RESUMO
BACKGROUND: Dietary nutrient intake and its metabolism by the gut microbiome have recently been implicated in cardiovascular disease (CVD) risk. In particular, trimethylamine N-oxide (TMAO), a metabolite of the gut microbiota, has been shown to be a predictor of incident CVD events. Elevated levels of branched-chain amino acids (BCAA) have also been associated with an increased propensity for insulin resistance. METHODS: To study the association of dietary intake with systemic TMAO, its nutrient precursors, and BCAA levels on fasting plasma levels of TMAO and its nutrient precursors and BCAA, we conducted an exploratory post-hoc analysis of 3 popular diets - high fat (Atkins), Mediterranean (South Beach), and very low fat (Ornish) - using plasma samples from a prior randomized, crossover study, with each isocaloric dietary phase lasting 4 weeks. Metabolites were quantified using stable isotope dilution HPLC with on-line tandem mass spectrometry. RESULTS: Compared to the low fat Ornish phase, the high fat Atkins dietary phase was characterized by increased levels of TMAO (3.3 vs. 1.8 µM, p = 0.01), and the BCAA valine (272.8 vs. 235.8 µM, p = 0.005) and leucine (105.9 vs. 96.4 µM, p = 0.01). The high fat Atkins dietary phase was also associated with higher levels of TMAO (3.3 vs 1.6 µM, p = 0.04), valine (272.8 vs. 240.7 µM, p = 0.004), and leucine (105.9 vs. 96.4 µM, p = 0.01) compared to baseline. CONCLUSIONS: These data suggest that over a 4-week interval, a saturated fat diet that is predominantly animal-based, compared to an isocaloric, low fat, predominantly plant-based diet, is associated with heightened risk for cardiometabolic derangements, as monitored by a higher plasma levels of both TMAO and BCAA.
Assuntos
Aminoácidos de Cadeia Ramificada/sangue , Bactérias/metabolismo , Doenças Cardiovasculares/etiologia , Dieta com Restrição de Gorduras , Dieta Hiperlipídica/efeitos adversos , Dieta Rica em Proteínas e Pobre em Carboidratos/efeitos adversos , Dieta Mediterrânea , Microbioma Gastrointestinal , Metilaminas/sangue , Biomarcadores/sangue , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/diagnóstico , Humanos , Ensaios Clínicos Controlados Aleatórios como Assunto , Medição de Risco , Fatores de Risco , Fatores de TempoRESUMO
Circulating cardiac troponins are markers of myocardial injury. We sought to determine whether cardiac troponin I (cTnI), measured by a sensitive assay, is associated with disease severity and prognosis in pulmonary arterial hypertension (PAH). cTnI was measured in 68 patients with PAH diagnostic category 1 in a research-based sensitive immunoanalyser with a lower limit of detection of 0.008 ng · mL(-1). The associations between cTnI and PAH severity and clinical outcomes were assessed using Chi-squared and Wilcoxon rank sum tests, Kaplan-Meier analysis and Cox regression models. cTnI was detected in 25% of patients. Patients with detectable cTnI had more advanced functional class symptoms, a shorter 6-min walk distance, more pericardial effusions, larger right atrial area, and higher B-type natriuretic peptide and C-reactive protein levels. 36-month transplant-free survival was 44% in patients with detectable cTnI versus 85% in those with undetectable cTnI. cTnI was associated with a 4.7-fold increased risk of death related to right ventricular failure or transplant (hazard ratio 4.74, 95% CI 1.89-11.89; p<0.001), even when adjusted individually for known parameters of PAH severity. Elevated plasma cTnI, even at subclinically detectable levels, is associated with more severe disease and worse outcomes in patients with PAH.
Assuntos
Hipertensão Pulmonar , Índice de Gravidade de Doença , Troponina I/sangue , Adulto , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Hipertensão Pulmonar Primária Familiar , Feminino , Humanos , Hipertensão Pulmonar/sangue , Hipertensão Pulmonar/diagnóstico , Hipertensão Pulmonar/mortalidade , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Peptídeo Natriurético Encefálico/sangue , Derrame Pericárdico/sangue , Derrame Pericárdico/diagnóstico , Derrame Pericárdico/mortalidade , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , Fatores de Risco , Sensibilidade e EspecificidadeRESUMO
AIMS: Trimethylamine N-oxide (TMAO), choline and betaine serum levels have been associated with metabolic diseases including type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). These associations could be mediated by insulin resistance. However, the relationships among these metabolites, insulin resistance and NAFLD have not been thoroughly investigated. Moreover, it has recently been suggested that TMAO could play a role in NAFLD by altering bile acid metabolism. We examined the association between circulating TMAO, choline and betaine levels and NAFLD in obese subjects. METHODS: Serum TMAO, choline, betaine and bile acid levels were measured in 357 Mexican obese patients with different grades of NAFLD as determined by liver histology. Associations of NAFLD with TMAO, choline and betaine levels were tested. Moreover, association of TMAO levels with non-alcoholic steatohepatitis (NASH) was tested separately in patients with and without T2D. RESULTS: TMAO and choline levels were significantly associated with NAFLD histologic features and NASH risk. While increased serum TMAO levels were significantly associated with NASH in patients with T2D, in non-T2D subjects this association lost significance after adjusting for sex, BMI and HOMA2-IR. Moreover, circulating secondary bile acids were associated both with increased TMAO levels and NASH. CONCLUSIONS: In obese patients, circulating TMAO levels were associated with NASH mainly in the presence of T2D. Functional studies are required to evaluate the role of insulin resistance and T2D in this association, both highly prevalent in NASH patients.
