioxidant. Here, we present an alternative perspective by showing that, when oxidized, urate propagates oxidative stress. Oxidation converts urate to the urate radical and the electrophilic products dehydrourate, 5-hydroxyisourate, and urate hydroperoxide, which eventually break down to allantoin. We investigated whether urate-derived electrophiles are intercepted by nucleophilic amino acid residues to form stable adducts on proteins. When urate was oxidized in the presence of various peptides and proteins, two adducts derived from urate (Mr 167 Da) were detected and had mass additions of 140 and 166 Da, occurring mainly on lysine residues and N-terminal amines. The adduct with a 140-Da mass addition was detected more frequently and was stable. Dehydrourate (Mr 166 Da) also formed transient adducts with cysteine residues. Urate-derived adducts were detected on human serum albumin in plasma of healthy donors. Basal adduct levels increased when neutrophils were added to plasma and stimulated, and relied on the NADPH oxidase, myeloperoxidase, hydrogen peroxide, and superoxide. Adducts of oxidized urate on serum albumin were elevated in plasma and synovial fluid from individuals with gout and rheumatoid arthritis. We propose that rather than acting as an antioxidant, urate's conversion to electrophiles contributes to oxidative stress. The addition of urate-derived electrophiles to nucleophilic amino acid residues, a process we call oxidative uratylation, will leave a footprint on proteins that could alter their function when critical sites are modified.

Identification of urate hydroperoxide in neutrophils: A novel pro-oxidant generated in inflammatory conditions

Uric acid is the final product of purine metabolism in humans and is considered to be quantitatively the main antioxidant in plasma. In vitro studies showed that the oxidation of uric acid by peroxidases, in presence of superoxide, generates urate free radical and urate hydroperoxide. Urate hydroperoxide is a strong oxidant and might be a relevant intermediate in inflammatory conditions. However, the identification of urate hydroperoxide in cells and biological samples has been a challenge due to its high reactivity. By using mass spectrometry, we undoubtedly demonstrated the formation of urate hydroperoxide and its corresponding alcohol, hydroxyisourate during the respiratory burst in peripheral blood neutrophils and in human leukemic cells differentiated in neutrophils (dHL-60). The respiratory burst was induced by phorbol myristate acetate (PMA) and greatly increased oxygen consumption and superoxide production. Both oxygen consumption and superoxide production were further augmented by incubation with uric acid. Conversely, uric acid significantly decreased the levels of HOCl, probably because of the competition with chloride by the catalysis of myeloperoxidase. In spite of the decrease in HOCl, the overall oxidative status, measured by GSH/GSSG ratio, was augmented in the presence of uric acid. In summary, the present results support the formation of urate hydroperoxide, a novel oxidant in neutrophils oxidative burst. Urate hydroperoxide is a strong oxidant and alters the redox balance toward a pro-oxidative environment. The production of urate hydroperoxide in inflammatory conditions could explain, at least in part, the harmful effect associated to uric acid.

P-57 - Urate hydroperoxide: A new reported oxidant generated in inflammatory conditions

Uric acid is the final product of purine metabolism in humans and is an alternative substrate for myeloperoxidase. The oxidation of uric acid by this enzyme generates urate free radical and urate hydroperoxide. This study investigated the formation of urate hydroperoxide in inflammatory cells and how it alters the redox balance in these cells. Human leukemic cells (HL-60) were differentiated in neutrophils (dHL-60) and blood peripheral neutrophils were isolated from healthy donors. Cells were activated with phorbol myristate acetate in presence or absence of uric acid. Oxygen consumption and superoxide production were increased by 7 and 89% in presence of uric acid. Both dHL-60 cells and blood neutrophils efficiently oxidized uric acid to urate hydroperoxide. This oxidation was dependent on myeloperoxidase activity and superoxide production. Uric acid decreased HOCl likely by the competition between chloride and uric acid by myeloperoxidase catalysis. Incubation with uric acid decreased GSH/GSSG ratio and increased peroxiredoxin oxidation. In spite of being considered the main antioxidant in plasma, uric acid can be oxidized in inflammatory conditions leading to an oxidative environment and contribute to tissue damage in inflammation.

