RESUMO
The ability of helminths to manipulate the immune system of their hosts to ensure their own survival is often credited with affecting responses to other pathogens. We undertook co-infection experiments in mice to determine how infection with the intestinal helminth Heligmosomoides polygyrus affected the parasitological, immunological and physiological outcomes of a primary infection with a distinct species of helminth; the lung migratory parasite Nippostrongylus brasiliensis. We found that migrating N. brasiliensis larvae were killed in the lungs of H. polygyrus-infected mice by a process involving IL-33-activated CD4+ T cells that released IL-5 and recruited activated eosinophils. The lung pathology normally associated with N. brasiliensis larval migration was also reduced. Importantly, lung immunity remained intact in mice cleared of prior H. polygyrus infection and also occurred during infection with another entirely enteric helminth, Trichuris muris. This study identifies a cross-mucosal immune mechanism by which intestinal helminths may protect their hosts against co-infection by a different parasite at a distal site, via circulation of activated CD4+ T cells that can be triggered to release effector cytokines and mount inflammatory responses by tissue damage-associated alarmins, such as IL-33.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Coinfecção , Eosinófilos/imunologia , Interleucina-5/metabolismo , Pulmão/imunologia , Nematospiroides dubius/fisiologia , Nippostrongylus/fisiologia , Infecções por Strongylida/imunologia , Tricuríase/imunologia , Trichuris/fisiologia , Animais , Antígenos de Helmintos/imunologia , Movimento Celular , Células Cultivadas , Citotoxicidade Imunológica , Feminino , Interações Hospedeiro-Parasita , Imunidade , Interleucina-33/metabolismo , Pulmão/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
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.
Assuntos
Ácido Úrico/química , Aminas/química , Sequência de Aminoácidos , Ativação Enzimática/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Humanos , Inflamação/metabolismo , Modelos Moleculares , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Conformação Proteica , Albumina Sérica/química , Albumina Sérica/metabolismo , Ácido Úrico/metabolismo , Ácido Úrico/farmacologiaRESUMO
Peroxidasin is a heme peroxidase that catalyses the oxidation of bromide by hydrogen peroxide to form an essential sulfilimine cross-link between methionine and hydroxylysine residues in collagen IV. We investigated cross-linking by peroxidasin embedded in extracellular matrix isolated from cultured epithelial cells and its sensitivity to alternative substrates and peroxidase inhibitors. Peroxidasin showed peroxidase activity as measured with hydrogen peroxide and Amplex red. Using a specific mass spectrometry assay that measures NADH bromohydrin, we showed definitively that the enzyme releases hypobromous acid (HOBr). Less than 1 µM of the added hydrogen peroxide was used by peroxidasin. The remainder was consumed by catalase activity that was associated with the matrix. Results from NADH bromohydrin measurements indicates that low micromolar HOBr generated by peroxidasin was sufficient for maximum sulfilimine cross-linking, whereas 100 µM reagent HOBr or taurine bromamine was less efficient. This implies selectivity for the enzymatic process. Physiological concentrations of thiocyanate and urate partially inhibited cross-link formation. 4-Aminobenzoic acid hydrazide, a commonly used myeloperoxidase inhibitor, also inhibited peroxidasin, whereas acetaminophen and a 2-thioxanthine were much less effective. In conclusion, HOBr is produced by peroxidasin in the extracellular matrix. It appears to be directed at the site of collagen IV sulfilimine formation but the released HOBr may also undergo other reactions.
Assuntos
Bromatos/química , Brometos/química , Proteínas da Matriz Extracelular/química , Matriz Extracelular/química , Peróxido de Hidrogênio/química , Peroxidase/química , Animais , Bromatos/análise , Linhagem Celular Tumoral , Colágeno Tipo IV/química , Proteínas da Matriz Extracelular/antagonistas & inibidores , Proteínas da Matriz Extracelular/genética , Técnicas de Inativação de Genes , Iminas/química , Espectrometria de Massas , Camundongos , NAD/química , Peroxidase/antagonistas & inibidores , Peroxidase/genética , PeroxidasinaRESUMO
Neutrophil-derived myeloperoxidase (MPO) is recognized as a major source of oxidative stress at the airway surface of a cystic fibrosis (CF) lung where, despite limited evidence, the antioxidant glutathione is widely considered to be low. The aims of this study were to establish whether oxidative stress or glutathione status are associated with bronchiectasis and whether glutathione deficiency is inherently linked to CF or a consequence of oxidative stress. MPO was measured by ELISA in 577 bronchoalveolar lavage samples from 205 clinically-phenotyped infants and children with CF and 58 children without CF (ages 0.2-6.92 years). Reduced glutathione (GSH), oxidized glutathione species (GSSG; glutathione attached to proteins, GSSP; glutathione sulfonamide, GSA) and allantoin, an oxidation product of uric acid, were measured by mass spectrometry. The odds of having bronchiectasis were associated with MPO and GSSP. GSH was low in children with CF irrespective of oxidation. Oxidized glutathione species were significantly elevated in CF children with pulmonary infections compared to uninfected CF children. In non-CF children, infections had no effect on glutathione levels. An inadequate antioxidant response to neutrophil-mediated oxidative stress during infections exists in CF due to an inherent glutathione deficiency. Effective delivery of glutathione and inhibition of MPO may slow the development of bronchiectasis.