Assuntos
Diabetes Mellitus Tipo 2 , Metilaminas/sangue , Hepatopatia Gordurosa não Alcoólica , Adulto , Betaína/sangue , Ácidos e Sais Biliares/sangue , Biomarcadores/sangue , Biópsia , Colina/sangue , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/epidemiologia , Feminino , Humanos , Resistência à Insulina , Fígado/patologia , Masculino , Americanos Mexicanos , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Obesidade/complicações , Obesidade/epidemiologiaRESUMO
BACKGROUND: Cerebral vasospasm is a significant cause of morbidity in patients after aneurysmal subarachnoid hemorrhage (aSAH). There are few effective treatments. The search for new treatments has focused predominantly on dilating cerebral blood vessels. Growing evidence supports a role for inflammation in its pathogenesis but no potential target for intervention has emerged. METHODS: CSF and clinical information from patients with aSAH were collected. Additionally, tyrosine modifications by stable isotope dilution HPLC with online tandem mass spectrometry were quantified in CSF samples. RESULTS: We report an association between neutrophil accumulation in the cerebrospinal fluid of patients with aSAH and the development of vasospasm. In particular, CSF neutrophil content of >62% on the third day after aSAH is an independent predictor of the later development of vasospasm (OR 6.8, 95% CI 2.0-23.3, P = 0.002). Further, activity of myeloperoxidase and NADPH oxidase is elevated in aSAH suggesting a role for modification of CSF proteins by reactive oxidant species. CONCLUSIONS: Neutrophil percentage is an independent predictor of vasospasm in aSAH patients, days prior to its onset suggesting a role of neutrophils in vasospasm. The activity of neutrophil enzymes is also increased suggesting a mechanism for blood vessel damage. Inflammation mediated by neutrophils is a potential target for therapies in vasospasm. More study is necessary to determine the mechanism by which neutrophils damage cerebral blood vessels.
Assuntos
Neutrófilos/metabolismo , Hemorragia Subaracnóidea/líquido cefalorraquidiano , Hemorragia Subaracnóidea/fisiopatologia , Vasoespasmo Intracraniano/líquido cefalorraquidiano , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/líquido cefalorraquidiano , Hemorragia Subaracnóidea/complicações , Espectrometria de Massas em Tandem , Tirosina/análogos & derivados , Tirosina/líquido cefalorraquidiano , Vasoespasmo Intracraniano/complicaçõesRESUMO
Oral supplementation with l-carnitine is a common therapeutic modality for mitochondrial disorders despite limited evidence of efficacy. Recently, a number of studies have demonstrated that a gut microbiota-dependent metabolite of l-carnitine, trimethylamine oxide (TMAO), is an independent and dose-dependent risk factor for cardiovascular disease (CVD). Given the limited data demonstrating efficacy with oral l-carnitine therapy and the newly raised questions of potential harm, we assessed plasma TMAO levels in patients with mitochondrial disease with and without oral l-carnitine supplementation. Nine subjects were recruited and completed the study. Eight out of 9 subjects at baseline had plasma TMAO concentrations <97.5th percentile (<15.5⯵M). One subject with stage 3 renal disease, had marked elevation in plasma TMAO (pre 33.98⯵m versus post 101.6⯵m). Following at least 3â¯months of l-carnitine supplementation (1000â¯mg per day), plasma TMAO levels were markedly increased in 7out of 9 subjects; overall, plasma TMAO significantly increased 11.8-fold (pâ¯<â¯0.001) from a baseline median level of 3.54⯵m (interquartile range (IQR) 2.55-8.72) to 43.26 (IQR 23.99-56.04) post supplementation. The results of this study demonstrate that chronic oral l-carnitine supplementation markedly increases plasma TMAO levels in subjects with mitochondrial disorders. Further studies to evaluate both the efficacy and long term safety of oral l-carnitine supplementation for the treatment of mitochondrial disorders are warranted.