Uric acid disrupts hypochlorous acid production and the bactericidal activity of HL-60 cells

Uric acid is the end product of purine metabolism in humans and is an alternative physiological substrate for myeloperoxidase. Oxidation of uric acid by this enzyme generates uric acid free radical and urate hydroperoxide, a strong oxidant and potentially bactericide agent. In this study, we investigated whether the oxidation of uric acid and production of urate hydroperoxide would affect the killing activity of HL-60 cells differentiated into neutrophil-like cells (dHL-60) against a highly virulent strain (PA14) of the opportunistic pathogen Pseudomonas aeruginosa. While bacterial cell counts decrease due to dHL-60 killing, incubation with uric acid inhibits this activity, also decreasing the release of the inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF- α). In a myeloperoxidase/Cl-/H2O2 cell-free system, uric acid inhibited the production of HOCl and bacterial killing. Fluorescence microscopy showed that uric acid also decreased the levels of HOCl produced by dHL-60 cells, while significantly increased superoxide production. Uric acid did not alter the overall oxidative status of dHL-60 cells as measured by the ratio of reduced (GSH) and oxidized (GSSG) glutathione. Our data show that uric acid impairs the killing activity of dHL-60 cells likely by competing with chloride by myeloperoxidase catalysis, decreasing HOCl production. Despite diminishing HOCl, uric acid probably stimulates the formation of other oxidants, maintaining the overall oxidative status of the cells. Altogether, our results demonstrated that HOCl is, indeed, the main relevant oxidant against bacteria and deviation of myeloperoxidase activity to produce other oxidants hampers dHL-60 killing activity.

Urate hydroperoxide oxidizes human peroxiredoxin 1 and peroxiredoxin 2

Urate hydroperoxide is a product of the oxidation of uric acid by inflammatory heme peroxidases. The formation of urate hydroperoxide might be a key event in vascular inflammation, where there is large amount of uric acid and inflammatory peroxidases. Urate hydroperoxide oxidizes glutathione and sulfur-containing amino acids and is expected to react fast toward reactive thiols from peroxiredoxins (Prxs). The kinetics for the oxidation of the cytosolic 2-Cys Prx1 and Prx2 revealed that urate hydroperoxide oxidizes these enzymes at rates comparable with hydrogen peroxide. The second-order rate constants of these reactions were 4.9 × 105 and 2.3 × 106 m-1 s-1 for Prx1 and Prx2, respectively. Kinetic and simulation data suggest that the oxidation of Prx2 by urate hydroperoxide occurs by a three-step mechanism, where the peroxide reversibly associates with the enzyme; then it oxidizes the peroxidatic cysteine, and finally, the rate-limiting disulfide bond is formed. Of relevance, the disulfide bond formation was much slower in Prx2 (k3 = 0.31 s-1) than Prx1 (k3 = 14.9 s-1). In addition, Prx2 was more sensitive than Prx1 to hyperoxidation caused by both urate hydroperoxide and hydrogen peroxide. Urate hydroperoxide oxidized Prx2 from intact erythrocytes to the same extent as hydrogen peroxide. Therefore, Prx1 and Prx2 are likely targets of urate hydroperoxide in cells. Oxidation of Prxs by urate hydroperoxide might affect cell function and be partially responsible for the pro-oxidant and pro-inflammatory effects of uric acid.