Assuntos
Bronquiectasia/metabolismo , Fibrose Cística/metabolismo , Glutationa/deficiência , Pulmão/metabolismo , Neutrófilos/enzimologia , Peroxidase/metabolismo , Idade de Início , Alantoína/metabolismo , Bronquiectasia/patologia , Líquido da Lavagem Broncoalveolar/química , Estudos de Casos e Controles , Criança , Pré-Escolar , Fibrose Cística/patologia , Feminino , Glutationa/análogos & derivados , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Humanos , Lactente , Inflamação , Pulmão/patologia , Masculino , Neutrófilos/metabolismo , Oxirredução , Estresse Oxidativo , Sulfonas/metabolismoRESUMO
BACKGROUND: In cystic fibrosis (CF) there is an urgent need for earlier diagnosis of pulmonary infections and inflammation using blood- and urine-based biomarkers. METHODS: Using mass spectrometry, oxidation products of glutathione and uric acid were measured in matched samples of bronchoalveolar lavage (BAL), serum and urine from 36 infants and children with CF, and related to markers of neutrophilic inflammation and infection in BAL. RESULTS: Oxidation products of glutathione (glutathione sulfonamide, GSA) and uric acid (allantoin), were elevated in BAL of children with pulmonary infections with Pseudomonas aeruginosa (PsA) compared to those without (p<0.05) and correlated with other markers of neutrophilic inflammation. Serum GSA was significantly elevated in children with PsA infections (p<0.01). Urinary GSA correlated with pulmonary GSA (r=0.42, p<0.05) and markers of neutrophilic inflammation. CONCLUSIONS: This proof-of-concept study demonstrates that urinary GSA but not allantoin shows promise as a non-invasive marker of neutrophilic inflammation in early CF lung disease.
Assuntos
Fibrose Cística , Dissulfeto de Glutationa , Inflamação/diagnóstico , Infecções por Pseudomonas/diagnóstico , Infecções Respiratórias/diagnóstico , Ácido Úrico , Biomarcadores/análise , Biomarcadores/sangue , Biomarcadores/urina , Líquido da Lavagem Broncoalveolar , Pré-Escolar , Fibrose Cística/diagnóstico , Fibrose Cística/microbiologia , Fibrose Cística/fisiopatologia , Diagnóstico Precoce , Feminino , Dissulfeto de Glutationa/análise , Dissulfeto de Glutationa/sangue , Dissulfeto de Glutationa/urina , Humanos , Lactente , Masculino , Neutrófilos/metabolismo , Reprodutibilidade dos Testes , Estatística como Assunto , Ácido Úrico/análise , Ácido Úrico/sangue , Ácido Úrico/urinaRESUMO
Duchenne Muscular Dystrophy (DMD) is a fatal skeletal muscle wasting disease presenting with excessive myofibre necrosis and increased inflammation and oxidative stress. In the mdx mouse model of DMD, homeostasis of the amino acid taurine is altered, and taurine administration drastically decreases muscle necrosis, dystropathology, inflammation and protein thiol oxidation. Since the severe pathology of the Golden Retriever Muscular Dystrophy (GRMD) dog model more closely resembles the human DMD condition, we aimed to assess the generation of oxidants by inflammatory cells and taurine metabolism in this species. In muscles of 8 month GRMD dogs there was an increase in the content of neutrophils and macrophages, and an associated increase in elevated myeloperoxidase, a protein secreted by neutrophils that catalyses production of the highly reactive hypochlorous acid (HOCl). There was also increased chlorination of tyrosines, a marker of HOCl generation, increased thiol oxidation of many proteins and irreversible oxidative protein damage. Taurine, which functions as an antioxidant by trapping HOCl, was reduced in GRMD plasma; however taurine was increased in GRMD muscle tissue, potentially due to increased muscle taurine transport and synthesis. These data indicate a role for HOCl generated by neutrophils in the severe dystropathology of GRMD dogs, which may be exacerbated by decreased availability of taurine in the blood. These novel data support continued research into the precise roles of oxidative stress and taurine in DMD and emphasise the value of the GRMD dogs as a suitable pre-clinical model for testing taurine as a therapeutic intervention for DMD boys.