RESUMO
Essentials Microbe-dependent production of trimethylamine N-oxide (TMAO) contributes to thrombosis risk. The impact of host flavin monooxygenase 3 (FMO3) modulation on platelet function is unknown. Genetic manipulation of FMO3 in mice alters systemic TMAO levels and thrombosis potential. Genetic manipulation of FMO3 is associated with alteration of gut microbial community structure. SUMMARY: Background Gut microbes play a critical role in the production of trimethylamine N-oxide (TMAO), an atherogenic metabolite that impacts platelet responsiveness and thrombosis potential. Involving both microbe and host enzymatic machinery, TMAO generation utilizes a metaorganismal pathway, beginning with ingestion of trimethylamine (TMA)-containing dietary nutrients such as choline, phosphatidylcholine and carnitine, which are abundant in a Western diet. Gut microbial TMA lyases use these nutrients as substrates to produce TMA, which upon delivery to the liver via the portal circulation, is converted into TMAO by host hepatic flavin monooxygenases (FMOs). Gut microbial production of TMA is rate limiting in the metaorganismal TMAO pathway because hepatic FMO activity is typically in excess. Objectives FMO3 is the major FMO responsible for host generation of TMAO; however, a role for FMO3 in altering platelet responsiveness and thrombosis potential in vivo has not yet been explored. Methods The impact of FMO3 suppression (antisense oligonucleotide-targeting) and overexpression (as transgene) on plasma TMAO levels, platelet responsiveness and thrombosis potential was examined using a murine FeCl3 -induced carotid artery injury model. Cecal microbial composition was examined using 16S analyses. Results Modulation of FMO3 directly impacts systemic TMAO levels, platelet responsiveness and rate of thrombus formation in vivo. Microbial composition analyses reveal taxa whose proportions are associated with both plasma TMAO levels and in vivo thrombosis potential. Conclusions The present studies demonstrate that host hepatic FMO3, the terminal step in the metaorganismal TMAO pathway, participates in diet-dependent and gut microbiota-dependent changes in both platelet responsiveness and thrombosis potential in vivo.
Assuntos
Plaquetas/fisiologia , Microbioma Gastrointestinal/fisiologia , Fígado/enzimologia , Metilaminas/metabolismo , Oxigenases/fisiologia , Trombofilia/enzimologia , Animais , Trombose das Artérias Carótidas/sangue , Trombose das Artérias Carótidas/induzido quimicamente , Artéria Carótida Primitiva , Cloretos/toxicidade , Compostos Férricos/toxicidade , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos Antissenso/farmacologia , Oxigenases/antagonistas & inibidores , Oxigenases/genética , Plasma Rico em Plaquetas , Ribotipagem , Risco , Trombofilia/microbiologia , TransgenesRESUMO
Oxidation of LDL may be of pivotal importance in atherogenesis, but the mechanisms that promote oxidation in vivo remain poorly understood. We have explored the possibility that one pathway involves myeloperoxidase, a heme protein secreted by phagocytes. Myeloperoxidase is the only human enzyme known to generate hypochlorous acid (HOCl), a potent oxidizing agent, at physiological halide concentrations. LDL exposed to the complete myeloperoxidase-H2O2-Cl- system underwent chlorination of its protein tyrosyl residues. Treatment of LDL with reagent HOCl resulted in 3-chlorotyrosine formation, implicating HOCl as an intermediate in the enzymatic reaction pathway. In contrast, 3-chlorotyrosine was undetectable in LDL oxidized by hydroxyl radical, copper, iron, hemin, glucose, peroxynitrite, horseradish peroxidase, lactoperoxidase, or lipoxygenase. These results indicate that 3-chlorotyrosine is a specific marker for LDL oxidation by myeloperoxidase. To address the role of myeloperoxidase in promoting LDL oxidation in vivo, we used stable isotope dilution gas chromatography-mass spectrometry to quantify 3-chlorotyrosine in human aortic tissue and in LDL isolated from atherosclerotic lesions. The level of 3-chlorotyrosine in atherosclerotic tissue obtained during vascular surgery was sixfold higher than that of normal aortic intima. Moreover, the level of 3-chlorotyrosine was 30-fold higher in LDL isolated from atherosclerotic intima compared with circulating LDL. The detection of 3-chlorotyrosine in human atherosclerotic lesions indicates that halogenation reactions catalyzed by the myeloperoxidase system of phagocytes constitute one pathway for protein oxidation in vivo. These findings raise the possibility that the myeloperoxidase-H2O2-Cl- system plays a critical role in converting LDL into an atherogenic form.