342 - Oxidation of Pericelullar Endothelial Protein Disulfide Isomerase by Urate Hydroperoxide: Implications on Vascular Homeostasis

Uric acid is associated with endothelial dysfunction and progression of cardiovascular disease. When oxidize by plasma peroxidases, uric acid is converted into urate free radical and urate hydroperoxide. Urate hydroperoxide oxidizes small antioxidants like glutathione and methionine and might target thiol groups from proteins in endothelial cell surface. The protein disulfide isomerase (PDI) is a redox sensitive protein that can be found (1 - 2 % of its total expression) at the external surface of the cell (PecPDI). PecPDI is involved in vascular remodeling, platelet adhesion and thrombus formation in a redox-dependent way. Our aim was to investigate whether PDI would be a target to urate hydroperoxide and if it would alter the endothelial cell function. When incubated with recombinant PDI, urate hydroperoxide was rapidly reduced to hydroxyisourate and the thiol groups from PDI were oxidized. The rate constant for this oxidation was 6 × 10 3 M -1 s -1 . After marking surface reduced proteins with the biotinylating reagent Na-(3-maleimidylpropionyl) biocytin, we observe that lower concentrations of urate hydroperoxide are already capable of increasing the extracellular pool of oxidized PDI in HUVECs. In addition, HUVECs exposed to increasing concentrations of urate hydroperoxide exhibited morphological changes - contracted and rounded - and became more sensitive to detachment from culture plates. Nevertheless, treatment with the oxidant did not result in severe cell death, as shown by the results of nuclear staining with specific dyes and detection of dehydrogenase lactate enzyme in supernatant. In conclusion, these results revealed that urate hydroperoxide is capable of causing disturbances in endothelial cells without leads them to death. The oxidation of PecPDI may be one of the mechanisms by which uric acid alters the vascular physiology and supports cardiovascular disease. Supported by: FAPESP CEPID-REDOXOMA 2013/07937-8 and 2011/18106-4, CNPq, CAPES, USP.

116 - Pro-Oxidant Effect of Uric Acid Metabolites in Inflammatory Cells

Uric acid is the final product of purine metabolism in humans and accumulates in plasma in the order of hundred micromolar. The one electron oxidation of uric acid by peroxidases generates urate free radical and urate hydroperoxide. Urate hydroperoxide is a strong oxidant and might contribute to oxidative imbalance in inflammatory states. Urate hydroperoxide efficiently reacts with glutathione and thiol proteins. In this study, we investigated the oxidative metabolism of uric acid in inflammatory cells and whether it contributes to an oxidative environment. Human leukemic (HL-60) were differentiated in neutrophils (dHL-60) and activated with phorbol myristate acetate (PMA, 100 ng/mL). The activation of cells greatly increased oxygen consumption and superoxide production. The pre-incubation with uric acid (0.05 – 0.5 mM) increased even further the oxygen consumption and superoxide production (51%). Conversely, uric acid (0.2 – 2 mM) significantly decreased hypochlorous acid (HOCl) production. The overall oxidative status of the cell was evaluated by GSH/GSSG ratio using LC-MS/MS. Uric acid significantly decreased GSH/GSSG ratio in activated dHL-60 cells, showing its pro-oxidant capability. Data by LC-MS/MS revealed that activated dHL-60 cells efficiently oxidized uric acid to urate hydroperoxide. This oxidation was dependent on the activity of myeloperoxidase and on the addition of superoxide to the urate free radical. These results show that uric acid increases superoxide but decreases HOCl formation in activated dHL-60 cells. The decrease in HOCl might be due to a competition between chloride and uric acid by myeloperoxidase catalysis. In agreement, uric acid was largely oxidized to urate hydroperoxide and significantly increased the oxidation of GSH to GSSG in these cells. Therefore, the oxidation of uric acid in inflammatory cells can lead to an oxidative environment and exacerbate tissue damage. Acknowledgment FAPESP, INCT/NAP/CEPID-Redoxoma

