RESUMEN
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.
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Artritis Reumatoide/metabolismo , Estrés Oxidativo/fisiología , Peroxidasa/metabolismo , Líquido Sinovial/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Peroxidasa/sangre , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Myeloperoxidase (MPO) is a prime candidate for promoting oxidative stress during inflammation. This abundant enzyme of neutrophils uses hydrogen peroxide to oxidize chloride to highly reactive and toxic chlorine bleach. We have identified 2-thioxanthines as potent mechanism-based inactivators of MPO. Mass spectrometry and x-ray crystal structures revealed that these inhibitors become covalently attached to the heme prosthetic groups of the enzyme. We propose a mechanism whereby 2-thioxanthines are oxidized, and their incipient free radicals react with the heme groups of the enzyme before they can exit the active site. 2-Thioxanthines inhibited MPO in plasma and decreased protein chlorination in a mouse model of peritonitis. They slowed but did not prevent neutrophils from killing bacteria and were poor inhibitors of thyroid peroxidase. Our study shows that MPO is susceptible to the free radicals it generates, and this Achilles' heel of the enzyme can be exploited to block oxidative stress during inflammation.
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Inhibidores Enzimáticos , Neutrófilos/enzimología , Estrés Oxidativo/efectos de los fármacos , Peritonitis/enzimología , Peroxidasa , Xantinas , Animales , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Inflamación/tratamiento farmacológico , Inflamación/etnología , Inflamación/microbiología , Inflamación/patología , Ratones , Neutrófilos/patología , Oxidación-Reducción/efectos de los fármacos , Peritonitis/tratamiento farmacológico , Peritonitis/patología , Peroxidasa/antagonistas & inhibidores , Peroxidasa/química , Peroxidasa/metabolismo , Xantinas/química , Xantinas/farmacologíaRESUMEN
We aimed to determine whether myeloperoxidase (MPO) is the main peroxidase present in the airways of children with cystic fibrosis (CF) and to assess which oxidants it produces and whether they are associated with clinical features of CF. Children with CF (n=54) and without CF (n=16) underwent bronchoscopy and bronchoalveolar lavage (BAL) for assessment of pulmonary infection and inflammation. BAL fluid was analyzed for MPO, halogenated tyrosines as markers of hypohalous acids, thiocyanate, and protein carbonyls. MPO was the only peroxidase detected in BAL samples from children with CF and its concentration was markedly higher than in controls. Levels of 3-chlorotyrosine and 3-bromotyrosine in proteins were higher in the CF group. They correlated with neutrophils and MPO. The concentration of thiocyanate in BAL samples was below 1µM. Protein carbonyl levels correlated with MPO and halogenated tyrosines in patients with CF. Levels of MPO and halogenated tyrosines were higher in children with infections, especially Pseudomonas aeruginosa, and in the presence of respiratory symptoms. They also correlated with the Kanga clinical score. Our findings suggest that MPO produces hypobromous acid as well as hypochlorous acid in the airways of children with CF and that these oxidants are involved in the early pathogenesis of CF.