Assuntos
Arteriosclerose/metabolismo , Lipoproteínas LDL/metabolismo , Peroxidase/metabolismo , Tirosina/análogos & derivados , Aorta/metabolismo , Aorta/cirurgia , Apolipoproteínas B/química , Biomarcadores , Cloro/metabolismo , Humanos , Ácido Hipocloroso/farmacologia , Lipoproteínas/química , Lipoproteínas LDL/análise , Lipoproteínas LDL/isolamento & purificação , Oxirredução , Peroxidase/fisiologia , Fagócitos/metabolismo , Tirosina/análise , Tirosina/isolamento & purificação , Tirosina/metabolismoRESUMO
Recent studies have demonstrated the existence of two members of a novel family of calcium-independent plasmalogen-selective phospholipases A2 in mammalian myocardium (Wolf, R. A., and R. W. Gross. 1985. J. Biol. Chem. 260:7295-7303; and Hazen, S. L., D. A. Ford, and R. W. Gross. 1991. J. Biol. Chem. 266:5629-5633). To examine the potential role of these calcium-independent phospholipases A2 in mediating membrane dysfunction during early myocardial ischemia, the temporal course of alterations in phospholipase A2 activity during global ischemia in Langendorf perfused rabbit hearts was quantified and compared with traditionally accepted markers of myocytic ischemic injury and anaerobic metabolism. We now report that membrane-associated calcium-independent plasmalogen-selective phospholipase A2 activity increased over 400% during 2 min of global ischemia (P less than 0.01), was near maximally activated (greater than 10-fold) after only 5 min of ischemia, and remained activated throughout the entire ischemic interval examined (2-60 min). Activation of membrane-associated plasmalogen-selective phospholipase A2 after 5 min of myocardial ischemia was rapidly reversible during reperfusion of ischemic tissue. Both the activation of phospholipase A2 and its reversibility during reperfusion were temporally correlated to alterations in myocytic anaerobic metabolism. Furthermore, activation of membrane-associated phospholipase A2 was essentially complete before electron microscopic evidence of cellular damage. Collectively, these results identify dynamic alterations in calcium-independent plasmalogen-selective phospholipase A2 activity during myocardial ischemia which precede irreversible cellular injury and demonstrate that activation of plasmalogen-selective phospholipase A2 is amongst the earliest biochemical alterations in ischemic myocardium.
Assuntos
Cálcio/farmacologia , Doença das Coronárias/enzimologia , Miocárdio/metabolismo , Fosfolipases A/análise , Plasmalogênios/farmacologia , Animais , Ativação Enzimática , Microssomos/enzimologia , Miocárdio/patologia , Fosfolipases A2 , CoelhosRESUMO
Recent studies have demonstrated the existence of a novel family of calcium-independent plasmalogen-selective phospholipases A2 in canine myocardium that have been implicated as enzymic mediators of ischemic membrane damage. We now report that human myocardium contains two functionally distinct isoforms of cytosolic calcium-independent phospholipase A2. The major cytosolic phospholipase A2 isoform preferentially hydrolyzes plasmalogen substrate, possesses a pH optimum of 7.0, and is chromatographically resolvable from a minor cytosolic calcium-independent phospholipase A2 isoform that hydrolyzes plasmenylcholine and phosphatidylcholine substrates at similar rates and possesses a pH optimum of 8.5. The major cytosolic calcium-independent phospholipase A2 isoform was identified as a 40-kD polypeptide after its 182,000-fold purification by sequential column chromatographies to a final specific activity of 67 mumol/mg.min. The purified 40-kD human myocardial phospholipase A2 preferentially hydrolyzes plasmalogens containing arachidonic acid at the sn-2 position. Both reverse-phase HPLC and fast atom bombardment mass spectroscopic analysis of human myocardial ethanolamine and choline glycerophospholipids demonstrated that plasmenylethanolamine and plasmenylcholine molecular species containing arachidonic acid at the sn-2 position are prominent constituents of human myocardium. Collectively, these results identify and characterize the major human myocardial cytosolic calcium-independent phospholipase A2 activity, demonstrate the presence of functionally distinct human myocardial cytosolic calcium-independent phospholipase A2 isoforms, and document the abundance of arachidonoylated plasmalogen molecular species in human myocardium that serve as substrates.
Assuntos
Citosol/enzimologia , Isoenzimas/isolamento & purificação , Miocárdio/enzimologia , Fosfolipases A/isolamento & purificação , Ácido Araquidônico/metabolismo , Cromatografia Líquida de Alta Pressão , Humanos , Cinética , Miocárdio/química , Fosfatidilcolinas/análise , Fosfatidiletanolaminas/análise , Fosfolipases A2 , Plasmalogênios/metabolismo , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Especificidade por SubstratoRESUMO
Reactive aldehydes derived from reducing sugars and lipid peroxidation play a critical role in the formation of advanced glycation end (AGE) products and oxidative tissue damage. We have recently proposed another mechanism for aldehyde generation at sites of inflammation that involves myeloperoxidase, a heme enzyme secreted by activated phagocytes. We now demonstrate that human neutrophils employ the myeloperoxidase-H202-chloride system to produce alpha-hydroxy and alpha,beta-unsaturated aldehydes from hydroxy-amino acids in high yield. Identities of the aldehydes were established using mass spectrometry and high performance liquid chromatography. Activated neutrophils converted L-serine to glycolaldehyde, an alpha-hydroxyaldehyde which mediates protein cross-linking and formation of Nepsilon-(carboxymethyl)lysine, an AGE product. L-Threonine was similarly oxidized to 2-hydroxypropanal and its dehydration product, acrolein, an extremely reactive alpha,beta-unsaturated aldehyde which alkylates proteins and nucleic acids. Aldehyde generation required neutrophil activation and a free hydroxy-amino acid; it was inhibited by catalase and heme poisons, implicating H202 and myeloperoxidase in the cellular reaction. Aldehyde production by purified myeloperoxidase required H202 and chloride, and was mimicked by reagent hypochlorous acid (HOCl) in the absence of enzyme, suggesting that the reaction pathway involves a chlorinated intermediate. Collectively, these results indicate that the myeloperoxidase-H202-chloride system of phagocytes converts free hydroxy-amino acids into highly reactive alpha-hydroxy and alpha,beta-unsaturated aldehydes. The generation of glycolaldehyde, 2-hydroxypropanal, and acrolein by activated phagocytes may thus play a role in AGE product formation and tissue damage at sites of inflammation.
