RESUMO
Hepatic disorders such as steatosis and alcoholic steatohepatitis are common diseases that affect thousands of people around the globe. This study aims to identify the main phenol compounds using a new HPLC-ESI+-MS/MS method, to evaluate some oxidative stress parameters and the hepatoprotective action of green dwarf coconut water, caffeic and ascorbic acids on the liver and serum of rats treated with ethanol. The results showed five polyphenols in the lyophilized coconut water spiked with standards: chlorogenic acid (0.18 µM), caffeic acid (1.1 µM), methyl caffeate (0.03 µM), quercetin (0.08 µM) and ferulic acid (0.02 µM) isomers. In the animals, the activity of the serum γ-glutamyltranspeptidase (γ-GT) was reduced to 1.8 I.U/L in the coconut water group, 3.6 I.U/L in the ascorbic acid group and 2.9 I.U/L in the caffeic acid groups, when compared with the ethanol group (5.1 I.U/L, p<0.05). Still in liver, the DNA analysis demonstrated a decrease of oxidized bases compared to ethanol group of 36.2% and 48.0% for pretreated and post treated coconut water group respectively, 42.5% for the caffeic acid group, and 34.5% for the ascorbic acid group. The ascorbic acid was efficient in inhibiting the thiobarbituric acid reactive substances (TBARS) in the liver by 16.5% in comparison with the ethanol group. These data indicate that the green dwarf coconut water, caffeic and ascorbic acids have antioxidant, hepatoprotective and reduced DNA damage properties, thus decreasing the oxidative stress induced by ethanol metabolism.
Assuntos
Ácido Ascórbico/farmacologia , Ácidos Cafeicos/farmacologia , Cocos/química , Dano ao DNA/efeitos dos fármacos , Etanol/farmacologia , Fígado/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Colesterol/sangue , Peroxidação de Lipídeos , Fígado/metabolismo , Masculino , Ratos Wistar , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Substâncias Reativas com Ácido Tiobarbitúrico , Fatores de Tempo , Triglicerídeos/sangue , Água/farmacologiaRESUMO
Urate hydroperoxide is a strong oxidant generated by the combination of urate free radical and superoxide. The formation of urate hydroperoxide as an intermediate in urate oxidation is potentially responsible for the pro-oxidant effects of urate in inflammatory disorders, protein degradation, and food decomposition. To understand the molecular mechanisms that sustain the harmful effects of urate in inflammatory and oxidative stress related conditions, we report a detailed structural characterization and reactivity of urate hydroperoxide toward biomolecules. Urate hydroperoxide was synthesized by photo-oxidation and by a myeloperoxidase/hydrogen peroxide/superoxide system. Multiple reaction monitoring (MRM) and MS(3) ion fragmentation revealed that urate hydroperoxide from both sources has the same chemical structure. Urate hydroperoxide has a maximum absorption at 308 nm, ε308nm = 6.54 ± 0.38 × 10(3) M(-1) cm(-1). This peroxide decays spontaneously with a rate constant of k = 2.80 ± 0.18 × 10(-4) s(-1) and a half-life of 41 min at 22 °C. Urate hydroperoxide undergoes electrochemical reduction at potential values less negative than -0.5 V (versus Ag/AgCl). When incubated with taurine, histidine, tryptophan, lysine, methionine, cysteine, or glutathione, urate hydroperoxide reacted only with methionine, cysteine, and glutathione. The oxidation of these molecules occurred by a two-electron mechanism, generating the alcohol, hydroxyisourate. No adduct between cysteine or glutathione and urate hydroperoxide was detected. The second-order rate constant for the oxidation of glutathione by urate hydroperoxide was 13.7 ± 0.8 M(-1) s(-1). In conclusion, the oxidation of sulfur-containing biomolecules by urate hydroperoxide is likely to be a mechanism by which the pro-oxidant and damaging effects of urate are mediated in inflammatory and photo-oxidizing processes.