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Líquido del Lavado Bronquioalveolar/química , Fibrosis Quística/enzimología , Neutrófilos/metabolismo , Peroxidasa/metabolismo , Pseudomonas aeruginosa/inmunología , Infecciones del Sistema Respiratorio/enzimología , Líquido del Lavado Bronquioalveolar/citología , Niño , Preescolar , Fibrosis Quística/complicaciones , Fibrosis Quística/fisiopatología , Progresión de la Enfermedad , Femenino , Humanos , Lactante , Inflamación , Masculino , Neutrófilos/patología , Oxidación-Reducción , Pseudomonas aeruginosa/patogenicidad , Infecciones del Sistema Respiratorio/complicaciones , Infecciones del Sistema Respiratorio/fisiopatología , Tirosina/análogos & derivados , Tirosina/análisisRESUMEN
BACKGROUND: Inhaled corticosteroids are widely used in the treatment of persistent asthma, usually combined with inhaled beta2-agonists. Previous research suggests that short-acting beta2-agonists (SABAs) may downregulate the anti-inflammatory effects of inhaled corticosteroids, thereby increasing asthma morbidity. OBJECTIVE: To determine whether 3-bromotyrosine and 3,5-dibromotyrosine levels, specific markers of eosinophil activation, reflect treatment effects on airway inflammation of inhaled corticosteroids and SABAs and support previous conclusions. METHODS: Levels of 3-bromotyrosine and 3,5-dibromotyrosine were measured in sputum supernatants using stable isotope dilution gas chromatography-mass spectrometry in a randomized, placebo-controlled, crossover study of treatment with terbutaline, budesonide, and their combination in patients with persistent asthma. Thirty-four individuals were randomized, and 28 completed the study. RESULTS: Treatment with budesonide lowered median 3-bromotyrosine levels compared with treatment with placebo, terbutaline, and budesonide-terbutaline (0.24 vs 0.64, 0.62, and 0.43 3-bromotyosine/tyrosine [mmol/mol]; P < .05) and lowered median 3,5-dibromotyrosine levels compared with placebo and terbutaline treatments (0.04 vs 0.11 and 0.07 3,5-dibromotyrosine/ tyrosine [mmol/mol], P < .05). Unlike eosinophil numbers, 3-bromotyrosine and 3,5-dibromotyrosine levels did not increase with terbutaline treatment compared with placebo treatment but were significantly raised when terbutaline was added to budesonide treatment. 3-Bromotyrosine levels correlated significantly with eosinophil cationic protein levels in all groups. CONCLUSIONS: 3-Bromotyrosine and 3,5-dibromotyrosine levels reflect treatment effects in asthma and support previous findings that SABAs impair the anti-inflammatory effects of inhaled corticosteroids. In addition to eosinophil numbers and eosinophil cationic protein levels, these modified tyrosine residues provide useful information about the inflammatory state of the airways.
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Agonistas Adrenérgicos beta/administración & dosificación , Asma/tratamiento farmacológico , Asma/metabolismo , Broncodilatadores/administración & dosificación , Budesonida/administración & dosificación , Esputo/metabolismo , Terbutalina/administración & dosificación , Tirosina/análogos & derivados , Adolescente , Adulto , Asma/inmunología , Estudios Cruzados , Método Doble Ciego , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pruebas de Función Respiratoria , Esputo/citología , Tirosina/metabolismo , Adulto JovenRESUMEN
MPO (myeloperoxidase) catalyses the oxidation of chloride, bromide and thiocyanate to their respective hypohalous acids. We have investigated the generation of HOBr by human neutrophils in the presence of physiological concentrations of chloride and bromide. HOBr was trapped with taurine and detected by monitoring the bromination of 4-HPAA (4-hydroxyphenylacetic acid). With 100 microM bromide and 140 mM chloride, neutrophils generated HOBr and it accounted for approx. 13% of the hypohalous acids they produced. Addition of SOD (superoxide dismutase) doubled the amount of HOBr detected. Therefore we investigated the reaction of superoxide radicals with a range of bromamines and bromamides and found that superoxide radicals stimulated the decomposition of these species, with this occurring in a time- and dose-dependent manner. The protection afforded by SOD against such decay demonstrates that these processes are superoxide-radical-dependent. These data are consistent with neutrophils generating HOBr at sites of infection and inflammation. Both HOBr and bromamines/bromamides have the potential to react with superoxide radicals to form additional radicals that may contribute to inflammatory tissue damage.