Assuntos
Inflamação/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Estresse Oxidativo , Animais , Biomarcadores , Modelos Animais de Doenças , Cães , Inflamação/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Proteínas Musculares/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/patologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Oxirredução , Peroxidase/metabolismo , Tirosina/metabolismoRESUMO
Calprotectin provides nutritional immunity by sequestering manganese and zinc ions. It is abundant in the lungs of patients with cystic fibrosis but fails to prevent their recurrent infections. Calprotectin is a major protein of neutrophils and composed of two monomers, S100A8 and S100A9. We show that the ability of calprotectin to limit growth of Staphylococcus aureus and Pseudomonas aeruginosa is exquisitely sensitive to oxidation by hypochlorous acid. The N-terminal cysteine residue on S100A9 was highly susceptible to oxidation which resulted in cross-linking of the protein monomers. The N-terminal methionine of S100A8 was also readily oxidized by hypochlorous acid, forming both the methionine sulfoxide and the unique product dehydromethionine. Isolated human neutrophils formed these modifications on calprotectin when their myeloperoxidase generated hypochlorous acid. Up to 90% of the N-terminal amine on S100A8 in bronchoalveolar lavage fluid from young children with cystic fibrosis was oxidized. Oxidized calprotectin was higher in children with cystic fibrosis compared to disease controls, and further elevated in those patients with infections. Our data suggest that oxidative stress associated with inflammation in cystic fibrosis will stop metal sequestration by calprotectin. Consequently, strategies aimed at blocking extracellular myeloperoxidase activity should enable calprotectin to provide nutritional immunity within the airways.
Assuntos
Ácido Hipocloroso/química , Complexo Antígeno L1 Leucocitário/química , Sequência de Aminoácidos , Pré-Escolar , Fibrose Cística/microbiologia , Humanos , Lactente , Complexo Antígeno L1 Leucocitário/fisiologia , Dados de Sequência Molecular , Neutrófilos/imunologia , Neutrófilos/microbiologia , Oxirredução , Estresse Oxidativo , Ligação Proteica , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
The physiological function of urate is poorly understood. It may act as a danger signal, an antioxidant, or a substrate for heme peroxidases. Whether it reacts sufficiently rapidly with lactoperoxidase (LPO) to act as a physiological substrate remains unknown. LPO is a mammalian peroxidase that plays a key role in the innate immune defense by oxidizing thiocyanate to the bactericidal and fungicidal agent hypothiocyanite. We now demonstrate that urate is a good substrate for bovine LPO. Urate was oxidized by LPO to produce the electrophilic intermediates dehydrourate and 5-hydroxyisourate, which decayed to allantoin. In the presence of superoxide, high yields of hydroperoxides were formed by LPO and urate. Using stopped-flow spectroscopy, we determined rate constants for the reaction of urate with compound I (k1 = 1.1 × 10(7) M(-1) s(-1)) and compound II (k2 = 8.5 × 10(3) M(-1) s(-1)). During urate oxidation, LPO was diverted from its peroxidase cycle because hydrogen peroxide reacted with compound II to give compound III. At physiologically relevant concentrations, urate competed effectively with thiocyanate, the main substrate of LPO for oxidation, and inhibited production of hypothiocyanite. Similarly, hypothiocyanite-dependent killing of Pseudomonas aeruginosa was inhibited by urate. Allantoin was present in human saliva and associated with the concentration of LPO. When hydrogen peroxide was added to saliva, oxidation of urate was dependent on its concentration and peroxidase activity. Our findings establish urate as a likely physiological substrate for LPO that will influence host defense and give rise to reactive electrophilic metabolites.