Assuntos
Peróxido de Hidrogênio/química , Luz , Peróxidos/química , Ácido Úrico/análogos & derivados , Ácido Úrico/química , Cromatografia Líquida , Glutationa/química , Cinética , Estrutura Molecular , Oxirredução , Estresse Oxidativo , Espectrometria de Massas por Ionização por Electrospray , Ácido Úrico/metabolismoRESUMO
Total absence of adipose tissue (lipoatrophy) is associated with the development of severe metabolic disorders including hepatomegaly and fatty liver. Here, we sought to investigate the impact of severe lipoatrophy induced by deletion of peroxisome proliferator-activated receptor gamma (PPARγ) exclusively in adipocytes on lipid metabolism in mice. Untargeted lipidomics of plasma, gastrocnemius and liver uncovered a systemic depletion of the essential linoleic (LA) and α-linolenic (ALA) fatty acids from several lipid classes (storage lipids, glycerophospholipids, free fatty acids) in lipoatrophic mice. Our data revealed that such essential fatty acid depletion was linked to increased: 1) capacity for liver mitochondrial fatty acid ß-oxidation (FAO), 2) citrate synthase activity and coenzyme Q content in the liver, 3) whole-body oxygen consumption and reduced respiratory exchange rate in the dark period, and 4) de novo lipogenesis and carbon flux in the TCA cycle. The key role of de novo lipogenesis in hepatic steatosis was evidenced by an accumulation of stearic, oleic, sapienic and mead acids in liver. Our results thus indicate that the simultaneous activation of the antagonic processes FAO and de novo lipogenesis in liver may create a futile metabolic cycle leading to a preferential depletion of LA and ALA. Noteworthy, this previously unrecognized cycle may also explain the increased energy expenditure displayed by lipoatrophic mice, adding a new piece to the metabolic regulation puzzle in lipoatrophies.
Assuntos
Fígado Gorduroso , Lipogênese , Animais , Camundongos , Ciclização de Substratos , Metabolismo dos Lipídeos , Fígado Gorduroso/metabolismo , Ácido alfa-Linolênico/metabolismoRESUMO
Maintaining islet cell viability in vitro, although challenging, appears to be a strategy for improving the outcome of pancreatic islet transplantation. We have shown that prolactin (PRL) leads to beta-cell cytoprotection against apoptosis, an effect mediated by heat shock protein B1 (HSPB1). Since the role of HSPB1 in beta-cells is still unclear and the hormone concentration used is not compatible with clinical applications because of all the side effects displayed by the hormone in other tissues, we explored the molecular mechanisms by which HSPB1 mediates beta-cell cytoprotection. Lysates from PRL- and/or cytokine-treated MIN6 beta-cells were subjected to HSPB1 immunoprecipitation followed by identification through mass spectrometry. PRL-treated cells presented an enrichment of several proteins co-precipitating with HSPB1. Of note were oxidative stress resistance-, protein degradation- and carbohydrate metabolism-related proteins. Wild type, HSPB1 silenced or overexpressing MIN6 cells were exposed to menadione and hydrogen peroxide and analysed for several oxidative stress parameters. HSPB1 knockdown rendered cells more sensitive to oxidative stress and led to a reduced antioxidant capacity, while prolactin induced an HSPB1-mediated cytoprotection against oxidative stress. HSPB1 overexpression, however, led to opposite effects. PRL treatment, HSPB1 silencing or overexpression did not change the expression nor activities of antioxidant enzymes, it also did not lead to a modulation of total glutathione levels nor G6PD expression. However, HSPB1 levels are related to a modulation of GSH/GSSG ratio, G6PD activity and NADPH/NADP + ratio. We have shown that HSPB1 is important for pro-survival effects against oxidative stress-induced beta-cell death. These results are in accordance with PRL-induced enrichment of HSPB1-interacting proteins related to protection against oxidative stress. Finally, our results outline the need of further studies investigating the importance of HSPB1 for beta-cell viability, since this could lead to the mitigation of beta-cell death through the up-regulation of an endogenous protective pathway.