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Bromatos/metabolismo , Bromuros/metabolismo , Neutrófilos/metabolismo , Superóxidos/metabolismo , Bromatos/química , Bromuros/química , Humanos , Fenilacetatos/química , Fenilacetatos/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Superóxido Dismutasa/metabolismo , Taurina/análogos & derivados , Taurina/metabolismoRESUMEN
Flavonoids are increasingly being ingested by the general population as chemotherapeutic and anti-inflammatory agents. They are potentially toxic because of their conversion to free radicals and reactive quinones by peroxidases. Little detailed information is available on how flavonoids interact with myeloperoxidase, which is the predominant peroxidase present at sites of inflammation. This enzyme uses hydrogen peroxide to oxidize chloride to hypochlorous acid, as well as to produce an array of reactive free radicals from organic substrates. We investigated how the flavonoid myricitrin is oxidized by myeloperoxidase and how it affects the activities of this enzyme. Myricitrin was readily oxidized by myeloperoxidase in the presence of hydrogen peroxide. Its main oxidation product was a dimer that underwent further oxidation. In the presence of glutathione, myricitrin was oxidized to a hydroquinone that was conjugated to glutathione. When myeloperoxidase oxidized myricitrin and related flavonoids it became irreversibly inactivated. The number of hydroxyl groups in the B ring of the flavonoids and the presence of a free hydroxyl m-phenol group in the A ring were important for the inhibitory effects. Less enzyme inactivation occurred in the presence of chloride. Neutrophils also oxidized myricitrin to dimers in a reaction that was partially dependent on myeloperoxidase. Myricitrin did not affect the production of hypochlorous acid by neutrophils. We conclude that myricitrin will be oxidized by neutrophils at sites of inflammation to produce reactive free radicals and quinones. It is unlikely to affect hypochlorous acid production by neutrophils.
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Flavonoides/metabolismo , Peroxidasa/metabolismo , Apoptosis/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Activación Enzimática/efectos de los fármacos , Flavonoides/química , Flavonoides/farmacología , Radicales Libres , Glutatión/metabolismo , Glutatión/farmacología , Humanos , Ácido Hipocloroso/química , Ácido Hipocloroso/metabolismo , Técnicas In Vitro , Inflamación/enzimología , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Oxidación-Reducción , Peroxidasa/antagonistas & inhibidores , Peroxidasa/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
OBJECTIVES: This study investigated relationships between plasma myeloperoxidase (MPO), protein oxidation markers, and clinical outcome retrospectively in patients after acute myocardial infarction (MI). BACKGROUND: Reactive oxidants are implicated in cardiovascular disease, and elevated plasma MPO is reported to predict adverse outcome in acute coronary syndromes. METHODS: Detailed demographic information, radionuclide ventriculography, neurohormone measurements, and clinical history were obtained for 512 acute MI patients at hospital admission. Plasma levels of MPO and protein carbonyls were measured in patients and 156 heart-healthy control subjects. 3-chlorotyrosine was measured in selected patients. Patient mortality was followed for 5 years. RESULTS: Plasma MPO and protein carbonyl concentrations were higher in MI patients 24 h to 96 h after admission than in control subjects (medians: MPO 55 ng/ml vs. 39 ng/ml, and protein carbonyls 48 pmol/mg vs. 17 pmol/mg protein, p < 0.001 for each). Both markers were significantly correlated with each other and with cardiovascular hormone levels. Chlorotyrosine was not elevated in patients with high MPO or carbonyl levels. Above-median levels of MPO but not protein carbonyls were independently predictive of mortality (odds ratio 1.8, 95% confidence interval 1.0 to 3.0, p = 0.034). Patients with above-median MPO levels in combination with above-median plasma amino-terminal pro-brain natriuretic peptide (NT-proBNP) or below-median left ventricular ejection fraction (LVEF) had significantly greater mortality compared with other patients. CONCLUSIONS: Myeloperoxidase and protein carbonyl levels are elevated in plasma after acute MI, apparently via independent mechanisms. High MPO is a risk factor for long-term mortality and adds prognostic value to LVEF and plasma NT-proBNP measurements.