341 - Plasma Allantoin Reveals Oxidative Status in Subclinical Atherosclerosis

The role of uric acid as an independent factor of risk for cardiovascular disease is a matter of intense debate. Uric acid can be oxidized by the inflammatory enzyme myeloperoxidase to urate free radical, urate hydroperoxide and allantoin. The present study investigated whether patients with subclinical atherosclerosis would be more prone to oxidation of uric acid than healthy individuals and the role of myeloperoxidase in this event. A total of 35 subjects with carotid intima-media thickness in the 75th percentile (c-IMT P>75, cut off 1 mm) and 35 healthy subjects (c-IMT P 30, glomerular filtration rate ≥ 70. The levels of xanthine/hypoxanthine, uric acid, allantoin and myeloperoxidase were higher in individuals with c-IMT P>75, but only allantoin levels were significantly different between groups. Retinol and beta-carotene were slightly decreased in c-IMT P>75 individuals. A simple logistic regression showed a positive and significant association between c-IMT and allantoin. Pearson’s correlation revealed that allantoin had a positive and significant correlation with the pro-inflammatory and pro-oxidant enzyme myeloperoxidase only for c-IMT P>75 patients. No correlation was found between allantoin and its precursor uric acid. A significant negative correlation was found between allantoin and the antioxidant retinol in plasma of healthy individuals. Uric acid was prompt oxidized to allantoin when purified myeloperoxidase and hydrogen peroxide were added to the plasma. This study demonstrated that uric acid is more prone to oxidation in patients with subclinical atherosclerosis. The vascular inflammation in these patients is likely to oxidize uric acid to urate free radical and urate hydroperoxide that, at last, breaks down to allantoin. The oxidation of uric acid by myeloperoxidase in subclinical atherosclerosis may propagate vascular damage and inflammation in these patients. The present study suggests that the measurement of allantoin and the inflammatory status would be more reliable than uric acid to the prognosis of subclinical atherosclerosis. Supported by: FAPESP CEPID-REDOXOMA 2013/07937-8 and 2011/18106-4, CNPq, USP.

Chemical Characterization of Urate Hydroperoxide, A Pro-oxidant Intermediate Generated by Urate Oxidation in Inflammatory and Photoinduced Processes.

Urate hydroperoxide is a strong oxidant generated by the combination of urate free radical and superoxide. The formation of urate hydroperoxide as an intermediate in urate oxidation is potentially responsible for the pro-oxidant effects of urate in inflammatory disorders, protein degradation, and food decomposition. To understand the molecular mechanisms that sustain the harmful effects of urate in inflammatory and oxidative stress related conditions, we report a detailed structural characterization and reactivity of urate hydroperoxide toward biomolecules. Urate hydroperoxide was synthesized by photo-oxidation and by a myeloperoxidase/hydrogen peroxide/superoxide system. Multiple reaction monitoring (MRM) and MS(3) ion fragmentation revealed that urate hydroperoxide from both sources has the same chemical structure. Urate hydroperoxide has a maximum absorption at 308 nm, ε308nm = 6.54 ± 0.38 × 10(3) M(-1) cm(-1). This peroxide decays spontaneously with a rate constant of k = 2.80 ± 0.18 × 10(-4) s(-1) and a half-life of 41 min at 22 °C. Urate hydroperoxide undergoes electrochemical reduction at potential values less negative than -0.5 V (versus Ag/AgCl). When incubated with taurine, histidine, tryptophan, lysine, methionine, cysteine, or glutathione, urate hydroperoxide reacted only with methionine, cysteine, and glutathione. The oxidation of these molecules occurred by a two-electron mechanism, generating the alcohol, hydroxyisourate. No adduct between cysteine or glutathione and urate hydroperoxide was detected. The second-order rate constant for the oxidation of glutathione by urate hydroperoxide was 13.7 ± 0.8 M(-1) s(-1). In conclusion, the oxidation of sulfur-containing biomolecules by urate hydroperoxide is likely to be a mechanism by which the pro-oxidant and damaging effects of urate are mediated in inflammatory and photo-oxidizing processes.