Assuntos
Lactoperoxidase/metabolismo , Tiocianatos/metabolismo , Ácido Úrico/metabolismo , Animais , Antibacterianos/metabolismo , Ligação Competitiva , Bovinos , Humanos , Imunidade Inata , Cinética , Lactoperoxidase/imunologia , Modelos Biológicos , Oxirredução , Pseudomonas aeruginosa/imunologia , Saliva/imunologia , Saliva/metabolismo , Especificidade por SubstratoRESUMO
OBJECTIVES: The aims of this study were to establish whether, in patients with gout, MPO is released from neutrophils and urate is oxidized to allantoin and if these effects are attenuated by allopurinol. METHODS: MPO, urate, allantoin and oxypurinol were measured in plasma from 54 patients with gout and 27 healthy controls. Twenty-three patients had acute gout, 13 of whom were receiving allopurinol, and 31 had intercritical gout, 20 of whom were receiving allopurinol. Ten additional gout patients had samples collected before and after 4 weeks of allopurinol. RESULTS: Plasma MPO and its specific activity were higher (P < 0.05) in patients with acute gout not receiving allopurinol compared with controls. MPO protein in patients' plasma was related to urate concentration (r = 0.5, P < 0.001). Plasma allantoin was higher (P < 0.001) in all patient groups compared with controls. In controls and patients not receiving allopurinol, allantoin was associated with plasma urate (r = 0.62, P < 0.001) and MPO activity (r = 0.45, P < 0.002). When 10 patients were treated with allopurinol, it lowered their plasma urate and allantoin (P = 0.002). In all patients receiving allopurinol, plasma allantoin was related to oxypurinol (r = 0.65, P < 0.0001). Oxypurinol was a substrate for purified MPO that enhanced the oxidation of urate. CONCLUSION: Increased concentrations of urate in gout lead to the release of MPO from neutrophils and the oxidation of urate. Products of MPO and reactive metabolites of urate may contribute to the pathology of gout and hyperuricaemia. At low concentrations, oxypurinol should reduce inflammation, but high concentrations may contribute to oxidative stress.
Assuntos
Alopurinol/uso terapêutico , Gota/metabolismo , Hiperuricemia/sangue , Estresse Oxidativo , Peroxidase/sangue , Ácido Úrico/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Ensaio de Imunoadsorção Enzimática , Feminino , Gota/tratamento farmacológico , Gota/etiologia , Supressores da Gota/uso terapêutico , Humanos , Hiperuricemia/complicações , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , OxirreduçãoRESUMO
Glutathione is an important antioxidant in the lungs but its concentration is low in the airways of patients with cystic fibrosis. Whether this deficit occurs from an early age or how oxidative stress contributes to lowering glutathione is unknown. We measured glutathione, its oxidation products, myeloperoxidase, and biomarkers of hypochlorous acid in bronchoalveolar lavage from children with cystic fibrosis and disease controls using mass spectrometry and immunological techniques. The concentration of glutathione was lower in bronchoalveolar lavage from children with cystic fibrosis, whereas glutathione sulfonamide, a specific oxidation product of hypochlorous acid, was higher. Oxidised glutathione and glutathione sulfonamide correlated with myeloperoxidase and a biomarker of hypochlorous acid. The percentage of glutathione attached to proteins was higher in children with cystic fibrosis than controls. Pulmonary infections in cystic fibrosis resulted in lower levels of glutathione but higher levels of oxidised glutathione and glutathione sulfonamide in bronchoalveolar lavage. The concentration of glutathione is low in the airways of patients with cystic fibrosis from an early age. Increased oxidation of glutathione by hypochlorous acid and its attachment to proteins contribute to this deficiency. Therapies targeted against myeloperoxidase may boost antioxidant defence and slow the onset and progression of lung disease in cystic fibrosis.