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Infarto del Miocardio/sangre , Infarto del Miocardio/mortalidad , Peroxidasa/sangre , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Estudios de Casos y Controles , Diabetes Mellitus Tipo 2/complicaciones , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Neurotransmisores/sangre , Nueva Zelanda/epidemiología , Pronóstico , Modelos de Riesgos Proporcionales , Carbonilación Proteica , Cintigrafía , Estudios Retrospectivos , Factores Sexuales , Volumen Sistólico , Tirosina/análogos & derivados , Tirosina/sangre , Disfunción Ventricular Izquierda/diagnóstico por imagenRESUMEN
Myeloperoxidase and eosinophil peroxidase use hydrogen peroxide to oxidize halides and thiocyanate to their respective hypohalous acids. Myeloperoxidase produces mainly hypochlorous acid and hypothiocyanite. Hypobromous acid and hypothiocyanite are the major products of eosinophil peroxidase. We have investigated the ability of myeloperoxidase to produce hypobromous acid in the presence of physiological concentrations of chloride and bromide. In accord with previous studies, between pH 5 and 7, myeloperoxidase converted about 90% of available hydrogen peroxide to hypochlorous acid and the remainder to hypobromous acid. Above pH 7, there was an abrupt rise in the yield of hypobromous acid. At pH 7.8, it accounted for 40% of the hydrogen peroxide. Bromide, at physiological concentrations, promoted a dramatic increase in bromination of human serum albumin catalyzed by myeloperoxidase. The level of 3-bromotyrosine increased to 16-fold greater than that for 3-chlorotyrosine. Chlorination of tyrosyl residues was not affected by bromide. With reagent hypohalous acids, bromination of tyrosyl residues was considerably more facile than chlorination. Hypochlorous acid promoted bromination to only a limited extent, which ruled out transhalogenation as a substantive route to 3-bromotyrosine. Chloramines and bromamines were also formed on albumin. Bromamines decayed much faster than chloramines and rapidly gave rise to protein carbonyls. We conclude that at physiological concentrations of chloride and bromide, hypobromous acid can be a major oxidant produced by myeloperoxidase. Its production in vivo will depend on pH and the concentration of bromide. Once produced, hypobromous acid will react with proteins to form bromamines, carbonyls, and brominated tyrosine residues. Consequently, 3-bromotyrosine should be considered as an oxidative product of myeloperoxidase and cannot be used as a specific biomarker for eosinophil peroxidase.
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Bromatos/química , Bromuros/química , Cloruros/química , Peróxido de Hidrógeno/química , Ácido Hipocloroso/química , Peroxidasa/química , Albúmina Sérica/química , Líquidos Corporales/química , Concentración de Iones de Hidrógeno , Oxidación-ReducciónRESUMEN
Cystic fibrosis (CF) is characterized by considerable oxidative stress. However, it is not known whether oxidative stress is an important feature early in this disease. We have investigated a group of infants and young children with CF to establish whether oxidants are produced in their airways. Bronchoalveolar lavage fluid (BALF) was assayed for myeloperoxidase as a measure of neutrophilic inflammation, and 3-chlorotyrosine as a biomarker of the potent oxidant hypochlorous acid, which is formed by myeloperoxidase. Protein carbonyls were also measured as a nonspecific indicator of reactive oxidant production. Myeloperoxidase and 3-chlorotyrosine levels in BALF from children with CF were 10- and fivefold higher, respectively, than in disease control subjects. There was a strong correlation between myeloperoxidase and 3-chlorotyrosine. Myeloperoxidase levels were fourfold higher in children with infections in their airways. Median protein carbonyls were elevated by only twofold compared with disease control subjects, but some children had extremely high levels of protein oxidation. We conclude that hypochlorous acid is produced early in CF and that it is a candidate for precipitating the fatal decline in lung function associated with this disease. Also, there must be other sourcesof oxidants because protein carbonyls were not related to either inflammation or infection.