Assuntos
Acetaldeído/análogos & derivados , Acroleína/metabolismo , Aldeídos/metabolismo , Aminoácidos/metabolismo , Cloretos/metabolismo , Peróxido de Hidrogênio/metabolismo , Hidroxiácidos/metabolismo , Inflamação/metabolismo , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Peroxidase/metabolismo , Acetaldeído/metabolismo , Catalase/farmacologia , Cromatografia Líquida de Alta Pressão , Reagentes de Ligações Cruzadas/metabolismo , Heme/metabolismo , Humanos , Ácido Hipocloroso/metabolismo , Lisina/análogos & derivados , Lisina/metabolismo , Espectrometria de Massas , Estrutura Molecular , Ativação de Neutrófilo , Oxirredução , Serina/metabolismo , Treonina/metabolismoRESUMO
Oxidized LDL is implicated in atherosclerosis; however, the pathways that convert LDL into an atherogenic form in vivo are not established. Production of reactive nitrogen species may be one important pathway, since LDL recovered from human atherosclerotic aorta is enriched in nitrotyrosine. We now report that reactive nitrogen species generated by the MPO-H2O2-NO2- system of monocytes convert LDL into a form (NO2-LDL) that is avidly taken up and degraded by macrophages, leading to massive cholesterol deposition and foam cell formation, essential steps in lesion development. Incubation of LDL with isolated MPO, an H2O2-generating system, and nitrite (NO2-)-- a major end-product of NO metabolism--resulted in nitration of apolipoprotein B 100 tyrosyl residues and initiation of LDL lipid peroxidation. The time course of LDL protein nitration and lipid peroxidation paralleled the acquisition of high-affinity, concentration-dependent, and saturable binding of NO2-LDL to human monocyte-derived macrophages and mouse peritoneal macrophages. LDL modification and conversion into a high-uptake form occurred in the absence of free metal ions, required NO2-, occurred at physiological levels of Cl-, and was inhibited by heme poisons, catalase, and BHT. Macrophage binding of NO2-LDL was specific and mediated by neither the LDL receptor nor the scavenger receptor class A type I. Exposure of macrophages to NO2-LDL promoted cholesteryl ester synthesis, intracellular cholesterol and cholesteryl ester accumulation, and foam cell formation. Collectively, these results identify MPO-generated reactive nitrogen species as a physiologically plausible pathway for converting LDL into an atherogenic form.
Assuntos
Arteriosclerose/metabolismo , Lipoproteínas LDL/metabolismo , Nitritos/metabolismo , Peroxidase/metabolismo , Animais , Ésteres do Colesterol/biossíntese , Humanos , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Dióxido de Nitrogênio/metabolismoRESUMO
Reactive oxidants generated by phagocytes are of central importance in host defenses, tumor surveillance, and inflammation. One important pathway involves the generation of potent halogenating agents by the myeloperoxidase-hydrogen peroxide-chloride system. The chlorinating intermediate in these reactions is generally believed to be HOCl or its conjugate base, ClO-. However, HOCl is also in equilibrium with Cl2, raising the possibility that Cl2 executes oxidation/ halogenation reactions that have previously been attributed to HOCl/ClO-. In this study gas chromatography-mass spectrometric analysis of head space gas revealed that the complete myeloperoxidase-hydrogen peroxide-chloride system generated Cl2. In vitro studies demonstrated that chlorination of the aromatic ring of free L-tyrosine was mediated by Cl2 and not by HOCl/ClO-. Thus, 3-chlorotyrosine serves as a specific marker for Cl2-dependent oxidation of free L-tyrosine. Phagocytosis of L-tyrosine encapsulated in immunoglobulin- and complement-coated sheep red blood cells resulted in the generation of 3-chlorotyrosine. Moreover, activation of human neutrophils adherent to a L-tyrosine coated glass surface also stimulated 3-chlorotyrosine formation. Thus, in two independent models of phagocytosis human neutrophils convert L-tyrosine to 3-chlorotyrosine, indicating that a Cl2-like oxidant is generated in the phagolysosome. In both models, synthesis of 3-chlorotyrosine was inhibited by heme poisons and the peroxide scavenger catalase, implicating the myeloperoxidase-hydrogen peroxide system in the reaction. Collectively, these results demonstrate that myeloperoxidase generates Cl2 and that human neutrophils use an oxidant with characteristics identical to those of Cl2 during phagocytosis. Moreover, our observations suggest that phagocytes exploit the chlorinating properties of Cl2 to execute oxidative and cytotoxic reactions at sites of inflammation and vascular disease.