Assuntos
Citoproteção , Proteínas de Choque Térmico/metabolismo , Células Secretoras de Insulina/citologia , Insulinoma/patologia , Chaperonas Moleculares/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Neoplasias Pancreáticas/patologia , Prolactina/farmacologia , Animais , Apoptose , Glutationa/metabolismo , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/genética , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Insulinoma/tratamento farmacológico , Insulinoma/metabolismo , Camundongos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Oxirredução , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Transporte Proteico , Proteólise , Células Tumorais CultivadasRESUMO
Uric acid is the final product of purine metabolism in humans and is considered to be quantitatively the main antioxidant in plasma. In vitro studies showed that the oxidation of uric acid by peroxidases, in presence of superoxide, generates urate free radical and urate hydroperoxide. Urate hydroperoxide is a strong oxidant and might be a relevant intermediate in inflammatory conditions. However, the identification of urate hydroperoxide in cells and biological samples has been a challenge due to its high reactivity. By using mass spectrometry, we undoubtedly demonstrated the formation of urate hydroperoxide and its corresponding alcohol, hydroxyisourate during the respiratory burst in peripheral blood neutrophils and in human leukemic cells differentiated in neutrophils (dHL-60). The respiratory burst was induced by phorbol myristate acetate (PMA) and greatly increased oxygen consumption and superoxide production. Both oxygen consumption and superoxide production were further augmented by incubation with uric acid. Conversely, uric acid significantly decreased the levels of HOCl, probably because of the competition with chloride by the catalysis of myeloperoxidase. In spite of the decrease in HOCl, the overall oxidative status, measured by GSH/GSSG ratio, was augmented in the presence of uric acid. In summary, the present results support the formation of urate hydroperoxide, a novel oxidant in neutrophils oxidative burst. Urate hydroperoxide is a strong oxidant and alters the redox balance toward a pro-oxidative environment. The production of urate hydroperoxide in inflammatory conditions could explain, at least in part, the harmful effect associated to uric acid.
Assuntos
Inflamação/sangue , Neutrófilos/metabolismo , Peróxidos/metabolismo , Espécies Reativas de Oxigênio/sangue , Ácido Úrico/análogos & derivados , Catálise , Linhagem Celular Tumoral , Radicais Livres/química , Radicais Livres/metabolismo , Humanos , Inflamação/patologia , Espectrometria de Massas , Neutrófilos/química , Oxirredução , Peroxidase/genética , Peroxidase/metabolismo , Peróxidos/química , Peróxidos/isolamento & purificação , Espécies Reativas de Oxigênio/isolamento & purificação , Superóxidos/química , Superóxidos/metabolismo , Ácido Úrico/química , Ácido Úrico/isolamento & purificação , Ácido Úrico/metabolismoRESUMO
Uric acid is the end product of purine metabolism in humans and is an alternative physiological substrate for myeloperoxidase. Oxidation of uric acid by this enzyme generates uric acid free radical and urate hydroperoxide, a strong oxidant and potentially bactericide agent. In this study, we investigated whether the oxidation of uric acid and production of urate hydroperoxide would affect the killing activity of HL-60 cells differentiated into neutrophil-like cells (dHL-60) against a highly virulent strain (PA14) of the opportunistic pathogen Pseudomonas aeruginosa. While bacterial cell counts decrease due to dHL-60 killing, incubation with uric acid inhibits this activity, also decreasing the release of the inflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF- α). In a myeloperoxidase/Cl-/H2O2 cell-free system, uric acid inhibited the production of HOCl and bacterial killing. Fluorescence microscopy showed that uric acid also decreased the levels of HOCl produced by dHL-60 cells, while significantly increased superoxide production. Uric acid did not alter the overall oxidative status of dHL-60 cells as measured by the ratio of reduced (GSH) and oxidized (GSSG) glutathione. Our data show that uric acid impairs the killing activity of dHL-60 cells likely by competing with chloride by myeloperoxidase catalysis, decreasing HOCl production. Despite diminishing HOCl, uric acid probably stimulates the formation of other oxidants, maintaining the overall oxidative status of the cells. Altogether, our results demonstrated that HOCl is, indeed, the main relevant oxidant against bacteria and deviation of myeloperoxidase activity to produce other oxidants hampers dHL-60 killing activity.