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Fibrosis Quística/metabolismo , Estrés Oxidativo , Peroxidasa/análisis , Líquido del Lavado Bronquioalveolar/química , Preescolar , Femenino , Humanos , Lactante , Masculino , Peroxidasa/metabolismo , Proteínas/metabolismoRESUMEN
Oxidative injury is implicated in the development of chronic lung disease in preterm infants with respiratory distress. However, direct evidence of a causal role is limited and the source of reactive oxidants has not been identified. We have previously shown that protein carbonyl levels in tracheal aspirates correlate positively with myeloperoxidase, suggesting that neutrophil oxidants could be the source of this protein injury. We have extended these observations by measuring 3-chlorotyrosine, a specific biomarker of the neutrophil oxidant, hypochlorous acid, in tracheal aspirate proteins (144 samples) from 69 infants with birth weight <1500 g. 3-Chlorotyrosine levels were higher in these infants than in larger infants without respiratory distress (median 83 compared with 13 micromol/mol tyrosine). They correlated strongly with myeloperoxidase activity (correlation coefficient 0.75, p < 0.0001) and to a lesser extent with protein carbonyls. 3-Chlorotyrosine levels (at 1 wk after birth) correlated negatively with birth weight or gestational age. They were significantly higher in infants who developed chronic lung disease (oxygen requirement at 36 wk postmenstrual age) than in those who did not (median 88 and 49 micromol/mol tyrosine, respectively) and correlated with days of supplemental oxygen. 3-Chlorotyrosine was also significantly higher in infants who had lung infection or were Ureaplasma urealyticum positive. Our results are the first evidence that chlorinated proteins are produced in the lungs of premature infants and that they are higher in infection. The higher 3-chlorotyrosine levels in infants who develop chronic lung disease suggest that neutrophil oxidants contribute to the pathology of this disease.
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Peroxidasa/metabolismo , Síndrome de Dificultad Respiratoria del Recién Nacido/metabolismo , Tráquea/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Biomarcadores , Enfermedad Crónica , Edad Gestacional , Humanos , Recién Nacido , Recien Nacido Prematuro , Recién Nacido de muy Bajo Peso , Neutrófilos/metabolismo , Estrés Oxidativo , Síndrome de Dificultad Respiratoria del Recién Nacido/microbiología , Infecciones por Ureaplasma/metabolismo , Ureaplasma urealyticumRESUMEN
Eosinophil peroxidase and myeloperoxidase use hydrogen peroxide to produce hypobromous acid and hypochlorous acid. These powerful oxidants may damage the lungs if they are produced as part of the inflammatory response in asthma. The aim of this study was to determine if peroxidases generate hypohalous acids in the airways of individuals with stable asthma, and if they affect lung function. Sputum was induced from patients with mild to moderate asthma and from healthy controls. Eosinophil peroxidase, myeloperoxidase, chlorinated and brominated tyrosyl residues, and protein carbonyls were measured in sputum supernatants. Eosinophil peroxidase protein was significantly elevated in asthmatic subjects whereas myeloperoxidase protein was not. There was significantly more 3-bromotyrosine (Br-Tyr) in proteins from the sputum of asthmatics compared to controls (0.79 vs. 0.23 mmol Br-Tyr/mol Tyr; medians p < .0001). Levels of 3-chlorotyrosine (0.23 vs. 0.14 mmol Cl-Tyr/mol Tyr; medians p = .11) and protein carbonyls (0.347 vs. 0.339 nmol/mg protein; medians p = .56) were not significantly increased in asthmatics. Levels of 3-bromotyrosine were strongly correlated with eosinophil peroxidase protein (r = 0.79, p < .0001). There were no significant correlations between the markers of oxidative stress and lung function. We conclude that eosinophil peroxidase produces substantial amounts of hypobromous acid in the airways of stable asthmatics. Although this highly reactive oxidant is a strong candidate for exacerbating inflammatory tissue damage in the lung, its role in asthma remains uncertain.