Assuntos
Cloro/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fagocitose , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Inflamação/metabolismo , Leucócitos/metabolismo , Ativação de Neutrófilo , Oxirredução , Peroxidase/metabolismo , Fagossomos/metabolismo , Tirosina/análogos & derivados , Tirosina/biossíntese , Tirosina/metabolismoRESUMO
Macrophage scavenger receptors have been implicated as key players in the pathogenesis of atherosclerosis. To assess the role of the class B scavenger receptor CD36 in atherogenesis, we crossed a CD36-null strain with the atherogenic apo E-null strain and quantified lesion development. There was a 76.5% decrease in aortic tree lesion area (Western diet) and a 45% decrease in aortic sinus lesion area (normal chow) in the CD36-apo E double-null mice when compared with controls, despite alterations in lipoprotein profiles that often correlate with increased atherogenicity. Macrophages derived from CD36-apo E double-null mice bound and internalized more than 60% less copper-oxidized LDL and LDL modified by monocyte-generated reactive nitrogen species. A similar inhibition of in vitro lipid accumulation and foam cell formation after exposure to these ligands was seen. These results support a major role for CD36 in atherosclerotic lesion development in vivo and suggest that blockade of CD36 can be protective even in more extreme proatherogenic circumstances.
Assuntos
Arteriosclerose/prevenção & controle , Antígenos CD36/fisiologia , Receptores Imunológicos/fisiologia , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/fisiologia , Arteriosclerose/etiologia , Antígenos CD36/genética , Células Cultivadas , Colesterol/sangue , Feminino , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneais/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/genética , Receptores Depuradores , Triglicerídeos/sangue , Aumento de PesoRESUMO
The oxidative conversion of LDL into an atherogenic form is considered a pivotal event in the development of cardiovascular disease. Recent studies have identified reactive nitrogen species generated by monocytes by way of the myeloperoxidase-hydrogen peroxide-nitrite (MPO-H(2)O(2)-NO(2)(-)) system as a novel mechanism for converting LDL into a high-uptake form (NO(2)-LDL) for macrophages. We now identify the scavenger receptor CD36 as the major receptor responsible for high-affinity and saturable cellular recognition of NO(2)-LDL by murine and human macrophages. Using cells stably transfected with CD36, CD36-specific blocking mAbs, and CD36-null macrophages, we demonstrated CD36-dependent binding, cholesterol loading, and macrophage foam cell formation after exposure to NO(2)-LDL. Modification of LDL by the MPO-H(2)O(2)-NO(2)(-) system in the presence of up to 80% lipoprotein-deficient serum (LPDS) still resulted in the conversion of the lipoprotein into a high-uptake form for macrophages, whereas addition of less than 5% LPDS totally blocked Cu(2+)-catalyzed LDL oxidation and conversion into a ligand for CD36. Competition studies demonstrated that lipid oxidation products derived from 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine can serve as essential moieties on NO(2)-LDL recognized by CD36. Collectively, these results suggest that MPO-dependent conversion of LDL into a ligand for CD36 is a likely pathway for generating foam cells in vivo. MPO secreted from activated phagocytes may also tag phospholipid-containing targets for removal by CD36-positive cells.
Assuntos
Antígenos CD36/metabolismo , Lipoproteínas LDL/metabolismo , Monócitos/metabolismo , Dióxido de Nitrogênio/metabolismo , Receptores Imunológicos/metabolismo , Receptores de LDL/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetinae , Glucose Oxidase/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Peroxidase/metabolismo , Receptores Depuradores , Fatores de TempoRESUMO
Eosinophils promote tissue injury and contribute to the pathogenesis of allergen-triggered diseases like asthma, but the chemical basis of damage to eosinophil targets is unknown. We now demonstrate that eosinophil activation in vivo results in oxidative damage of proteins through bromination of tyrosine residues, a heretofore unrecognized pathway for covalent modification of biologic targets in human tissues. Mass spectrometric studies demonstrated that 3-bromotyrosine serves as a specific "molecular fingerprint" for proteins modified through the eosinophil peroxidase-H(2)O(2) system in the presence of plasma levels of halides. We applied a localized allergen challenge to model the effects of eosinophils and brominating oxidants in human lung injury. Endobronchial biopsy specimens from allergen-challenged lung segments of asthmatic, but not healthy control, subjects demonstrated significant enrichments in eosinophils and eosinophil peroxidase. Baseline levels of 3-bromotyrosine in bronchoalveolar lavage (BAL) proteins from mildly allergic asthmatic individuals were modestly but not statistically significantly elevated over those in control subjects. After exposure to segmental allergen challenge, lung segments of asthmatics, but not healthy control subjects, exhibited a >10-fold increase in BAL 3-bromotyrosine content, but only two- to threefold increases in 3-chlorotyrosine, a specific oxidation product formed by neutrophil- and monocyte-derived myeloperoxidase. These results identify reactive brominating species produced by eosinophils as a distinct class of oxidants formed in vivo. They also reveal eosinophil peroxidase as a potential therapeutic target for allergen-triggered inflammatory tissue injury in humans.