PSS337 - Underlying the Pro-Oxidant Effects of Urate Hydroperoxide on Redox Modulated Proteins: a Pivotal Role in Inflammation

PSS337 - Underlying the Pro-Oxidant Effects of Urate Hydroperoxide on Redox Modulated Proteins: a Pivotal Role in Inflammation

Characterization of Peroxides Formed by Riboflavin and Light Exposure of Milk. Detection of Urate Hydroperoxide as a Novel Oxidation Product

Characterization of peroxides by size exclusion chromatography (SEC) of milk following exposure to riboflavin and light showed that hydrogen peroxide was the most abundant peroxide formed since it could be removed by catalase. Formation of peroxides after separation by SEC showed that hydrogen peroxide formation was primarily increased in the presence of caseins and ascorbate, although whey proteins also were found to contribute. Caseins and beta-lactoglobulin also formed catalase-resistant peroxides, presumably protein hydroperoxides. A catalase-resistant and unstable peroxide was observed in fractions containing urate. Experiments performed with pure urate suggested that urate radicals reacted further with superoxide leading to a urate hydroperoxide. Electron paramagnetic resonance spectroscopy using spin-traps showed that the presence of oxygen was required for urate radical formation, which could be assigned as nitrogen-centered radicals. These results suggest a new route during light-induced oxidation sensitized by flavins, in effect making urate pro-oxidative.

Podagra, Uric Acid, and Cardiovascular Disease

Hippocrates would not be surprised to read that persons with gout had higher mortality, and particularly cardiovascular-related mortality, than persons without gout.1 On the other hand, he might well have been bemused, nearly 2500 years after he described the syndrome, that this made news. Article p 894 He should not have been. All too often, rigorous examination debunks conventional wisdom. Reassuringly, in this case, Drs Choi and Curhan found evidence to support long-standing belief. These dedicated and productive students of the pathogenesis and natural history of gout have here reported empiric data from the 51 000 male nonphysician health professionals who participated in the Health Professions Follow-up Study. From initial examination in 1986 through nearly 600 000 person-years of follow-up, 5825 deaths occurred, of which 2132 were cardiovascular and 1576 were ascribed to coronary heart disease, rates that attest to the generally good health of the group. The ≈6% prevalence of gout, at baseline, in men in their mid-50s, who mirror the general US population in body mass index, reported history of hypertension, hypercholesterolemia, and diabetes mellitus, was higher than expected. Interim biennial questionnaires identified incident cases of gout. Participants with gout were both more likely to report coronary heart disease (CHD) at baseline and, regardless of CHD status, more likely than gout-free persons to have cardiovascular disease (CVD) risk factors. Results were presented after stratification by CHD status at baseline. As expected, the incidence of all-cause and CHD mortality was much greater in those with than in those without prevalent CHD. The adjusted increased relative risk of those with gout (compared with those without) for all-cause, CVD, and CHD mortality was ≈25%. The single exception was a near doubling of increased relative risk of CHD mortality among those free of CHD at baseline. Neither the inclusion of incident …

Evidence for Urate Hydroperoxide as an Intermediate in the Urate Oxidase Reaction

Evidence for Urate Hydroperoxide as an Intermediate in the Urate Oxidase Reaction

Spectroscopic Characterization of Intermediates in the Urate Oxidase Reaction

The oxidation of urate catalyzed by soybean urate oxidase was studied under single-turnover conditions using stopped-flow absorbance and fluorescence spectrophotometry. Two discrete enzyme-bound intermediates were observed; the first intermediate to form had an absorbance maximum at 295 nm and was assigned to a urate dianion species; the second intermediate had an absorbance maximum at 298 nm and is believed to be urate hydroperoxide. These data are consistent with a catalytic mechanism that involves formation of urate hydroperoxide from O2 and the urate dianion, collapse of the peroxide to form dehydrourate, and hydration of dehydrourate to form the observed product, 5-hydroxyisourate. The rate of formation of the first intermediate was too fast to measure accurately at 20 degreesC; the second intermediate formed with a rate constant of 32 s-1 and decayed with a rate constant of 6.6 s-1. The product of the reaction, 5-hydroxyisourate, is fluorescent, and its release from the active site occurred with a rate constant of 31 s-1.