Assuntos
Fibrose Cística/metabolismo , Glutationa/química , Oxigênio/química , Antioxidantes/química , Lavagem Broncoalveolar , Líquido da Lavagem Broncoalveolar/química , Estudos de Casos e Controles , Criança , Pré-Escolar , Glutationa/análogos & derivados , Glutationa/metabolismo , Humanos , Ácido Hipocloroso/química , Inflamação , Pulmão/metabolismo , Espectrometria de Massas , Neutrófilos/metabolismo , Estresse Oxidativo , Peroxidase/química , Radiografia Torácica , Sistema Respiratório/metabolismo , Estudos Retrospectivos , Sulfonas/metabolismo , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: Chlorine bleach, or hypochlorous acid, is the most reactive two-electron oxidant produced in appreciable amounts in our bodies. Neutrophils are the main source of hypochlorous acid. These champions of the innate immune system use it to fight infection but also direct it against host tissue in inflammatory diseases. Neutrophils contain a rich supply of the enzyme myeloperoxidase. It uses hydrogen peroxide to convert chloride to hypochlorous acid. SCOPE OF REVIEW: We give a critical appraisal of the best methods to measure production of hypochlorous acid by purified peroxidases and isolated neutrophils. Robust ways of detecting it inside neutrophil phagosomes where bacteria are killed are also discussed. Special attention is focused on reaction-based fluorescent probes but their visual charm is tempered by stressing their current limitations. Finally, the strengths and weaknesses of biomarker assays that capture the footprints of chlorine in various pathologies are evaluated. MAJOR CONCLUSIONS: Detection of hypochlorous acid by purified peroxidases and isolated neutrophils is best achieved by measuring accumulation of taurine chloramine. Formation of hypochlorous acid inside neutrophil phagosomes can be tracked using mass spectrometric analysis of 3-chlorotyrosine and methionine sulfoxide in bacterial proteins, or detection of chlorinated fluorescein on ingestible particles. Reaction-based fluorescent probes can also be used to monitor hypochlorous acid during phagocytosis. Specific biomarkers of its formation during inflammation include 3-chlorotyrosine, chlorinated products of plasmalogens, and glutathione sulfonamide. GENERAL SIGNIFICANCE: These methods should bring new insights into how chlorine bleach is produced by peroxidases, reacts within phagosomes to kill bacteria, and contributes to inflammation. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.
Assuntos
Ácido Hipocloroso/análise , Inflamação/metabolismo , Neutrófilos/metabolismo , Animais , Humanos , Peroxidase/metabolismoRESUMO
The neutrophil enzyme myeloperoxidase (MPO) promotes oxidative stress in numerous inflammatory pathologies by producing hypohalous acids. Its inadvertent activity is a prime target for pharmacological control. Previously, salicylhydroxamic acid was reported to be a weak reversible inhibitor of MPO. We aimed to identify related hydroxamates that are good inhibitors of the enzyme. We report on three hydroxamates as the first potent reversible inhibitors of MPO. The chlorination activity of purified MPO was inhibited by 50% by a 5 nm concentration of a trifluoromethyl-substituted aromatic hydroxamate, HX1. The hydroxamates were specific for MPO in neutrophils and more potent toward MPO compared with a broad range of redox enzymes and alternative targets. Surface plasmon resonance measurements showed that the strength of binding of hydroxamates to MPO correlated with the degree of enzyme inhibition. The crystal structure of MPO-HX1 revealed that the inhibitor was bound within the active site cavity above the heme and blocked the substrate channel. HX1 was a mixed-type inhibitor of the halogenation activity of MPO with respect to both hydrogen peroxide and halide. Spectral analyses demonstrated that hydroxamates can act variably as substrates for MPO and convert the enzyme to a nitrosyl ferrous intermediate. This property was unrelated to their ability to inhibit MPO. We propose that aromatic hydroxamates bind tightly to the active site of MPO and prevent it from producing hypohalous acids. This mode of reversible inhibition has potential for blocking the activity of MPO and limiting oxidative stress during inflammation.
Assuntos
Inibidores Enzimáticos/farmacologia , Hidrocarbonetos Aromáticos/farmacologia , Ácidos Hidroxâmicos/farmacologia , Peroxidase/química , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Humanos , Hidrocarbonetos Aromáticos/síntese química , Hidrocarbonetos Aromáticos/química , Ácidos Hidroxâmicos/química , Cinética , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Neutrófilos/enzimologia , Peroxidase/antagonistas & inibidores , Peroxidase/metabolismo , Ligação ProteicaRESUMO
Neutrophils ingest Mycobacteria tuberculosis (Mtb) in the lungs of infected individuals. During phagocytosis they use myeloperoxidase (MPO) to catalyze production of hypochlorous acid (HOCl), their most potent antimicrobial agent. Isoniazid (INH), the foremost antibiotic in the treatment of tuberculosis, is oxidized by MPO. It rapidly reduced compound I of MPO [k = (1.22 ± 0.05) × 10(6) M(-1) s(-1)] but reacted less favorably with compound II [(9.8 ± 0.6) × 10(2) M(-1) s(-1)]. Oxidation of INH by MPO and hydrogen peroxide was unaffected by chloride, the physiological substrate for compound I, and the enzyme was partially converted to compound III. This indicates that INH is oxidized outside the classical peroxidation cycle. In combination with superoxide dismutase (SOD), MPO oxidized INH without exogenous hydrogen peroxide. SOD must favor reduction of oxygen by the INH radical to give superoxide and ultimately hydrogen peroxide. In both oxidation systems, an adduct with methionine was formed and it was a major product with MPO and SOD. We show that it is a conjugate of an acyldiimide with amines. INH substantially inhibited HOCl production by MPO and neutrophils below pharmacological concentrations. The reversible inhibition is explained by diversion of MPO to its ferrous and compound III forms during oxidation of INH. MPO, along with SOD released by Mtb, will oxidize INH at sites of infection and their interactions are likely to limit the efficacy of the drug, promote adverse drug reactions via formation of protein adducts, and impair a major bacterial killing mechanism of neutrophils.