Assuntos
Asma/imunologia , Asma/metabolismo , Bromo/metabolismo , Eosinófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Alérgenos/administração & dosagem , Asma/etiologia , Líquido da Lavagem Broncoalveolar/química , Estudos de Casos e Controles , Humanos , Técnicas In Vitro , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Neutrófilos/metabolismo , Tirosina/metabolismoRESUMO
Myeloperoxidase (MPO), a heme enzyme secreted by activated phagocytes, generates an array of oxidants proposed to play critical roles in host defense and local tissue damage. Both MPO and its reaction products are present in human atherosclerotic plaque, and it has been proposed that MPO oxidatively modifies targets in the artery wall. We have now generated MPO-deficient mice, and show here that neutrophils from homozygous mutants lack peroxidase and chlorination activity in vitro and fail to generate chlorotyrosine or to kill Candida albicans in vivo. To examine the potential role of MPO in atherosclerosis, we subjected LDL receptor-deficient mice to lethal irradiation, repopulated their marrow with MPO-deficient or wild-type cells, and provided them a high-fat, high-cholesterol diet for 14 weeks. White cell counts and plasma lipoprotein profiles were similar between the two groups at sacrifice. Cross-sectional analysis of the aorta indicated that lesions in MPO-deficient mice were about 50% larger than controls. Similar results were obtained in a genetic cross with LDL receptor-deficient mice. In contrast to advanced human atherosclerotic lesions, the chlorotyrosine content of aortic lesions from wild-type as well as MPO-deficient mice was essentially undetectable. These data suggest an unexpected, protective role for MPO-generated reactive intermediates in murine atherosclerosis. They also identify an important distinction between murine and human atherosclerosis with regard to the potential involvement of MPO in protein oxidation.
Assuntos
Arteriosclerose/etiologia , Peroxidase/fisiologia , Tirosina/análogos & derivados , Animais , Candida albicans/imunologia , Humanos , Ácido Hipocloroso/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/enzimologia , Oxirredução , Peroxidase/deficiência , Peroxidase/genética , Fagócitos/metabolismo , Tirosina/análiseRESUMO
Protein nitration and lipid peroxidation are implicated in the pathogenesis of atherosclerosis; however, neither the cellular mediators nor the reaction pathways for these events in vivo are established. In the present study, we examined the chemical pathways available to monocytes for generating reactive nitrogen species and explored their potential contribution to the protein nitration and lipid peroxidation of biological targets. Isolated human monocytes activated in media containing physiologically relevant levels of nitrite (NO(2)(-)), a major end product of nitric oxide ((*)NO) metabolism, nitrate apolipoprotein B-100 tyrosine residues and initiate LDL lipid peroxidation. LDL nitration (assessed by gas chromatography-mass spectrometry quantification of nitrotyrosine) and lipid peroxidation (assessed by high-performance liquid chromatography with online tandem mass spectrometric quantification of distinct products) required cell activation and NO(2)(-); occurred in the presence of metal chelators, superoxide dismutase (SOD), and scavengers of hypohalous acids; and was blocked by myeloperoxidase (MPO) inhibitors and catalase. Monocytes activated in the presence of the exogenous (*)NO generator PAPA NONOate (Z-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2- diolate) promoted LDL protein nitration and lipid peroxidation by a combination of pathways. At low rates of (*)NO flux, both protein nitration and lipid peroxidation were inhibited by catalase and peroxidase inhibitors but not SOD, suggesting a role for MPO. As rates of (*)NO flux increased, both nitrotyrosine formation and 9-hydroxy-10,12-octadecadienoate/9-hydroperoxy-10,12-octadecadieno ic acid production by monocytes became insensitive to the presence of catalase or peroxidase inhibitors, but they were increasingly inhibited by SOD and methionine, suggesting a role for peroxynitrite. Collectively, these results demonstrate that monocytes use distinct mechanisms for generating (*)NO-derived oxidants, and they identify MPO as a source of nitrating intermediates in monocytes.