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Asma/enzimología , Bromatos/metabolismo , Pulmón/enzimología , Peroxidasas/metabolismo , Adulto , Anciano , Asma/metabolismo , Bromo/metabolismo , Cloro/metabolismo , Peroxidasa del Eosinófilo , Femenino , Radicales Libres , Halógenos/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Estrés Oxidativo , Oxígeno/metabolismo , Peroxidasa/metabolismo , Esputo/metabolismoRESUMEN
Free radicals have been hypothesized to play a key role in the evolution of periventricular leukomalacia, although direct evidence of oxidative injury in the human infant is lacking. This case report is the first to demonstrate a marked elevation in the levels of lipid and protein oxidative products in the cerebrospinal fluid during the evolution of periventricular leukomalacia in a premature infant with meningitis.
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Recien Nacido Prematuro , Leucomalacia Periventricular/líquido cefalorraquídeo , Peroxidación de Lípido , Meningitis Bacterianas/líquido cefalorraquídeo , Estrés Oxidativo , Biomarcadores , Femenino , Humanos , Recién Nacido , Leucomalacia Periventricular/microbiología , Leucomalacia Periventricular/patología , Imagen por Resonancia Magnética , Meningitis Bacterianas/complicacionesRESUMEN
Oxidative modification of low-density lipoprotein is thought to promote arterial lipid accumulation and atherogenesis. Previous studies reported on the presence of certain lipid or protein oxidation products in lesions, although a systematic investigation measuring several oxidation parameters and the accumulation of nonoxidized lipids and antioxidants at various stages of atherosclerosis has not been performed in the same tissue. Using the intimal lipoprotein-containing fraction of human aortic lesions, we demonstrate here that cholesterol accumulated with lesion development and that this increase was already significant at the fatty streak stage. By comparison, cholesterylesters increased significantly only in fibro-fatty and more complex lesions that also contained significantly increased amounts of cholesterylester hydro(pero)xides and 27-hydroxycholesterol. Cholesterylester hydroxides were the major lipid oxidation product detected. Despite accumulation of oxidized lipid, alpha-tocopherol was also present and maintained at a comparable level over the disease process. Of the oxidized protein moieties measured only o,o-dityrosine increased with disease, although chlorotyrosines were present at relatively high levels in all lesions compared to healthy vessels. Our data show that accumulation of nonoxidized lipid precedes that of oxidized lipid in human aortic lesions.
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Aorta/metabolismo , Arteriosclerosis/metabolismo , Metabolismo de los Lípidos , Proteínas/metabolismo , Adulto , Anciano , Antioxidantes/metabolismo , Aorta/patología , Arteriosclerosis/patología , Colesterol/metabolismo , Ésteres del Colesterol/metabolismo , Progresión de la Enfermedad , Humanos , Persona de Mediana Edad , Oxidación-Reducción , Fenilalanina/metabolismo , Tirosina/metabolismoRESUMEN
Myeloperoxidase is proposed to play a central role in bacterial killing by generating hypochlorous acid within neutrophil phagosomes. However, it has yet to be demonstrated that these inflammatory cells target hypochlorous acid against bacteria inside phagosomes. In this investigation, we treated Staphylococcus aureus with varying concentrations of reagent hypochlorous acid and found that even at sublethal doses, it converted some tyrosine residues in their proteins to 3-chlorotyrosine and 3,5-dichlorotyrosine. To determine whether or not ingested bacteria were exposed to hypochlorous acid in neutrophil phagosomes, we labeled their proteins with [(13)C(6)]tyrosine and used gas chromatography with mass spectrometry to identify the corresponding chlorinated isotopes after the bacteria had been phagocytosed. Chlorinated tyrosines were detected in bacterial proteins 5 min after phagocytosis and reached levels of approximately 2.5/1000 mol of tyrosine at 60 min. Inhibitor studies revealed that chlorination was dependent on myeloperoxidase. Chlorinated neutrophil proteins were also detected and accounted for 94% of total chlorinated tyrosine residues formed during phagocytosis. We conclude that hypochlorous acid is a major intracellular product of the respiratory burst. Although some reacts with the bacteria, most reacts with neutrophil components.