Assuntos
Aminas/química , Isoniazida/farmacologia , Peroxidase/antagonistas & inibidores , Peroxidase/metabolismo , Superóxido Dismutase/metabolismo , Benzidinas , Cromatografia Líquida de Alta Pressão , Humanos , Peróxido de Hidrogênio , Ácido Hipocloroso/metabolismo , Imunidade Inata/efeitos dos fármacos , Isoniazida/análogos & derivados , Isoniazida/química , Espectrometria de Massas , Estrutura Molecular , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , OxirreduçãoRESUMO
OBJECTIVE: To determine whether MPO contributes to oxidative stress and disease activity in RA and whether it produces hypochlorous acid in SF. METHODS: Plasma and where possible SF were collected from 77 RA patients while 120 healthy controls supplied plasma only. MPO and protein carbonyls were measured by ELISAs. 3-Chlorotyrosine in proteins and allantoin in plasma were measured by mass spectrometry. RESULTS: Plasma MPO concentrations were significantly higher in patients with RA compared with healthy controls [10.8 ng/ml, inter-quartile range (IQR): 7.2-14.2; P<0.05], but there was no significant difference in plasma MPO protein concentrations between RA patients with high disease activity (HDA; DAS-28 >3.2) and those with low disease activity (LDA; DAS-28 ≤ 3.2) (HDA 27.9 ng/ml, 20.2-34.1 vs LDA 22.1 ng/ml, 16.9-34.9; P>0.05). There was a significant relationship between plasma MPO and DAS-28 (r=0.35; P=0.005). Plasma protein carbonyls and allantoin were significantly higher in patients with RA compared with the healthy controls. MPO protein was significantly higher in SF compared with plasma (median 624.0 ng/ml, IQR 258.4-2433.0 vs 30.2 ng/ml, IQR 25.1-50.9; P<0.0001). The MPO present in SF was mostly active. 3-Chlorotyrosine, a specific biomarker of hypochlorous acid, was present in proteins from SF and related to the concentration of MPO (r=0.69; P=0.001). Protein carbonyls in SF were associated with MPO protein concentration (r=0.40; P=0.019) and 3-chlorotyrosine (r=0.66; P=0.003). CONCLUSION: MPO is elevated in patients with RA and promotes oxidative stress through the production of hypochlorous acid.
Assuntos
Artrite Reumatoide/metabolismo , Estresse Oxidativo/fisiologia , Peroxidase/metabolismo , Líquido Sinovial/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peroxidase/sangue , Espécies Reativas de Oxigênio/metabolismoRESUMO
Allantoin is the major oxidation product of urate in humans and is a potential biomarker of oxidative stress. Several methods are used to measure allantoin in biological samples but they have inherent issues that can include lack of specificity and sensitivity, difficulty in sample preparation, or artefactual generation of allantoin. We have developed a method for measuring allantoin using hydrophilic liquid chromatography with stable isotope dilution tandem mass spectrometry (HILIC-MS/MS). It was validated for measuring allantoin in plasma, synovial fluid and urine from human subjects. The limit of quantification was determined to be 10 fmol and the assay displayed excellent linearity for the wide range of concentrations found in clinical samples. Relative standard deviations were <5% for between-day and <7% for within-day variation. Accuracy was between 100% and 104%. Concentrations of allantoin in plasma of healthy controls (2.0 µM; interquartile range 1.4-3.6 µM, n=35) was significantly lower (p<0.001) than that in plasma from patients with rheumatoid arthritis (3.7 µM; IQR 3.0-5.6 µM, n=43) and in synovial fluid of patients with gout (3.3 µM; IQR 2.8-5.8 µM, n=10). This newer HILIC-MS/MS method is a simple and highly sensitive assay for detection of allantoin. It can be used to assess the level of oxidative stress in human pathologies.