Assuntos
Monócitos/metabolismo , Óxido Nítrico/metabolismo , Oxidantes/metabolismo , Peroxidase/metabolismo , Apolipoproteína B-100 , Apolipoproteínas B/metabolismo , Humanos , Peróxidos Lipídicos/metabolismo , Lipoproteínas LDL/metabolismo , Nitratos/metabolismo , Tirosina/metabolismoRESUMO
A family of extremely reactive electrophiles, isolevuglandins (isoLGs), is generated in vivo by free radical-induced lipid oxidation and rearrangement of endoperoxide intermediates of the isoprostane pathway. Protein adducts of two different oxidized lipids, isoLGE(2) and iso[4]LGE(2), and the corresponding autoantibodies are present in human blood. Western blot analysis of a polyacrylamide gel electrophoresis gel detects several immunoreactive plasma proteins. Only a minor fraction of the isoLG-protein modifications is associated with low density lipoprotein since mean levels were decreased only 20-22% by immunoprecipitation of apolipoprotein B (apoB). Mean levels of both isoLGE(2) and iso[4]LGE(2)-protein adducts in plasma from patients with atherosclerosis (AS) (n=16) or end-stage renal disease (RD) (n=8) are about twice those in healthy individuals (n=25). These elevated levels are not related to variations in age, total cholesterol or apoB. A linear correlation (r=0.79) between plasma isoLGE(2) and iso[4]LGE(2)-protein adduct levels in all 49 individuals is consistent with a common free radical-induced mechanism for the production of both oxidized lipids in vivo. The correlation is even stronger (r=0.86) for patients with AS or RD. That isoLG-protein adduct levels are more strongly correlated with disease than are total cholesterol or apoB suggests an independent defect that results in an abnormally high level of oxidative injury associated with AS and RD.
Assuntos
Ácidos Graxos Insaturados/metabolismo , Hemocianinas/metabolismo , Prostaglandinas E/metabolismo , Prostaglandinas H/metabolismo , Adulto , Animais , Apolipoproteínas B/metabolismo , Arteriosclerose/sangue , Autoanticorpos/sangue , Autoanticorpos/imunologia , Colesterol/metabolismo , Radicais Livres , Humanos , Falência Renal Crônica/sangue , Metabolismo dos Lipídeos , Pessoa de Meia-Idade , Estrutura Molecular , Oxirredução , Prostaglandina H2 , Prostaglandinas E/sangue , Coelhos , EstereoisomerismoRESUMO
OBJECTIVE: The objective of this study was to investigate changes in volatile organic compounds (VOCs) in exhaled breath in overweight/obese children compared with their lean counterparts. STUDY DESIGN: Single exhaled breath was collected and analyzed per protocol using selective ion flow tube mass spectrometry (SIFT-MS). RESULTS: Sixty overweight/obese children and 55 lean controls were included. Compared with the lean group, the obese group was significantly older (14.1 ± 2.8 vs. 12.1 ± 3.0 years), taller (164.8 ± 10.9 vs. 153.3 ± 17.1 cm) and more likely to be Caucasian (60% vs. 35.2%); P < 0.05 for all. A comparison of the SIFT-MS results of the obese group with the lean group revealed differences in concentration of more than 50 compounds. A panel of four VOCs can identify the presence of overweight/obesity with excellent accuracy. Further analysis revealed that breath isoprene, 1-decene, 1-octene, ammonia and hydrogen sulfide were significantly higher in the obese group compared with the lean group (P value < 0.01 for all). CONCLUSION: Obese children have a unique pattern of exhaled VOCs. Changes in VOCs observed in this study may help to gain insight into pathophysiological processes and pathways leading to the development of childhood obesity.
Assuntos
Testes Respiratórios/métodos , Fígado/fisiopatologia , Obesidade Infantil/metabolismo , Magreza/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Adolescente , Biomarcadores/metabolismo , Criança , Colesterol/biossíntese , Expiração , Estudos de Viabilidade , Humanos , Espectrometria de Massas , Estresse Oxidativo , Obesidade Infantil/fisiopatologia , Valor Preditivo dos Testes , Magreza/fisiopatologia , Estados UnidosRESUMO
Atherosclerosis is a chronic inflammatory process where oxidative damage within the artery wall is implicated in the pathogenesis of the disease. Mononuclear phagocytes, an inflammatory cell capable of generating a variety of oxidizing species, are early components of arterial lesions. Their normal functions include host defense and surveillance through regulated generation of diffusible radical species, reactive oxygen or nitrogen species, and HOCl (hypochlorous acid). However, under certain circumstances an excess of these oxidizing species can overwhelm local antioxidant defenses and lead to oxidant stress and oxidative tissue injury, processes implicated in the pathogenesis of atherosclerosis. This review focuses on oxidation reactions catalyzed by myeloperoxidase (MPO), an abundant heme protein secreted from activated phagocytes which is present in human atherosclerotic lesions. Over the past several years, significant evidence has accrued demonstrating that MPO is one pathway for protein and lipoprotein oxidation during the evolution of cardiovascular disease. Multiple distinct products of MPO are enriched in human atherosclerotic lesions and LDL recovered from human atheroma. However, the biological consequences of these MPO-catalyzed reactions in vivo are still unclear. Here we discuss evidence for the occurrence of MPO-catalyzed oxidation reactions in vivo and the potential role MPO plays in both normal host defenses and inflammatory diseases like atherosclerosis.