Assuntos
Neutrófilos/metabolismo , Peróxidos/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Ácido Úrico/análogos & derivados , Ácido Úrico/metabolismo , Catálise , Diferenciação Celular/genética , Radicais Livres/metabolismo , Glutationa/metabolismo , Células HL-60/metabolismo , Células HL-60/microbiologia , Humanos , Peróxido de Hidrogênio/metabolismo , Ácido Hipocloroso/química , Neutrófilos/microbiologia , Oxidantes/metabolismo , Oxirredução/efeitos dos fármacos , Peróxidos/química , Pseudomonas aeruginosa/patogenicidade , Ácido Úrico/químicaRESUMO
Coconut water (CW) is a natural nutritious beverage, which contains several biologically active compounds that are traditionally used in the treatment of diarrhea and rehydration. Several works with CW have been related with antioxidant activity, which is very important in the diabetic state. To evaluate the hypoglycemic and nephroprotective activities of CW, alloxan-induced diabetic rats were pre- and post-treated by gavage with CW (3 mL/kg), caffeic acid (CA) (10 and 15 mg/kg), and acarbose (Acb) (714 µg/kg) during a period of 16 days. Body weight, blood glucose, glycated hemoglobin (HbA1c), and Amadori products in plasma and kidney homogenates were evaluated in all groups and used as parameters for the monitoring of the diabetic state. The results showed that rats of the CW+diabetic group had maintenance in blood glucose compared with the control group (P<.05) in addition to a decrease of HbA1c levels and increase of body weight when compared with the diabetic group rats (P<.05). The animals of the CA and CA+diabetic groups did not have significant variation of body weight (P<.05) during the experiment; however, they showed decrease in their HbA1c and urea levels in plasma as well as Amadori products in kidney homogenates when compared with the diabetic group (P<.05). Our results indicate that CW has multiple beneficial effects in diabetic rats for preventing hyperglycemia and oxidative stress caused by alloxan.
Assuntos
Bebidas , Cocos/química , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/prevenção & controle , Hipoglicemiantes/administração & dosagem , Acarbose/administração & dosagem , Aloxano , Animais , Antioxidantes/administração & dosagem , Ácidos Cafeicos/administração & dosagem , Hemoglobinas Glicadas/análise , Hiperglicemia/prevenção & controle , Masculino , Estresse Oxidativo/efeitos dos fármacos , Fitoterapia , Ratos , Ratos Wistar , Ureia/sangueRESUMO
ABSTRACT Hepatic disorders such as steatosis and alcoholic steatohepatitis are common diseases that affect thousands of people around the globe. This study aims to identify the main phenol compounds using a new HPLC-ESI+-MS/MS method, to evaluate some oxidative stress parameters and the hepatoprotective action of green dwarf coconut water, caffeic and ascorbic acids on the liver and serum of rats treated with ethanol. The results showed five polyphenols in the lyophilized coconut water spiked with standards: chlorogenic acid (0.18 µM), caffeic acid (1.1 µM), methyl caffeate (0.03 µM), quercetin (0.08 µM) and ferulic acid (0.02 µM) isomers. In the animals, the activity of the serum γ-glutamyltranspeptidase (γ-GT) was reduced to 1.8 I.U/L in the coconut water group, 3.6 I.U/L in the ascorbic acid group and 2.9 I.U/L in the caffeic acid groups, when compared with the ethanol group (5.1 I.U/L, p<0.05). Still in liver, the DNA analysis demonstrated a decrease of oxidized bases compared to ethanol group of 36.2% and 48.0% for pretreated and post treated coconut water group respectively, 42.5% for the caffeic acid group, and 34.5% for the ascorbic acid group. The ascorbic acid was efficient in inhibiting the thiobarbituric acid reactive substances (TBARS) in the liver by 16.5% in comparison with the ethanol group. These data indicate that the green dwarf coconut water, caffeic and ascorbic acids have antioxidant, hepatoprotective and reduced DNA damage properties, thus decreasing the oxidative stress induced by ethanol metabolism.