Assuntos
Alantoína/análise , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Humanos , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
A comparison of the efficacy of the copper chelator, trientine, with combined renin angiotensin system (RAS) blockade on the progression of glomerular pathology in the diabetic (mREN-2)27 rat is reported. Animals were treated for 2 months with trientine, combined RAS blockers, combined trientine plus RAS blockers or none. Treatments began after inducing diabetes with streptozotocin. Physiological data were recorded monthly and light microscopic glomerular features were scored. Plasma allantoin and both plasma and renal protein carbonyls were measured as markers of oxidative stress. Trientine and RAS blockade decreased proteinuria and albuminuria and prevented an increase in creatinine clearance and kidney weight. Both reduced the diabetes-related glomerular features of mesangiolysis and glomerular segmental hypocellularity and trientine prevented severe tuft-to-capsule adhesion and reduced tubularization. Hypertension-related severe mesangial matrix expansion and global hypercellularity were increased by both treatments, which may reflect repair of mesangiolysis. Trientine reduced plasma but not renal protein carbonyls or plasma allantoin. In this model, trientine prevented the development of many diabetes-specific features similarly to RAS blockade. Amelioration of oxidative stress and features commonly observed in human diabetic nephropathy (DN), support a diabetes-related defect in copper (Cu) metabolism. The addition of Cu(II) chelation may improve current DN therapy.
Assuntos
Quelantes/farmacologia , Nefropatias Diabéticas/patologia , Rim/efeitos dos fármacos , Sistema Renina-Angiotensina/efeitos dos fármacos , Trientina/farmacologia , Alantoína/sangue , Animais , Cobre/metabolismo , Creatinina/metabolismo , Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/induzido quimicamente , Nefropatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Heterozigoto , Humanos , Hipertensão/complicações , Rim/patologia , Rim/fisiopatologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Glomérulos Renais/fisiopatologia , Masculino , Estresse Oxidativo , Proteinúria/prevenção & controle , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Sistema Renina-Angiotensina/fisiologia , EstreptozocinaRESUMO
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, which decayed to predominantly allantoin. The redox intermediates of MPO were reduced by urate with rate constants of 4.6 × 10(5) M(-1) s(-1) for compound I and 1.7 × 10(4) M(-1) s(-1) for compound II. Urate competed with chloride for oxidation by MPO and at hyperuricemic levels is expected to be a substantive substrate for the enzyme. Oxidation of urate promoted super-stoichiometric consumption of glutathione, which indicates that it is converted to a free radical intermediate. In combination with superoxide and hydrogen peroxide, MPO oxidized urate to a reactive hydroperoxide. This would form by addition of superoxide to the urate radical. Urate also enhanced MPO-dependent consumption of nitric oxide. In human plasma, stimulated neutrophils produced allantoin in a reaction dependent on the NADPH oxidase, MPO and superoxide. We propose that urate is a physiological substrate for MPO that is oxidized to the urate radical. The reactions of this radical with superoxide and nitric oxide provide a plausible link between urate and MPO in cardiovascular disease.
Assuntos
Peróxido de Hidrogênio/metabolismo , Hiperuricemia/enzimologia , Neutrófilos/enzimologia , Peroxidase/metabolismo , Superóxidos/metabolismo , Alantoína/biossíntese , Alantoína/química , Doenças Cardiovasculares/enzimologia , Humanos , Peróxido de Hidrogênio/química , Inflamação , NADPH Oxidases/química , NADPH Oxidases/metabolismo , Oxirredução , Peroxidase/química , Especificidade por Substrato , Superóxidos/química , Ácido ÚricoRESUMO
During inflammatory events, neutrophils and platelets interact to release a variety of mediators. Neutrophils generate superoxide and hydrogen peroxide, and also discharge the haem enzyme myeloperoxidase. Among numerous other mediators, platelets liberate serotonin (5-hydroxytryptamine), which is a classical neurotransmitter and vasoactive amine that has significant effects on inflammation and immunity. In the present study, we show that serotonin is a favoured substrate for myeloperoxidase because other physiological substrates for this enzyme, including chloride, did not affect its rate of oxidation. At low micromolar concentrations, serotonin enhanced hypochlorous acid production by both purified myeloperoxidase and neutrophils. At higher concentrations, it almost completely blocked the formation of hypochlorous acid. Serotonin was oxidized to a dimer by myeloperoxidase and hydrogen peroxide. It was also converted into tryptamine-4,5-dione, especially in the presence of superoxide. This toxic quinone was produced by stimulated neutrophils in a reaction that required myeloperoxidase. In plasma, stimulated human neutrophils oxidized serotonin to its dimer using the NADPH oxidase and myeloperoxidase. We propose that myeloperoxidase will oxidize serotonin at sites of inflammation. In doing so, it will impair its physiological functions and generate a toxic metabolite that will exacerbate inflammatory tissue damage. Consequently, oxidation of serotonin by myeloperoxidase may profoundly influence inflammatory processes.
Assuntos
Indolquinonas/metabolismo , Peroxidase/metabolismo , Serotonina/metabolismo , Superóxidos/metabolismo , Triptaminas/metabolismo , Catálise/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Citotoxinas/metabolismo , Dimerização , Inibidores Enzimáticos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Humanos , Peróxido de Hidrogênio/metabolismo , Ácido Hipocloroso/metabolismo , Espectrometria de Massas , Neutrófilos/citologia , Neutrófilos/metabolismo , Oxirredução/efeitos dos fármacos , Peroxidase/antagonistas & inibidores , Serotonina/química , Especificidade por SubstratoRESUMO
During infection and inflammation, neutrophils and eosinophils produce hypochlorous acid, hypobromous acid, chloramines, and bromamines. These reactive halogen species preferentially oxidize methionine and thiols. It is commonly assumed that they convert methionine to methionine sulfoxide. However, iodine and organic chloramines are known to convert methionine to dehydromethionine, which is a cyclic azasulfonium salt. The potential for this reaction to occur in biologically relevant situations has so far been neglected. Therefore, we investigated the oxidation of methionine and N-terminal methionine residues by biologically relevant reactive halogen species and neutrophils. When hypochlorous acid reacted with methionine, two major products in addition to methionine sulfoxide were formed. They both had molecular masses two mass units lower than that of methionine and were identified as the diastereomers of dehydromethionine. Hypochlorous acid and chloramines converted methionine to a mixture of approximately 25% dehydromethionine and 75% methionine sulfoxide. Hypobromous acid and bromamines produced upward of 50% dehydromethionine. When methionine was present on the N-termini of peptides, reactive halogen species oxidized them to dehydromethionine with yields as high as 80%. Formylated N-terminal methionines and non-N-terminal methionine residues gave stoichiometric production of the corresponding sulfoxides only. Purified myeloperoxidase used hydrogen peroxide and chloride to catalyze the oxidation of N-terminal methionines to dehydromethionine. Neutrophils oxidized extracellular methionine to 30% dehydromethionine and 70% methionine sulfoxide. They also oxidized their intracellular methionine to dehydromethionine as well as methionine sulfoxide. We propose that reactive halogen species will produce dehydromethionine and form azasulfonium cations on the N-termini of peptides and proteins during inflammatory events.
Assuntos
Halogênios/química , Metionina/química , Neutrófilos/metabolismo , Tiazóis/química , Halogênios/metabolismo , Humanos , Metionina/metabolismo , Oxirredução , Peptídeos/química , Peptídeos/metabolismo , Peroxidase/metabolismo , Tiazóis/metabolismoRESUMO
BACKGROUND: Discrepancies have been reported between estimates of the prevalence of riboflavin deficiency based on intakes of riboflavin and estimates based on measures of riboflavin status. One reason for this may be an overestimate of the bioavailability of riboflavin from foods, about which relatively little is known. OBJECTIVE: We aimed to quantify the bioavailability of riboflavin from milk and spinach by using stable-isotope labels and a urinary monitoring technique and by a plasma appearance method based on kinetic modeling. DESIGN: Twenty healthy women aged 18-65 y were recruited for a randomized crossover study performed with extrinsically labeled (13C) milk and intrinsically labeled (15N) spinach as sources of riboflavin. An intravenous bolus of labeled riboflavin was administered with each test meal to assess the apparent volume of distribution of riboflavin in plasma. RESULTS: No significant differences were noted in riboflavin absorption from the spinach meal and from the milk meal according to either the urinary monitoring technique (60 +/- 8.0% and 67 +/- 5.4%, respectively; P = 0.549) or the plasma appearance method (20 +/- 2.8% and 23 +/- 5.3%, respectively; P = 0.670). CONCLUSIONS: A large fraction of newly absorbed riboflavin is removed by the liver on "first pass." The plasma appearance method therefore underestimates riboflavin bioavailability and should not be used to estimate riboflavin bioavailability from foodstuffs. Urinary monitoring suggests that riboflavin from spinach is as bioavailable as is riboflavin from milk.